<div>Rare circulating cells are becoming a subject of interest due to their potential clinical applications to replace invasive procedures. Due their low presence in blood (as low as 1 in 1 ml of blood) various platforms are developed to capture and isolate them. Common limitations of current platforms include the inability to process large volumes of blood without an initial volume reduction step such as centrifugation, reliance on a single antibody for the capture, and the difficulty of releasing and retrieving the captured cells with high purity. A rare cell retrieval platform with high throughput operation and high purity retrieval is needed to capture these rare cells by processing large volumes of blood.</div><div><br></div><div>In this thesis study, we have developed a two-tiered microchip system to capture and retrieve rare cells from blood samples with high purity. The first module of the system is a high throughput microfluidic interface that is used to immunomagnetically isolate targeted rare cells from whole blood, and discard > 99.999% of the unwanted leukocytes. The second module is a microwell array that furthers the purification by magnetically guiding each cell into a separate well concurrently, and allows individual retrieval of each cell. Even though the system we have developed is applicable to many fields pertaining to rare cell capture, here we demonstrate the proof-of-concept using model cell lines that represent circulating fetal trophoblasts. We describe the design, operation as well as the experimental characterization of the system. Our characterization results show that the process can be completed within 145 minutes from the very beginning till the retrieval of a target cell, and can provide efficiencies and purities that are as high as 100%. </div><div><br></div><div>In order to demonstrate a real-world use case for our device, we present preliminary experiments done with blood samples from pregnant women. We show that we are able to retrieve candidate fetal cells under 167 minutes. Future work will be focused on sequencing the candidate fetal cells retrieved from maternal samples to confirm their fetal origin as well as enhancing system performance in maximizing the number of cells captured.</div><div><br></div>

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.

Spiral-artery (SA) remodeling is a fundamental process during pregnancy that involves the action of cells of the initial vessel, such as vascular smooth-muscle cells (VSMCs) and endothelial cells, but also maternal immune cells and fetal extravillous trophoblast cells (EVTs). Mast cells (MCs), and specifically chymase-expressing cells, have been identified as key to a sufficient SA remodeling process in vivo. However, the mechanisms are still unclear. The purpose of this study is to evaluate the effects of the MC line HMC-1 and recombinant human chymase (rhuCMA1) on human primary uterine vascular smooth-muscle cells (HUtSMCs), a human trophoblast cell line (HTR8/SV-neo), and human umbilical-vein endothelial cells (HUVEC) in vitro. Both HMC-1 and rhuCMA1 stimulated migration, proliferation, and changed protein expression in HUtSMCs. HMC-1 increased proliferation, migration, and changed gene expression of HTR8/SVneo cells, while rhuCMA treatment led to increased migration and decreased expression of tissue inhibitors of matrix metalloproteinases. Additionally, rhuCMA1 enhanced endothelial-cell-tube formation. Collectively, we identified possible mechanisms by which MCs/rhuCMA1 promote SA remodeling. Our findings are relevant to the understanding of this crucial step in pregnancy and thus of the dysregulated pathways that can lead to pregnancy complications such as fetal growth restriction and preeclampsia.

La tumeur des cellules de la granulosa (GCT) représente 5% des cas de cancers ovariens chez la femme. Bien que considérées comme peu malignes, la mort survient dans 80% des cas suite à une recrudescence de la maladie. En dépit de ces statistiques sinistres, peu d’études ont été portées sur ce type de cancer. Le premier objectif de cette étude consistait à élucider les mécanismes moléculaires causant les GCT en démontrant l’implication de la voie de signalisation PI3K/AKT dans leur étiologie. Pour ce faire, nous avons employé la technologie Cre-Lox afin de cibler le gène Pten (antagoniste de cette voie) spécifiquement dans les cellules de la granulosa chez la souris. Ces souris (Ptenflox/flox;Amhr2cre/+) ont occasionnellement développé des GCT, soutenant notre hypothèse de l’importance de la voie PI3K/AKT dans leur étiologie. La voie WNT/CTNNB1 est une autre voie de signalisation qui a récemment été impliquée dans le développement des GCT. Dans le cadre de ce projet, nous avons également testé l’existence possible d’une synergie fonctionnelle entre les voies WNT/CTNNB1 et PI3K/AKT dans le développement de la maladie. Pour ce faire, nous avons créé le modèle transgénique Ptenflox/flox;Ctnnb1flox(ex3)/+;Amhr2cre/+, chez lequel les cellules de la granulosa présentant non seulement une désinhibition de la voie PI3K/AKT, mais aussi une suractivation de la voie WNT/CTNNB1. Tel que prédit, les souris Ptenflox/flox;Ctnnb1flox(ex3)/+;Amhr2cre/+ ont développé une forme de GCT beaucoup plus agressive que celle observée chez les femelles Ptenflox/flox;Amhr2cre/+. Spécifiquement, le développement des tumeurs se déclenchait plus tôt, leur croissance était beaucoup plus rapide, nous avons pu observer des métastases pulmonaires et la dissémination des cellules tumorales dans la cavité péritonéale, et la maladie était invariablement fatale avant l’âge de 8 semaines. Le modèle Ptenflox/flox;Ctnnb1flox (ex3)/+;Amhr2cre/+ a donc servi à démontrer l'existence d'une synergie entre les voies WNT/CTNNB1 et PI3K/AKT dans le développement de la GCT. De façon inattendue, les souris Ptenflox/flox;Amhr2cre/+ ont aussi présenté un phénotype de sous-fertilité qui n’était pas d’origine ovarienne. Il a récemment été démontré que la souche Amhr2cre dirige l’expression de Cre non seulement aux cellules de la granulosa, mais aussi au stroma utérin et au myomètre. Le second objectif de ce travail était donc de démontrer si et comment le phénotype d’infertilité chez les souris Ptenflox/flox;Amhr2cre/+ pouvait découler d’un défaut utérin. Lors de l'implantation, les cellules du stroma utérin se différencient en cellules déciduelles pour former la décidua maternelle (DM), qui se régresse ensuite par apoptose afin de faciliter l’invasion des cellules trophoblastiques. De plus, la DM, en collaboration avec le tissu foetal, recrute des uNKs dont le rôle est de remodeler les artères spiralées pour augmenter l’apport sanguin maternel vers le foetus en développement. Nous avons pu démontrer que l'utérus des femelles gestantes Ptenflox/flox;Amhr2cre/+ présentait une DM anormalement résistante à l'apoptose, moins de uNKs et des artères spiralées non-remodelées. Par conséquent, l’invasion des cellules du trophoblaste était restreinte, compromettant le développement et la survie de l'embryon. Nous avons donc établi pour la première fois l’importance de Pten lors de la décidualisation et de l’invasion du trophoblaste. / The granulosa cell tumor (GCT) represents up to 5% of all ovarian cancer cases in women. Although GCT is characterized as a low-grade malignancy, death occurs in approximately 80% of cases following to the disease’s recurrence. Despite these statistics, very few studies have been dedicated to increasing our understanding of the molecular mechanisms responsible for the development of GCT. The first objective of this work was to elucidate some of these molecular mechanisms by proving the involvement of the PI3K/AKT signaling pathway in the etiology of GCT. To this end, we used Cre-LoxP technology to conditionally target Pten (a PI3K/AKT signaling antagonist) in granulosa cells. These transgenic mice (Ptenflox/flox;Amhr2cre/+) occasionally developed GCT, supporting our hypothesis on the importance of the PI3K/AKT pathway in the etiology of this disease. The WNT/CTNNB1 signaling pathway has also recently been implicated in GCT development. We therefore postulated that a functional synergy may exist between the WNT/CTNNB1 and PI3K/AKT pathways in development of GCT. To test this, we created a second transgenic model (Ptenflox/flox;Ctnnb1flox(ex3)/+;Amhr2cre/+), in which the granulosa cells featured not only a disinhibition of the PI3K/AKT pathway but also a constitutive activation of the WNT/CTNNB1 pathway. As predicted, the Ptenflox/flox;Ctnnb1flox(ex3)/+;Amhr2cre/+ mice developed a form of GCT that was much more aggressive than the one observed in the Ptenflox/flox;Amhr2cre/+ model. Specifically, the tumors developed sooner, their growth was faster, pulmonary metastases and the seeding of tumor cells in the abdominal cavity were observed, and the disease was invariably fatal by 8 weeks of age. The Ptenflox/flox;Ctnnb1flox(ex3)/+;Amhr2cre/+ model therefore served to demonstrate a synergy between the WNT/CTNNB1 and PI3K/AKT pathways in GCT development. Unexpectedly, the majority of the Ptenflox/flox;Amhr2cre/+ females presented an infertility phenotype that was not caused by an ovarian defect. Recently, it has been shown that the Amhr2cre strain can direct the Cre activity not only to the granulosa cells but also to the uterine stroma and myometrium. The second objective of this work was therefore to determine if and how the infertility observed in Ptenflox/flox;Amhr2cre/+ mice could be caused by a uterine defect. During implantation, uterine stromal cells differentiate into decidual cells and form the maternal decidua (MD), which subsequently regresses by apoptosis to facilitate trophoblast invasion. In collaboration with the fetal tissue, the MD also recruits uNKs, which are responsible for spiral artery remodeling that increases maternal blood flow to the developing fetus. We were able to demonstrate that the uteri of pregnant Ptenflox/flox;Amhr2cre/+ females had a MD that was resistant to apoptosis, less uNKs, and spiral arteries that were not properly remodeled. Consequently, the trophoblast cell invasion was restricted, thereby compromising the development and survival of the embryo. We have therefore established for the first time the importance of Pten during the decidualization of the uterine stroma and trophoblast invasion.

Experimentos anteriores realizados em nosso laboratório indicam que o sucesso gestacional é afetado pela poluição atmosférica. O presente estudo teve como objetivo avaliar os efeitos biológicos associados a uma curva dose resposta das partículas de exaustão do diesel (PED) sobre o desenvolvimento embrionário inicial e o potencial de implantação, utilizando-se como modelo a fertilização in vitro e o cultivo embrionário de camundongos. No Experimento 1, encontrou-se um efeito negativo dose-dependente sobre o desenvolvimento embrionário inicial, o processo de eclosão, a alocação das células e a morfologia da massa celular interna (MCI) dos blastocistos. A análise post-hoc revelou que o desenvolvimento precoce do embrião não foi afetado pelas concentrações de 0,2 µg/cm2 ou 2 µg/cm2, mas foi significativamente afetado pela concentração de 20 µg/cm2 de PED. O processo de eclosão foi prejudicado pelas concentrações de 2 µg/cm2 e 20 µg/cm2. A alocação das células da MCI e a relação entre as células da MCI e do trofectoderma foram significativamente afetadas por todas as concentrações. Adicionalmente, observou-se um efeito negativo sobre a morfologia da MCI para as concentrações de 2 µg/cm2 e 20 µg/cm2. O Experimento 2, apesar de não mostrar efeito significativo sobre o potencial de implantação, evidenciado pela capacidade de adesão dos blastocistos e crescimento trofoblástico, revelou que a morfologia da MCI no dia 8 de cultivo, as taxas de viabilidade e de apoptose celular e a expressão de Oct4 e Cdx2 foram significativamente afetadas. O teste HSD-Tukey demonstrou que a presença de PED (0,2 µg/cm2 e 2 µg/cm2) durante o desenvolvimento embrionário aumentou significativamente a taxa de células em apoptose dos embriões tanto no dia 5 quanto no dia 8 de cultivo e, embora a proporção de células viáveis no dia 8 tenha sido prejudicada por ambas as concentrações, apenas a exposição a 2 µg/cm2 de PED diminuiu a viabilidade celular no dia 5. Por outro lado, tanto a concentração de 0,2 µg/cm2 como a de 2 µg/cm2 tiveram um efeito negativo significativo sobre a qualidade da MCI no dia 8 e a taxa de expressão de Oct4 nos blastocistos e aumentaram a porcentagem de células desses blastocistos expressando Cdx2, adicionalmente, a razão Oct4/Cdx2 dos embriões expostos a 0,2 µg/cm2 e 2 µg/cm2 foi significativamente menor. Frente a esses resultados, presumi-se que as PED poderiam estar envolvidas nos mecanismos que levariam à diminuição do sucesso reprodutivo observado em camundongos expostos à poluição atmosférica ambiental / Previous experiments conducted in our laboratory demonstrate that successful pregnancy is affected by air pollution. The aim of this study was to evaluate the biological effects associated with a dose-response curve of the diesel exhaust particles (DEP) on early embryonic development and implantation potential, using mice in vitro fertilization and culture embryo as model. In Experiment 1, we found a negative dose-dependent effect on the embryonic development, hatching process, cell allocation and morphology of inner cell mass (ICM) of blastocysts. A post-hoc analysis revealed that the early development of the embryo was not affected by concentrations of 0.2 g/cm2 or 2g/cm2, but was significantly affected by the concentration of 20 g/cm2 of DEP. The hatching process was impaired by concentrations of 2 g/cm2 and 20 g/cm2. Cell allocation of ICM and the ratio between cells of ICM and trophectoderm were significantly affected by all concentrations. Addicionaly, we observed a negative effect on ICM morphology was observed for the 2 µg/cm2 and the 20 µg/cm2 concentrations. Experiment 2, despite showing no significant effect on implantation potential, as evidenced by the adhesion ability and trophoblast outgrowth, revealed that ICM morphology on day 8 of culture, rates of cell viability and apoptosis, and expression of Oct4 and Cdx2 were significantly affected. The Tukey HSD test showed that presence of DEP (0.2 g/cm2 and 2 g/cm2) during embryonic development increased significantly the rate of apoptotic cells in embryos as on day 5 as on day 8 of culture, although the proportion of viable cells on day 8 was impaired by both concentrations, only exposure to 2 g/cm2 PED decreased cell viability on day 5. On the other hand, both the concentration of 0.2 g/cm2 such as 2 g/cm2 had a significant negative effect on the quality of ICM on the day 8 and the rate of expression of Oct4 on blastocysts, and increased the percentage of cells from these embryos expressing Cdx2, also, Oct4/Cdx2 ratio were significantly lower in the blastocysts derived from embryos exposed to 0.2 g/cm2 and 2 g/cm2¬ concentrations. Given these results, the suggestion is that DEP could be involved in the mechanisms that lead to decreased reproductive success observed in mice exposed to environmental pollution

Uterine NK (uNK) cells are a distinct subset of NK cells in the decidua of humans and rodents during pregnancy, which are essential for remodelling of the spiral arteries supplying the feto-placental unit. Similarly to peripheral NK cells, uNK cells express Natural Killer receptors (NKRs) that engage MHC class I molecules. Evidence from human genetic association studies suggests that, in the presence of allogeneic cognate paternal MHC class I ligands, inhibitory uterine NKRs are associated with disorders of pregnancy arising from impaired decidual vascular remodelling. Conversely, enhancement of human uNK cell activity through activating NKRs is associated with high birth weight. Evidence from mouse models corroborates that uNK cell activity is modulated by interactions between NKRs and MHC class I, but has largely focussed on the effect of paternal MHC. In this study, the contribution of maternal immunogenetic regulation of NK cell function to reproductive outcome was assessed independently of parental MHC disparity in mice. To evaluate the role of NKR genes in isolation, I used congenic B6.BALB-TC1 (TC1) mice that differ from C57BL/6 (B6) mice only within the region of chromosome six encoding NKRs that recognise MHC class I. Absence of a major inhibitory NKR for self-MHC, Ly49I, in TC1 mice causes a compensatory shift in the NKR repertoire expressed and preserves a majority subpopulation of educated NK cells. B6 and TC1 splenic and uterine NK cells are similarly functionally reactive and mature, and no significant differences could be detected in spiral arterial remodelling or fetal growth between these strains in MHC-syngeneic matings. This supports data from human immunogenetic studies showing that maternal uterine NKRs are not associated with differences in pregnancy outcome in the absence of novel paternal MHC class I ligands, and highlights the importance of maternal and paternal co-regulation of uNK cell activity during pregnancy. No mouse models of uNK cell activation are currently available with which to corroborate human immunogenetic associations between activating uterine NKRs and high birth weight. Male m157-transgenic (m157-Tg) mice, which ubiquitously express viral m157 glycoprotein ligands for the activating NKR Ly49H, were mated with B6 females. Exclusive expression of m157 glycoprotein by trophoblast improved placental efficiency, but did not enhance fetal growth. Some fertility clinics surmise that uNK cell activation initiates the pathogenesis of spontaneous abortion. It has been suggested that this may occur due to reduced expression by human uNK cells of miR-483-3p, which stimulates endogenous insulin-like growth factor (IGF)-1 production and uNK cell cytotoxicity in vitro. It is demonstrated here that neither miR-483-3p nor IGF-1 regulate murine NK cell development, maturation or function. No discernible reproductive phenotype is evident in miR-483 deficient females. It can be inferred that post-transcriptional control by miR-483 is not biologically relevant to murine NK cell function. Although m157-Tg mice may provide an interesting model to further study uNK cell-mediated placental adaptations, it remains important to identify a murine model of enhanced uNK cell function to corroborate human immunogenetic associations with high birth weight and to challenge the supposition that uNK cell activation is harmful to pregnancy.

La tumeur des cellules de la granulosa (GCT) représente 5% des cas de cancers ovariens chez la femme. Bien que considérées comme peu malignes, la mort survient dans 80% des cas suite à une recrudescence de la maladie. En dépit de ces statistiques sinistres, peu d’études ont été portées sur ce type de cancer. Le premier objectif de cette étude consistait à élucider les mécanismes moléculaires causant les GCT en démontrant l’implication de la voie de signalisation PI3K/AKT dans leur étiologie. Pour ce faire, nous avons employé la technologie Cre-Lox afin de cibler le gène Pten (antagoniste de cette voie) spécifiquement dans les cellules de la granulosa chez la souris. Ces souris (Ptenflox/flox;Amhr2cre/+) ont occasionnellement développé des GCT, soutenant notre hypothèse de l’importance de la voie PI3K/AKT dans leur étiologie. La voie WNT/CTNNB1 est une autre voie de signalisation qui a récemment été impliquée dans le développement des GCT. Dans le cadre de ce projet, nous avons également testé l’existence possible d’une synergie fonctionnelle entre les voies WNT/CTNNB1 et PI3K/AKT dans le développement de la maladie. Pour ce faire, nous avons créé le modèle transgénique Ptenflox/flox;Ctnnb1flox(ex3)/+;Amhr2cre/+, chez lequel les cellules de la granulosa présentant non seulement une désinhibition de la voie PI3K/AKT, mais aussi une suractivation de la voie WNT/CTNNB1. Tel que prédit, les souris Ptenflox/flox;Ctnnb1flox(ex3)/+;Amhr2cre/+ ont développé une forme de GCT beaucoup plus agressive que celle observée chez les femelles Ptenflox/flox;Amhr2cre/+. Spécifiquement, le développement des tumeurs se déclenchait plus tôt, leur croissance était beaucoup plus rapide, nous avons pu observer des métastases pulmonaires et la dissémination des cellules tumorales dans la cavité péritonéale, et la maladie était invariablement fatale avant l’âge de 8 semaines. Le modèle Ptenflox/flox;Ctnnb1flox (ex3)/+;Amhr2cre/+ a donc servi à démontrer l'existence d'une synergie entre les voies WNT/CTNNB1 et PI3K/AKT dans le développement de la GCT. De façon inattendue, les souris Ptenflox/flox;Amhr2cre/+ ont aussi présenté un phénotype de sous-fertilité qui n’était pas d’origine ovarienne. Il a récemment été démontré que la souche Amhr2cre dirige l’expression de Cre non seulement aux cellules de la granulosa, mais aussi au stroma utérin et au myomètre. Le second objectif de ce travail était donc de démontrer si et comment le phénotype d’infertilité chez les souris Ptenflox/flox;Amhr2cre/+ pouvait découler d’un défaut utérin. Lors de l'implantation, les cellules du stroma utérin se différencient en cellules déciduelles pour former la décidua maternelle (DM), qui se régresse ensuite par apoptose afin de faciliter l’invasion des cellules trophoblastiques. De plus, la DM, en collaboration avec le tissu foetal, recrute des uNKs dont le rôle est de remodeler les artères spiralées pour augmenter l’apport sanguin maternel vers le foetus en développement. Nous avons pu démontrer que l'utérus des femelles gestantes Ptenflox/flox;Amhr2cre/+ présentait une DM anormalement résistante à l'apoptose, moins de uNKs et des artères spiralées non-remodelées. Par conséquent, l’invasion des cellules du trophoblaste était restreinte, compromettant le développement et la survie de l'embryon. Nous avons donc établi pour la première fois l’importance de Pten lors de la décidualisation et de l’invasion du trophoblaste. / The granulosa cell tumor (GCT) represents up to 5% of all ovarian cancer cases in women. Although GCT is characterized as a low-grade malignancy, death occurs in approximately 80% of cases following to the disease’s recurrence. Despite these statistics, very few studies have been dedicated to increasing our understanding of the molecular mechanisms responsible for the development of GCT. The first objective of this work was to elucidate some of these molecular mechanisms by proving the involvement of the PI3K/AKT signaling pathway in the etiology of GCT. To this end, we used Cre-LoxP technology to conditionally target Pten (a PI3K/AKT signaling antagonist) in granulosa cells. These transgenic mice (Ptenflox/flox;Amhr2cre/+) occasionally developed GCT, supporting our hypothesis on the importance of the PI3K/AKT pathway in the etiology of this disease. The WNT/CTNNB1 signaling pathway has also recently been implicated in GCT development. We therefore postulated that a functional synergy may exist between the WNT/CTNNB1 and PI3K/AKT pathways in development of GCT. To test this, we created a second transgenic model (Ptenflox/flox;Ctnnb1flox(ex3)/+;Amhr2cre/+), in which the granulosa cells featured not only a disinhibition of the PI3K/AKT pathway but also a constitutive activation of the WNT/CTNNB1 pathway. As predicted, the Ptenflox/flox;Ctnnb1flox(ex3)/+;Amhr2cre/+ mice developed a form of GCT that was much more aggressive than the one observed in the Ptenflox/flox;Amhr2cre/+ model. Specifically, the tumors developed sooner, their growth was faster, pulmonary metastases and the seeding of tumor cells in the abdominal cavity were observed, and the disease was invariably fatal by 8 weeks of age. The Ptenflox/flox;Ctnnb1flox(ex3)/+;Amhr2cre/+ model therefore served to demonstrate a synergy between the WNT/CTNNB1 and PI3K/AKT pathways in GCT development. Unexpectedly, the majority of the Ptenflox/flox;Amhr2cre/+ females presented an infertility phenotype that was not caused by an ovarian defect. Recently, it has been shown that the Amhr2cre strain can direct the Cre activity not only to the granulosa cells but also to the uterine stroma and myometrium. The second objective of this work was therefore to determine if and how the infertility observed in Ptenflox/flox;Amhr2cre/+ mice could be caused by a uterine defect. During implantation, uterine stromal cells differentiate into decidual cells and form the maternal decidua (MD), which subsequently regresses by apoptosis to facilitate trophoblast invasion. In collaboration with the fetal tissue, the MD also recruits uNKs, which are responsible for spiral artery remodeling that increases maternal blood flow to the developing fetus. We were able to demonstrate that the uteri of pregnant Ptenflox/flox;Amhr2cre/+ females had a MD that was resistant to apoptosis, less uNKs, and spiral arteries that were not properly remodeled. Consequently, the trophoblast cell invasion was restricted, thereby compromising the development and survival of the embryo. We have therefore established for the first time the importance of Pten during the decidualization of the uterine stroma and trophoblast invasion.

Experimentos anteriores realizados em nosso laboratório indicam que o sucesso gestacional é afetado pela poluição atmosférica. O presente estudo teve como objetivo avaliar os efeitos biológicos associados a uma curva dose resposta das partículas de exaustão do diesel (PED) sobre o desenvolvimento embrionário inicial e o potencial de implantação, utilizando-se como modelo a fertilização in vitro e o cultivo embrionário de camundongos. No Experimento 1, encontrou-se um efeito negativo dose-dependente sobre o desenvolvimento embrionário inicial, o processo de eclosão, a alocação das células e a morfologia da massa celular interna (MCI) dos blastocistos. A análise post-hoc revelou que o desenvolvimento precoce do embrião não foi afetado pelas concentrações de 0,2 µg/cm2 ou 2 µg/cm2, mas foi significativamente afetado pela concentração de 20 µg/cm2 de PED. O processo de eclosão foi prejudicado pelas concentrações de 2 µg/cm2 e 20 µg/cm2. A alocação das células da MCI e a relação entre as células da MCI e do trofectoderma foram significativamente afetadas por todas as concentrações. Adicionalmente, observou-se um efeito negativo sobre a morfologia da MCI para as concentrações de 2 µg/cm2 e 20 µg/cm2. O Experimento 2, apesar de não mostrar efeito significativo sobre o potencial de implantação, evidenciado pela capacidade de adesão dos blastocistos e crescimento trofoblástico, revelou que a morfologia da MCI no dia 8 de cultivo, as taxas de viabilidade e de apoptose celular e a expressão de Oct4 e Cdx2 foram significativamente afetadas. O teste HSD-Tukey demonstrou que a presença de PED (0,2 µg/cm2 e 2 µg/cm2) durante o desenvolvimento embrionário aumentou significativamente a taxa de células em apoptose dos embriões tanto no dia 5 quanto no dia 8 de cultivo e, embora a proporção de células viáveis no dia 8 tenha sido prejudicada por ambas as concentrações, apenas a exposição a 2 µg/cm2 de PED diminuiu a viabilidade celular no dia 5. Por outro lado, tanto a concentração de 0,2 µg/cm2 como a de 2 µg/cm2 tiveram um efeito negativo significativo sobre a qualidade da MCI no dia 8 e a taxa de expressão de Oct4 nos blastocistos e aumentaram a porcentagem de células desses blastocistos expressando Cdx2, adicionalmente, a razão Oct4/Cdx2 dos embriões expostos a 0,2 µg/cm2 e 2 µg/cm2 foi significativamente menor. Frente a esses resultados, presumi-se que as PED poderiam estar envolvidas nos mecanismos que levariam à diminuição do sucesso reprodutivo observado em camundongos expostos à poluição atmosférica ambiental / Previous experiments conducted in our laboratory demonstrate that successful pregnancy is affected by air pollution. The aim of this study was to evaluate the biological effects associated with a dose-response curve of the diesel exhaust particles (DEP) on early embryonic development and implantation potential, using mice in vitro fertilization and culture embryo as model. In Experiment 1, we found a negative dose-dependent effect on the embryonic development, hatching process, cell allocation and morphology of inner cell mass (ICM) of blastocysts. A post-hoc analysis revealed that the early development of the embryo was not affected by concentrations of 0.2 g/cm2 or 2g/cm2, but was significantly affected by the concentration of 20 g/cm2 of DEP. The hatching process was impaired by concentrations of 2 g/cm2 and 20 g/cm2. Cell allocation of ICM and the ratio between cells of ICM and trophectoderm were significantly affected by all concentrations. Addicionaly, we observed a negative effect on ICM morphology was observed for the 2 µg/cm2 and the 20 µg/cm2 concentrations. Experiment 2, despite showing no significant effect on implantation potential, as evidenced by the adhesion ability and trophoblast outgrowth, revealed that ICM morphology on day 8 of culture, rates of cell viability and apoptosis, and expression of Oct4 and Cdx2 were significantly affected. The Tukey HSD test showed that presence of DEP (0.2 g/cm2 and 2 g/cm2) during embryonic development increased significantly the rate of apoptotic cells in embryos as on day 5 as on day 8 of culture, although the proportion of viable cells on day 8 was impaired by both concentrations, only exposure to 2 g/cm2 PED decreased cell viability on day 5. On the other hand, both the concentration of 0.2 g/cm2 such as 2 g/cm2 had a significant negative effect on the quality of ICM on the day 8 and the rate of expression of Oct4 on blastocysts, and increased the percentage of cells from these embryos expressing Cdx2, also, Oct4/Cdx2 ratio were significantly lower in the blastocysts derived from embryos exposed to 0.2 g/cm2 and 2 g/cm2¬ concentrations. Given these results, the suggestion is that DEP could be involved in the mechanisms that lead to decreased reproductive success observed in mice exposed to environmental pollution

Le placenta humain est un organe indispensable au maintien de la grossesse et au développement foetal. Son unité structurale et fonctionnelle est la villosité choriale constituée principalement de trophoblastes qui se différencient selon la voie villeuse endocrine ou extravilleuse invasive. Ces deux populations trophoblastiques sécrètent de l'hormone chorionique gonadotrope humaine (hCG), hormone indispensable à la grossesse. C'est une glycoprotéine constituée de deux sous-unités: la sous-unité alpha commune avec la LH, FSH et la TSH et la sous-unité beta, spécifique à chaque hormone, codée par un cluster de gênes regroupés en type I (gêne beta 7) et type II (gênes beta 3, 5 et 8). L'hCG est sécrétée dans le compartiment maternel où elle joue un rôle endocrine essentiel au maintien de la grossesse en stimulant la production de progestérone par l'ovaire. L'hCG joue également un rôle localement en stimulant la différenciation de chaque type de trophoblaste. Elle présente, dans le sang maternel, un pic de sécrétion à 10-12 semaines d'aménorrhée (SA), période ou le statut oxydatif placentaire change. En effet, les bouchons trophoblastiques obstruant la lumière des artères spiralées utérines se délitent à cette période, permettant l'entrée progressive du sang maternel dans la chambre intervilleuse. La pression en oxygène augmente de 18 mm/Hg (8-9 SA, 1er trimestre précoce) à 60 mm/Hg (12-14 SA, 1er trimestre tardif). Dans mon travail de thèse, j'ai cherché à mettre en évidence in situ et in vitro l'impact de ce changement de statut oxydatif sur la différenciation des trophoblastes villeux du 1er trimestre, et plus particulièrement sur l'expression des hCG beta de type I et de type II. J'ai ainsi mis en évidence que les trophoblastes villeux mononucléés du 1er trimestre précoce sécrétaient plus d'hCG beta de type I et II, fusionnaient plus rapidement et exprimaient un panel de facteurs de transcription différents par rapport aux trophoblastes villeux du 1er trimestre tardif. Dans un deuxième temps, j'ai comparé in vitro l'expression et la régulation des deux types d'hCG beta entre les trophoblastes villeux et extravilleux. J'ai montré que: 1) les trophoblastes villeux expriment plus d'hCG beta de type I et II que les trophoblastes extravilleux, 2) dans les deux cas l'hCG beta de type II est majoritaire et 3) PPAR gamma régule de façon opposée ces deux types d'hCG entre les trophoblastes villeux et extravilleux. Enfin j'ai mis en évidence que l'expression de ces deux types d'hCG était dérégulée dans la pré-éclampsie et le RCIU. L'étude des mécanismes impliqués dans la régulation des gênes codants pour l'hCG représente un enjeu important pour la compréhension de la différenciation du trophoblaste humain, du développement précoce du placenta et des pathologies de la grossesse. / The human placenta is an essential organ to maintain pregnancy and for foetal growth. Its structural and functional unit is the chorionic villous, which is mainly composed of cytotrophoblasts that follow two differentiation pathways: the endocrine villous and the invasive extravillous trophoblasts. These two trophoblastic subtypes secrete the human chorionic gonadotropin hormone (hCG), an essential hormone for trophoblast differentiation, placental development and pregnancy. hCG is a glycoprotein composed of two subunits: the alpha subunit, which is common to LH, FSH and TSH, and the beta subunit that confers hormone specificity. A gene cluster encodes the beta subunit, type I (CGB7) and type II (CGB3, 5 and 8), that code for two different proteins. hCG is detected in the maternal blood from the first week of pregnancy, with a peak level at 10-12 weeks of gestation (WG). During the first trimester the oxygen concentration in the intervillous space changes from about 2% (prior to 10 WG) to approximately 6-8% (after 12 WG) due to development of blood flow to the placenta. During my PhD work, I studied in situ and in vitro the impact of these different environments during the first trimester on villous cytotrophoblast differentiation, and more specifically on the type I and type II beta hCG gene expression. I showed that type I and type II beta hCG are more expressed in early first trimester cytotrophoblasts and that these cells exibit more fusion features and express a different panel of transcription factors compare to cells from late first trimester. In the second part of my work, I compared the expression and the regulation in vitro of the two types of beta hCG between villous and extravillous cytotrophoblasts. I demonstrated: 1) villous trophoblast express more type I and type II beta hCG compared to the extravillous trophoblast, 2) in both case type II hCG beta is the major form of beta hCG and 3) PPAR gamma differentially regulates type I and type II beta hCG expression in villous and extravillous trophoblasts. Lastly I showed that the expression of type I and type II beta hCG is deregulated in pre-eclampsia and FGR. The study of the mechanisms involved in hCG regulation represents an important issue for the understanding of human trophoblast differenciation and pregnancy pathophysiology.

ROS are frequently associated with deleterious effects caused by oxidative stress. Despite the harmful effects of non-specific oxidation, ROS also function as signal transduction molecules that regulate various biological processes, including stem cell proliferation and differentiation. Here we show that mitochondrial ROS level determines cell fate during differentiation of the pluripotent stem cell line P19. As stem cells in general, P19 cells are characterized by a low respiration activity, accompanied by a low level of ROS formation. Nevertheless, we found that P19 cells contain fully assembled mitochondrial electron transport chain supercomplexes (respirasomes), suggesting that low respiration activity may serve as a protective mechanism against ROS. Upon elevated mitochondrial ROS formation, the proliferative potential of P19 cells is decreased due to longer S phase of the cell cycle. Our data show that besides being harmful, mitochondrial ROS production regulates the differentiation potential of P19 cells: elevated mitochondrial ROS level favours trophoblast differentiation, whereas preventing neuron differentiation. Therefore, our results suggest that mitochondrial ROS level serves as an important factor that directs differentiation towards certain cell types while preventing others.