Dissertations / Theses on the topic 'Placenta – Physiologie'

Le placenta est un organe provisoire joignant la mère et le fœtus qui transfère l’oxygène et des nutriments de la mère au fœtus et permet l’évacuation de l’anhydride carbonique et des produits du métabolisme du fœtus. Le but de notre travail était d’étudier la fonction de transfert des tissus placentaires selon son développement en se basant sur les images ultrasonores. Nous avons développé au cours de ce travail une nouvelle approche de la classification du développement placentaire en ultrasons par des techniques de traitement d’images avancées basée sur une représentation par réseau neuronal. Le modèle réalisé par la transformée en ondelettes basé sur le réseau neuronal MLP représente donc un outil efficace et rapide répondant à nos critères et bien adapté à nos applications concernant l’étude de la maturation placentaire. L’application du modèle réalisée en cas de traitement d’images placentaires ouvre des portes intéressantes en terme de classification des grades placentaires afin d’identifier des stades de maturation autorisant la définition d’une maturation normale et de classes à risque
The placenta is a temporary organ joins the mother and the fœtus, which transfers oxygen from the mother to the foetus, allows the evacuation of the carbon dioxide and the products of foetus metabolism. The goal of our work is to study the transfer function of placental development using ultrasound images. A new approach is developed during this work to classify the placental development by image processing techniques based on supervised neural network. The realized model by the wavelet transform based on MLP neural network, represents an effective tool answering our criteria and adapted to our applications concerning the study of placental maturation. The realized model application in the event of placental image processing opens interesting doors in terms of placental grades classification in order to identify the stages of maturation, authorizing the definition of a normal maturation and an abnormal maturation

La prééclampsie (PE) est une pathologie obstétricale complexe associée à un défaut de placentation. Selon la littérature, le placenta anormalement développé libèrerait des facteurs qui induiraient une dysfonction endothéliale maternelle. Nous avons émis comme hypothèse qu’une accumulation de sorbitol, un sucre hyperosmotique produit par la voie des polyols, pourrait induire la libération par le placenta de sFlt-1, un facteur anti-angiogénique antagoniste du VEGF en PE. Nous avons comparé les niveaux d’expression d’ARNm et de protéines de AKR1B1 et SORD, les deux enzymes impliquées dans la voie des polyols, dans les placentas de mères ayant eu une grossesse normotensive (groupe témoin) ou prééclamptique par RT-PCR quantitatif et par immunobuvardage respectivement. Nous les avons ensuite localisées par immunohistochimie. Nos résultats suggèrent que la voie des polyols serait altérée au niveau de la membrane amniochorionique des placentas issus de grossesses prééclamptiques, ce qui pourrait favoriser une accumulation de sorbitol à l’interface fœto-maternelle.
Preeclampsia (PE) is a complex obstetrical pathology associated to a defective placentation. The abnormally developed placenta is believed to release factors causing a maternal endothelial dysfunction. We hypothesized that an accumulation of sorbitol, a hyperosmotic sugar produced through the polyol pathway, could induce the release by the placenta of sFlt-1, an antagonist of the angiogenic factor VEGF in PE. We compared mRNA and protein expression levels of AKR1B1 and SORD, the two enzymes of the polyol pathway, in placentas from normotensive (control) and PE pregnancies by quantitative RT-PCR and immunoblotting respectively. Then, we localized the two enzymes by immunohistochemistry. Our results suggest that polyol pathway is altered in amniochorionic membranes from PE pregnancies, and that this phenomenon would promote sorbitol accumulation at the foeto-maternal interface.

La naissance est une période à risque qui met en jeux de multiples mécanismes qui permettent une transition de la vie fœtale et la vie extra-utérine. Chaque année, dans les conséquences d'une mauvaise adaptation à la vie extra-utérine et la persistance de résistances vasculaires pulmonaires trop élevées, 1 million de nouveau-né décède dans les 24 premières heures de vie. Dix pourcents des nouveau-nés requièrent une assistance médicale en salle de naissance. Le clampage du cordon ombilical retardé entre 60 et 180 secondes après la naissance est désormais recommandé pour toutes les situations où le nouveau-né, à terme comme prématuré, s'adapte bien à son nouvel environnement diminuant entre autres, le risque d'anémie ferriprive des premiers mois de vie. La hernie de coupole diaphragmatique (HCD) est une malformation cardio-pulmonaire secondaire à un défaut de fermeture du muscle diaphragmatique. Elle entraine une mortalité élevée et responsable d'un trouble de l'adaptation à la vie extra-utérine. Dans les situations de réanimation en salle de naissance, du fait du manque de données physiologiques et cliniques, il n'est pas encore recommandé de maintenir les échanges fœto-placentaires en parallèle de l'initiation de la réanimation. Dans ce travail de thèse, nous posons l'hypothèse que le placenta puisse participer à l'oxygénation et à la décarboxylation du nouveau-né le temps que la circulation cardio-pulmonaire du nouveau-né se mette en place. Le but de ce travail est d'étudier la physiologie de l'hémodynamique et des échanges gazeux transplacentaires lors d'une réanimation à cordon intact (RCI) dans un modèle d'agneau sain et dans un modèle d'agneau porteur de hernie diaphragmatique. Les objectifs étaient (1) de présenter l'étude clinique « CHIC » évaluant l'impact de la RCI chez le nouveau-né porteur de HCD, (2) mettre en place le modèle expérimental d'agneau HCD, (3) explorer la faisabilité et la durée maximale d'une réanimation à cordon intact chez l'agneau, (4) étudier l'évolution de l'hémodynamique et des échanges gazeux transplacentaires au cours d'une RCI dans un modèle d'agneau sain et porteur d'une HCD. Nous avons démontré que l'hémodynamique fœto-placentaire pouvait était stable (débits veineux ombilicaux, résistances vasculaire transplacentaires) jusque 1 heure après la mise en place d'une RCI. Dans un modèle d'agneau hernie diaphragmatique, où l'échangeur pulmonaire ne permet pas d'assurer normalement une augmentation rapide de la pression partielle artérielle en oxygène (PaO2), le placenta permettait d'assurer une oxygénation et une décarboxylation tout au long de la réanimation avec un apport en oxygène par le placenta stable pendant 1 heure (2,7 [2,2-3,3] ml/kg/min). A l'inverse, dans un modèle physiologique, le maintien d'une circulation placentaire est associé à une diminution de la pression artérielle systémique de l'ordre de 20% comparée au groupe hernie diaphragmatique (p<0,05). L'augmentation de la PaO2 dans ce groupe est associée avec une diminution des apports en oxygène par le placenta. Le clampage du cordon entraine dans ce groupe une élévation de la PaO2 et une diminution de la capnie. L'ensemble de ces travaux apporte une base physiologique essentielle à la pratique d'une réanimation à cordon intact et souligne l'importance de stratégies de réanimation individualisées en fonction des conditions cliniques spécifiques
Birth is a critical period during which numerous mechanisms are engaged to enable the transition from fetal to extrauterine life. Each year, due to poor adaptation to this transition and the persistence of elevated pulmonary vascular resistance, 1 million newborns die within the first 24 hours of life. Ten percent of newborns require medical assistance in the delivery room. Delayed umbilical cord clamping, between 60 and 180 seconds after birth, is now recommended in all situations where the newborn, whether full-term or premature, adapts well to the new environment. This practice notably reduces the risk of iron deficiency anemia in the first months of life.Congenital diaphragmatic hernia (CDH) is a cardiopulmonary malformation caused by a defect in the closure of the diaphragm, leading to high mortality and impairing adaptation to extrauterine life. In delivery room resuscitation scenarios, the lack of physiological and clinical data has not yet allowed for the recommendation of maintaining feto-placental circulation alongside the initiation of resuscitation.In this thesis, we hypothesize that the placenta could contribute to oxygenation and decarboxylation of the newborn until the cardio-pulmonary circulation is established. The aim of this work is to study the physiology of hemodynamics and transplacental gas exchange during intact cord resuscitation (ICR) in a healthy lamb model and in a lamb model with CDH. The specific objectives were: (1) to present the clinical study “CHIC” evaluating the impact of ICR in newborns with CDH; (2) to establish an experimental lamb model of congenital diaphragmatic hernia; (3) to explore the feasibility and maximum duration of intact cord resuscitation in this model; and (4) to study the evolution of hemodynamics and transplacental gas exchange during ICR in both healthy and CDH lamb models.We demonstrated that feto-placental hemodynamics (umbilical venous flow, transplacental vascular resistance) remained stable up to one hour after the initiation of ICR. In the lamb model with diaphragmatic hernia, where the pulmonary exchange system cannot adequately increase arterial partial oxygen pressure (PaO2), the placenta provided sufficient oxygenation and decarboxylation throughout the resuscitation, with stable placental oxygen delivery for one hour (2.7 [2.2-3.3] ml/kg/min). Conversely, in the physiological model, maintaining placental circulation was associated with a 20% decrease in systemic arterial pressure compared to the CDH group (p<0.05). The increase in PaO2 in this group was associated with a decrease in placental oxygen delivery. Cord clamping in this group led to an increase in PaO2 and a decrease in carbon dioxide levels. These findings provide an essential physiological basis for the practice of intact cord resuscitation and highlight the importance of individualized resuscitation strategies based on specific clinical conditions

The placenta is a transient organ, essential for the growth and development of the fetus throughout pregnancy. This temporary organ brings maternal and fetal blood circulation into close proximity, which allows for the exchange of oxygen, carbon dioxide, waste, and other essential nutrients. Despite constant influences by internal and external factors, the human placenta has a defined life span of approximately 280 days. From conception, through to term, the placenta undergoes chronological aging, which is regulated by a range of cellular processes. Advanced placental aging and cellular senescence have been known to contribute to the pathophysiology of preterm birth, fetal growth restriction and may cause an increased risk of stillbirth. However, the molecular mechanisms behind placental aging are still poorly understood. As a key regulator of cell homeostasis, mitochondria have been recognised as an important mediator of age-related disease processes through the production of reactive oxygen species which activate mechanisms that induce cellular senescence. Currently, we do not understand the molecular link between cellular aging processes and the role of mitochondria in chronological and pathological placental aging. Therefore, this research aimed to, 1) identify key areas of mitochondrial physiology that change with placental development, 2) characterise a set of markers that define aging in the human placenta, 3) assess the role of the mitochondria in the placenta as it develops throughout a healthy pregnancy, 4) to measure the chosen markers of aging in placentas complicated by pregnancy pathologies. Chapter 1 presents a comprehensive review of the literature to date and highlights key gaps in our current knowledge. It sets the scene for the experimental chapters to follow. Chapter 2 provides the details of the methods and materials that have been used in the laboratory to generate the data presented in the results chapters. Chapter 3 explored the molecular changes within healthy and pathological placentas, through analysing large publicly available datasets. This chapter aimed to establish an understanding of placental aging and mitochondrial physiology through measuring mitochondrial biogenesis, dynamics, mitophagy, apoptosis and senescence transcripts. Furthermore, this chapter measured altered transcripts in an additional validation cohort consisting of placentas affected by term, preterm, post-term and FGR pregnancies. This study was a large-scale investigation across multiple datasets that identified altered TOMM2020, MFN1, and MFN2 expression throughout preterm, post-term and FGR pregnancies, which may be a contributing factor to placental insufficiency. It established key markers that influence mitochondrial physiology in placental aging, which informed future studies in this thesis. Chapter 4 focused on understanding healthy aging by measuring mitochondrial and senescent changes in term and post-term placenta. Post-term placentae from healthy pregnancies selectively retain highly functioning mitochondria through increases in mitochondrial dynamics proteins MFN1, MFN2 and mitochondrial complex specific proteins. This study directly associated mitochondrial adaptions with increases in cellular senescence in the placenta and may be the reason why some post-term pregnancies are healthy, whilst others turn pathological. These findings have helped to expand our knowledge of the role of mitochondria and healthy aging in the placenta. The current literature on placental aging has focused on comparing the differences between two timepoints in pregnancy, or healthy and complicated pregnancies. The reason being that it is nearly impossible to ethically collect healthy placental tissue from early in pregnancy. Even when these samples are collected, they are inherently impacted by factors which lead to early termination. Therefore, Chapter 5 used placental samples from an established rodent ontogeny model that were collected between mid- and term gestation, without pathologies. These placentas were used to understand the role of mitochondrial biology, senescence, and the ER and in the developing placenta. Markers associated with mitochondrial biogenesis, dynamics and senescence were differentially altered in healthy placentas collected throughout gestation, which was different to what was identified in previous chapters. Therefore, throughout different stages of pregnancy, mitochondria function differently compared to placentas from post-term and growth restricted pregnancies. Lastly, Chapter 6 measured the most differentially expressed genes from previous chapters in placentas complicated by preterm, term, post-term, fetal growth restriction (FGR), preterm preeclampsia (PE), FGR/ PE pregnancies. The aim of this chapter was to utilise MetaboAnalyst data software to identify relationships between genes related to mitochondria, ER and cellular senescence. This study revealed that placentas complicated by pathologies, PE and FGR have tremendously different transcription patterns, compared to the healthy controls. Although this study only investigated a small number of genes in a relatively small sample size, it revealed that the TOMM20/PARK2 ratio is a promising marker to discriminate between healthy placenta and placenta that have been affected by pregnancy pathologies. Overall, the findings in this thesis highlight the importance of mitochondrial alterations and cellular senescence within chronological and pathological aging of the placenta. Whilst the exact mechanisms of mitochondrial aging in the placenta still requires further investigation, MFN1, MFN2, TOMM20 and PARK2 are promising markers of placental aging and should be investigated in further models of placental insufficiency. This work provides the foundation for future work in mitochondrial aging within the human placenta.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Pharmacy & Med Sci
Griffith Health
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Le Bisphénol A (BPA) est un perturbateur endocrinien dont les effets développementaux observés chez les rongeurs soulèvent la question du risque pour la santé humaine relatif à une exposition fœtale au BPA. L’objectif de cette thèse est de déterminer les mécanismes toxicocinétiques impliqués dans l’exposition fœtale au BPA. La caractérisation in vivo dans un modèle intégratif ovin des expositions maternelles et fœtales au BPA et à ses métabolites ont permis d’identifier le transfert placentaire et le métabolisme fœto-placentaire comme les déterminants majeurs de l’exposition fœtale au BPA. Le transfert bidirectionnel du BPA à travers le placenta humain se fait par diffusion passive conduisant à un rapport maximal des concentrations plasmatiques de BPA libre entre le fœtus et sa mère de 1. En revanche, la perméabilité placentaire du BPA-G est très limitée, en particulier dans le sens materno-fœtal. Les activités de conjugaison hépatique du BPA ont été faibles chez le fœtus ovin à un stade précoce de gestation et ont augmenté au cours du développement. Par ailleurs la réactivation des conjugués du BPA mise en évidence ex vivo dans les gonades fœtales ovines pourrait conduire à une exposition locale au BPA actif. L’ensemble de ces données suggère que le début de la gestation pourrait représenter une fenêtre critique d’exposition au BPA
Bisphenol A (BPA) an endocrine disruptor interfering with developmental processes in rodents, raises the question of risk for human health related to fetal exposure to BPA. The goal of this work was to determine the toxicokinetic mechanisms involved in fetal exposure to BPA. The disposition of BPA and its metabolites in the maternal-placental-fetal unit in an in vivo ovine model enabled us to identify the placental transfer and the fetal-placental metabolism as the major determining factors of fetal exposure to BPA. Bidirectional placental transfer of BPA occurs by passive diffusion leading to a ratio of free BPA between the fetal and maternal plasma concentrations of about 1. By contrast, the permeability of BPA-G is very limited, particularly in materno-to-fetal direction. The hepatic conjugation activities were very low in ovine fetus at an early stage of development and increased throughout pregnancy. Hydrolysis of BPA conjugates observed ex vivo into fetal ovine gonads could lead to local exposure to native BPA. Altogether, these results suggest that the early stage of pregnancy is a critical window of exposure for the developing fetus

Placenta is a transient association of the fetal and maternal tissues, that develops during pregnancy, in most viviparous animals. The evolution of placenta ensured the development of the fetus inside the womb of the mother, providing a protected environment for the development of the fetus, and preventing the loss of progeny due to unfavorable environmental conditions. Because it is strategically poised at the maternal and fetal interface, the placenta is ideally suited to carry out alimentary, respiratory and excretory functions for the developing fetus. In addition, it serves as an immunological barrier preventing the rejection of the fetal semi-allograft, by the maternal immune system. Furthermore, the placenta elaborates a variety of protein, polypeptide and steroid hormones. These include growth factors, growth factor receptors, neuropeptides, opioids, progesterone and estrogen, whose secretion is dependent on the gestational age of the placenta and its differentiation status. The human placenta, adapts itself remarkably to cater to the changing requirements of the developing fetus. For instance, during the first trimester of pregnancy, the placenta is an actively dividing, a highly invasive and a rapidly differentiating organ; while near term, it represents a fully differentiated and a non-invasive unit. Furthermore, the placenta of the first trimester and that at term differ in their hormone profiles, extents of apoptosis, expression of several transcription factors, etc. This dramatic change in the phenotype of the human placenta can be considered to be the outcome of an intrinsically programmed pattern of differentiation, which may be referred to as the functional differentiation of the placenta. It may be hypothesized therefore, that this functional differentiation could be brought about by the differential expression of genes in the first trimester and the term placenta. The objectives of the present study were: 1. To gain an insight into this process of " functional differentiation” by investigating the differential expression of genes in the two developmentally distinct stages during gestation, viz. during the first trimester and at term. 2. To understand the functional relevance of the differentially expressed genes. A general introduction of the human placenta, describing the importance of differential expression in modulating placental function, is discussed in chapter 1. The functions of the human placenta along with a brief description of its development and differentiation are also briefly described. A Differential Display RT-PCR-based (DD RT-PCR) approach, using total RNA from the first-trimester and term placental villi, was employed to display the differentially expressed genes in the first trimester and the term placenta. The display so generated was used to identify a few differentially expressed cDNAs. This study was aimed at understanding the functional significance of the transcripts which were identified from the display, rather than just concentrate on documenting the differences in the gene expression patterns in the first trimester and the term placental tissue. A detailed description of the methodology adopted for performing DD-PCR using placental tissue, discussing the advantages and disadvantages of using differential display PCR, is described in chapter2. The use of DD-PCR for studying differential gene expression in the human placenta was validated by the finding that one of the cDNAs that was differentially expressed in the first trimester placental tissue, is a fragment of β-hCG cDNA. It is well documented that the differential expression of the β-subunit of hCG (human chorionic gondatrophin) during the first eight weeks of gestation is the rate limiting step in the synthesis and secretion of the functional hormone, which comprises the α and the β-subunits. Furthermore, the use of the model system viz., the first trimester and term placental tissue, was also validated for carrying out DD-PCR by ensuring that all placental samples used for DD analysis were free of endometrial contamination. A detailed description of optimization and validation of DD-PCR in human placental tissues is given in chapter 2. Cloning and sequencing of yet another cDNA from the first trimester differential display revealed that it is T-Plastin. T-Plastin is a member of a family of proteins that are involved in actin-bundling. Northern blot analysis and immunohistochemical studies using an antibody generated to a peptide corresponding to human T-Plastin, confirmed its differential expression and localization in the first trimester placenta. Considering the fact that several carcinomas show enhanced expression of T-Plastin, we tested the hypothesis that its differential expression is correlated with the proliferative potential of the first-trimester placenta It was observed that the first-trimester tissue expressed high levels of beta-actin as compared to the term placental tissue. This is in agreement with the up-regulation of beta-actin following mitogenic stimulation/proliferation and during neoplastic transformation or transformation-associated invasive behaviour of cells, two characteristic features shared by the early placenta with cancerous tissues. Based on our studies and available information in the literature, it is proposed that T-Plastin expression in the first trimester placenta is a growth-associated phenomenon which is partially responsible for the tumor-like phenotype of the first trimester tissue. Studies carried out with the partial T-Plastin cDNA clone that was isolated from the first trimester differential display, are presented in chapter 3. Sequencing of yet another cDNA clone identified from the term placental differential display, T-18 revealed that it had no homology to any known sequence in the nucleotide or est databases. The sequence corresponding to this clone was submitted to the GenBank and was assigned an accession number- AF089811. The differential expression of T-18 was confirmed by Northern blot analysis and RT-PCR analysis. Attempts were made to isolate the full-length cDNA corresponding to T-18 from a commercially available library from Clontech. However, repeated trials to identify the clone corresponding to T-18 did not yield any positive results. However, a genome database search revealed that T-18 was a portion of a large contig contained in chromosome 15. Analysis of the annotated gene sequences in and around the region in which T-18 is located in chromosome 15, revealed that there are very few ests reported in this contig and quite a few repeat sequences reported. Interestingly, it was observed that 6 kb downstream of the region in which T-18 is located, there was an est that had homology to a Bcl-2 precursor protein (an evolutionarily conserved, anti-apoptotic protein, capable of conferring protection against death-inducing signals) and the death adaptor protein, CRADD {Caspase and RIP adapter with death domain). Further updating of the ests in the database might probably be of help in the identification of the full-length cDNA corresponding to T-18 and confirm as to whether T-18 is a part of the gene/gene cluster that comprises the afore-mentioned est. An account of the identification and cloning of T-18 from the term placenta and the attempts to isolate the full-length cDNA clone corresponding to T-18 from a term placental cDNA library, is described in chapter 4. In the absence of any information on the identity of T-18, a study to understand the functional significance of T-18 expression was carried out. Since it was not possible to carry out studies pertaining to the temporal expression of T-18 throughout gestation on the human placenta for ethical reasons, alternate animal/organ models were employed to study T-18 expression. Rat placenta and rat Corpus Luteum (CL) were chosen as alternate models for studying T-18 expression as these two organs/tissues underwent dynamic changes in their function throughout pregnancy. For instance, it is well known that CL is the primary source of progesterone for maintaining pregnancy in the rat and that the progesterone secreting capacity of the luteal cells peak on day 16 of gestation and decline thereafter. Interestingly, a common feature among all the tissues that were chosen for investigating the regulation of T-18 expression, is the fact that they underwent apoptosis with increase in gestational age. The expression of T-18, in tissues exhibiting increased incidence of apoptosis suggested that T-18 maybe an apoptosis-associated gene. Using an explant culture model it was demonstrated that placental villi when cultured in vitro underwent spontaneous apoptosis and that the levels of T-18 message increased, under these conditions. Furthermore, this spontaneous induction of apoptosis in explant cultures could be blocked when villi were cultured in the presence of superoxide dismutase, a free radical scavenging enzyme. In addition, the expression of T-18 was shown to be modulated following treatment with SOD, or in response to oxidative stress. These studies clearly indicate a role for T-18 in placental apoptosis and moreover, implicate the usefulness of explant culture to examine the molecular mechanisms involved in placental apoptosis. Furthermore, the expression of the anti- and pro-apoptotic genes, bcl-x and bax respectively, were investigated, in an attempt to elucidate the signalling pathway(s) that led to the activation of an important downstream protease, caspase-3, in placental apoptosis. The present study revealed that induction of apoptosis in the placenta in vitro involved a bcl/bax independent, caspase-3 dependant pathway. The validation of an explant culture model for studying placental apoptosis and data pertaining to the role of T-18, bcl-x, bax and CPP32 in placental apoptosis, in response to oxidative stress, are presented in chapter 5. The last section titled general discussion summarizes the work carried out in this study and proposes a model for the apoptotic mechanism(s) that may be operating in placenta In conclusion, the present study has led to the identification of two developmentally-regulated factors, T-Plastin and T-18 in the first trimester and term placenta, respectively. The differential expression of these genes, in addition to several other molecular players, is proposed to be responsible for the overall functional differentiation of the placenta through the course of gestation.

Placenta is a transient association of the fetal and maternal tissues, that develops during pregnancy, in most viviparous animals. The evolution of placenta ensured the development of the fetus inside the womb of the mother, providing a protected environment for the development of the fetus, and preventing the loss of progeny due to unfavorable environmental conditions. Because it is strategically poised at the maternal and fetal interface, the placenta is ideally suited to carry out alimentary, respiratory and excretory functions for the developing fetus. In addition, it serves as an immunological barrier preventing the rejection of the fetal semi-allograft, by the maternal immune system. Furthermore, the placenta elaborates a variety of protein, polypeptide and steroid hormones. These include growth factors, growth factor receptors, neuropeptides, opioids, progesterone and estrogen, whose secretion is dependent on the gestational age of the placenta and its differentiation status. The human placenta, adapts itself remarkably to cater to the changing requirements of the developing fetus. For instance, during the first trimester of pregnancy, the placenta is an actively dividing, a highly invasive and a rapidly differentiating organ; while near term, it represents a fully differentiated and a non-invasive unit. Furthermore, the placenta of the first trimester and that at term differ in their hormone profiles, extents of apoptosis, expression of several transcription factors, etc. This dramatic change in the phenotype of the human placenta can be considered to be the outcome of an intrinsically programmed pattern of differentiation, which may be referred to as the functional differentiation of the placenta. It may be hypothesized therefore, that this functional differentiation could be brought about by the differential expression of genes in the first trimester and the term placenta. The objectives of the present study were: 1. To gain an insight into this process of " functional differentiation” by investigating the differential expression of genes in the two developmentally distinct stages during gestation, viz. during the first trimester and at term. 2. To understand the functional relevance of the differentially expressed genes. A general introduction of the human placenta, describing the importance of differential expression in modulating placental function, is discussed in chapter 1. The functions of the human placenta along with a brief description of its development and differentiation are also briefly described. A Differential Display RT-PCR-based (DD RT-PCR) approach, using total RNA from the first-trimester and term placental villi, was employed to display the differentially expressed genes in the first trimester and the term placenta. The display so generated was used to identify a few differentially expressed cDNAs. This study was aimed at understanding the functional significance of the transcripts which were identified from the display, rather than just concentrate on documenting the differences in the gene expression patterns in the first trimester and the term placental tissue. A detailed description of the methodology adopted for performing DD-PCR using placental tissue, discussing the advantages and disadvantages of using differential display PCR, is described in chapter2. The use of DD-PCR for studying differential gene expression in the human placenta was validated by the finding that one of the cDNAs that was differentially expressed in the first trimester placental tissue, is a fragment of β-hCG cDNA. It is well documented that the differential expression of the β-subunit of hCG (human chorionic gondatrophin) during the first eight weeks of gestation is the rate limiting step in the synthesis and secretion of the functional hormone, which comprises the α and the β-subunits. Furthermore, the use of the model system viz., the first trimester and term placental tissue, was also validated for carrying out DD-PCR by ensuring that all placental samples used for DD analysis were free of endometrial contamination. A detailed description of optimization and validation of DD-PCR in human placental tissues is given in chapter 2. Cloning and sequencing of yet another cDNA from the first trimester differential display revealed that it is T-Plastin. T-Plastin is a member of a family of proteins that are involved in actin-bundling. Northern blot analysis and immunohistochemical studies using an antibody generated to a peptide corresponding to human T-Plastin, confirmed its differential expression and localization in the first trimester placenta. Considering the fact that several carcinomas show enhanced expression of T-Plastin, we tested the hypothesis that its differential expression is correlated with the proliferative potential of the first-trimester placenta It was observed that the first-trimester tissue expressed high levels of beta-actin as compared to the term placental tissue. This is in agreement with the up-regulation of beta-actin following mitogenic stimulation/proliferation and during neoplastic transformation or transformation-associated invasive behaviour of cells, two characteristic features shared by the early placenta with cancerous tissues. Based on our studies and available information in the literature, it is proposed that T-Plastin expression in the first trimester placenta is a growth-associated phenomenon which is partially responsible for the tumor-like phenotype of the first trimester tissue. Studies carried out with the partial T-Plastin cDNA clone that was isolated from the first trimester differential display, are presented in chapter 3. Sequencing of yet another cDNA clone identified from the term placental differential display, T-18 revealed that it had no homology to any known sequence in the nucleotide or est databases. The sequence corresponding to this clone was submitted to the GenBank and was assigned an accession number- AF089811. The differential expression of T-18 was confirmed by Northern blot analysis and RT-PCR analysis. Attempts were made to isolate the full-length cDNA corresponding to T-18 from a commercially available library from Clontech. However, repeated trials to identify the clone corresponding to T-18 did not yield any positive results. However, a genome database search revealed that T-18 was a portion of a large contig contained in chromosome 15. Analysis of the annotated gene sequences in and around the region in which T-18 is located in chromosome 15, revealed that there are very few ests reported in this contig and quite a few repeat sequences reported. Interestingly, it was observed that 6 kb downstream of the region in which T-18 is located, there was an est that had homology to a Bcl-2 precursor protein (an evolutionarily conserved, anti-apoptotic protein, capable of conferring protection against death-inducing signals) and the death adaptor protein, CRADD {Caspase and RIP adapter with death domain). Further updating of the ests in the database might probably be of help in the identification of the full-length cDNA corresponding to T-18 and confirm as to whether T-18 is a part of the gene/gene cluster that comprises the afore-mentioned est. An account of the identification and cloning of T-18 from the term placenta and the attempts to isolate the full-length cDNA clone corresponding to T-18 from a term placental cDNA library, is described in chapter 4. In the absence of any information on the identity of T-18, a study to understand the functional significance of T-18 expression was carried out. Since it was not possible to carry out studies pertaining to the temporal expression of T-18 throughout gestation on the human placenta for ethical reasons, alternate animal/organ models were employed to study T-18 expression. Rat placenta and rat Corpus Luteum (CL) were chosen as alternate models for studying T-18 expression as these two organs/tissues underwent dynamic changes in their function throughout pregnancy. For instance, it is well known that CL is the primary source of progesterone for maintaining pregnancy in the rat and that the progesterone secreting capacity of the luteal cells peak on day 16 of gestation and decline thereafter. Interestingly, a common feature among all the tissues that were chosen for investigating the regulation of T-18 expression, is the fact that they underwent apoptosis with increase in gestational age. The expression of T-18, in tissues exhibiting increased incidence of apoptosis suggested that T-18 maybe an apoptosis-associated gene. Using an explant culture model it was demonstrated that placental villi when cultured in vitro underwent spontaneous apoptosis and that the levels of T-18 message increased, under these conditions. Furthermore, this spontaneous induction of apoptosis in explant cultures could be blocked when villi were cultured in the presence of superoxide dismutase, a free radical scavenging enzyme. In addition, the expression of T-18 was shown to be modulated following treatment with SOD, or in response to oxidative stress. These studies clearly indicate a role for T-18 in placental apoptosis and moreover, implicate the usefulness of explant culture to examine the molecular mechanisms involved in placental apoptosis. Furthermore, the expression of the anti- and pro-apoptotic genes, bcl-x and bax respectively, were investigated, in an attempt to elucidate the signalling pathway(s) that led to the activation of an important downstream protease, caspase-3, in placental apoptosis. The present study revealed that induction of apoptosis in the placenta in vitro involved a bcl/bax independent, caspase-3 dependant pathway. The validation of an explant culture model for studying placental apoptosis and data pertaining to the role of T-18, bcl-x, bax and CPP32 in placental apoptosis, in response to oxidative stress, are presented in chapter 5. The last section titled general discussion summarizes the work carried out in this study and proposes a model for the apoptotic mechanism(s) that may be operating in placenta In conclusion, the present study has led to the identification of two developmentally-regulated factors, T-Plastin and T-18 in the first trimester and term placenta, respectively. The differential expression of these genes, in addition to several other molecular players, is proposed to be responsible for the overall functional differentiation of the placenta through the course of gestation.

The placenta is a unique example of a complex organ that has evolved independently more than 115 times in amniotes (birds, reptiles, and mammals). Placentae exchange materials including respiratory gasses, nutrients, and hormones, between the mother and embryo. I aimed to identify the physiological and ecological factors that contribute to the evolution of placentation in vertebrates, focusing on the genetic mechanisms underpinning the evolution of placental functions, and the role of parent-offspring conflict in the evolution of placental traits. Given placentation has evolved independently many times, I assessed whether convergent evolution of placental functions is underpinned by the same genes in mammals and reptiles. I used high throughput sequencing to identify the gene expression patterns that facilitate placental functions in the placentotrophic southern grass skink (Pseudemoia entrecasteauxii).I show that hormone production occurs in the embryonic membranes of amniotes, and the production of growth factors by embryonic placental tissues is an exaptation of ancestrally expressed genes. I show that embryonic hormone production is a mechanism by which embryos can manipulate the function of the placenta, and regulate placental nutrient transport. By investigating the physiology and genetic underpinning of placental functions in reptiles, I provide fundamental data for understanding the evolution of viviparity and placentation in a lineage independent of viviparous mammals. I identify key similarities and differences between reptile and mammal pregnancy that outline the limits to which comparisons between the two lineages can be made. Finally, I show that parent offspring conflict is unlikely to play a role in the evolution of nutrient transport mechanisms in the placenta, but may be a driving force in the shift from lecithotrophic (a reliance on egg yolk for embryonic development) to placentotrophic nutrient provisioning through pregnancy.

La surexpression de protéines d’efflux, et tout particulièrement la P-glycoprotéine, est impliquée dans la multidrug résistance. Dans cette thèse, nous démontrons que le vandetanib, inhibiteur de tyrosine kinase, est à la fois substrat et inhibiteur de la P-glycoprotéine et qu’il est capable de réverser in vitro la résistance à la doxorubicine liée à la surexpression de la P-glycoprotéine.Nous nous sommes également intéressés à l’étude du passage transplacentaire du vandetanib et nous montrons que ce médicament traverse la barrière placentaire
Overexpression of ABC transporters, especially P-glycoprotein, is involved in multidrug resistance. In this study, we demonstrate that vandetanib, a tyrosine kinase inhibitor, is both substrate and inhibitor of P-glycoprotein and is able to reverse in vitro resistance to doxorubicin, linked to overexpression of P-glycoprotein.We also studied the placental transfer of vandetanib and we show that this drug crosses the placenta

Mycotoxin contamination in food commodities is an age-old problem. Due to the detrimental impact of mycotoxins on human health, exposure to mycotoxins and their health implications have been increasingly recognized. Ochratoxin A (OTA), one of the mycotoxins, has been found to cause diverse toxicities in animals, with potential impact on human health. OTA has been reported to be teratogenic and interfere with steroidogenesis in vivo. Chronic exposure of pregnant women to OTA may be hazardous for the human foetus, especially when endocrine and developmental toxicities are taken into consideration. Accordingly, in the first part of this project, I hypothesized that OTA may interfere with enzymes involved in human placental steroidogenesis. By evaluation of human placental 3β–hydroxysteroid dehydrogenase/isomerase (3β-HSD) at both mRNA and protein (hormonal) levels, my results showed that OTA could up-regulate 3β-HSD1 expression in human placental cells with concentration relevant to human exposure. This study is the first to report the endocrine disruption potential of OTA in human placental cells. As several mycotoxins have been demonstrated previously to cross human placental barrier and OTA has been associated with developmental toxicity in vivo, I further hypothesized that OTA may be transferred through human placenta and accumulate in foetal compartment. In the second part of this project, human perfused placenta was used to investigate the placental toxicokinetics of OTA using concentrations found in serum of pregnant women. Findings from this study clearly showed that the transfer of OTA through term human placenta was minimal, contradicting the existing epidemiological studies reporting higher foetal OTA levels than maternal. This is the first study where transplacental kinetics of OTA has been studied in human perfused placenta. To assess the relevance of the study findings, it is very important to provide information on maternal OTA exposure during pregnancy. Currently there is limited information regarding OTA exposure of pregnant women. The third part of this project aimed at evaluating the frequency and level of exposure to OTA in pregnant women from Egypt, where exposure to dietary mycotoxins is common due to the environmental conditions. Biomonitoring of both serum and urinary OTA levels showed that more than 70% of pregnant women were exposed to OTA with a geometric mean of 0.27 ng/ml in serum and 37.21 pg/mg creatinine in urine indicating frequent exposure of this subpopulation. As an ultimate aim, maternal-foetal risk assessment served as a conclusive part of this project to predict and evaluate both maternal and foetal risk of exposure to OTA during pregnancy. Data from the exposure of pregnant women in Egypt to OTA were further ultilized to conduct maternal-foetal risk assessment in relation to OTA exposure. Based on the refined Klaassen equation for exposure estimation during pregnancy and the benchmark dose approach for risk assessment, this subpopulation of pregnant women generally was not exposed to OTA in a high-risk manner. However, considering the suspected chronic exposure beginning from early pregnancy with high foetal susceptibility and diverse toxic effects, and in particular the potential endocrine disruption of OTA, keeping OTA exposure to a minimum is recommended.
published_or_final_version
Biological Sciences
Doctoral
Doctor of Philosophy

The effects of various steroid hormones on human chorionic gonadotropin (hCG) production and placental viability were investigated using an explant culture model of human placenta.
Placental hCG production was assessed using two different methods: (a) hCG concentrations in media recovered from cultures were measured by radioimmunoassay and (b) tissue levels of hCG in cultured placentae were determined immunohistochemically; both were evaluated before and after exposure to steroid hormones. In first trimester placentae, progesterone and dehydroepiandrosterone (DHEA) increased hCG concentrations both in collected medium and levels in cultured placentae. Estradiol increased the levels of hCG in tissues but not in media. Cortisol increased concentrations in media but did not alter tissue levels. Testosterone decreased hCG levels in media, but had no effect on hCG placental content. In third trimester cultures, progesterone and DHEA were the only hormones studied which increased concentrations of hCG in media; estradiol, cortisol and testosterone had no effect. Progesterone, estradiol and DHEA, alone or in combination, extended the viability of first trimester placental explant cultures from approximately 7 to 30 days. There was a significant relationship between placental viability and tissue hCG levels (r = 0.73, P $<$ 0.001). The concentrations of hCG, progesterone and estradiol in human placentae were determined at various times through gestation. These studies suggest that a temporal relationship exists between the placental levels of hCG and these steroids, and that they may be significant determinants of growth and differentiation of the placenta in vivo. Furthermore, these investigations support the hypothesis that hCG production by the placenta is subject to paracrine regulation by steroid hormones.

Production of hormones in the ovary is controlled by endocrine, paracrine, autocrine and intracrine influences. Similar controls may exist in the placenta. I wished to investigate the involvement of second messengers in the action of hormones in control of hormonogenesis in rat ovary and human placenta. The second messengers involved in the action of gonadotropin-releasing hormone (GnRH) and prostaglandin (PG) F₂[formula omitted] were investigated in rat granulosa and luteal cells. As well, the endocrine role of GnRH in the placenta and the possible second messengers involved were investigated. Monolayer cultures of rat granulosa and luteal cells and human placental cells were prepared. Rat granulosa cells were mechanically dispersed; rat luteal cells were enzymatically dispersed with collagenase and DNase. Rat granulosa cells were treated during the first 24 hours in culture; rat luteal cells were treated up to 3 days after dispersion. Radioimmunoassay of medium was used to determine the effect of treatments on hormone production. Studies which examined the effect of hormones on the intracellular free calcium concentration ([Ca²⁺]i) in single cells using the calcium sensitive fluorescent dye, Fura-2, were done in monolayer rat granulosa and luteal cell cultures. Human placental cells, from first trimester and term placentae, were dispersed using trypsin-DNase or collagenase-DNase. Cells were cultured for 2 days prior to treatment. The effects of treatments on production of steroid (progesterone and estrogen), glycoprotein (human chorionic gonadotropin; hCG) and protein (human placental lactogen; hPL) hormones were determined by radioimmunoassay of the medium. In rat granulosa and luteal cell cultures, I examined the effect of a number of hormones and second messengers. Effects of follicle-stimulating hormone (FSH), luteinizing hormone (LH), cyclic adenosine monophosphate (cAMP), GnRH and PGF₂[formula omitted] on ovarian hormonogenesis have been previously reported. Changes in cytosolic free calcium concentrations ([Ca²⁺]i) in response to PGF₂[formula omitted] were measured in single rat granulosa and luteal cells. I found that in 34% of granulosa cells, and 53% of luteal cells, there was a 3 to 4 fold increase in resting [Ca²⁺]i within 30 seconds of administration of PGF₂[formula omitted]. Many cells which responded to PGF₂[formula omitted] also responded to GnRH (39% of granulosa cells; 67% of luteal cells). The immediate source of the increased [Ca²⁺]i appeared to be common intracellular stores. No change in hormone production in response to GnRH in placental cell cultures was seen. Trypsin dispersion may have damaged cell surface receptors, therefore the effect of second messengers on hormone production in these cultures was examined. In term and first trimester trophoblast cultures, I observed the following effects with 8-bromo-cyclic adenosine monophosphate (8-br-cAMP): inhibited estrogen production from the supplied androgen precursors; stimulated hCG production; stimulated hPL production in first trimester placental cell cultures (hPL was not measured in enough term cultures to determine the effect of 8-br-cAMP), and stimulated progesterone production. I also investigated the effects of activators and inhibitors of the phosphoinositide (PtdIns(4,5)P₂) breakdown second messenger pathway (TPA, A23187, arachidonic acid); no effects of these agents were seen. Other hormones suspected of having endocrine, paracrine or autocrine effects in the placenta were tested without effect. I conclude that GnRH and PGF₂[formula omitted] cause increases in [Ca²]i in rat ovarian cells, from common intracellular stores of calcium, and that the production of hormones by the human placenta may be under regulation of an agent or agents which induce production of cAMP.
Medicine, Faculty of
Obstetrics and Gynaecology, Department of
Graduate

Bibliography: 161-199.
[xxvi], 199, [151] leaves, [7] leaves of plates : ill. (some col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Characterises the normal otogeny of the cellular composition and structure of placentomes in sheep, their relationship to the macroscopic parameters of placentome size and morphology, and the effect of experimental and natural restriction of implantation on the growth and development of placentomes between mid and late gestation.
Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 1995

The hypothalamic component of HPA–axis CRH is also produced by the placenta and it has been postulated to control a “placental clock” during pregnancy which determines the onset of parturition. However, the role of CRH in early trophoblast biology is currently unknown. In this thesis we investigated the role of CRH/CRH receptor in early placentation using the fusigenic choriocarcinoma cell line BeWo. These cells expressed functional CRH-R coupled to MAPK signalling cascades. In this cellular model, CRH was shown to promote survival and to induce cell proliferation, however in the presence of differentiation-inducing signals (such as forskolin) CRH further augmented up-regulation of fusogenic gene expression and hCG secretion induced by forskolin. Therefore, placental CRH appears to influence placenta biology. Finally, it was shown for the first time that CRH actions were sensitive to TLR4 activation raising the possibility that the innate immune system is an important regulator of placental CRH biological actions.

These studies have quantified human placental development throughout 10-41 weeks of `normal' gestation, and compared the structure of term organs delivered at low (400m) and high (3600m) altitudes in Bolivia. Quantitative analyses of histological preparations were performed using stereological techniques. Salient structural parameters were then combined with physicochemical constants (gleaned from the literature) to estimate the oxygen conductances of the maternal and fetal erythrocytes and plasmas, the trophoblast and the stroma. These were combined to yield Dp, the total morphometric diffusing capacity for oxygen (in ml O₂/min/torr). During the third trimester, villous trees expand predominantly by the elaboration of terminal villi. This increases the surface areas and decreases the thicknesses of the trophoblast and the stroma (the two compartments which offer the greatest resistance to oxygen diffusion). The sources of expansion of the villous trees may lie in the transformation of immature intermediate villi into stem and mature intermediate villi. Hypoxically stimulated capillary elongation within mature intermediate villi may be involved in initiating the production of new terminal villi. It is interesting that the total length and surface area of terminal villi were maintained in highland organs while those of larger diameter villi were significantly lower than in lowland controls. Total villous surface area was significantly reduced in highland organs while capillary surface was comparable to lowland estimates. However, reductions in harmonic mean trophoblast and stromal thicknesses helped to conserve the conductance of the trophoblast and to increase that of the stroma above lowland values. Dp increased significantly but specific Dp remained constant throughout gestation. Although the main stressor at high altitude is hypobaric hypoxia, no significant altitudinal differences in Dp were found. The intrauterine growth retardation consistently observed and high altitudes therefore occurs despite placental adaptations which maintain Dp at lowland values.

Little is known about the factors stimulating placental progesterone (P4) production at the time of the luteo-placental shift (6-8 weeks post-conception). To explore the regulatory mechanism, the effects of various steroids and peptides on the production of P4 by placental explants were studied.
In early placental explant culture P4 production was stimulated by 19-nortestosterone (19-NT), androstenedione (A-dione), 5$ alpha$-androstane-3$ alpha,$17$ beta$ diol (3$ alpha$-diol) and 5$ alpha$-androstane-3$ beta,$17$ beta$ diol (3$ beta$-diol). Of all the compounds tested, 19-NT had maximal effect. At term, P4 production was stimulated only by 3$ beta$-diol. 19-NT and A-dione were poorly aromatized in early placental explants compared to another androgen (Androst-5-ene-3$ beta,$17$ beta$ diol).
In accord with the above observations, placental levels of 19-NT and A-dione were higher in early gestation while the diols were higher in late gestation.
19-NT stimulated P4 production in early placenta by effects on the conversion of P4 both from 25-hydroxycholesterol and from pregnenolone. The stimulatory influences of A-dione and 3$ alpha$-diol were mediated by increasing the P450scc activity. The specific increase of the conversion of P4 from pregnenolone accounted for the P4 stimulation observed by 3$ beta$- diol treatment of culture.
Cyloheximide (CH) treatment abolished the stimulatory influences of the aforementioned steroids on P4 production except for the initial phase of P4 stimulation by 19-NT, suggesting that all but the latter are dependent on protein synthesis.
P4 production was also stimulated and prolonged to 30 days in the presence of human maternal serum (HMS); a good correlation (r = 0.74, P $<$ 0.05) was seen between the histological appearance of the explants and P4 production. The stimulatory activity of HMS was heat labile, non-dialyzable and non-extractable into an organic solvent, suggesting that it is protein in nature.
In conclusion, this study suggests that 19-NT and A-dione are important for placental P4 production at the time of the luteo-placental shift. For in vitro study of placental hormonal regulation, HMS is a better nutrient supplement than fetal bovine serum.

[Truncated abstract] The placenta forms a barrier regulating the transfer of gases, nutrients and wastes between the mother and the developing conceptus, and also produces hormones affecting both the fetus and the mother. This barrier is formed by the differentiation of the outer layer of the blastocyst- the trophoblast- to facilitate implantation and subsequent invasion of the uterus. The trophoblast consists of an underlying proliferative pool of cytotrophoblasts, which differentiate to replenish the overlying continuous, multi-nucleated syncytiotrophoblast that forms the barrier between the mother and fetus. Moreover, the location of the syncytiotrophoblast directly in contact with the maternal circulation suggests an endothelial role for the trophoblast regulating blood flow, thrombosis and immune cell adhesion. Disruption to the function of the human trophoblast may result in preeclampsia, a maternally manifested disorder of pregnancy characterised by hypertension and proteinurea. Blood flow to preeclamptic placentae is reduced and the cytotrophoblast pool is diminished; however the exact cause (or causes) remains elusive. Many potential causes are hypothesised, including endothelial damage, premature remodelling of maternal spiral arteries, increased oxidative stress and impaired trophoblast differentiation and apoptosis. Caspase-14 is an unusual caspase in that it is not involved in apoptosis. Furthermore, it possesses a limited, predominantly epithelial, tissue distribution. In the epidermis, caspase-14 is expressed in the apical differentiating layers. Here it cleaves profilaggrin to stabilise intracellular keratin intermediate filaments, and indirectly provides natural hydration and UV protection to the corneocytes. Thus, caspase-14 is vital to the maintenance of the barrier function of the skin. ... As differentiation-associated genes were elevated in the absence of caspase-14, this implies that caspase-14 suppresses biochemical trophoblast differentiation. The cytoskeletal keratin network was also examined following RNA Interference. The synthesis of cytokeratin 18 was significantly enhanced after caspase-14 suppression during BeWo differentiation, linking caspase-14 with keratin homeostasis. Therefore caspase-14 suppresses trophoblast differentiation, potentially through modulation of the cytoskeletal keratin filament network. The precise mechanism remains to be elucidated, however the identification of pathways regulated by caspase-14 advances our knowledge of trophoblast differentiation and potential causes of disorders of pregnancy. In summary, caspase-14 appears to be involved in the suppression of differentiation in the human trophoblast. As disorders of pregnancy such as preeclampsia often feature disturbed differentiation and a diminished cytotrophoblast pool, a greater understanding of caspase-14 biology in the human placenta could lead potential therapies for various disorders of pregnancy.

Pregnancy; a natural insulin resistant state; becomes exaggerated when complicated by obesity and gestational diabetes (GDM). Both obesity and GDM are associated with severe maternal and fetal complications as well as with increased risks of obesity in the offspring later in life. Little work has been performed on the levels of adipokines in lean, obese and diabetic pregnancy. This study aimed to explore the roles of three adipokines; namely, Adipsin, Acylation stimulating protein and Fibroblast Growth Factor-21, all of which are involved in insulin resistant and dysmetabolic states such as obesity and type 2 DM. We hypothesized that these adipokines might play a role in pregnancy. A cohort of Caucasian pregnant women undergoing elective caesarean section was studied. Clinical parameters were assayed as well as circulating maternal and fetal levels of adipsin, ASP and FGF21. Paired samples of fat and placental tissue were taken for explant studies to measure secreted Adipsin, ASP and FGF21 levels. Cord levels of adipsin and ASP were significantly elevated in the offspring of obese and diabetic mothers compared to their lean controls. Plasma FGF21 levels were significantly higher in GDM compared to lean controls. FGF21 levels in cerebrospinal fluid (CSF) were also measured and a CSF/Plasma ratio calculated. I have identified the human placenta as a source of adipsin, ASP and FGF21. More specifically, I have shown that placental Hofbauer cells (macrophages) produce adipsin and ASP. This is the first time secretion of adipsin and ASP by Hofbauer cells has been demonstrated. I conjecture a role of these macrophages in lipid metabolism at the materno-fetal interface. Also, I describe that GDM mothers have higher CSF FGF21 as compared to controls but the CSF:plasma ratio of FGF21 was lower in GDM mothers, potentially suggesting an alternative reason for and contributing to hyperglycaemia in GDM.

Reproductive physiology in production animals is a key economic component of longevity and profitability of animal farming. There are several components that can benefit or compromise adequate pregnancy periods. Sheep production is not only a very important economic activity for farmers around the United States, but sheep are also an important medical and surgical model to study human diseases. Our findings suggest that estradiol-17 beta could be involved in acute increased plasma volume early in gestation which can benefit overall gestation. We report that umbilical blood flow decreases upon nutrient restriction in adolescent ewes and does not recover upon realimentation. Finally, we suggest that a similar umbilical blood flow, placental development and plasma volume expansion in twins and singleton pregnancies could be enough to obtain similar birthweights in singletons and twins.

Thyroid hormones (THs) are important for fetoplacental development. Human intrauterine growth restriction (IUGR) is associated with malplacentation and reduced fetal circulating concentration of THs. The expression of the plasma membrane TH transporters MCT8, MCT10, LAT1, LAT2, OATP1A2 and OATP4A1 was characterised in human placental biopsies across gestation. The protein expression of MCT8 was increased in samples from severe IUGR compared with normal pregnancies. In vitro, triiodothyronine (T\(_3\)) decreased survival and increased apoptosis of IUGR compared with normal cytotrophoblasts, which was associated with increased MCT8 expression. In normal cytotrophoblasts, MCT8 upregulation decreased survival, whilst MCT8 silencing increased survival independently of T\(_3\). In the extravillous trophoblast-like cell line, SGHPL-4, T\(_3\) resulted in a significant increase in cell invasion when MCT8 was upregulated. Contrary to cytotrophoblasts, silencing MCT8 decreased apoptosis in SGHPL-4s independently of T\(_3\). In mice, fetal to placental weight ratio was decreased in male MCT8-null compared with wild-type embryos. These findings support the hypothesis that THs have an important role in fetoplacental development and that IUGR is associated with changes in TH transport and responsiveness of the placenta. Furthermore, they highlight the importance of MCT8, which impacts on placental cells via both T\(_3\)- dependent and independent mechanisms.

Abstract The first aim of this study was to investigate the relationship between Doppler ultrasonographic parameters and biochemical markers of human fetal cardiac dysfunction and myocardial cell damage in pregnancies complicated by placental insufficiency and/or fetal growth restriction. Our second aim was to examine fetal central and peripheral hemodynamic characteristics associated with retrograde aortic isthmus net blood flow. Fetuses with significant myocardial cell damage (cTnT > 0.10 ng/ml) had increased pulsatility in the blood velocity waveforms of ductus venosus, left hepatic vein and inferior vena cava, and had more often atrial pulsations in the umbilical vein. Their umbilical artery NT-proANP concentrations were higher than in fetuses without myocardial cell damage. The proportion of left ventricular cardiac output of the combined cardiac output was greater and the corresponding proportion of the right ventricle was less than in fetuses with only increased NT-proANP levels ( > 1145 pmol/l). Tricuspid regurgitation was present more often and the right ventricular fractional shortening was less in fetuses with myocardial cell damage than in fetuses with normal umbilical artery cTnT levels. In fetuses with placental insufficiency and/or growth restriction (n = 48), umbilical artery NT-proANP concentrations showed a significant positive correlation with ductus venosus, left hepatic vein and inferior vena cava pulsatility index values for veins. Fetuses with placental insufficiency and antegrade aortic isthmus net blood flow demonstrated a shift in their right ventricular cardiac output from the pulmonary to the systemic circulation, and foramen ovale volume blood flow made up the majority of the left ventricular cardiac output. Fetuses with retrograde aortic isthmus net blood flow failed to demonstrate these changes, and they had signs of increased left atrial pressure. In addition, right ventricular fractional shortening was decreased and the pulsatility in the ductus venosus blood velocity waveforms was increased. In conclusion, human fetal myocardial cell damage was associated with a rise in systemic venous pressure, a change in the distribution of cardiac output towards the left ventricle and a rise in right ventricular afterload. Fetuses with retrograde aortic isthmus net blood flow failed to rearrange the distribution of the cardiac output and they had signs of increased left atrial pressure. In addition, right ventricular afterload and pulsatility in the ductus venosus blood velocity waveforms were increased.

The present studies were conducted to gain a better understanding of the effects of pregnenolone (P₅), human chorionic gonadotropin (hCG) and 3' 5', cyclic adenosine monophosphate ( cAMP) on porcine placental and endometrial production of progesterone (P₄), testosterone (T) and estrone (E₁) at 30, 60 and 90 days of gestation. Duplicate 300 mg samples of placenta, endometrium or both (co-incubation) were incubated in medium199 containing either no P₅, P₅, P₅ + hCG or P₅ + cAMP for either zero (control), .5, 1 or 2 h. The first study compared P₄ and E₁ production with or without addition of P₅. At d 30, 60 and 90, respectively, P₄ production (ng/g) increased significantly in the presence (vs absence) of P₅ in the incubation medium of placental (13.2 vs 7.5, 73.9 vs 42.7, 137.4 vs 113.5, respectively) coincubation (14.5 vs 10.0, 33.6 vs 22.3, 77.9 vs 49.4, respectively) and endometrial (16.0 vs 13.3, 23.0 vs 16.0, 17.1 vs 6.7, respectively) tissue. Presence of P₅ increased E₁ production in d 60 (1.3 vs .7 ng/g) and d 90 (51.7 vs 34.6 ng/g) placental tissue and d 90 endometrial tissue (9.8 vs 8.0 ng/g). In a second study, P₅ + cAMP increased (vs P₅ alone) P₄ in placental tissue at d 30 (11.6 vs 8.7 ng/g) and coincubation tissue at d 90 (103.7 vs 75.3 ng/g). Cyclic AMP stimulated increased P₄ synthesis ( vs P₅ alone), throughout the incubation period in d 60 and d 90 tissue. E₁ production by endometrial tissue at d 30 (4.1 vs 2.9 ng/g), and placental tissue at d 60 ( 1. 2 vs . 9 ng/g). Presence of hCG in the incubation medium had no overall effect on either P₄ or E₁ accumulation. Only trace amounts of T were detected in either study, suggesting rapid aromatization of C₁₉ steroids to estrogens.
M.S.

Abstract Knowledge of the effects of maternally administered vasopressors on human fetal and placental haemodynamics is sparse and limited to elective Caesarean deliveries in uncomplicated pregnancies. We hypothesized that, after short-term fetal hypoxaemia, which activates fetal cardiovascular compensatory mechanisms, treatment of maternal hypotension with ephedrine or phenylephrine results in divergent responses in fetal and placental haemodynamics. Chronically instrumented near-term sheep fetuses with either normal placental function or increased placental vascular resistance following placental embolization were exposed to two subsequent periods of decreased fetal oxygenation caused by maternal hypoxaemia and epidural-induced hypotension. The fetuses that underwent placental embolization were also chronically hypoxaemic. Fetal and placental haemodynamics were assessed by invasive techniques and by noninvasive Doppler ultrasonography. Our results show that umbilical artery blood flow velocity waveforms cannot be used to derive information of fetal cardiac function. Furthermore, the changes in placental volume blood flows and vascular resistances caused by maternal vasopressor treatment cannot be reliably recognized based on uterine and umbilical artery pulsatility index values. In response to acute hypoxaemia, a fetus with normal placental function redistributes its right ventricular cardiac output from the pulmonary to the systemic circulation and is able to increase its combined cardiac output, with a concomitant relative decrease in the net forward flow through the aortic isthmus. However, fetal haemodynamic responses to subsequent hypoxaemic insults may vary. Furthermore, the compensatory responses of fetuses with increased placental vascular resistance differ from those of normal fetuses. In these fetuses, repeated episodes of a further decrease in oxygenation lead to lactataemia. The effects of ephedrine on uteroplacental and umbilicoplacental circulations were more favourable than those of phenylephrine. Ephedrine restored the changes in fetal cardiovascular haemodynamics caused by maternal hypotension to the baseline conditions in both embolized and nonembolized fetuses. Phenylephrine did not reverse fetal pulmonary vasoconstriction or the relative decrease in the net forward flow through the aortic isthmus. Moreover, fetal left ventricular function was impaired by phenylephrine. Although no significant differences in fetal acid-base status were observed in fetuses with normal placental function, the lactate concentrations of the embolized fetuses increased further when maternal hypotension was treated with phenylephrine.

This thesis will be organized into two chapters discussing the placental expression of two proteins, pyruvate kinase M2 (PKM2) and heat shock protein 27 (HSP 27), in human placentas. Understanding the mechanisms of placental metabolism in healthy and diseased placentas helps us understand how placenta disorders occur and how we can treat these disorders. The goal is to investigate these proteins to gain an understanding of their roles in placental disorders and help decrease maternal and fetal mortality rates. Chapter one covers the background of pyruvate kinase M2 (PKM2) in cancer and embryonic tissues, and the expression of PKM2 in the human placenta. Cancer PKM2 has been studied extensively, but little is know about the role of placental PKM2. Expression of PKM2 is confirmed in normal human placenta samples and described in preeclamptic and intrauterine growth restriction (IUGR) affected human placentas. Proteins associated with elevated PKM2 in cancer are also associated with elevated PKM2 in human placentas. Comparing normal and diseased placenta samples helps understand the similarities between cancer PKM2 and placental PKM2. Understanding the mechanisms of placental metabolism and PKM2 expression in the human placenta will clarify how the placenta is affected by preeclampsia and IUGR and the role placental PKM2 plays in each of these diseases. Chapter two will cover a paper that I wrote on the expression of phosphorylated heat shock protein 27 (HSP27) in the human placenta. Heat shock proteins are involved in the stress response and help inhibit apoptosis. The object of the study was to look for correlations between p-HSP27 and apoptosis in human and ovine placenta samples. P-HSP27 was quantified in human placenta samples and in placenta sampled collected from ovine models. Pregnant control and hyperthermic sheep models were used to quantify expression of p-HSP27 across gestation. This study showed similarities between human IUGR and our ovine IUGR model, suggesting a link between decreased p-HSP27 and increased apoptosis in IUGR.

Abstract Abnormal placentation is associated with preeclampsia and placental insufficiency, both of which increase the risk for fetal growth restriction. So far the early recognition of the risk population for preeclampsia has been problematic. The first hypothesis of this study was that in preeclampsia, the maternal serum proteomic profile is different from that in uncomplicated pregnancies, and this difference is detectable already in early pregnancy. The findings of this study demonstrate that in clinical preeclampsia the maternal serum proteomic profile is different from that in uncomplicated pregnancies with increased levels of placental proteins and antiangiogenic factors in pregnancies with clinical preeclampsia. Furthermore, the early pregnancy maternal serum proteomic profile in women who later develop preeclampsia revealed a distinct and different pattern compared with the profile in clinical preeclampsia. In early pregnancy, the differentially expressed proteins belong to placental proteins, vascular and/or transport proteins and matrix and/or acute phase proteins, while angiogenic and antiangiogenic proteins were not significantly expressed in early pregnancy. Preeclampsia, placental insufficiency, fetal growth restriction and type-1 diabetes may have an impact on fetal cardiovascular hemodynamics. The second hypothesis in this thesis was that in placental insufficiency, abnormalities in fetal cardiovascular status correlate with biochemical markers of cardiac dysfunction and chronic hypoxia. In placental insufficiency, increases in fetal N-terminal pro-atrial (NT-proANP) and pro-B-type natriuretic peptide (NT-proBNP) and in fetal erythropoietin concentrations were related to increased pulsatility in the fetal umbilical artery and descending aorta. In addition, these fetuses demonstrated increased pulsatility in their systemic venous blood velocity waveforms. Thus, in placental insufficiency, biochemical markers of cardiac dysfunction and chronic hypoxia are associated with signs of increased fetal cardiac afterload and systemic venous pressure. Increased NT-proANP and NT-proBNP levels were also detected in fetuses of type-1 diabetic mothers with normal umbilical artery velocimetry. In these pregnancies, NT-proANP and NT-proBNP levels were related to poor maternal glycemic control during early pregnancy.

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

Bien que le rôle du P1GF soit encore débattu, des études ont mis en évidence le rôle du P1GF et de son récepteur, le VEGFR-1 dans la croissance tumorale et l'angiogenèse tumorale. Une étude récente a démontré q'un anticorps monoclonal anti-P1GF (aP1GF) inhibe la croissance tumorale et l'angiogenèse tumorale, y compris dans les modèles résistants aux anti-VEGF. La néo-vascularisation tumorale étant caractérisée par des vaisseaux anormaux, nous avons émis l'hypothèse que l'axe P1GFNEGFR-1 pouvait jouer un rôle dans le remodelage vasculaire anormal et que la signalisation du VEGFR-1 pouvait induire in vitro D114 et la forme active de Notch4 dans les HUVECs Notre première étude a montré, dans un modèle de CHC murin, (i) que les taux de P1GF et de VEGFR-1 étaient significativement supérieurs à ceux du VEGF-A et du VEGFR-2, aux stades précoces du CHC coïncidant avec l'initiation des anomalies vasculaires, (ii) que seules les CEs sinusoïdales tumorales expriment le VEGFR-1 et Notch4 et (iii) que la signalisation du VEGFR-1 par le P1GF et le VEGF-A induit l'expression de D114 et de Notch4 activé dans les HUVECs. Dans notre seconde étude, en collaboration avec Peter Carmeliet, nous avons montré dans 2 modèles différents de CHC que le blocage du P1GF par siRNA ou aP1GF inhibant la croissance tumorale et les anomalies vasculaires décrits dans le CHC à savoir l'artérialisation et la capillarisation des sinusoïdes. Dans notre troisième étude, nous avons montré qu'une statine diminuait la croissance tumorale et prolongeait la survie des animaux en diminuant l'angiogenèse et les anomalies vasculaires du CHC. En conclusion, nous mettons en évidence le rôle prépondérant de l'axe P1GFNEGFR-1 dans l'initiation des anoamlies vasculaires du CHC et le rôle de la signalisation du VEGFR-1 dans l'induction de D114 et de Notch4 activé dans les HUVECs
Even if the role of P1GF is still debated, studies have highlighted the role of P1GF and its receptor, VEGFR-1, in tumor growth and angiogenesis. A recent study has shown that a monoclonal antibody, anti-P1GF, inhibits tumor growth and angiogenesis, including models resistant to anti-VEGF treatments. Since tumor neovascularization is characterized by abnormal blood vessels, we hypothesized that the P1GFNEGFR-1 axis could play a role in abnormal vascular remodelling, and that VEGFR-1 signaling could induce D114 and the active form of Notch4 in HUVECs in vitro. Chu- first study carried out in a mutine model of HCC showed that: (i) the level of P1GF and VEGFR-1 were significantly higher than those of VEGF-A and VEGFR-2 at early stages of HCC coincided with the initiation of vascular anomalies, (ii) only the sinusoidal tumor endothelial cells express VEGFR-1 and Notch4 and (iii) VEGFR-1 signaling by P1GF and VEGF-A induces the expression of D114 and the active form of Notch4 in HUVECs. In our second study performed in collaboration with Peter Carmeliet, we have shown in two different models of HCC, that P1GF blockade, using siRNA or anti-P1GF antibodies, inhibited tumor growth and vascular anomalies described in HCC namely arterialization and capillarization of sinusoids. In our third study, we showed that statins decreased tumor growth and prolonged survival of animais by decreasing angiogenesis and abnormal vasculature in HCC. In conclusion, our results evidence the role of P1GFNEGFR-1 axis in the initiation of vascular abnormalities in HCC and the role of VEGFR-1 signaling in the induction of D114 and activated Notch4 in HUVECs

Estudo longitudinal, prospectivo, comparativo de avaliação dopplervelocimétrica das artérias uterinas direita e esquerda em 37 gestantes normais em três períodos da gestação, acompanhadas na Clínica Obstétrica do HCFMUSP. Análise da relação S/D, índice de pulsatilidade e índice de resistência, relação com o número de incisuras e com posição da placenta. Foi observada redução nos valores dos índices durante a gestação. A relação S/D AUTE com placenta a direita apresentou média superior. A incidência de incisura foi maior no período entre a 16ª e a 24ª semana de gestação. A análise da relação entre os índices dopplervelocimétricos e a presença de incisura não teve resultados significativos
Prospective, longitudinal, comparative study of Doppler evaluation of the right and left uterine arteries of 37 healthy women with singleton pregnancies, performed in three periods of pregnancy, at the Pre-Natal Care Unit HCFMUSP. S/D ratio, pulsatility index and resistance index, number of notches and placental position were analysed. A decrease in the indices was observed with advancing gestation. When placenta was on the right side, the left uterine artery S/D ratio showed increased mean values. The incidence of uterine notch was higher between the 16th and 24th week of gestation in both arteries. No correlation was found between the presence of uterine notch and the placental position

With differences in fetal growth patterns observed across racial demographics, interest in possible biological causes for these differences has increased. Prior works have studied the effects of insulin resistance in pregnancy on fetal fat deposition and growth in utero. One proposed mechanism to explain the physiological decrease in insulin sensitivity observed in normal pregnancy is the release of epigenetic factors such as placental miRNAs that have downstream effects on nutrient availability and fetal growth. The aim of this study was to identify placental miRNAs in women of different races and to establish expression patterns between these groups, specifically if expression patterns were related to measures of fetal growth and insulin resistance. Untargeted RNAseq and targeted RT-qPCR techniques were utilized for miRNA expression analysis and validation, respectively. Statistical modeling was used to interpret relationships between miRNA expression and maternal and neonatal body composition variables. qPCR results validated RNA sequencing data of differential miRNA expression between maternal racial groups. While miRNA-34c-5p and 192-5p fold changes were not correlated with maternal insulin resistance (as measured by HOMA-IR) in both non-Hispanic black and non-Hispanic white women, both miRNA-34c-5p and miRNA-192-5p were both positively correlated with neonatal body composition measures in neonates born to NHB women only. Further analysis of more miRNAs from additional RNAseq is planned.
2022-06-08T00:00:00Z

Bibliography: 161-199.
[xxvi], 199, [151] leaves, [7] leaves of plates : ill. (some col.) ; 30 cm.
Characterises the normal otogeny of the cellular composition and structure of placentomes in sheep, their relationship to the macroscopic parameters of placentome size and morphology, and the effect of experimental and natural restriction of implantation on the growth and development of placentomes between mid and late gestation.
Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 1995

Kwan Pui-Chun.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.
Includes bibliographical references (leaves 140-155).
Abstracts in English and Chinese.
Chapter Chapter 1 --- Introduction
Chapter 1.1 --- Overview --- p.1
Chapter 1.2 --- Iron homeostasis --- p.5
Chapter 1.3 --- Natural Resistance Associated Marcophage Protein (Nramp) Family --- p.15
Chapter 1.4 --- Divalent Metal Transporter 1 (DMT1) --- p.18
Chapter 1.5 --- Iron Responsive Element (IRE) and Iron Regulatory Protein (IRP) --- p.23
Chapter 1.6 --- Expression and localization of DMT-1 in human --- p.27
Chapter 1.7 --- Iron and the developing feus --- p.31
Chapter 1.8 --- Objectives of the study --- p.36
Chapter Chapter 2 --- Materials and Method
Chapter 2.1 --- Study population --- p.37
Chapter 2.2 --- Procedure of surgical termination of pregnancy --- p.38
Chapter 2.3 --- Tissues collection and preparation --- p.39
Chapter 2.4 --- Semi-quantitative Reverse Transcription-Polymerase Chain Reaction --- p.44
Chapter 2.5 --- Immunohistochemistry --- p.49
Chapter 2.6 --- Statistical analysis --- p.55
Chapter Chapter 3 --- Results
Chapter 3.1 --- Description of subjects --- p.56
Chapter 3.2 --- Existence of human DMT-1 isoforms at early pregnancy --- p.58
Chapter 3.3 --- Relative expression of DMT-1 isoforms to β -actin mRNA expression at different week gestation --- p.67
Chapter 3.4 --- Cellular localization of DMT-1 isoforms at early pregnancy --- p.91
Chapter 3.5 --- Relative expression of DMT-1 proteins at early pregnancy --- p.101
Chapter Chapter 4 --- Discussion
Chapter 4.1 --- Existence of DMT-1 at early pregnancy --- p.116
Chapter 4.2 --- Expression of DMT-1 isoforms at early pregnancy at gene level --- p.118
Chapter 4.3 --- Expression of DMT-1 isoforms at early pregnancy at protein level --- p.120
Chapter 4.4 --- "Comparison expression of DMT-1 between human fetus, human adult and animal studies" --- p.121
Chapter 4.5 --- Functional importance of DMT-1 at developing fetus at early pregnancy --- p.130
Chapter 4.6 --- Conclusion --- p.138
Chapter 4.7 --- Further study --- p.139
Chapter Chapter 5 --- Reference --- p.140
Appendix I: Calculation of EM --- p.156

Pre-eclampsia, often described as toxaemia of pregnancy, historically represents one of the most widely investigated conditions relating to human reproduction. To date no firm cure has been found and a clear, well defined mechanism has not been ascribed to the pathogenesis of the disease. Researchers seem to focus on single pathways in isolation of others. The disease rather represents a multitude of possible underlying pathologies nvolving genetics, immune dysregulation, vascular maladaptation, and sociobiological factors thus complicating the approach to treatment. However, a central theme is the presence of reduced placental perfusion resulting in a hypoxic and/or ischaemic placenta and the subsequent secretion of various factors that initiate the maternal syndrome. It is within this context that we examine how an intervention such as increasing placental perfusion may represent a promising treatment strategy for this disease. We sought to manipulate the vasodilatory mechanisms of the uterine vasculature using sildenafil citrate and a flavonoid extracted from Eriosema kraussianum (Kr2), in Sprague-Dawley rats that exhibited preeclampsia-like manifestations. Both treatment regimens improved fetal outcomes and reduced blood pressure amplification and proteinuria. They also reduced the plasma concentrations of the two anti-angiogenic factors; sFlt1 and sEng. Only sildenafil citrate improved nitric oxide levels which was expected, suggesting that Kr2 causes vasodilation by some other mechanism. Nevertheless, both compounds improved both pup and placental weights, suggesting that they also improve utero-placental perfusion. These findings that selective uterine vascular dilation improves placental perfusion may be promising in averting possible death to mothers and their babies from pre-eclampsia especially in low resource environments.
Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

Bibliography: p. 190-234.
276 p. : ill. ; 30 cm.
Aims to directly test the hypothesis that restricting placental delivery of oxygen and nutrients to the fetus restricts fetal growth, in part by reducing endogenous production of insulin like growth factor-I
Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 1999?

Although maternal low density lipoprotein (LDL) is the primary source of cholesterol substrate for progesterone biosynthesis in the primate placental syncytiotrophoblast, the hypothesis to be tested is that the baboon trophoblast may derive significant amounts of substrate from sources secondary to the classical LDL pathway during early pregnancy or when faced with a paucity of lipoprotein-cholesterol. Expression of LDL receptor mRNA in syncytiotrophoblasts, assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), was greater (P $<$ 0.05) in late than in early baboon pregnancy, although no difference was observed in whole villous tissue. Divergently, both enzyme activity and protein levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in syncytiotrophoblast cells were higher (P $<$ 0.01) in early than in late pregnancy, while mRNA concentrations were unchanged with advancing gestation. Abundance of scavenger receptor-I (SR-I) mRNA was low in baboon placenta. Scavenger receptor-II (SR-II) mRNA concentrations in syncytiotrophoblast cells were two-fold higher than in whole villous tissue, in contrast to LDL receptor related protein (LRP) mRNA concentrations, which were higher in whole villous tissue than in syncytiotrophoblasts Cholesterol yielding pathways were also investigated in pregnant baboons via treatment with 4-aminopyrazolo (3-4-d) pyrimidine (4-APP, an inhibitor of hepatic lipoprotein production). Thus, LDL-cholesterol concentrations were dramatically lower (P $<$ 0.005) and commensurate progesterone levels also lower (P $<$ 0.03) in 4-APP treated baboons than in untreated baboons. A potential enhancement of LDL receptor (mRNA) and HMG-CoA reductase (mRNA, protein and enzyme activity), as well as a decline in acyl-coenzyme A:cholesterol acyl transferase (mRNA) in the baboon syncytiotrophoblast, probably resulted from 4-APP induced lipoprotein withdrawal and constitute the first demonstration of this compensatory mechanism in the primate placenta In conclusion, secondary cholesterol-yielding mechanisms in the syncytiotrophoblast may provide significant amounts of cholesterol substrate for progesterone production during early baboon pregnancy and when the availability of lipoprotein-cholesterol substrate is reduced. Further, mechanisms responsible for cholesterol homeostasis in the steroidogenically active syncytiotrophoblast are regulated in a divergent manner from those in proliferative nonendocrine components of the placenta
acase@tulane.edu

Leptin, the product of the LEP gene, may play an important role in pregnancy, specifically in the regulation of placental endocrinology and angiogenesis. In trophoblasts collected early in gestation, leptin increases hormone biosynthesis and metalloproteinase secretion and activation, suggesting leptin's involvement in placental function and development. Therefore, we hypothesize that: (1) estrogen regulates the expression of the leptin receptor in the placental syncytiotrophoblast and leptin, in turn, modulates the secretion of human chorionic gonadotropin (hCG), human growth hormone (hGH), and progesterone; (2) leptin, in combination with factors previously known to modulate placental angiogenesis and vascular permeability, regulates placentation by enhancing vascular endothelial cell proliferation Studies in the baboon (Papio sp) demonstrated leptin receptor (Ob-R) expression. Furthermore, use of in situ hybridization demonstrated the localization of two leptin receptor isoforms to the leptin-producing, placental syncytiotrophoblast, suggesting the possibility for autocrine/paracrine action. Subsequently, leptin's effect upon placental endocrinology was assessed in vitro using third trimester human syncytiotrophoblast cultures. Leptin did not affect hCG, progesterone or hGH elaboration, and estrogen administration had no effect on leptin secretion or Ob-R transcript abundance in this cell type. Collectively, these results differed from studies using first trimester cells, suggesting that leptin's role(s) in early pregnancy differs from that in late gestation. Leptin may potentially play roles in processes that are pertinent in early pregnancy such as angiogenesis and implantation After validating leptin's angiogenic potential using a murine corneal pocket model assay, cultured human umbilical vein endothelial cells (HUVECs) were utilized to test whether leptin affected HUVEC proliferation induced by angiogenic factors pertinent to placentation. Despite Ob-R expression on the cell membrane of HUVECs, leptin did not stimulate cell proliferation. Vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor alpha induced HUVEC proliferation, but leptin failed to enhance or decrease the effects of any of these factors. These results indicate that the angiogenic effects of leptin may be mediated by a mechanism other than the induction of vascular endothelial cell proliferation. Overall, early in gestation, leptin may mediate placental endocrinology and angiogenesis, a role that may change later in pregnancy
acase@tulane.edu

The prepartum surge in fetal plasma cortisol, essential for the maturation of organs in mammals and the normal timing of parturition in some species, including sheep, may result from an increase in the molar ratio of adrenocorticotropin (ACTH) to pro-opiomelanocortin (POMC) in the fetal circulation. Related to this, the cleavage of POMC to ACTH by the enzyme, prohormone convertase 1 (PC1), may be influenced by corticotrophin releasing hormone (CRH) stimulation. Accumulating evidence suggests that the capacity of individual corticotrophs to process POMC to ACTH may vary and individual corticotrophs are differentially responsive to CRH. It is not known, however, if there are separate corticotroph subpopulations in the fetal sheep pituitary which can be identified by differential colocalisation of POMC, ACTH and the CRH receptor 1, CRHR₁, nor if changes in the relative proportions of such subpopulations play a role in the molecular mechanisms underlying the overall changes in pituitary function described previously during gestation and in response to suboptimal uterine environments. To investigate these hypotheses, it was first necessary to develop novel methods for the simultaneous immunohistochemical labelling of POMC, ACTH and CRHR₁ in individual cells on sections of fetal sheep pituitary. In addition, I developed and validated an automated method to categorise and count individual cells to increase the quantitative power of this study. Pituitary tissue was collected from control fetuses at 53-55 (n=6), 63-85 (n=6), 110 (n=4), 139-141 (n=4) and 144-145 (n=6) days gestation. Two animal models, known to alter pituitary function in the fetal sheep, were used to investigate corticotrophic adaptations to suboptimal uterine environments. For the maternal periconceptional undernutrition (PCUN) model, maternal feed was reduced to 70% of maintenance requirements from at least 45 days before to 7 days after mating and fetal tissues were collected at 53-55 days gestation (n=7). For the placental restriction (PR) model, the majority of the placental attachment sites were removed in five ewes before mating and fetal tissues were collected at 140 (n=4) and 144 (n=4) days gestation. Pituitary sections were simultaneously labelled with antisera raised against full length POMC, ACTH and CRHR₁ and the proportions of pituitary cells with combinations of antisera were quantified. Four subpopulations of corticotrophs were identified, which expressed either: POMC+ACTH+CRHR₁, ACTH+CRHR₁, POMC+ CRHR₁ or POMConly. There was a significant decrease in the proportion of pituitary cells expressing POMC+ACTH+CRHR₁ between 53-55 and 65-85 days gestation, before an increase at 110 days gestation and a further marked decrease between 139-141 and 144-145 days gestation. In fetuses from the PCUN group, the proportion of pituitary cells expressing POMC+ACTH+CRHR₁ in early gestation was reduced. PR resulted in a significantly higher proportion of corticotrophs expressing POMC+ACTH+CRHR₁ during the prepartum period. This work represents the discovery of the differential expression of POMC, ACTH and CRHR₁ in individual corticotrophs of the fetal sheep pituitary and the first insights into the pituitary adaptations to periconceptional nutrient restriction and placental restriction at the level of individual corticotrophs.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008