Introdução: Alterações no ambiente fetal conferem um risco aumentado para doenças crônicas como obesidade, doença cardiovascular, hipertensão arterial e diabetes tipo 2. As evidências sugerem que a restrição de crescimento intrauterino (RCIU) pode programar de forma persistente as preferências alimentares, e acredita-se que esse tipo de alteração comportamental, pode explicar, pelo menos em parte, o aumento do risco para essas doenças em indivíduos que sofreram RCIU. Portanto, torna-se importante entender os fatores associados e mecanismos envolvidos nesse comportamento. O objetivo deste trabalho foi investigar o efeito da RCIU no comportamento alimentar em animais e humanos, assim como os possíveis mecanismos envolvidos na sua programação. Métodos: Ratas Sprague Dawley prenhes foram randomizadas para o grupo controle (Adlib), que recebeu dieta padrão ad libitum ou grupo restrição 50% (FR), que recebeu 50% do consumo habitual de genitoras alimentadas ad libitum. As dietas foram oferecidas a partir do dia 10 de gestação até o dia 21 de lactação. Em até 24h após o nascimento, foi realizada a adoção cruzada formando os grupos: Adlib_Adlib, FR_Adlib, FR_FR, Adlib_FR. O consumo de ração padrão foi comparado entre todos os grupos. A preferência alimentar, a preferência condicionada por lugar induzida por alimento palatável, assim como a fosforilação da enzima tirosina hidroxilase e os níveis de receptores D2 no núcleo acumbens foram comparados entre os grupos de interesse (Adlib_Adlib e FR_Adlib). Nos humanos, 75 jovens, classificados quanto à RCIU, participaram de avaliação antropométrica, bioquímica e de comportamento alimentar (teste de escolha alimentar, no qual todos recebiam um valor monetário para compra de um lanche, e Dutch Eating Behaviour Questionnaire, DEBQ). Dados de neuroimagem funcional em repouso entre regiões relacionadas à recompensa de 28 indivíduos foram processados e analisados, de um total de 43 exames realizados. Resultados: No estudo experimental, viu-se que o consumo de ração padrão não foi diferente entre os grupos. Ratos restritos apresentaram preferência pela dieta palatável, mas menor condicionamento de preferência ao lugar associado ao alimento palatável. A fosforilação da tirosina hidroxilase no núcleo acumbens foi maior nestes animais no estado basal, mas após exposição ao doce essa diferença entre os grupos permaneceu apenas nos machos. A RCIU também se associou a menores níveis de receptores D2 no núcleo acumbens. No estudo clínico, encontrou-se que a menor razão de crescimento fetal (indicativo de maior RCIU) e alto índice de massa corporal predizem um estilo alimentar restritivo visto pelo DEBQ. Pessoas nascidas com RCIU também usaram menor quantidade do um recurso financeiro oferecido no teste de escolha alimentar após um período de jejum. Os dados de neuroimagem funcional sugerem que os indivíduos restritos apresentam um padrão de conectividade em repouso alterado entre o córtex orbito-frontal, o estriado ventral/dorsal e a amígdala. Conclusão: A RCIU esteve associada com preferência por alimentos palatáveis e alterações no sistema dopaminérgico no estudo experimental e alterações da conectividade em repouso entre áreas do sistema mesocorticolímbico no estudo clínico. As alterações observadas no sistema dopaminérgico dos animais restritos indicam que esse sistema estaria envolvido na programação da preferência alimentar nesses indivíduos. Além disso, o padrão de conectividade em repouso observado nos indivíduos restritos sugere que alterações em determinadas regiões do sistema de recompensa poderiam estar associadas com mudanças no comportamento alimentar. As alterações neurocomportamentais observadas confirmam a existência de programação fetal do comportamento alimentar pela RCIU, apontando modificações persistentes no sistema de recompensa do cérebro, o que pode ser visto como um fator de risco para o desenvolvimento de obesidade e suas comorbidades. / Introduction: Fetal environment changes can lead to adaptations that are associated with increased risk for obesity, cardiovascular disease, hypertension and diabetes in adult life. Evidence suggests that intrauterine growth restriction (IUGR) can persistently program the subject’s preference for palatable foods. It is believed that feeding behavior alterations can explain, at least in part, the increased risk for chronic diseases in IUGR individuals. Therefore, it becomes important to understand the factors and mechanisms involved in this behavior. The aim of this study was to explore how IUGR affects feeding behavior of animals and humans, as well as to verify the potential mechanisms related to this behavioral programming. Methods: Time-mated pregnant Sprague-Dawley rats were randomly allocated to Control (receiving standard chow ad libitum) or 50% food restricted (FR), receiving 50% of the ad libitum-fed dam’s habitual intake. These diets were provided from day 10 of pregnancy throughout day 21 of lactation. Within 24 hours after birth, pups were crossfostered, forming four groups: Adlib_Adlib, FR_Adlib, FR_FR, Adlib_FR. Standard chow consumption was compared between all groups. Food preference, conditioned place preference to a palatable diet, and the nucleus accumbens tyrosine hydroxylase phosphorylation and D2 receptor levels were analyzed focusing on two groups of interest (Adlib_Adlib and FR_Adlib). In humans, 75 youths were classified regarding IUGR and had anthropometric data, biochemical data, and feeding behavior (food choice task, in which everyone received a monetary value to purchase a snack, and Dutch Eating Behaviour Questionnaire) assessed. Forty three neuroimaging exams were performed and resting state functional connectivity between brain regions related to reward of 28 individuals were processed and analyzed. Results: In the experimental study, standard chow consumption was not different between groups. IUGR adult rats had increased preference for palatable food, but showed less conditioned place preference to a palatable diet compared to controls. At baseline, the accumbal tyrosine hydroxylase phosphorylation was increased in IUGR rats compared to controls. After sweet food exposure, the difference between groups remained only in males. Accumbal D2 receptors levels were decreased in IUGR rats. In the clinical study, it was found that low birth weight ratio (indicative of higher IUGR) and high body mass index predict a restrained eating style as seen by the DEBQ. IUGR individuals used a smaller quantity of a financial resource offered in the food choice task after a fasting period. Resting state functional connectivity data suggest that IUGR individuals had an altered pattern of connectivity between the orbitofrontal cortex, the ventral/dorsal striatum and the amygdala. Conclusion: IUGR was associated with a preference for palatable foods and alterations in the dopaminergic system in the experimental study, as well as changes in the resting state functional connectivity between regions of the mesocorticolimbic pathway in the clinical study. Alterations in the mesolimbic dopaminergic system observed in IUGR rats indicate an important role in the programming of food preferences. Moreover, the IUGR pattern of brain connectivity observed suggests that alterations in certain regions involved in reward processing and evaluation could be associated with changes in eating behavior. Neurobehavioral changes observed confirmed the existence of a fetal programming of feeding behavior associated with IUGR, pointing out to persistent modifications in the brain reward system, which can be seen as a risk factor for the development of obesity and its comorbidities.

Evidências epidemiológicas tem sugerido que a desnutrição intra-uterina pode ter papel importante no desenvolvimento de hipertensão arterial na fase adulta. No presente trabalho, foi estudada a influência da desnutrição intra-uterina na reatividade vascular e na pressão arterial de animais Wistar e SHR (ambos os sexos). Para tanto, ratas prenhes foram submetidas à restrição alimentar equivalente a 50% da dieta normal, durante toda a gestação. Curvas concentração-efeito à noradrenalina (NA), acetilcolina (ACh) e ao nitroprussiato de sódio (NPS) foram realizadas em anéis de aorta de ratos Wistar e SHR nutridos e desnutridos (ambos os sexos). A desnutrição intra-uterina elevou a pressão arterial em animais Wistar, machos e fêmeas, até níveis considerados de hipertensão. Além disso, acarretou alterações da reatividade à NA e à ACh, mas não ao NPS. Em animais SHR, de ambos os sexos, os efeitos deletérios da desnutrição intra-uterina foram capazes de exacerbar a hipertensão e a disfunção endotelial já existentes nesses animais. / Epidemiological studies suggest that intrauterine undernutrition can play an important role in the development of arterial hypertension in adulthood (FERRARI et al., 2000). Furthermore, maternal malnutrition during organ developmental stage impairs fetal growth and is believed to alter permanently the metabolism and physiology of the developing tissues. The aim of the present study was to examine the effects of intrauterine undernutrition in the arterial blood and vascular reactivity pressure of male and female normotensive (Wistar) and spontaneously hypertensive (SHR) rats offspring. Female pregnant rats (Wistar and SHR) were fed either normal or 50% of the normal intake diets, during the whole gestational period. Arterial blood pressure and the norepinephrine (NE), acetylcholine (ACh) and sodium nitroprusside (SNP) dose-response curves in isolated aortic rings of their offspring (male and female – when they reached adulthood) were determined. In Wistar rats, the intrauterine undernutrition induced an increase in the arterial blood pressure, leading to hypertension. Dietary restriction during pregnancy of Wistar rats altered the vascular reactivity to NE and ACh, whereas the response to SNP remained unaltered in the offspring. In the SHR offspring (male and female) the intrauterine undernutrition exacerbates the already existing hypertension and endothelial dysfunction. In summary, this study has shown that intrauterine undernutrition increased the arterial blood pressure and altered the vascular reactivity of male and female normotensive and SHR offspring.

The purpose of this research project was to elucidate the molecular interactions and detail the signaling pathways in obstetric pathologies. This work first seeks to understand inflammation related complications relevant to obstetrics. Prior research in our lab identified the implications of the receptor of advanced glycation end products (RAGE) during inflammatory response in the placenta. Current work identified the presence of DNA double-strand breaks (DNA-DSBs) in inflammation associated pregnancy complications of preeclampsia (PE) and preterm labor (PTL) and demonstrated the positive role of RAGE in repairing the damage. The confluent relevance of disrupted mitochondrial function and inflammation has been recognized in the etiology of numerous chronic diseases. Our current studies aim to understand the connections between energy metabolism and inflammation in pathologies of pregnancy complications. Previous research conducted in our laboratory has demonstrated the mediation of the Gas6/Axl pathway on the mechanistic target of rapamycin (mTOR), an important metabolic molecule. We observed the negative regulation of Gas6 treatment on the mTOR pathway and its negative effects on trophoblast cell invasion. In the current study looking at the aspect of energy regulation, we identified the activation of placental mTOR in gestational diabetes mellitus (GDM) and its decrease during PE and intrauterine growth restriction (IUGR). We further evaluated the regulation of mTOR on its downstream effector pyruvate kinase M2 (PKM2). We found that inhibition of mTOR decreased PKM2 activation; while PKM2 activation positively regulated trophoblastic invasion and rescued negative effects observed in our second-hand smoke IUGR murine model. Our work has opened a new direction of placental research, especially in pregnancy complications stemming from genomic instability. We also clarified details of mTOR and PKM2 meditated metabolic signaling that are crucial for future investigation on the dynamic metabolic regulation during pregnancy.

Résumé : Le sFlt1 semble être un bon marqueur de la prééclampsie (PE). Nous avons évalué dans le cadre d’un essai randomisé contrôlé (MOMA), l’impact sur l’issue de grossesse d’un suivi rapproché de femmes identifiées comme « à risque » par un taux élevé de sFlt1 plasmatique entre 24 et 29 SA. Dans cet essai 939 nullipares ont été incluses en 2 bras (sflt1 connu ou non connu). Nos résultats ne montrent pas d’amélioration de l’issue de grossesse et suggèrent que l’inefficience de l’intervention (suivi rapproché) en est la cause la plus probable. Aussi la mesure du sFlt1 pour prédire la survenue d'une PE ne semble pas utile tant qu’un traitement efficace n’est pas à disposition. Des anomalies de la stéroïdogénèse placentaire ont été retrouvées associées à la PE. 90 échantillons issus de la cohorte MOMA répartis en trois groupes (25 PE, 25 retard de croissance intra utérin (RCIU) sans PE et 40 contrôles) ont été utilisés pour évaluer le profil stéroïdes par la technique fiable de GC/MS (entre 24 et 29 SA) - soit avant les signes cliniques -. Nous avons mis en évidence dans le groupe PE un défaut d'aromatisation des androgènes par le calcul du ratio estradiol/Δ4-Dione (sang) et un défaut d’expression de l’aromatase (placenta). Tout comme les taux significativement bas de sulfate de prégnénolone retrouvés, ce défaut d’aromatisation semble spécifique de la PE car ils n’ont pas été observés dans le groupe RCIU. Ces modifications s'inscrivent dans une dérégulation du profil stéroïdien global. Nous avons en effet observé dans les groupes PE et RCIU, un excés de 20α-dihydro-progestérone (20α-DHP) et un ratio 20α-DHP/Progestérone significativement élevé. Les stéroïdes sexuels partagent des voies de signalisation communes avec les facteurs angiogéniques, faisant inscrire aisément l’hypothèse « anomalies de la stéroïdogénèse » dans les concepts actuels de la physiopathologie de la PE. Les liens entre perturbations de la stéroïdogénèse et la prééclampsie sont discutés. / Abstract : sFlt1 appears to be a good biomarker of preeclampsia (PE). The impact on pregnancy outcomes of close monitoring of women identified as "at risk" at 24 and 29 weeks of gestation (weeks) by a high level of plasma sFlt1, was evaluated in a randomized controlled trial (MOMA). 939 nulliparous women were included in 2 arms (sFlt1 known or unknown). Our results do not show any improvement of pregnancy outcomes and suggest that the inefficiency of the intervention (close follow-up) is the most likely cause. Thus, routine sFlt1 screening to predict the occurrence of PE does not seem useful until effective treatment is available. Abnormalities of placental steroidogenesis have been associated with PE. 90 samples from the MOMA cohort divided into three groups (25 intra-uterine growth retardation (IUGR) without PE, 25 PE and 40 controls) were used to assess by the reliable GC / MS technique, the steroid profile (between 24 and 29 weeks)before the development of clinical signs. In the PE group, we showed abnormal androgen aromatization, by calculating the estradiol/Δ4-Dione ratio (blood), and a lack of expression of placental aromatase. Similarly to the significantly low levels of pregnenolone sulfate found, this lack of aromatization seems specific to PE as they are not found in the IUGR group. These modifications are part of a deregulation of the overall steroid profile. In the PE and IUGR groups, we observed an excess of 20α-dihydroprogesterone (20α-DHP) and a significantly high 20α-DHP/Progesterone ratio. Sex steroids share common signaling pathways with angiogenic factors which easily integrate the "steroidogenesis abnormalities" hypothesis in the current concepts in the pathophysiology of PE. Links between disturbances of steroidogenesis and preeclampsia are discussed.

Introduction: Actually 0.7-1 percent of all deliveries can be followed by secondary uterine bleeding. There is a residual trophoblastic tissue diagnosed in most of these cases and it is often managed by repeat intrauterine interventions. These operations are connected with high risk of formation of intrauterine adhesions and their early diagnosis and management can be important for next fertility. Material and methods: There were generally 188 patients included into the study. All patients underwent ultrasound examination in 6 weeks after delivery and ambulant hysteroscopy after next 2 months without anesthesia. Described intrauterine pathologies (residual tissue and adhesions) were managed in one step. Results: In cases with suspect ultrasound finding, the retained trophoblastic tissue was diagnosed by hysteroscopy in 66 percent vs. in 96 percent with sensitivity 85 percent and specificity 85 percent. Patients with intrauterine adhesions had normal ultrasound finding in 74 percent and it did not recognized patients with severe adhesions in 94 percent. Clinical signs had generally 72 percent of patients with diagnosed left residual tissue. Number of severe residual tissue is increasing with delay of instrumental evacuation from delivery (10 vs. 30 percent). When is necessary to repeat the operation...

Philosophiae Doctor - PhD / Infertility is currently a serious problem that is escalating, not only in South Africa, but also worldwide. In Cape Town, a culturally diverse, urban community of approximately 1000 couples are referred to the Groote Schuur Hospital Infertility Clinic annually. Although infertility is primarily regarded as a medical condition, the treatments have emotional effects on infertile couples due to the recurring highs and lows that often accompany treatments. This study aimed to qualitatively explore and understand the emotional and psychological experiences of infertility and its treatments (specifically In Vitro Fertilisation and Embryo Transfer). Social constructionism is based on the premise that realities are not constructed in a vacuum but rather undergo a process whereby the subjective and inter-subjective experiences over time and through cultural processes come to be regarded as truths. These truths become internalised and function as lenses through which we see ourselves, compose and invent ourselves accordingly, making sense of what would otherwise have been chaotic and meaningless experiences. Additional aims were to examine women's experiences of infertility care whilst undergoing treatment and describe their experiences of coping with infertility and In Vitro Fertilisation and Embryo Transfer (IVF-ET). Semi-structured, in-depth individual interviews were conducted with 21 women presenting with primary infertility at a fertility clinic. This study utilised an ethnographic case study design. The results of the study suggested that women perceived themselves as not conforming to a dominant belief system that promotes motherhood as the most important role for women. The women described their 'failure' to fulfill socio-cultural expectations as emotionally turbulent. Some of the psychological responses to infertility included feelings of disappointment, shock, denial, devastation, anger, frustration, sadness, inadequacy, poor self-image and self-esteem. The women's personal accounts of their experiences of In Vitro Fertilisation and Embryo Transfer (IVF-ET) revealed that they found the treatment to be highly stressful, with emotional bouts of anxiety, nervousness, excitement and optimism. A psychological synopsis of infertility and IVF-ET is infertility and IVF-ET presents. The fmdings in this study suggest the need for the incorporation of presented. This diagrammatic representation shows the intensity of the emotional rollercoaster that psychosocial intervention into infertility management. Greater attention to the psychological and emotional repercussions of infertility treatment could lead to a more personalised approach which, in turn, would optimise patient satisfaction and also prepare couples for the demands of the program by informing them about better ways of coping.

This thesis will be organized into two chapters discussing the role of hypoxia in the human placenta. The goal of this thesis is to characterize pyruvate kinase M2, mammalian target of rapamycin, mitochondrial function, and cell invasion in hypoxic conditions in the trophoblast. Understanding the mechanisms of placental metabolism can lead to further treatments for placental diseases. Chapter one covers the background of intrauterine growth restriction, hypoxia, placental metabolism, and pyruvate kinase M2 (PKM2). Little is currently understood about the role of the mitochondria in placental diseases. Expression of PKM2, trophoblast cell invasion, and mitochondrial function is shown to be inhibited by hypoxia. PKM2 inhibition decreases trophoblast cell invasion and nuclear expression of PKM2, but increases mitochondrial function. Studying how hypoxia affects the placenta during placental diseases can help clarify the mechanisms by which these diseases occur. Chapter two further characterizes the background of intrauterine growth restriction and hypoxia. It also covers the background of mammalian target of rapamycin. The objective of this chapter was to assess activated mTOR in the trophoblast in hypoxia. Decreased placental and fetal weights, as well as trophoblast cell invasion were observed in hypoxia. A decrease in the activation of mTOR was also found in the hypoxic placenta. This study could provide insight into the physiological relevance of the pathways and could be targeted to help alleviate placental diseases.

Cold shock Y-box binding protein-1 (YB-1) coordinates several molecular processes between the nucleus and the cytoplasm and plays a crucial role in cell function. Moreover, it is involved in cancer progression, invasion, and metastasis. As trophoblast cells share similar characteristics with cancer cells, we hypothesized that YB-1 might also be necessary for trophoblast functionality. In samples of patients with intrauterine growth restriction, YB-1 mRNA levels were decreased, while they were increased in preeclampsia and unchanged in spontaneous abortions when compared to normal pregnant controls. Studies with overexpression and downregulation of YB-1 were performed to assess the key trophoblast processes in two trophoblast cell lines HTR8/SVneo and JEG3. Overexpression of YB-1 or exposure of trophoblast cells to recombinant YB-1 caused enhanced proliferation, while knockdown of YB-1 lead to proliferative disadvantage in JEG3 or HTR8/SVneo cells. The invasion and migration properties were affected at different degrees among the trophoblast cell lines. Trophoblast expression of genes mediating migration, invasion, apoptosis, and inflammation was altered upon YB-1 downregulation. Moreover, IL-6 secretion was excessively increased in HTR8/SVneo. Ultimately, YB-1 directly binds to NF-κB enhancer mark in HTR8/SVneo cells. Our data show that YB-1 protein is important for trophoblast cell functioning and, when downregulated, leads to trophoblast disadvantage that at least in part is mediated by NF-κB.

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Maternell immunisering är ett sjukdomstillstånd där moderns försvarsmekanism förstör fostrets röda blodkroppar och orsakar anemi hos fostret. Den primära behandlingsmetoden är intrauterina blodtransfusioner. Undersökning av foster under graviditeten utförs i stor utsträckning med hjälp av ultraljud. Ultraljudsundersökning begränsas av att fostret rör på sig och har en hög hjärtfrekvens. Målet med arbetet var att undersöka deformationen av myokardiet hos två grupper av foster. Den ena bestod av nio foster som genomgår intrauterina blodtransfusioner, den andra bestod av nio normala foster i motsvarande graviditetsvecka. Mätningar utfördes i programmet EchoPAC med 2D STE metoden, en tvådimensionell metod där speckle från myokardiet markeras och observeras över tid för att studera förändringen i hjärtats segment, hjärtdeformationen. Detta gjordes som en pilotundersökning primärt i syfte att bedöma genomförbarheten samt möjliga svårigheter med undersökningarna samt undersöka om metoden skulle kunna påvisa skillnader i deformationen mellan foster som behandlas med intrauterina blodtransfusioner och normala foster. Metoden visades vara genomförbar. Resultaten visar att deformationen i hjärtat hos foster som genomgår intrauterina blodtransfusioner är en aning högre än hos normala foster. Skillnaden i hjärtdeformationen mellan grupperna har visat sig vara signifikant. / Maternal immunization is a condition in which the mother's defense mechanism destroys the red blood cells in the fetus, which causes anemia. The primary treatment method is intrauterine blood transfusions. Ultrasound is one of the most common tools used to assess fetuses. Examination with ultrasound is limited by the fetal movements and a high fetal heart rate. The aim of this project was to pilot an investigation of myocardial strain within two groups of fetuses. One group consisted of nine fetuses undergoing intrauterine blood transfusions, the other consisted of nine normal fetuses in the corresponding week of pregnancy. Measurements were performed in the program EchoPAC with the 2D STE method, a two-dimensional method where speckles from the myocardium are marked and observed over time to study the change in the heart segment, the strain. This was done primarily to investigate if measurements were feasible and what problems could occur, and secondarily whether the method could show a difference in strain between fetuses treated with intrauterine blood transfusions and normal fetuses. The results show that the measurements were feasible. Results show that the strain of the heart in fetuses undergoing intrauterine blood transfusions is higher than in normal fetuses. The difference in the strain between the groups has shown to be significant