Obese pregnant women have increased risk of a number of pregnancy complications, including poor maternal health, fetal growth restriction (FGR) and fetal demise. The success of pregnancy is dependent on precise regulation of the immune response within the utero-placental environment. Rats as a model for human related pregnancy complications are beginning to be widely used because of the similarities between these species in terms of trophoblast invasion and spiral artery remodeling. However our knowledge of immune cells and cytokine localization in the rat utero-placental tissue relating to these processes is limited. Therefore our first aim was to characterize the immune cell populations, such as uterine natural killer (uNK) cells, neutrophils and macrophages in the rat utero-placental unit at two crucial gestational ages relevant to trophoblast invasion and spiral artery remodeling, gestational day (GD) 15 and GD18. In addition, we characterized the cytokine distribution of TNFα, IFNγ and IL-10 in the utero-placental tissue at both above mentioned gestational ages. Our study has demonstrated co-localization of TNFα and IFNγ with uNK cells in the perivascular region of the spiral arteries in the rat mesometrial triangle. Neutrophils were localized at the maternal fetal interface and in the spiral artery lumen of the rat mesometrial triangle at both gestational ages. TNFα and IL-10 demonstrated a temporal change in the localization from GD15 to GD18 which coincides with the leading edge of trophoblast invasion into the mesometrial triangle. The results of the current study furthers our knowledge of the localization and temporal expression of uterine immune cells and relevant cytokines, and provides a base to research the function of these immune cells and cytokines during rat pregnancy as a model to study human pregnancy and complications related to immune functions. Since obesity is associated with a peripheral and systemic pro-inflammatory state in humans, our second objective was to investigate whether maternal obesity could alter the utero-placental and systemic immune response in the rats. To characterize maternal obesity induced changes in uterine immune state we used pregnant rats fed a control diet (normal weight; CD) or a high fat diet (obese; HFD) at GD15 and GD18. We performed immunohistochemistry to localize TNFα and IL-10, and quantified the levels of TNFα, IL-1β and IL-10 in the uterine tissue by immunoassay. To assess the systemic immune state, circulating levels of pro-inflammatory cytokine MCP-1 were assessed by immunoassay. We demonstrated an increased concentration of the pro-inflammatory marker TNFα and a reduced anti-inflammatory IL-10-positive cell distribution in the rat mesometrial triangle in response to a HFD. In addition increased circulating MCP-1 was observed in the HFD-fed dams at both gestation ages. HFD induced obesity in our rat model leads to an increase in uterine and systemic pro-inflammatory markers. These markers have demonstrated the potential to alter utero-placental development. Pregnancy complications such as FGR and fetal demise have been shown to be associated with impaired placental development as a result of altered trophoblast invasion and aberrant maternal spiral artery remodeling. Therefore, our third aim was to compare these parameters between the CD-fed rats and HFD-fed rats at GD15 and GD18. Early trophoblast invasion was increased by approximately 2-fold in HFD-fed dams with a concomitant increase in the expression of matrix metalloproteinase-9 protein, a mediator of tissue remodeling and invasion. By late gestation reduced trophoblast invasion was observed in HFD-fed dams. Furthermore, we also observed in late gestation significantly higher levels of smooth muscle actin surrounding the uterine spiral arteries of HFD-fed dams, suggesting impaired spiral artery remodeling. We also determined the impact of human serum from obese mothers on trophoblast invasion. We compared the invasion of HTR-8/SVneo cells treated with pooled first-trimester serum from obese women with or without fetal growth restriction vs. cells treated with serum from normal-weight women with or without fetal growth restriction. First-trimester serum from obese pregnant women reduced invasion of the trophoblast cell line HTR8/SVneo compared to serum from normal-weight pregnant women. Taken together, the results of this study suggest that maternal obesity can negatively influence crucial utero-placental development processes resulting in the poor pregnancy outcomes and increased fetal demise. To summarize, the HFD increased the pro-inflammatory marker TNFα which was associated with altered trophoblast invasion profiles and impaired vascular remodeling. These disturbances in utero-placental development were also associated with decreased birth weights (indication of FGR) and increased rates of stillbirths in our obese rat model. In conclusion, we have made progress in defining the influence of maternal obesity (HFD) on utero-placental development. The importance of these studies is evident since FGR represents a leading cause of perinatal morbidity and mortality. Furthermore, FGR fetuses have an increased risk of becoming obese in their lifetime as a result of fetal programming, therefore resulting in the propagation of a transgenerational obesity cycle. Therefore by understanding the mechanisms by which maternal obesity influences utero-placental development leading to FGR, we may be able to impact short term morbidity and prevent the programming of obesity in future generations. In addition, characterization of maternal obesity’s influence on utero-placental development will also help in the search for therapeutics or intervention strategies to help optimize fetal growth and improve pregnancy outcomes in obese women.

Affecting 6-8% of all pregnancies, preeclampsia is the leading cause of maternal morbidity in the western world and is charactensed by hypertension, proteinuria, edema and platelet aggregation. Despite its prevalence and severity, no comprehensive theory or single factor has been suggested to explain the pathophysiology of this multi system disorder of pregnancy, with the only therapies being bed rest, pharmacological symptom management and if necessary early delivery. Oxidative stress plays an important role in the pathophysiology of preeclampsia, resulting from defective trophoblast invasion, reductions in placental perfusion and placental hypoxia/reoxygenation. The inability of endogenous antioxidant systems up regulated in normal pregnancy, to control increased levels of oxidative stress, is suggested as a possible factor in the feed forward generation of reactive oxygen species and placental oxidative stress. That in turn may stimulate increased syncytiotrophoblast apoptosis, endothelial cell activation and the maternal hyper immune response characteristic of preeclampsia. Analysis of the research literature revealed that previous evaluations of placental oxidation and antioxidant enzyme activity in preeclampsia were by no means comprehensive, and exhibited significant inter-study variations. It was the aim of this thesis to clarify the placental oxidative state and the endogenous antioxidant activity of glutathione peroxidase, thioredoxin reductase, thioredoxin and superoxide dismutase in human placentae in an attempt to determine if variations in antioxidant function were due to changes in gene expression or protein oxidation. The findings reported in this thesis indicate the presence of increased levels of oxidative stress in the preeclamptic placenta, associated with significant reductions in antioxidant enzyme capacity. Quantitative real-time PCR analysis of placental samples revealed that deceases in antioxidant capacity in the placenta are more likely to be related to the significant oxidative burden within the tissue rather than reductions in gene expression. A number of animal models exist to investigate components of preeclampsia pathophysiology, however the ability of these models to mimic the oxidative and antioxidant features of preeclampsia remains unclear. The exposure of pregnant rats to N(G)-nitro-L-arginine methyl ester is a widely used model of endothelial cell dysfunction during preeclampsia. It was the aim of this thesis to determine the biochemical characteristics of this model in an attempt to assess its effectiveness in mimicking oxidative changes in the preeclamptic placenta. Although this model is capable of producing a syndiome in rats similar to the disorder in terms of physiology, this is not manifest in terms of placental biochemistry. The importance of selenium in the synthesis of selenobased antioxidants such as glutathione peroxidase and thioredoxin reductase is well documented. Increasing demand for selenium by the developing fetus may be linked to reductions in selenium status during pregnancy. Considering preeclampsia is associated with significant reductions in selenium status it may be hypothesised that reductions in antioxidant function may be linked to selenium inadequacy. The modulation of dietary selenium in pregnant rats was used to determine the importance of selenium during pregnancy and its effect on antioxidant function and placental oxidative stress. The results of this analysis revealed that selenium deficiency causes a pregnancy specific condition similar to preeclampsia. This condition was found to be associated with increased placental oxidative stress and significant reductions in the systemic activity of selenobased antioxidants that could be modified through selenium supplementation. In summary, data obtained in this thesis indicate that placental oxidative stress and reduced antioxidant enzyme activity play a significant role in the pathogenesis of preeclampsia. These studies support the hypothesis that antioxidant sufficiency is crucial in the maintenance of oxidative balance and that antioxidant dysfunction may result in damage to the placenta and the progression of the disease. These novel data further our understanding of the pathophysiology of preeclampsia and provide new insight into the pathogenesis of clinical complications exhibited in this condition, suggesting antioxidant therapy as a possible means for improving the health outcomes of both mother and baby.

O sucesso da gestação depende da adequada comunicação materno-fetal, que em algumas espécies têm um contato mais íntimo devido à capacidade migratória de populações de células trofoblásticas. Nos bovinos esse mecanismo é realizado pelas células trofoblásticas gigantes (CTGs), com invasão limitada até a lâmina basal do epitélio materno. Apesar dessa leve invasão das CTGs, é possível encontrar células fetais circulantes no sangue periférico da vaca gestante, levando ao microquimerismo fetal. Além de toda uma sinalização local e sistêmica e mudanças conformacionais, a migração das CTGs também é dependente da tolerância imunológica do epitélio materno que possui uma baixa expressão de MHC de classe I. Em contrapartida, o trofoblasto expressa MHC de classe Ib para impedir a ativação das células natural killers uterinas (uNK) contra ele mesmo. Neste contexto, o objetivo desse trabalho foi estudar a ocorrência e contribuir para o entendimento dos mecanismos da migração celular na placenta bovina, com marcadores exclusivos do cromossomo Y e de um modelo de clone transgênico expressando a proteína GFP. A hipótese testada foi que o microquimerismo fetal observado mediante a detecção do gene TSPY no sangue periférico da vaca gestante de embrião macho, e de GFP nos tecidos placentários maternos, associado à expressão de MHC classe 1b (Qa2) na interface materno-fetal. Para tanto, 153 embriões produzidos por fertilização in vitro (FIV) foram transferidos, resultando em 34 embriões machos e 31 fêmeas no dia 62 de gestação, quando foi realizada a coleta de sangue periférico da receptora. Dentre estas gestações, foram selecionadas de 25 machos, 4 fêmeas e 5 perdas gestacionais (confirmadas no D39 por ultrassonografia) para detecção de TSPY. Também foram produzidas gestações de clones transgênicos, expressando GFP com 30, 60 e 90 dias que foram utilizadas para a detecção de mRNA e a proteína GFP. Nas gestações de FIV 60% dos embriões machos, 50% das fêmeas e 40% das perdas gestacionais foram positivos para TSPY. A detecção de TSPY nas gestações de fêmeas possivelmente é resultante da persistência do microquimerismo de gestações anteriores. Nas gestações de clones transgênicos, observou-se a presença de mRNA e proteína GFP no endométrio, também indicando migração nesta região ou o transporte da GFP, e outros conteúdos do trofoblasto, para o epitélio materno. Nos placentônios, usando anticorpo anti-GFP pode-se ver a marcação positiva tanto no trofoblasto como no epitélio materno, possivelmente decorrente de liberação das CTGs no estroma endometrial após a fusão. As CTGs, quando em formação sincicial, têm a sua expressão de GFP diminuída, o que também foi observado, utilizando-se anticorpo anti-Qa2 (antígeno murino para MHC classe Ib). O epitélio materno e o trofoblástico também foram marcados para Qa2. Mediante as técnicas utilizadas, observamos que o microquimerismo pôde ser identificado nas gestações analisadas com o uso dos marcadores TSPY no sangue e o GFP nos tecidos placentários maternos. Este estudo mostra que na placenta bovina ocorre uma migração de células fetais além do epitélio materno e abre novas perspectivas para estudos das características da interação materno-fetal ainda pouco explorada nos bovinos. / The pregnancy success depends of adequate materno-fetal communication, that in some species are have a more intimate contact due migratory capacity of trophoblastic cells populations. In bovines, this mechanism is realized by trophoblast giant cells (TGC) with limited invasion until basal lamina of maternal epithelium. Besides this light invasion of TGCs, is possible to encounter circulate fetal cells in peripheral blood of pregnant cow, leading to fetal microchimerism. Beyond local and systemic sinalization and conformational changes, TGC migration is also dependent of immunologic tolerance of maternal epithelium that possess a downregulation of classe I MHC. In complement, the trophoblast express classe Ib MHC to inhibit natural killers cells activation. In this context, the objective of this work was to study the occurrence and contribute for knowledge cellular migration mechanisms in bovine palcenta, with Y-specific markers and a model of transgenic clone expressing GFP. The tested hypothesis was that fetal microchimerism observed by detection od TSPY gene in peripheral blood of cow pregnant of male embryo, and of GFP in maternal placental tissues associated by expression of class Ib MHC (Qa2) in materno-fetal interface. For this, 153 embryos produced by in vitro fertilization (IVF) were trasnfered, resulting in 34 male embryos and 31 female in day 62 of pregnancy, when recipient peripheral blood was collected. Among these pregnancies, 25 males, 4 females and 5 pregnancy losses (confirmed at 39 days of pregnancy by ultrassonography) was selected for TSPY detection. Also were produced pregnancies of transgenic cloned, embryos expressing GFP, with 30, 60 and 90 days of pregnancy that utilized for GFP mRNA and protein detection. In IVF pregnancies, 60% of male embryos, 50% of females and 40% of pregnancy losses were positive for TSPY. The detection of TSPY in female pregnancies possible is resultant of persistence of microchimerism of anterior pregnancies. In transgenic cloned pregnancies, was observed presence of GFP mRNA and protein in endometrium, also indicating migration in this region or GFP transport, and another trophoblast content, to maternal epithelium. In placentomes, using anti-GFP antibody, could be observed positive immunolabeling in trophoblast and maternal epithelium, possible due CTGs liberation in endometrial stroma after fusion. The CTGs, in syncytium formation, have a downregulation of GFP, that also be observed, utilizing anti-Qa2 antibody (murine antigen of classe Ib MHC). The maternal and trophoblastic epithelium also was Qa2 immunolabed. By utilized techniques, microchimerism could be indentified in analyzed pregnancies with use of markers for TSPY in maternal blood and GFP in maternal placental tissues. This study show that in bovine placenta occurs fetal cell migration further maternal epithelium and show new perspectives for studies of materno-fetal interaction characteristics until under explored in bovines.

O sucesso da gestação depende da adequada comunicação materno-fetal, que em algumas espécies têm um contato mais íntimo devido à capacidade migratória de populações de células trofoblásticas. Nos bovinos esse mecanismo é realizado pelas células trofoblásticas gigantes (CTGs), com invasão limitada até a lâmina basal do epitélio materno. Apesar dessa leve invasão das CTGs, é possível encontrar células fetais circulantes no sangue periférico da vaca gestante, levando ao microquimerismo fetal. Além de toda uma sinalização local e sistêmica e mudanças conformacionais, a migração das CTGs também é dependente da tolerância imunológica do epitélio materno que possui uma baixa expressão de MHC de classe I. Em contrapartida, o trofoblasto expressa MHC de classe Ib para impedir a ativação das células natural killers uterinas (uNK) contra ele mesmo. Neste contexto, o objetivo desse trabalho foi estudar a ocorrência e contribuir para o entendimento dos mecanismos da migração celular na placenta bovina, com marcadores exclusivos do cromossomo Y e de um modelo de clone transgênico expressando a proteína GFP. A hipótese testada foi que o microquimerismo fetal observado mediante a detecção do gene TSPY no sangue periférico da vaca gestante de embrião macho, e de GFP nos tecidos placentários maternos, associado à expressão de MHC classe 1b (Qa2) na interface materno-fetal. Para tanto, 153 embriões produzidos por fertilização in vitro (FIV) foram transferidos, resultando em 34 embriões machos e 31 fêmeas no dia 62 de gestação, quando foi realizada a coleta de sangue periférico da receptora. Dentre estas gestações, foram selecionadas de 25 machos, 4 fêmeas e 5 perdas gestacionais (confirmadas no D39 por ultrassonografia) para detecção de TSPY. Também foram produzidas gestações de clones transgênicos, expressando GFP com 30, 60 e 90 dias que foram utilizadas para a detecção de mRNA e a proteína GFP. Nas gestações de FIV 60% dos embriões machos, 50% das fêmeas e 40% das perdas gestacionais foram positivos para TSPY. A detecção de TSPY nas gestações de fêmeas possivelmente é resultante da persistência do microquimerismo de gestações anteriores. Nas gestações de clones transgênicos, observou-se a presença de mRNA e proteína GFP no endométrio, também indicando migração nesta região ou o transporte da GFP, e outros conteúdos do trofoblasto, para o epitélio materno. Nos placentônios, usando anticorpo anti-GFP pode-se ver a marcação positiva tanto no trofoblasto como no epitélio materno, possivelmente decorrente de liberação das CTGs no estroma endometrial após a fusão. As CTGs, quando em formação sincicial, têm a sua expressão de GFP diminuída, o que também foi observado, utilizando-se anticorpo anti-Qa2 (antígeno murino para MHC classe Ib). O epitélio materno e o trofoblástico também foram marcados para Qa2. Mediante as técnicas utilizadas, observamos que o microquimerismo pôde ser identificado nas gestações analisadas com o uso dos marcadores TSPY no sangue e o GFP nos tecidos placentários maternos. Este estudo mostra que na placenta bovina ocorre uma migração de células fetais além do epitélio materno e abre novas perspectivas para estudos das características da interação materno-fetal ainda pouco explorada nos bovinos. / The pregnancy success depends of adequate materno-fetal communication, that in some species are have a more intimate contact due migratory capacity of trophoblastic cells populations. In bovines, this mechanism is realized by trophoblast giant cells (TGC) with limited invasion until basal lamina of maternal epithelium. Besides this light invasion of TGCs, is possible to encounter circulate fetal cells in peripheral blood of pregnant cow, leading to fetal microchimerism. Beyond local and systemic sinalization and conformational changes, TGC migration is also dependent of immunologic tolerance of maternal epithelium that possess a downregulation of classe I MHC. In complement, the trophoblast express classe Ib MHC to inhibit natural killers cells activation. In this context, the objective of this work was to study the occurrence and contribute for knowledge cellular migration mechanisms in bovine palcenta, with Y-specific markers and a model of transgenic clone expressing GFP. The tested hypothesis was that fetal microchimerism observed by detection od TSPY gene in peripheral blood of cow pregnant of male embryo, and of GFP in maternal placental tissues associated by expression of class Ib MHC (Qa2) in materno-fetal interface. For this, 153 embryos produced by in vitro fertilization (IVF) were trasnfered, resulting in 34 male embryos and 31 female in day 62 of pregnancy, when recipient peripheral blood was collected. Among these pregnancies, 25 males, 4 females and 5 pregnancy losses (confirmed at 39 days of pregnancy by ultrassonography) was selected for TSPY detection. Also were produced pregnancies of transgenic cloned, embryos expressing GFP, with 30, 60 and 90 days of pregnancy that utilized for GFP mRNA and protein detection. In IVF pregnancies, 60% of male embryos, 50% of females and 40% of pregnancy losses were positive for TSPY. The detection of TSPY in female pregnancies possible is resultant of persistence of microchimerism of anterior pregnancies. In transgenic cloned pregnancies, was observed presence of GFP mRNA and protein in endometrium, also indicating migration in this region or GFP transport, and another trophoblast content, to maternal epithelium. In placentomes, using anti-GFP antibody, could be observed positive immunolabeling in trophoblast and maternal epithelium, possible due CTGs liberation in endometrial stroma after fusion. The CTGs, in syncytium formation, have a downregulation of GFP, that also be observed, utilizing anti-Qa2 antibody (murine antigen of classe Ib MHC). The maternal and trophoblastic epithelium also was Qa2 immunolabed. By utilized techniques, microchimerism could be indentified in analyzed pregnancies with use of markers for TSPY in maternal blood and GFP in maternal placental tissues. This study show that in bovine placenta occurs fetal cell migration further maternal epithelium and show new perspectives for studies of materno-fetal interaction characteristics until under explored in bovines.

No description available.

It was postulated that 3D cell culture models more accurately reflect the complex tissue physiology and morphology in comparison to 2D cell monolayers. Currently, there is a shortage of well-characterized and easily maintainable high-throughput experimental models of the human placenta. Here, we characterized three different 3D cultures (e.g., spheroids) derived from trophoblast cell lines and studied their functionality in comparison to primary fetal trophoblasts and placental tissue. The spheroid growth rates of JEG3, BeWo and HTR8/SVneo cell lines were similar among each other and were significantly larger in comparison to primary trophoblast spheroids. All spheroids exhibited migratory properties and shortest distances were registered for JEG3 spheroids. Even though all spheroids displayed invasive capabilities, only the invasive features of HTR8/SVneo spheroids resulted in specific branching. This was in agreement with the invasive properties of the spheroids obtained from primary trophoblasts. Human chorionic gonadotropin production was highest in JEG3 spheroids and only increased when stimulated with cAMP and forskolin in BeWo, but not HTR8/SVneo spheroids. The gene expression analysis confirmed that 3D trophoblast cell cultures and especially HTR8/SVneo spheroids showed considerable similarities with the gene expression profile of primary placental tissue. This study offers a broad characterization of 3D trophoblast spheroids that, in turn, can help in selecting the best model depending on the scientific question that needs to be answered.

Le placenta humain est un organe indispensable au bon déroulement de la grossesse. La villosité choriale est l’unité structurale et fonctionnelle du placenta. Elle est constituée essentiellement de cellules trophoblastiques. Les cytotrophoblastes extra-villeux (CTEV) présentent des propriétés invasives et assurent l’ancrage du placenta dans l’endomètre maternel. De plus, une apoptose physiologique assure le renouvellement des cytotrophoblastes tout au long de la grossesse.Le Facteur Préimplantatoire (PIF) est un peptide de 15 acides aminés, sécrété par des embryons viables. Le PIF exerce un effet autocrine positif sur le développement embryonnaire. Le PIF est également impliqué dans le contrôle de l’immunité et de l’inflammation dans divers types cellulaires.Au cours de ce travail de thèse, nous nous sommes intéressés aux rôles du PIF dans le développement placentaire humain. Dans un premier temps, nous avons caractérisé l’expression protéique du PIF dans des placentas humains de 1er et 3ème trimestre de grossesse.Nous avons montré que i) l’expression du PIF diminue au cours de la grossesse et ii) le PIF est majoritairement exprimé dans les CTEV.Dans un second temps, nous avons mis en évidence que le PIF i) favorise l’invasion trophoblaste et ii) inhibe l’apoptose des CTEV en régulant la voie de signalisation de p53.Par ailleurs, des altérations de l’invasion et de l’apoptose trophoblastiques sont associées à des pathologies de la grossesse telles que la pré-éclampsie et le retard de croissance intra-utérin. Ainsi, dans un dernier temps nous avons montré que l’expression du PIF est diminuée dans des placentas humains de 3ème trimestre issus de grossesses pathologiques par comparaison avec des grossesses normales.L’ensemble de ces résultats démontrent que le PIF est un nouvel acteur de la placentation humaine. De plus, le PIF pourrait être considéré comme un nouveau biomarqueur des pathologies de la grossesse. / Human placentation is a critical step in the establishment of a successful pregnancy. The chorionic villus constitutes the structural and functional unit of the placenta. The extravillous trophoblast (EVT) is a placental cell type that differentiates from the highly proliferative cytotrophoblast located at the base of the anchoring villous. EVT have invasive properties, essential for placenta anchoring in the endometrium and uterine artery remodeling. Moreover, programmed cell death is an active process required for normal trophoblastic cell turnover during pregnancy.PreImplantation Factor (PIF) is a 15-amino-acid peptide secreted by developing embryos. PIF exerts autotrophic and protective effects on the embryo. PIF is also implicated in the control of immune and inflammatory processes in various cell types.In this work, we aimed to determine the direct effects of PIF on human placental development.In a first part, we characterized PIF protein expression in first and third trimester human placentas. We showed that PIF protein expression i) decreased over the course of the pregnancy and ii) was higher in EVT compared to villous trophoblast.In a second part, we showed that PIF i) enhanced pro-invasive capacities and ii) prevented cell death by regulating p53 signaling pathway in human EVT.Dysregulation of trophoblastic invasion and apoptosis have been associated with pregnancy pathologies. Thereby, in a last part, we showed that PIF protein expression was lower in placentas from preeclampsia and intra-uterine growth restriction as compared with non-pathological placentas. Altogether, we highlighted for the first time, that PIF is a new positive regulator of placental functions. PIF could be considered as a novel biomarker of a favorable outcome of pregnancy.