O exercício físico é intervenção de primeira linha e parece melhorar os sintomas da síndrome dos ovários policísticos (SOP). Porém, os mecanismos dessas alterações ainda são pouco entendidos. O objetivo desse estudo foi avaliar os efeitos do exercício voluntário sobre os aspectos metabólicos e reprodutivos de modelos murinos de SOP induzida por dihidrotestosterona (DHT) ou letrozol (LET). Péletes de DHT ou LET foram utilizados para a indução de SOP experimental em camundongas pré-puberes. Os camundongos controles receberam péletes sem substância ativa. Cinco semanas após a exposição aos compostos teve início o exercício voluntário em rodas de corrida. Foram avaliados peso, composição corporal, tolerância à insulina e à glicose e o ciclo estral. Os grupos experimentais foram: controle (n=9), DHT (n=10), LET (n=9), DHT+exercício (EX) (n=9) e LET+EX (n=10). Após 4-5 semanas de exercício, os animais foram eutanasiados e os tecidos adiposos inguinal e mesentérico foram utilizados para análises morfológicas e moleculares. O grupo DHT apresentou aumento do peso corporal, aumento da proporção de grandes adipócitos mesentéricos e ciclo estral anormal. O grupo LET apresentou aumento do peso corporal e massa gorda, diminuição da sensibilidade à insulina, aumento da população de adipócitos pequenos, aumento da expressão de genes relacionados à lipólise na gordura mesentérica e ciclo estral anormal. O exercício reduziu a massa gorda e a expressão de genes da via NOTCH no tecido adiposo inguinal e mesentérico, e restaurou a morfologia alterada dos adipócitos mesentéricos, e a expressão de genes da via do NOTCH foi negativamente correlacionada com a expressão de genes da via de browning (transformação da gordura branca em gordura de fenótipo marrom) nos grupos DHT+EX e LET+EX. Em conclusão, o exercício restaurou a morfologia dos adipócitos mesentéricos em modelos animais de SOP induzida por DHT e LET, melhorou a sensibilidade à insulina, aumentou a expressão mesentérica de genes relacionados à lipólise e melhorou o ciclo estral em modelo animal de SOP induzida por LET. Esses benefícios podem ser atribuídos, pelo menos em parte, pela inibição de genes da via do NOTCH e pela modulação das vias de browning e lipólise no tecido adiposo / Exercise is the first line treatment and it seems to improve the symptoms of polycystic ovary syndrome (PCOS), but the mechanisms of these alterations are still poorly understood. The objective of this study was to evaluate the effects of voluntary exercise on metabolic and reproductive aspects of PCOS mouse models induced by dihydrotestosterone (DHT) or letrozole (LET). DHT or LET pellets were used for the induction of experimental PCOS in prepubertal mice. Control mice received placebo pellets. Five weeks after exposure to the substances, voluntary exercise on running wheels began. Body weight, body composition, insulin and glucose tolerance, and estrous cycle were evaluated. The experimental groups were control (n = 9), DHT (n = 10), LET (n = 9), DHT+exercise (EX) (n = 9) and LET+EX (n = 10). After 4-5 weeks of exercise, the animals were euthanized and the inguinal and mesenteric fat depots were collected for morphological and molecular analyzes. The DHT group showed increased body weight, increased proportion of large mesenteric adipocytes and abnormal estrous cycle. The LET group presented increased body weight and fat mass, decreased insulin sensitivity, increased population of small adipocytes, increased expression of genes related to lipolysis in mesenteric fat and abnormal estrous cycle. Exercise reduced fat mass and NOTCH pathway gene expression in inguinal and mesenteric adipose tissue, and restored the altered morphology of mesenteric adipocytes, and NOTCH pathway gene expression was negatively correlated with gene expression of the browning (transformation from white fat to brown-like fat) markers in both DHT+EX and LET+EX groups. In conclusion, the exercise restored the mesenteric adipocyte morphology in the DHT- and LET-induced PCOS mouse models, and the insulin sensitivity, the mesenteric expression of genes related to lipolysis, and improved the estrous cycle in the LET-induced PCOS mouse model. These benefits may be attributed, at least in part, to the inhibition of NOTCH pathway genes and the modulation of browning and lipolysis pathways in the adipose tissue

VIEIRA, TMB. Qualidade de Vida em Mulheres Obesas e com Síndrome dos Ovários Policísticos. 2010. 65 fls. Dissertação (Mestrado) - Faculdade de Medicina de Ribeirão Preto. Universidade de São Paulo. Ribeirão Preto. 2010. A síndrome dos ovários policísticos (SOP) é uma das desordens endócrinas mais comuns na idade reprodutiva, sendo a sua prevalência de 6 a 10% na população feminina. A SOP associada à obesidade pode agravar a saúde física e mental com um impacto negativo no funcionamento bio-psico-social. Os distúrbios metabólicos e as manifestações clínicas do hiperandrogenismo, comuns em mulheres com SOP tem levado ao aumento dos distúrbios psicológicos e redução na qualidade de vida (QV). Alguns estudos têm mostrado uma piora da QV na SOP, mas não fazem distinção sobre os efeitos que a própria obesidade pode acarretar a QV, uma vez que mais de 50% das pacientes SOP apresentam sobrepeso e obesidade. O presente estudo objetivou avaliar se há diferença na QV de pacientes com SOP obesas comparadas a controles obesas com ciclos ovulatórios. Foram incluídas 35 obesas com SOP e 28 obesas com ciclos ovulatórios, com idade entre 20 e 40 anos, IMC 30 e que aceitaram assinar o TCLE. Utilizou-se para a caracterização da população o questionário sócio-demográfico, médico, reprodutivo e somatométrico e para a avaliação da qualidade de vida, o questionário SF-36, que avalia os componentes da saúde física e os componentes da saúde mental. As pacientes com SOP mostraram melhor escore no que diz respeito ao domínio da capacidade funcional (73,43±20,57 vs 59,46±22,08) (p=0,01). Para os demais domínios não houve diferenças significativas entre os grupos. Nossos resultados sugerem que o aumento de androgênios comum em obesas com SOP possa estar associado à melhor capacidade funcional. / VIEIRA, TMB. Quality of Life of Obese Women With Polycystic Ovary Syndrome. 2010. 65 fls. Dissertation (Master). Faculdade de Medicina de Ribeirão Preto. Universidade de São Paulo, Ribeirão Preto, 2010. Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders occurring during reproductive age, with a prevalence of 6 to 10% in the female population. PCOS associated with obesity can affect physical and mental health, with a negative impact on biopsychosocial functioning. The metabolic disorders and clinical manifestations of hyperandrogenism, common among women with PCOS, have led to an increase in psychological disorders and a reduction of quality of life (QL). Some studies have shown a worsening of QL in PCOS but without distinguishing between the effects that obesity itself may have on QL, since more than 50% of patients with PCOS are overweight or obese. The objective of the present study was to determine whether there is a difference in the QL of obese patients with PCOS compared to obese controls with ovulatory cycles. The study was conducted on 35 obese women with PCOS and 28 obese women with ovulatory cycles aged 20 to 40 years, with a BMI 30, who gave written informed consent to participate. A sociodemographic, medical, reproductive and somatometric questionnaire was used to characterize the population and QL was assessed with the SF-36 questionnaire, which evaluates the components of physical and mental health. The patients with PCOS had a better score regarding the functional capacity domain (73,43±20,57 vs 59,46±22,08) (p=0,01), whereas no significant differences were observed beween groups regarding the remaining domains. The present results suggest that the increase in androgens commonly occurring among obese women with PCOS may be associated with their better functional capacity.

Este trabalho tem por objetivo descrever as características anatômicas, macroscópicas e microscópicas, dos órgãos reprodutores femininos da ema (Rhea americana). O material consistiu nos órgãos reprodutores femininos (ovário, oviduto e cloaca) de 24 fêmeas, das quais, 04 filhotes (15 dias) e 20 adultos (12 e 36 meses), oriundas da Cooperativa Emas do Brasil LTDA. Os órgãos foram fixados em formol 10% tamponado e em glutaraldeído 2,5%, PBS 0,1 M, pH 7,4, para microscopia de luz e eletrônica de varredura, respectivamente. Os resultados mostram que o ovário esquerdo ocupa a porção dorsal da cavidade celomática, em contato com a porção cranial do rim e com a glândula suprarenal, sendo sustentado na cavidade pelo mesovário. Na superfície livre do ovário observam-se folículos em diversas fases de desenvolvimento, variando de 1 a 90 mm e atrésicos com 27,6 ± 3 mm de diâmetro (n=10), unidos à superfície ovariana por um pedúnculo folicular e apresentam uma cinta esbranquiçada contornando sua superfície, o estigma folicular. Histologicamente o ovário é constituído de uma medula e um córtex e a parede dos folículos pelas tecas externa e interna, extrato granuloso e zona radiada. O oviduto possuiu comprimento médio de 1,22 ± 0,23m (n=4) e foi composto pelo infundíbulo, magno, istmo, útero e vagina, revestidos por epitélio colunar ciliado com células secretoras recobrindo as pregas luminais. O infundíbulo apresentou uma abertura cranial com fímbrias delgadas e longas. No magno estas pregas são mais espessas e volumosas, preenchidas por glândulas tubulares, representando a maior porção do oviduto. O istmo curto apresentou pregas menos volumosas e com menor quantidade de glândulas tubulares. O útero pregueado em forma de bolsa, teve uma região cranial com pregas delgadas e uma outra caudal com pregas mais ramificadas e com poucas glândulas. A vagina apresentou pregas luminais longas e espessamento da muscular, formando o músculo esfíncter vaginal. O ovário e o oviduto da ema possuem características comuns à morfologia das aves domésticas. A cloaca teve os mesmos compartimentos que outras aves, diferenciando-se pela presença da prega entre o reto e o coprodeu, permitindo separação das fezes da urina / The aim of this work was to describe the morphological characteristics of the feminine reproductive organs of rhea (Rhea americana). The material consisted of ovary, oviduct and cloaca from 24 females, 04 young chick (15 days) and 20 adult ones (12 to 36 months), from Cooperativa Emas do Brasil LTDA. Fragments of each organ were fixed in phormaldehyde 10% and glutaraldehyde 2.5%, 0.1 PBS M, pH 7.4, for light and scanning electron microscopy, respectively. Results demonstrated that left ovary occupied the dorsal portion of the celomatic cavity in contact with the cranial portion of the kidney and with the suprarenal gland, being supported in the cavity for the mesovary. On the free surface of the ovary, follicles in different phases of development were observed measuring between 1 to 90 mm and atresic, measuring 27.6 ± 3 mm of diameter (n=10), linked to the ovarian surface by one follicular stalk and presents a white band surrounding its surface, the stigma folliculare. Histologically, the ovary is constituted by the medulla and the cortex, and the follicular wall by the theca externa and theca interna, stratum granulosum and the zona radiata. The oviduct possess average length of 1.20 ± 0.23m (n=4) and was composed by infundibulum, magnum, isthmus, uterus and vagina, covered by a ciliated columnar epithelium on luminal folds. The infundibulum presents a cranial opening with long and thin fimbriae. The magnum represent the bigger part of oviduct and have a increased thickness of the mucosa with big folds, composed by tubular glands and with a pseudostratifed ciliated epithelium. The isthmus is short with small luminal folds and less tubular glands. The uterus was folded with global form, it had a cranial region with thin folds and a caudal part with folds branched and a few tubular glands. The vagina had long luminal folds and the muscular layer was thicker forming the musculus sphincter vaginae. The ovary and oviduct of the rhea possessed similar characteristics to the one from domestic birds. The cloaca had the same compartments cited in other birds, with the fold between coprodeu and rectum, which make the feces and urine separation, as difference

Grande parte das perdas embrionárias ocorrem nas três primeiras semanas após a inseminação e grande parte pode-se atribuir a baixas concentrações de progesterona. Desta maneira, o objetivo do presente experimento foi avaliar os efeitos e a interação entre o escore de condição corporal (ECC) e condição ovariana (EO) em vacas suplementadas com progesterona de longa ação (P4) após IATF sobre a taxa de prenhez em vacas de corte. Neste experimento foram utilizados 1573 vacas de corte, lactantes entre 30 e 60 dias pós-parto. As vacas tiveram a ovulação sincronizada iniciando-se no dia - 10 e foram inseminadas no dia 0. No dia 4, os animais foram blocados de acordo com ECC e EO e ordem de parto e divididos em dois tratamentos: (P4, n = 786) e (Controle, n = 787). Foram feitas avaliações ultrassonograficas dos ovários nos dias -10, 0 e 4, para mensurações de tamanho de folículo e corpo lúteo e no dia -10 foi feita a avaliação do ECC. A taxa de prenhez (P/IA) entre os grupos P4 vs CONTROLE não apresentou diferenças P=0,49. Vacas com ECC > 2,75 tiveram melhora P/IA (P<0,05) comparadas com vacas com ECC ≤ 2,5. Vacas com presença de folículo ≥ 8 mm no dia -10 apresentou melhor P/IA (P<0,05) comparadas com vacas com presença de CL ou folículo < 8 mm. Vacas com folículo ≥8 mm e CL apresentaram maior tamanho de folículo, maior área de CL, no entanto, os animais com presença de folículo ≥ 8 mm apresentaram maior taxa de concepção comparado com os animais com folículo < 8 mm e com presença de CL. Concluimos que a suplementação com P4 após a IATF não melhora as taxas de prenhez, independente do ECC e do EO, no entanto, o ECC e o EO apresentam influências nas taxas de prenhez, onde os animais com melhor ECC no início do protocolo apresentaram melhor EO e maior taxa de prenhez. / Embryo loss commonly occurs in the first three weeks after insemination due to low progesterone (P4) concentrations. Therefore, this study evaluates the effects of body condition score (BCS) and ovarian condition (OC) on pregnancy rates (P/AI) in beef cows supplemented with long-acting P4 after timed artificial insemination (TAI). We used 1573 lactating Nellore cows at 30-60 days postpartum. The animals were pre-synchronized on Day -10 and inseminated on Day 0. On Day 4, they were evaluated for BCS, OC and parturition order and divided into two treatment groups: P4 supplementation (N= 786) and control group (N = 787). Ultrasound exams were performed on Days -10 and 0 to measure follicle diameter and on Day 4 to evaluate corpus luteum (CL) area. BCS was determined on Day -10. P/AI did not vary between P4 supplementation and control treatments (P = 0.49). Cows with BCS > 2.75 had better P/AI than cows with BCS ≤ 2.5 (P < 0.05). Cows without CL and with follicle diameter ≥ 8 mm on Day -10 presented better P/AI than cows with follicle < 8 mm or CL (P < 0.05). In conclusion, P4 supplementation after TAI did not improve P/AI. However, BCS and OC affected P/AI and the animals with better BCS at the beginning of the experiment had better OC and P/AI.

Este estudo teve como objetivos avaliar o metabolismo de quilomícrons utilizando a metodologia da cinética plasmática de uma emulsão de quilomícrons artificiais em pacientes com síndrome dos ovários policísticos (SOP), assim como o impacto da obesidade nesta cinética. Foram estudadas 43 mulheres adultas jovens, subdivididas em 4 grupos, sendo 8 pacientes com SOP e índice de massa corporal normal [SOP-N (IMC = 22,7 ± 1,9 Kg/m2)], e 15 com IMC >=30 kg/m2 [SOP-O (IMC = 33,8 ± 3,3 kg/m2)], pareadas com 20 mulheres controles, sendo 10 com IMC normal [Controle-N (IMC = 21 ± 1,76 kg/m2)] e 10 com IMC obeso [Controle-O (IMC = 33,7± 3,1 kg/m2)]. Quando os grupos foram comparados entre si, com relação às características antropométricas, perfil lipídico e apolipoproteínas; detectou-se diferença estatisticamente significante entre IMC (P < 0,001), circunferência abdominal (CA) (P < 0,001), colesterol total (P = 0,042), HDL-colesterol (P < 0,001), LDL-colesterol (P = 0,009), triglicérides (TG) (P < 0,001) e apolipoproteína B (P < 0,001). As médias destas variáveis foram maiores nos grupos Controle- O e SOP-O, não havendo diferenças entre eles. Com relação à apolipoproteína A1 e ácidos graxos livres não houve diferença entre os grupos. A média da apolipoproteína E foi significativamente maior no grupo Controle-N, não havendo diferença ao compararmos os outros três grupos entre si. Com relação à concentração dos hormônios, as pacientes com SOP tiveram médias significativamente maiores para a testosterona total e testosterona livre (TL) (P < 0,001, P = 0,001), respectivamente. O estradiol foi menor nas pacientes com SOP (P = 0,039), não havendo o impacto da obesidade nestas variáveis hormonais. A média da globulina ligadora dos esteróides (SHBG) foi significativamente maior no grupo Controle-N, não havendo diferença ao compararmos os outros três grupos entre si. Com relação ao modelo homeostático de resistência à insulina (HOMA-IR), houve um impacto significativo da obesidade e da SOP. A média do HOMA-IR foi significativamente maior nas mulheres obesas (Controle e SOP), e nas pacientes com SOP, ao compararmos com as controles pareadas para o IMC. Com relação à cinética plasmática de emulsão de quilomícrons artificiais, não houve diferença estatisticamente significante entre os grupos, da taxa fracional de remoção plasmática de 3H-triglicérides (TFR-TG), que avalia indiretamente a lipólise das partículas de triglicérides dos quilomícrons pela lipase lipoprotéica. Com relação à média da taxa fracional de remoção plasmática de 14C- éster de colesterol (TFR-EC), houve diferenças estatisticamente significantes, sendo as médias das pacientes com SOP menores que as médias das mulheres controles (P = 0,004), sem impacto da obesidade nesta variável. Após a análise de regressão multivariada, não se observou influência de nenhuma das variáveis estudadas na TFR-EC das pacientes com SOP. Na análise de Correlação de Pearson, observamos nas pacientes com SOP, uma correlação direta entre IMC e TG (r = 0,480; P = 0,020), IMC e HOMA-IR (r = 0,687; P < 0,001), CA e TG (r = 0,574; P = 0,004), CA e HOMA-IR (r = 0,634; P = 0,001), HDL e SHBG (r = 0,481; P = 0,020), e correlação inversa entre IMC e SHBG (r = - 0,581; P = 0,004), CA e SHBG (r = - 0,629; P = 0,001), CA e HDL (r = - 0,464; P = 0,016), SHBG e TG (r = - 0,414; P = 0,050), SHBG e HOMA-IR (r = - 0.528; P = 0,010), TL e SHBG (r = - 0.510; P = 0,013). A diminuição da recaptação de remanescentes de quilomícrons, demonstrada através da diminuição da TFR-EC, é compatível com níveis circulantes maiores destes remanescentes, assim como um tempo de permanência maior na circulação, facilitando e progredindo o processo de aterosclerose. A diminuição da TFR-EC está presente na SOP, independentemente do IMC, sendo mais um fator de risco cardiovascular nas portadoras desta síndrome. / The aims of this study were to evaluate the chylomicrons metabolism using the method of plasma kinetics of an emulsion of artificial chylomicrons in patients with polycystic ovary syndrome (PCOS), as well as the impact of obesity in this kinetics. Forty-three young adult women were studied , subdivided into 4 groups: 8 of them, with PCOS and normal body mass index [ PCOS-N (BMI = 22.7 ± 1.9 kg/m2)], and 15 with BMI >=30 kg/m2 [PCOS-O (BMI = 33.8 ± 3.3 kg/m2 )] , pairwise matched with 20 controls, being 10 with normal BMI [ Control-N (BMI =21 ± 1.76 kg/m2 )] and 10 with obese BMI [Control-O (BMI = 33.7 ± 3.1 kg/m2 )]. When the groups were compared among themselves, in relation to the antropometric features, lipid profile and apolipoproteins; it was detected a statistically significant difference among BMI (P < 0.001), waist circunference (WC) (P < 0.001), total cholesterol (P = 0.042), HDL-cholesterol (P < 0.001), LDL-cholesterol (P = 0.009), triglycerides (TG) (P < 0.001) and apolipoprotein B (P < 0.001). The means of these variables were higher in the Control-O and PCOS groups and there were no differences among them. In relation to apolipoprotein A1 and to free fatty acids, there was no difference among the groups. The means of apolipoprotein E was significantly higher in the Control-N group and there was no difference when we compared the other three groups among themselves. In relation to hormone concentration, the PCOS patients had means significantly higher for total testosterone and free testosterone (P < 0.001, P = 0.001), respectively. Estradiol was lower in PCOS patients (P = 0.039), and there was no obesity impact in these hormonal variables. The means of sex hormone-binding globulin (SHBG) was significantly higher in the Control-N group, and there was no difference when we compared the other three groups among themselves. In relation to the homeostasis model assessment of insulin resistance (HOMA-IR), there was a significant impact of obesity and of PCOS. The means of HOMA-IR was significantly higher in obese women (Controls and PCOS), and in PCOS patients when compared with pairwise matched controls for BMI. In relation to the plasma kinetics of artificial chylomicrons emulsion, there was no statistically significant difference among the groups, of the plasma 3H-triglyceride fractional clearance rate (TFR-TG) , which evaluates indirectly the lipolysis of triglycerides particles of chylomicrons by the lipoprotein lipase. In relation to the means of plasma fractional clearance rate of 14C-cholesterol ester (TFR-EC), there were statistically significant differences, being the means of PCOS patients, lower than the means of controls (P = 0.004), without obesity impact in this variable. After the multivariate regression analysis, it was not observed influence of any of the variables studied in TFR-EC of PCOS patients. Using the Pearson\'s Correlation analysis, we observed in PCOS patients, a direct correlation between BMI and TG (r = 0.480; P = 0.020), BMI and HOMA-IR (r = 0.687; P < 0.001), CA and TG (r = 0.574; P = 0.004), CA and HOMA-IR (r = 0.634; P = 0.001); HDL and SHBG (r = 0.481; P = 0.020) and inverse correlation between BMI and SHBG ( r = - 0.581; P = 0.004), CA and SHBG (r = - 0.629; P = 0.001), CA and HDL (r = -0.464; P = 0.016), SHBG and TG ( r = - 0.414; P = 0.050), SHBG and HOMA-IR ( r = - 0.528; P = 0.010), TL and SHBG ( r = - 0.510; P = 0.013). The decrease of the uptake of chylomicrons remnants shown through the decrease of TFR-EC, is compatible with higher circular levels of these remnants, as well as a prolonged duration in the circulation, facilitating and proceeding to the atherosclerosis process. The decrease of TFR-EC is present in PCOS, independently of BMI, and it is one more cardiovascular risk factor for PCOS patients.

INTRODUÇÃO: A matriz extracelular no endométrio fornece vasta gama de sinais envolvidos em diferentes processos celulares, tais como morte celular e proliferação. Neste sentido, os glicosaminoglicanos e os proteoglicanos, juntamente com os fatores de crescimento, modulam várias etapas da angiogênese, proliferação celular e remodelação do estroma, o que pode ser importante para o fluxo menstrual regular e a redução dos processos proliferativos. Além disso, são importantes para a adequada interação maternofetal. OBJETIVO: Avaliar a concentração de ácido hialurônico, das enzimas de biossíntese do ácido hialurônico - hialurônico sintases (HAS1, HAS2 e HAS3) e dos pequenos proteoglicanos ricos em leucina (decorim, biglicam, lumicam e fibromodulina) no endométrio de pacientes com síndrome dos ovários policísticos (SOP) e de mulheres eumenorreicas. MÉTODOS: Foram analisadas 20 amostras de endométrio, 10 provenientes de pacientes com SOP e 10 de mulheres com ciclos menstruais regulares na fase proliferativa do ciclo, com idade variando entre 20 e 35 anos, atendidas na Divisão de Clínica Ginecológica do Hospital das Clínicas da FMUSP (HC-FMUSP). A determinação do perfil e da concentração do ácido hialurônico foi efetuada por método bioquímico de ensaio fluorimétrico (ELISA-like). A sua localização no tecido endometrial, assim como as dosagens das enzimas sintases (HAS1, HAS2 e HAS3) e dos pequenos proteoglicanos ricos em leucina (decorim, biglicam, lumicam e fibromodulina e), foi feita por imunoistoquímica e \"western blotting\". Para a análise dos resultados foi utilizado o teste t de student (p<=0,05). Os cálculos foram realizados com auxílio do programa SPSS versão13 (SPSS, Chicago, IL). RESUTADOS: Houve maior concentração de ácido hialurônico no endométrio de mulheres eumenorreicas na fase proliferativa do ciclo menstrual do que no das com síndrome dos ovários policísticos. Com relação às sintases do ácido hialurônico, observou-se maior reatividade de HAS1 e HAS2 e menor reatividade de HAS3 no endométrio de mulheres com SOP em relação às mulheres com ciclos menstruais regulares na fase proliferativa. Quanto aos pequenos proteoglicanos ricos em leucina notou-se maior imunorreatividade de decorim e lumicam no endométrio de pacientes com SOP. CONCLUSÕES: As pacientes com SOP apresentam menor concentração de ácido hialurônico e maior reatividade ao decorim e lumicam em relação às mulheres com ciclos regulares na fase proliferativa. Esses dados sugerem que nas pacientes com SOP, o endométrio seria menos receptivo e teria mecanismos para evitar a proliferação excessiva / INTRODUCTION: Endometrium extracellular matrix provides wide range of signals at different cellular levels like cell death and proliferation. In this regard, glycosaminoglycans and proteoglycans, along with growth factors, modulate various stages of angiogenesis, cell proliferation and remodeling of the stroma, which can be important for regulating menses and reducing the proliferative processes. Additionally, it is important for proper fetal-maternal interactions. OBJECTIVE: Evaluate hyaluronic acid concentration, the enzymes of hyaluronic acid synthases (HAS1, HAS2 and HAS3) and small leucine-rich proteoglycans (decorin, lumican, fibromodulim and biglycan) in the endometrium of patients with polycystic ovary syndrome (PCOS) and eumenorrheic women. METHODS: A total of 20 endometrial samples from 10 patients with PCOS and 10 women with regular menstrual cycles in the proliferative phase, with ages ranging between 20 and 35 years, attended at Gynecology Division of Clinical Hospital of the FMUSP (HC-USP). Profile determination and the concentration of hyaluronic acid were performed by biochemical method of fluorimetric assay (ELISA-like). Its location in the endometrial tissue as well as the dosage of enzymes synthases (HAS1, HAS2 and HAS3) and small leucine-rich proteoglycans (decorin, lumican, fibromodulim and biglycan) was done by immunohistochemistry and western blotting. To analyze the results Student t test was used (p < 0.05). The calculations were performed with software SPSS version 13. RESULTS: A higher concentration of hyaluronic acid in eumenorrheic women endometrium in proliferative phase when compared with polycystic ovary syndrome. Regarding hyaluronic acid synthases, there was a higher HAS1 and HAS2 reactivity and lower HAS3 reactivity in the PCOS endometrium compared to women with regular menstrual cycles in the proliferative phase. Decorin and lumican showed higher immunoreactivity in PCOS endometrium. CONCLUSIONS: PCOS patients have a lower concentration of hyaluronic acid and greater reactivity to decorin and lumican than eumenorrheic women in proliferative phase. These data suggest that in patients with PCOS, the endometrium would be less receptive and have mechanisms to prevent excessive proliferation

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.

OBJECTIVE: To examine the likelihood of self-reporting a diagnosis for PCOS with the presentation of menstrual irregularity in a diverse, multiethnic population, based on data collected between August 9th, 2017 and October 24th, 2017 for the pilot of the Ovulation and Menstruation (OM) Study at Boston University School of Medicine. BACKGROUND: Polycystic ovary syndrome, or PCOS, is the most common endocrine disorder among reproductive age women. It is typically diagnosed by variable combinations of menstrual irregularity, clinical or biochemical hyperandrogenism, and polycystic ovaries on ultrasound. An alternative is its diagnosis as one of exclusion due to similarities in presentation to other endocrine disorders. As a result, PCOS may often be misdiagnosed and mismanaged in the course of a patient’s care, further exacerbated by a poor understanding of the syndrome, a lack of easily available resources, and patient frustration with clinician interactions. The early identification of key hallmarks of the disorder, such as menstrual irregularity, and awareness of its linkage to PCOS, could lead to early diagnosis and intervention. METHODS: 248 participants enrolled and participated in the Ovulation and Menstruation (OM) Health Study’s as members of its pilot cohort. Inclusion criteria were women ages 18-45 currently experiencing menstrual periods without a history of chemotherapy, radiation, or surgical menopause. Participants completed the relevant sections of the OM Study survey related to demographics, menstrual cycle patterns, and history of PCOS. Demographic questions pertained to the age, race/ethnicity, country of birth, and education levels of the participants. The menstrual cycle questions provided information regarding the age of menarche, length and pattern of menses and the menstrual cycle overall. The questions regarding history of PCOS ascertained the presence of an official or self-diagnosis for PCOS for the participant, and the age at which this was determined. The descriptive measures were presented for comparison before determining the concurrence of the presence of menstrual irregularity and the diagnosis of PCOS across demographic categories and calculating an associated prevalence ratio. RESULTS: Among women reporting a history of menstrual irregularity for 3 months or greater, PCOS was the second-highest self-reported cause for menstrual irregularity, with 20.7% of participants endorsing it as the cause for their irregularity. The presence of menstrual irregularity for 3 or more months was also more likely to be present in concurrence with a clinician diagnosis, or to a lesser extent, a self-diagnosis, for PCOS. Participants were also far more likely to have a clinician diagnosis for PCOS if they were White, US-born, young, or educated. The same applied for the likelihood of a self-diagnosis with the exception of age. CONCLUSIONS: The association between menstrual cycle irregularities and likelihood of being diagnosed with PCOS is supported by the data and appears to be influenced by demographic factors such as race/ethnicity, age, and education.

Fetal germ cell development is a key stage of female reproductive life. The DAZ family proteins (DAZ, DAZL and BOLL) are RNA-binding proteins with critical roles in murine germ cell development but their expression and potential targets in the human are largely unknown. The studies in this Thesis investigated the expression and function of DAZL and BOLL in human fetal ovary. Both DAZL and BOLL mRNA are increased dramatically at the time of entry into meiosis. Immunohistochemical analysis with specific meiotic markers suggested that DAZL and BOLL have distinct spatial-temporal expression patterns, with minimal co-expression – BOLL expression was transient prior to follicle formation. This pattern was shown not to be present in the mouse fetal ovary, where Dazl and Boll are co-expressed, indicating a limitation of the mouse for exploring the function of Boll. Two human cell lines, embryonic kidney derived HEK293 cells and germ cell tumour derived TCam-2 cells were used as models to identify the mRNA targets of DAZL and BOLL after transfection of DAZL or BOLL vectors. In HEK293 cells, TEX19 and TEX14 were confirmed as potential targets of both DAZL and BOLL, and CDC25A as a potential DAZL target. Further experiments indicated that DAZL and BOLL did not increase target mRNA transcription but increased stabilisation. A DAZL/GFP co-transfection-FACS system for TCam-2 cells was established as this cell line has very low transfection efficiency. TEX14 and SYCP3 significantly increased in GFP+ve-DAZL+ve cells when compare to the GFP-ve-DAZL-ve cells, whilst SOX17 and DNMT3L significantly decreased in the GFP+ve-DAZL+ve cells. A 3'-UTR luciferase assay confirmed regulation of TEX14 and SOX17 by DAZL through their 3'-UTR. RNA immunoprecipitation further demonstrated direct binding between human TEX14, TEX19, SYCP3, SOX17 mRNA and DAZL protein, and that TEX14 binding is through its 3'-UTR. Dual fluorescence immunohistochemistry showed that SOX17 and DMNT3L are expressed in early germ cells with DAZL, and are later down-regulated co-incident with that of DAZL, consistent with the novel repressive effect of human DAZL on these two potential targets. These studies indicate that DAZL and BOLL are associated with different key meiotic stages of germ cell development in human fetal ovary. Several potential mRNA targets of DAZL and BOLL, and a novel repression function of human DAZL on its mRNA targets were identified giving further insight into the role of these factors in human ovarian development.

The longstanding belief that women are born with a finite ovarian reserve has been debated for over a decade, ever since the discovery, and subsequent isolation, of purported oogonial stem cells (OSCs) from adult mammalian ovaries. This rare cell population has now been reported in the mouse, rat, pig, rhesus macaque monkey and humans and, although a physiological role for the cells has not been proven, they do appear to generate oocytes when cultured in specific environments, resulting in live offspring in rodents. The primary aim of this thesis was to verify independently the existence of OSCs in human ovary and determine whether they could be isolated from a large animal model, the cow. The secondary aim was to investigate the cells’ in vitro potential, both to undergo neo-oogenesis and as a model for germ cell development. Putative bovine and human OSCs were isolated from disaggregated adult ovarian cortex using a previously validated fluorescence-activated cell sorting (FACS)-based technique, with cells sorted for externally expressed DDX4 (VASA). Freshly isolated and cultured cells were characterised by analysing their expression of pluripotency and germline markers, using RT-PCR, immunocytochemistry and Western blotting. The in vitro neo-oogenesis potential of the cells was explored by injecting fluorescently labelled cells into fragments of adult ovarian cortex and by forming aggregated artificial “ovaries” with putative OSCs and fetal ovarian somatic cells. Germ cell model experiments comprised treatment of cultured cells with BMP4 and/or retinoic acid (RA), with subsequent quantitative RT-PCR and immunocytochemistry analysis for downstream BMP4- and RA-response genes, and liposomal-mediated transfection of cells with a DAZL overexpression plasmid to assess their meiosis-related gene response. Scarce populations of putative OSCs were retrieved from 5 human samples (aged 13- 40 years) and 6 bovine samples. The cells were cultured long-term for up to 7 months and demonstrated consistent expression of several pluripotency-associated and germline markers at the mRNA and protein level, including LIN28, NANOG, POU5F1 (OCT4), IFITM3 (fragilis), STELLA, PRDM1 (BLIMP1), and C-KIT, indicating their early germline nature. Investigation of neo-oogenesis potential revealed that putative human OSCs were associated rarely with fetal somatic cells in primordial follicle-like structures, but could not be confirmed to have undergone oogenesis. However, like early germ cells, putative bovine and human OSCs were BMP4 and RA responsive, with both species demonstrating significant upregulation of expression of ID1 and bovine cells exhibiting a significant increase in MSX1, MSX2 and the meiotic marker SYCP3 in response to BMP4 and/or RA treatment. Cells could be successfully transfected to overexpress DAZL; however, no significant downstream gene expression changes were observed. This is the first report of putative bovine OSC isolation and corroborates a previous report showing putative human OSC isolation. Although the expression of both stem cell and germline markers indicates the cells have characteristics of OSCs, their capacity to enter meiosis and form functional oocytes has yet to be determined. Putative bovine OSCs, however, show promise as a novel model for investigating germ cell development. If their potential can be harnessed, then OSCs may have a role in clinical applications, for example in fertility preservation, in the future. Future experiments will examine the neo-oogenesis capabilities of the cells further and explore novel cell delivery systems for clinical use.