Preeclampsia (PE), a hypertensive disease of pregnancy, occurring at or after gestational week 20. PE can have life threating consequences for both the mother and the baby. PE is a highly heterogenous disease which makes it challenging to identify any effective therapeutic interventions. We previously discovered three molecular subclasses of PE disease. One of these subclasses is characterized by heightened placental inflammation (inflammation-driven PE). Since it is a newly identified form of PE, we currently do not know about the molecular mechanisms driving this inflammation-driven form of PE. Interestingly, we have observed that placentas from this inflammatory PE subclass uniquely express higher levels of NAD+ consuming enzymes- PARPs - and thus exhibit a decrease in NAD+ content. NAD+ is a regulator of cellular energy metabolism and mitochondrial function. Several studies in the non-pregnant populations suggested that pro-inflammatory disease conditions can trigger hyperactivation of NAD+ consuming enzymes causing a depletion in total NAD+ content, leading to mitochondrial dysfunction and organ failure. Thus, we tested the hypothesis that NAD+ depletion causes placental mitochondrial dysfunction in the inflammatory subclass of PE and that boosting NAD+ could prevent development of this form of placental disease. We aimed to profile PARP activity, NAD+ availability, and mitochondrial health in human cases of all three PE subclasses. We examined the causal relationship between inflammation and dysregulated NAD+ signalling in both an in vitro human trophoblast culture model and in a rodent model of inflammation-driven PE. We also evaluated the therapeutic potential of NAD+ booster, nicotinamide riboside (NR) to improve placental health and function in the rodent model of inflammation-driven PE. Our results suggest that along with increased activity of PARP enzymes and decreased NAD+ levels, human inflammatory PE placentas also exhibit decreased levels of mitochondrial proteins and increased oxidative DNA damage. Using an in vitro human placental (HTR8 cell line) inflammation model we showed that increasing NAD+ under an inflammatory condition improved trophoblast mitochondrial and cellular function. Using an in vivo LPS induced rat model of inflammation-driven PE, we demonstrated that NAD+ boosting during pregnancy improved placental mitochondrial function, reduced inflammation and oxidative stress. This subsequently resulted in improved pregnancy outcomes demonstrated by reduced maternal blood pressure, increased placental/fetal weights and increased fetal survival in the LPS model. Overall, this study identifies targeting NAD+ signaling as a promising intervention for PE. NAD+ boosting through NR has been tested in non-pregnant human populations and found to be safe and effective in enhancing NAD+ levels. Thus, findings of this thesis lay the ground to test NAD+ boosting strategies in PE patients in near future.

No description available.

Introduction: The widespread presence of plastics in our environment poses a growing concern as they may pose risks to human and environmental health. Microplastics (MPs) are small particles generated through fragmentation of larger plastic items. The presence of MPs particles has been reported in the human placenta, an organ essential for pregnancy and fetal development. The presence of MP contamination of the womb raises the possibility of adverse effects on the developing fetus with potential life-long consequences. This thesis seeks to investigate this issue through: 1) A review aimed to examine the current state of knowledge on the effects of exposure to MP on maternal and fetal health within the DOHaD framework; 2) A study conducted to confirm and further the reports of microplastics in human placentas through a study, in a Canadian setting, comparing MPs exposure to delivery methods. Methods: 1) A review was conducted of the current literature on microplastic contamination in human reproductive tissues, and the resulting reproductive consequences of exposure. 2) Placentas (n=10) were collected from singleton, uncomplicated pregnancies. Placentas were collected from vaginal (n=5) and cesarean section (n=5) deliveries within a plastics-reduced clinical setting. Placental tissue biopsies were micro-dissected under plastic-reduced conditions - from basal plate, chorionic villous and chorionic plate. Samples were chemically digested and filtered through glass microfiber filters and the retained particles were identified and characterized using Raman microspectroscopy. iii Results: 1) The review reports multiple lines of evidence that suggest that MP-exposure prior to or during pregnancy can contaminate various internal tissues (including those of the fetus) and may result in potential adverse effects on fertility, fetal development and long-term health of the exposed fetus. More importantly, the available evidence is limited and several significant gaps in knowledge were identified. 2) Microplastics composed of various polymer types were detected in placentas from both delivery types (vaginal or caesarian), with polyethylene being the most common. In addition, non-plastic foreign particles including graphite, lead oxide and black carbon were observed in a higher frequency than microplastics. Notably, both microplastic and non-microplastic particles were found in all placentas sampled with variations in the number of particles. Particles both plastic and non-plastic were observed in placenta regions of maternal and fetal circulation suggesting that these can pass through the placenta into fetal tissues. Conclusion: This thesis provides evidence that the human placenta can serve as a reservoir for the accumulation of a variety of foreign particles during pregnancy. The potential human health impacts of such particles in general or on fetal development, in particular, are unknown but is a critical question for future work to understand the health consequences of plastic pollution.

Among all organs, lungs are the last ones to grow and develop fully. As a result, extreme premature neonates may suffer from respiratory failure due to their immature lungs and will require respiratory support in the form of mechanical ventilation or extracorporeal membrane oxygenation (ECMO). In addition, extreme prematurity is recognized as the primary cause of neonatal morbidity and mortality. The conventional standard of care for respiratory support of preterm neonates with respiratory failure are invasive and may lead to long-term morbidities and complications. Hence, a non-invasive respiratory support technique named “Artificial Placenta” has been developed to address the issues and challenges associated with the current technologies. An artificial placenta type device is one designed to provide required oxygenation in room air via non-invasive access to the umbilical vessels without the need of any external pump. In this thesis, microfluidic and microfabrication technologies have been employed in the development of a pumpless neonatal lung assist device (LAD) for preterm neonates in two approaches: 1) design and develop novel microfabrication techniques to fabricate advanced microfluidic blood oxygenators with high gas exchange capacity and reduced form factor and 2) design and construct several modular LADs based on the oxygenators that were developed to fulfill the required gas transfer needs for these babies. The new microfluidic blood oxygenators with double-sided gas transfer channels were found to enhance oxygenation up to 343 % in room air and be easily scaled-up to achieve higher gas exchange capacities without a noticeable increase in priming volume. Furthermore, this microfabrication method has been utilized to make the largest all PDMS ultra-thin double-sided blood oxygenator with higher gas exchange capabilities. Also, a novel composite material made of PDMS and PTFE was introduced that conferred high flexibility to the oxygenator to decrease the form factor of such devices. This device was one of the first microfluidic blood oxygenators with enough flexibility to be deformed, bent, or rolled without limitation and losing its functionality. In order to satisfy the gas transfer need of these preterm neonates, few microfluidic-based modular LADs were constructed to support different birth weights up to 2 kg. The main design criteria for such a LAD in this research was low pressure drops (capable of being operated by a baby’s heart), an oxygen transfer of 1.3 – 1.9 mL min-1 kg-1 of body weight (or an increase in oxygen saturation level from ~ 75 % to ~ 100 % and ideally in room air), and low priming volume (less than 10 % of the total blood volume of a baby). These LADs first were evaluated in vitro to measure their gas exchange capacities and those which could meet needed oxygenation would be tested in vivo. For the first time, it was shown that a pumpless microfluidic-based LAD could support a newborn piglet and provide adequate oxygenation in room air or the oxygen-rich environment. The application of these microfluidic blood oxygenators was not only limited to preterm neonates but also can be used to develop LADs for adult patients. / Thesis / Doctor of Philosophy (PhD)

The etiology of many gestational disorders is still unknown. However, insufficient trans-placental passage of nutrients and wastes due to poor placentation is characteristic of several pathologies and may be due, in part, to altered function of placental mitochondria. Mitochondrial activity is essential in pregnancy because it sustains the metabolic activity of the placenta throughout gestation. Exposure to stressors that perturb processes governing placentation, including maternal drug use, can negatively impact fetal development. Cannabis use is prevalent during pregnancy. The psychoactive constituent, delta-9-tetrahydrocannbinol (THC), can cross the placenta to affect placental and fetal physiology. Importantly, cannabinoid receptors have been reported on trophoblast cells, and on mitochondria which are abundant in placentae. It has been reported that THC may target the mitochondria in various tissue types, including placental tissue, and alter its function. However, few studies have addressed the physiological control of mitochondria within the placenta, an organ that is critical for fetal growth and pregnancy maintenance. I investigated the role of mitochondria in trophoblast differentiation and syncytialization using rotenone, a complex I inhibitor. Subsequently, I investigated the role of THC on two important aspects of placentation – invasion and syncytialization – using placental trophoblast cells HTR8/SVneo and BeWo, respectively. In response to rotenone and THC, there was increased ROS production, oxidative stress, and altered transcriptional markers favouring mitochondrial fragmentation. Treatment with 20µM THC for 48 hours led to reduced mitochondrial respiration, ATP production and loss of mitochondrial membrane polarity. Critically, these THC-induced mitochondrial changes occurred concomitant with evidence of reduced trophoblast invasion and syncytialization. Furthermore, THC exposure reduced levels of human chorionic gonadotropin, human placental lactogen and insulin-like growth factor 2, which are growth factors necessary for fetal development. Placental mitochondrial dysfunction, particularly when THC-induced, may be critical in a range of gestational disorders which have important implications for maternal and fetal/offspring health. / Dissertation / Doctor of Philosophy (Medical Science) / Cannabis is commonly used by pregnant women. Fetal exposure to cannabis and its components can impair fetal growth and neurological development. These negative fetal outcomes may be the result of poor placental formation, due to placental cell exposure to cannabis and its psychoactive component, delta-9-tetrahydrocannabinol (THC). Importantly, THC can also target intracellular organelles, like the mitochondria which are known as the “powerhouses” of the cell. Few studies have investigated the direct effects of THC on placental development. The purpose of this study was to determine how THC exposure to placental cells may alter their function. We found that THC impaired processes that allow placental attachment to the uterus and form a protective barrier, and compromised mitochondrial function, which are important for placental formation. These findings serve to inform scientists and doctors, thus stimulating the creation of new ideas and methods to further explore the impact of THC on pregnancy outcomes.

People living near coal mines have raised concerns on how coal mining affects surrounding communities. Coal mining is a well-documented source of selenium inputs into the environment, and while there is considerable evidence demonstrating adverse effects of excess selenium on reproductive outcomes in fish, selenium toxicity in mammals is less understood. Studies in humans showed a correlation between high levels of selenium and increased adverse pregnancy outcomes, but the mechanisms behind this association are unclear. Importantly, many of the observed adverse pregnancy outcomes associated with high levels of selenium are linked to placental dysfunction. Mechanistically, supraphysiological concentrations of selenium have been shown to cause dysregulation of cortisol and induce ER stress. Balancing the amount of cortisol and ER stress during placental development is important, as a deficiency or surplus of either can cause aberrant placental development and/or placental dysfunction. Given that exposure to excess cortisol has been shown to induce ER stress, and ER stress has been shown to cause aberrant invasion and migration, which are important processes during placental development, the objective of my thesis is to test the hypothesis that excess selenium exposure impacts invasion and migration in first-trimester trophoblasts, and that these effects are mediated by the glucocorticoid and ER stress pathways. HTR-8/SVneo cells (human first-trimester trophoblasts) were exposed to environmentally relevant concentrations of sodium selenite (NaSe) for 24 or 48h. Cortisol was measured via ELISA, migration was measured via a wound-healing assay, and steady-state mRNA expression of genes involved in glucocorticoid homeostasis, ER stress, and invasion, migration, and angiogenesis were measured by qPCR. NaSe treatment caused increased cortisol and induced genes that are indicative of glucocorticoid receptor activation. NaSe also induced genes involved in ER stress as well as the regulation of invasion, migration and angiogenesis. NaSe also decreased migration as measured in the wound healing assay. When cells were co-treated with NaSe and either 1) metyrapone, an inhibitor of the enzyme responsible for synthesizing cortisol (CYP11B1), or 2) mifepristone, an antagonist of glucocorticoid receptor, the genes associated with increased cortisol did not decrease in the cells, suggesting that selenium may be activating the glucocorticoid pathway through alternate means. When the cells were co-treated with NaSe and ER stress inhibitor TUDCA, there was an attenuation of ER stress-related and invasion, migration and angiogenesis-related genes, as well as partial restoration of migration. Selenium treatment appears to have an impact on glucocorticoid activation, ER stress, and migration. While these results do not definitively identify the role that glucocorticoids play in the impact of selenium on migration, the results support the hypothesis that ER stress induced by selenium exposure partially affects migration in first-trimester trophoblasts cells. / Thesis / Master of Science (MSc)

La prééclampsie est une pathologie de la deuxième moitié de la grossesse d'étiologie encore méconnue et ne possédant aucun traitement efficace, mis à part l'accouchement provoqué. Il est reconnu que la cause première de cette pathologie provient du placenta, celui-ci relâchant des facteurs qui amènent une dysfonction des cellules endothéliales entraînant un stress oxydatif. Nous croyons que les glutathion peroxydases (GPx), qui sont une classe majeure d'enzymes antioxydantes contrôlant la peroxydation lipidique pourraient être impliquées dans le développement et le maintien de cette maladie. Dans cette étude, nous avons analysé l'expression des GPx dans les placentas de femmes normotendues et prééclamptiques. Nous avons découvert quatre formes de GPx dans le placenta humain et avons démontré que leurs expressions (ARNm) spatiales et tissulaires diffèrent. Nous avons aussi montré la localisation cellulaire de la protéine GPx-4 au niveau placentaire. Nous avons donc trouvé que les GPx sont différentiellement régulées dans le placenta suggérant un rôle complexe de celles-ci dans le contrôle de l'homéostasie placentaire.

Each year, 7.9 million babies are born with birth defects. Seventy percent of those could be prevented, ameliorated, or repaired; yet 3.2 million children still die by the age of three (March of Dimes Global Report 2006). We have found that non-specific maternal immune stimulation with the cytokine interferon-gamma (IFN-gamma) can successfully ameliorate some of these defects in the C57BL/6N mouse model. We have observed a reduction in the distal limb malformations syndactyly, polydactyly, and webbing by 47%, 100%, and 63% respectively when IFN-gamma is given 2 days prior to MNU administration. We have also observed that IFN-gamma works at the placental level to protect against MNU-induced damage. Trophoblast loss and associated cytokine alterations occur in gestation day (GD) 14 placenta following GD9 MNU exposure, showing that fetal-maternal communication can be hindered due to MNU. In the labyrinthine layer of the placenta, we observed multifocal fibrinous necrosis of endothelial cells due to MNU, however IFN-gamma almost completely protected the trophoblast and endothelial cells when given to the dam as an immune stimulant. To determine the genes participating in these processes, gene microarray studies were conducted. Hepatocyte growth factor (HGF), interleukin 1 beta (IL1Β), and insulin-like growth factor 2 (IGF2) were elucidated as genes that were significantly expressed in GD12 placenta. These genes are similar in that they are all connected to the Jak-Stat signaling pathway. These findings provide a possible mechanism for birth defect reduction by maternal immune stimulation with IFN-gamma in MNU-challenged mice. / Master of Science

Under år 2003-2009 utgjorde hypertensiva sjukdomar, såsom eklampsi och preeklampsi, 14,0% av värdens mödradödlighet. Preeklampsi kännetecknas vanligtvis av kliniska observationer av hypertoni och signifikant proteinuri i graviditetens andra trimester. Inom diagnostiken används en riskbedömningsprogramvara som kan beräkna vilken sannolikhetsgrad den havande kvinnan har för att utveckla preeklampsi. Förutom mätning av blodtryck och proteinuri har biomarkören placenta growth factor 1 (PlGF-1) visat ett högt prediktivt värde vid bedömningen. Studien syftade till att kartlägga och fördjupa sig i metoderna som analyserar biomarkören samt verifiera metoden för PlGF på instrumentet Brahms Kryptor compact plus. Fördjupningen baserades på granskning av vetenskapliga artiklar och resulterade i två manuella och tre automatiserade metoder. Metoden Quantikine användes i 47% av artiklarna och konstaterades som studiens golden standard. Vid jämförelse av metoderna sågs en lägre bakgrundsstörning, en högre sensitivitet samt en kortare analystid hos de automatiserade metoderna. Den laborativa verifieringen innefattade bestämning av överensstämmelse med externt laboratorium, beräkning av instrumentets provsmitta mellan höga och låga prov samt kvantifiering av inomserie- och mellanliggande precision. Verifieringen resulterade i en god överensstämmelse (r=0,953, p=0,327) med det externa laboratoriet, en konstaterad provsmitta på 0,04% samt en god precision inom leverantörens angivelser. / Between 2003-2009 hypertensive disorders as eclampsia and preeclampsia constituted 14.0% of the world’s maternal mortality. Preeclampsia characterize as clinical observations of hypertension and significant proteinuria in the second trimester of pregnancy. In diagnostics a risk assessment software is normally used to estimate the probability of developing the disorder. Besides calculating the blood pressure and proteinuria, the placenta growth factor 1 (PlGF-1) has proven to possess a high predictive value. The study’s aim was to chart the different methods used to quantify the biomarker and verify the method for PlGF on Brahms Kryptor compact plus. The recess was based on review of scientific articles and resulted in the findings of two manual and three automated methods. The method Quantikine was used in 47% of the articles and was seen as the golden standard of the study. When comparing the methods a lower signal to noise-ratio, a higher sensitivity and a shorter assay time was observed in the automated methods. The verification contained determination of compliance with an external laboratory, calculation of carry over and quantification of inter-assay and intra-assay precision. The verification resulted in a good compliance (r=0.953, p=0,327) with the external laboratory, a carry over at 0,04% and a good precision within the providers indication.

Le placenta assure les échanges materno-fœtaux et possède une fonction endocrine autonome. Les hormones placentaire lactogène (hPL) et chorionique gonadotrope (hCG) sont synthétisées par le syncytiotrophoblaste. A ce jour, les mécanismes impliqués dans le contrôle de la sécrétion de ces deux hormones ne sont pas connus. In vitro, l’influx d’ions Ca2+ entraîne une augmentation immédiate et soutenue de la libération d’hPL et d’hCG à partir d’explants de placentas à terme. En outre, l’élévation de la concentration extracellulaire en albumine, principale protéine maternelle circulante en contact direct avec le trophoblaste, stimule de manière immédiate et transitoire la libération d’hPL et d’hCG. L’objectif de nos travaux a été de vérifier la spécificité de l’activité sécrétagogue de l’albumine au niveau du placenta, de caractériser les messagers cellulaires potentiellement impliqués dans la libération d’hPL et d’hCG, et de définir l’interaction entre l’albumine et le trophoblaste, en utilisant des explants provenant de placentas humains à terme. Nos travaux démontrent que la riposte sécrétoire à l’albumine (5%, m/v) est largement mimée par d’autres agents colloïdaux (dextran et polygéline). Cette stimulation colloïdale de la libération d’hPL et d’hCG impliquerait une mobilisation de Ca2+ à partir de réserves intracellulaires. L’intervention de 3 messagers cellulaires a été envisagée: les IPs/DAG, l’AMPc, et le GMPc. Le fluorure de sodium, la forskoline, ou le nitroprussiate sodique, activateurs connus de la production respective des IPs, de l’AMPc, et du GMPc, augmentent de manière significative les taux placentaires de chacun de ces messagers, sans toutefois affecter la libération d’hPL ou d’hCG. De plus, l’élévation de la concentration extracellulaire en albumine (5%, m/v) ne modifie pas les taux des IPs, de l’AMPc et du GMPc dans les explants placentaires, tandis qu’elle stimule la sécrétion hormonale. Ces systèmes de signalisation, bien que fonctionnels au niveau du trophoblaste, ne joueraient donc pas un rôle majeur dans la régulation de la libération d’hPL et d’hCG. Nos résultats mettent en évidence une internalisation rapide d’albumine marquée, avec de l’125I ou de la fluorescéïne, dans le syncytiotrophoblaste. Une large fraction de cette albumine est recyclée, intacte, vers la circulation maternelle selon un processus sensible à l’abaissement de la température et indépendant du cytosquelette. L’albumine marquée restant dans les explants placentaires est partiellement dégradée. Trois mécanismes ont été envisagés pour expliquer ces mouvements d’entrée et de sortie de l’albumine au sein du placenta humain: l’endocytose médiée par l’albondine via les caveolae, le système des coated pits clathrine-dépendant, et l’endocytose médiée par la mégaline. Par immunohistochimie, nous avons montré que, dans le tissu placentaire, la caveoline-1, protéine caractéristique des caveolae, est localisée uniquement dans l’endothelium des capillaires fœtaux. La clathrine, au niveau des coated pits, et la mégaline se trouvent au contraire dans le syncytiotrophoblaste. La méthyl-b-cyclodextrine et l’hydrochlorure de chlorpromazine, inhibiteurs d’une endocytose dépendant de la clathrine, réduisent significativement l’internalisation placentaire de l’albumine marquée. Par contre, le DIDS ou le NPPB, susceptibles de perturber l’endocytose médiée par la mégaline, n’affectent pas la captation d’albumine marquée par les explants placentaires. L’albumine pénétrerait donc dans le syncytiotrophoblaste principalement par un processus clathrine-dépendant. La mégaline ne jouerait ici qu’un rôle mineur dans l’entrée de la protéine. Un tel processus de recyclage de l’albumine pourrait être similaire à celui décrit pour les immunoglobulines G au niveau du syncytiotrophoblaste. Ces mouvement d’entrée et de sortie de l’albumine ne semblent pas associés à la stimulation de la libération d’hPL et d’hCG par l’albumine. Ils pourraient par contre participer significativement, étant donné leur ampleur, à la nutrition fœtale. L’albumine est en effet un transporteur notoire d’ions et d’acides gras, molécules qui pourraient être acheminées au fœtus via le phénomène de recyclage placentaire de l’albumine mis en évidence par ce travail. / The human placenta is the site of all maternal-fetal exchanges, and is also an active endocrine organ. Placental lactogen (hPL) and chorionic gonadotrophin (hCG) hormones are synthesized by the syncytiotrophoblast. So far, the mechanisms involved in the regulation of both hormones secretion remain elusive. In vitro, calcium inflow causes an immediate and sustained rise in the hPL and hCG releases from human term placenta explants. Moreover, increasing the extracellular concentration of albumin, the major maternal plasma protein in direct contact with the human trophoblast, stimulates the hPL and hCG releases in an immediate and transient way. Our study have aimed to check the specificity of this secretory effect of albumin, to investigate the potential cellular messengers involved in the hPL and hCG releases, and to define the interaction between albumin and the throphoblast layer, using human term placenta explants. Our results indicate that the triggering effect of albumin (5%, w/v) is largely mimicked by two other colloidal agents (dextran and polygelin). This “colloidal” stimulation of the hPL and hCG releases would involve the mobilization of calcium from intracellular pools. Three cellular messengers have been considered to mediate this process: the IPs/DAG, the cAMP, and the cGMP. Sodium fluoride, forskolin, or sodium nitroprusside, known activators of respectively the IPs, cAMP, and cGMP production, significantly increase the placental content of each of those messengers, without modifying the hPL and hCG releases. In addition, raising the extracellular concentration of albumin does not cause any change in the placental level of IPs, cAMP, and cGMP, while stimulating the hormonal release. These three signaling pathways are thus functional in human term trophoblast but do not appear to significantly modulate the hPL and hCG secretions. Our findings show that albumin, labeled with 125I or with fluorescein, is rapidly internalized into the syncytiotrophoblast. Thereafter, the intact protein is largely recycled to the maternal circulation, through a temperature-sensitive and cytoskeleton-independent process. The labeled albumin remaining in placental explants is partially degraded. Three different mechanisms could participate to the albumin entry into the human placenta: the albondin-mediated endocytosis via the caveolae, the clathrin-dependent coated pits system, and the megalin-mediated endocytosis. Using immunohistochemistry, caveolin-1, marker of the caveolae, is localized in the endothelium of the fetal capillaries and not in the syncytiotrophoblast. By contrast, clathrin and megalin are observed only in the syncytiotrophoblast. Methyl-b-cyclodextrin, and chlorpromazine hydrochloride, known inhibitors of the clathrin-dependent endocytotic process, significantly reduce the placental uptake of labeled albumin. On the other hand, DIDS or NPPB, able to perturb the megalin-mediated endocytosis, do not affect the labeled albumin uptake. Thus, albumin seems to be internalized into the syncytiotrophoblast mainly through a clathrin-dependent mechanism. Megalin would only play a minor role in this process. Such movements of albumin in the human placenta may be similar to the recycling process reported for IgG at that site. The placental apical recycling of albumin is not associated to the albumin triggering effect on the hPL and hCG releases. This quantitatively significant internalization process may participate to the fetus’ nutrition. Indeed, Albumin carries ions and fatty acid, which could be brought to the fetus via the protein recycling evidenced by our study.