The intrauterine environment induces developmental adaptations that impact health and disease risk later in life. Reproductive abnormalities are now included in the long list of health complications seen in offspring exposed to early life adversity including poor prenatal nutrition. Previous work has shown using a rat model, that offspring born to mothers that were nutrient restricted (UN) during pregnancy are growth restricted, enter puberty early, and as adults, display characteristics of early ovarian aging with reduced follicle number. This present study aimed to investigate whether key proteins involved in ovarian follicle recruitment and growth, including the PI3K/Akt pathway, may be impaired as a result of early life nutritional adversity. Maternal UN resulted in irregular estrous cyclicity due to persistent estrus, a significant decrease in young adult ovarian antral follicles, corpora lutea, and a significant increase in atretic follicles. A decrease in growing follicles in UN offspring appears to be due to lowered expression of granulosa-cell secreted growth factor IGF-1 in antral follicles, and increased expression of pro-apoptotic factor Casp3 in secondary follicles of young adult offspring born to UN dams. Changes in follicle signalling pathways were apparent before observing altered ovarian function. In UN prepubertal offspring, expression levels of IGF1-R and FSHR were lowered in secondary and antral follicles respectively. These growth factors may contribute to a decrease in PI3K/Akt activation as immunohistochemical staining revealed a decrease in pAkt immunolocalization in prepubertal antral follicles. Moreover, neonatal ovaries of UN offspring show decreased levels of immunopositive staining for AMHRII, a regulatory receptor of the ovarian reserve. This study demonstrates that maternal UN during pregnancy impacts ovarian function in female offspring as early as P65. Findings from this study provides a model of understanding mechanisms of follicle loss and reproductive dysfunction as a result of nutrient restriction during fetal life. / Thesis / Master of Science (MSc)

Untersuchungen haben gezeigt, dass unterdurchschnittlich leichte Neugeborene für zahlreiche Erkrankungen ein erhöhtes Risiko tragen. Beschrieben ist unter anderem das vermehrte Auftreten psychosozialer Probleme sowie metabolischer und kardiovaskulärer Spätfolgen. Inzwischen sind zahlreiche mögliche Ursachen einer intrauterinen und postnatalen Wachstumsretardierung beschrieben worden. Unter diesen Ursachen finden sich auch genetische Veränderungen von Proteinen der endokrinologischen Wachstumsregulierung. So wurden Mutationen im GH1 Gen, in Entwicklungsgenen von GH produzierenden Zellen, im IGF-I Gen und schließlich auch im IGF-I Rezeptor Gen identifiziert. Mutationen im letztgenannten Gen stellen den neuesten Forschungszweig dar und wurden bisher weltweit bei lediglich 19 Patienten festgestellt. Mit dieser Arbeit wird ein weiterer Patient mit einer heterozygoten IGF-I Rezeptormutation beschrieben. Neben einer ausführlichen klinischen Beschreibung war die Analyse der Kausalzusammenhänge von Mutation und klinischem Bild Hauptziel dieser Studie. Über eine ausgeprägte intrauterine und postnatale Wachstumsretardierung hinaus präsentierte die betroffene Patientin eine mentale Entwicklungsverzögerung. Durch verschiedene molekularbiologische Methoden konnte eine gestörte intrazelluläre Prozessierung des veränderten Rezeptorproteins nachgewiesen werden. Beobachtet wurde eine fehlende Zelloberflächenexpression aufgrund einer Retention von Rezeptorvorstufen im Endoplasmatischen Retikulum. Damit wurde ein neuer Mechanismus der IGF-I Resistenz beschrieben.

Die prospektive Studie untersuchte den Verlauf von Hochrisikoschwangerschaften mit zwischen 24/0 und 34/0 Schwangerschaftswochen erstmals diagnostiziertem ARED-Flow in der Arteria umbilicalis hinsichtlich ihres fetal outcome im Vergleich zu einer nach Gestationsalter gematchten Kontrollgruppe. Während der Studiendauer von 1995-1999 wurden 60 Feten aus Einlingsschwangerschaften mit ARED-Flow in der Umbilikalarterie in die Beobachtung einbezogen. Feten mit Malformationen und Chromosomenanomalien wurden ausgeschlossen. Die Beobachtung erfolgte mit wiederholten Doppler-Messungen der arteriellen und venösen Gefäße, mit CTG und mütterlichen Parametern. Eine Entbindung wurde eingeleitet, wenn sich entweder die fetalen Parameter verschlechterten (68%), wie ein Reverse Flow im Ductus venosus oder späte Dezelerationen im CTG oder eine mütterliche Indikation gegeben war (28%), wie das Auftreten einer Präeklampsie. Gemäß Protokoll wurden 50% der Feten mit ARED-Flow 6 Tage nach Erstdiagnose entbunden. Bei mütterlicher Indikation war dies bereits nach 4 Tagen, bei fetaler Indikation nach 7 Tagen der Fall. Im Ergebnis zeigte sich eine Mortalität von 38% mit 23 Verstorbenen (16 intrauteriner Fruchttod und 7 postnataler Tod), die meisten intrauterinen Fruchttode (14) traten bei einem Schwangerschaftsalter unter 29/0 SSW auf. 44 Feten wurden lebend geboren. Die lebendgeborenen Feten wurden in drei Gruppen A, B und C nach dem bei der Entbindung erreichten Schwangerschaftsalter eingeteilt. Zwischen 24/0 und 28/6 SSW (Gruppe A) betrug die Mortalität 36%, zwischen 29/0 und 31/6 SSW (Gruppe B) 10% und ab 32/0 SSW (Gruppe C) 8%. Signifikante Unterschiede zwischen den drei Gruppen zeigten sich bezüglich des Auftretens von periventrikulärer Leukomalazie (nur A: 36%), von neurologischen Auffälligkeiten (A: 73%, B: 45%, C: 8%), von Atemnotsyndrom (A: 100%, B: 35%, C: 8%) und hinsichtlich der Apgar-Werte nach 5 und 10 Minuten. Die 44 lebendgeborenen Feten mit ARED-Flow wurden einer nach Schwangerschaftswochen gematchten Kontrollgruppe normgewichtiger Frühgeborener gegenübergestellt. Zwischen den Feten mit ARED-Flow und der Kontrollgruppe konnten verschiedene signifikante Unterschiede festgestellt werden: In der ARED-Gruppe fanden sich niedrigere pH-Werte (p=0,001), ein geringeres Geburtsgewicht (p=0.0001), häufigeres Auftreten von bronchopulmonaler Dysplasie (p=0.002) und von Darmkomplikationen (p=0.01). Weitere beobachtete Parameter, wie z.B. peri- und intraventrikuläre Hämorrhagien, neurologische Auffälligkeiten, waren nicht signifikant verschieden. Allerdings war das Risiko postnatal zu versterben in der ARED-Gruppe 8-fach erhöht. Es ergibt sich der Rückschluss, dass die Diagnose ARED-Flow eine Gruppe ernsthaft hypotropher hypoxämischer Feten umschreibt, die eine hohe Mortalitäts- und Morbiditätsrate aufweist. Die Mortalität ist tendenziell bei niedrigem Schwangerschaftsalter bei der Entbindung (vor 29/0 SSW), bei extrem niedrigem Geburtsgewicht (unterhalb der 3. Perzentile), bei Vorliegen von spät auftretenden Dopplerveränderungen (wie Reverse Flow in der Arteria umbilicalis oder pathologischem venösen Doppler) erhöht. Obwohl die Inzidenz peri- und intraventrikulärer Hämorrhagien (16%) und neurologischer Defizite (40%) hoch war, erwies sich dieser Unterschied gegenüber den Frühgeborenen der Kontrollgruppe als nicht signifikant. Es kann angenommen werden, dass eine Verlängerung der Schwangerschaft unter venöser Dopplerkontrolle das fetal outcome verbessert. / The study examined prospectively the progression of high-risk pregnancies in cases of ARED flow in the Arteria umbilicalis detected between 24/0 and 34/0 weeks gestation in respect of their fetal outcome compared with a gestational age-matched control group. During the 1995-99 study period, 60 fetuses from singleton pregnancies with ARED flow in the umbilical artery were included in the study. Fetuses with malformations and anoiploidy were excluded. Surveillance was performed by repeated Doppler measurements of arterial and venous vessels, CTG and maternal parameters. Delivery was induced when either fetal parameters deteriorated (68%), as in the case of reverse flow in the Ductus venosus or late decelerations in CTG, or when maternal indication (28%) was given, as when pre-eclampsia occurred. Following our protocol, 50% of fetuses with ARED flow were delivered 6 days after the first diagnosis. In the event of maternal indication, this took place after 4 days, with fetal indication after 7 days. The results showed a mortality rate of 38% with 23 deaths (16 intrauterine and 7 postnatal). The majority of intrauterine deaths (14) occurred under 29/0 weeks of term. 44 fetuses were born live. The fetuses born live were divided into Groups A, B and C in line with gestational age on delivery. Mortality between 24/0 and 28/6 weeks pregnancy (Group A) was 36%, between 29/0 and 31/6 weeks (Group B) 10%, and 8% from 32/0 weeks onward (Group C). Significant variations between the three groups was indicated in the incidence of periventricular leukomalacia (only A:36%), neurological development (A:73%, B:45%, C:8%), respiratory syndrome (A:100%, B:35%, C:8%), and in relation to the Apgar values after 5 and 10 minutes. The 44 fetuses with ARED flow born live were compared with a control group of preterm neonates of average weight and similar gestational age. Various significant differences could be shown between ARED flow fetuses and the control group: the ARED group showed lower pH values (p=0.001), lower birth weight (p=0.0001), and a higher incidence of broncho-pulmonal dysplasia (p=0.002) and of intestinal complications (p=0.01). Other observed parameters such as peri- and intraventricular haemorrhage and neurological development were not significantly different. Nevertheless, the risk of postnatal death was 8 times higher in the ARED group. It has to be concluded that diagnosis of ARED flow encompasses a group of severely hypotrophic, acidemic fetuses showing high rates of mortality and morbidity. Mortality tends to increase in the case of low gestational age on delivery (up to 29/0 weeks), in cases of extremely low birth weight (less than 3%), and in incidences of late Doppler changes (such as reverse flow in the umbilical artery or pathological venous Doppler). Although the incidence of peri- and intraventricular haemorrhages (16%) and neurological defects (40%) was high, compared with the neonates of the control group it was insignificant; It can be assumed that prolongation of pregnancy using venous Doppler improves the fetal outcome.

Untersuchungen haben gezeigt, dass unterdurchschnittlich leichte Neugeborene für zahlreiche Erkrankungen ein erhöhtes Risiko tragen. Beschrieben ist unter anderem das vermehrte Auftreten psychosozialer Probleme sowie metabolischer und kardiovaskulärer Spätfolgen. Inzwischen sind zahlreiche mögliche Ursachen einer intrauterinen und postnatalen Wachstumsretardierung beschrieben worden. Unter diesen Ursachen finden sich auch genetische Veränderungen von Proteinen der endokrinologischen Wachstumsregulierung. So wurden Mutationen im GH1 Gen, in Entwicklungsgenen von GH produzierenden Zellen, im IGF-I Gen und schließlich auch im IGF-I Rezeptor Gen identifiziert. Mutationen im letztgenannten Gen stellen den neuesten Forschungszweig dar und wurden bisher weltweit bei lediglich 19 Patienten festgestellt. Mit dieser Arbeit wird ein weiterer Patient mit einer heterozygoten IGF-I Rezeptormutation beschrieben. Neben einer ausführlichen klinischen Beschreibung war die Analyse der Kausalzusammenhänge von Mutation und klinischem Bild Hauptziel dieser Studie. Über eine ausgeprägte intrauterine und postnatale Wachstumsretardierung hinaus präsentierte die betroffene Patientin eine mentale Entwicklungsverzögerung. Durch verschiedene molekularbiologische Methoden konnte eine gestörte intrazelluläre Prozessierung des veränderten Rezeptorproteins nachgewiesen werden. Beobachtet wurde eine fehlende Zelloberflächenexpression aufgrund einer Retention von Rezeptorvorstufen im Endoplasmatischen Retikulum. Damit wurde ein neuer Mechanismus der IGF-I Resistenz beschrieben.

Le placenta humain est un organe indispensable au maintien de la grossesse et au développement fœtal. Son unité structurale et fonctionnelle est la villosité choriale, constituée principalement de cytotrophoblastes qui se différencient selon la voie villeuse endocrine ou extravilleuse invasive. Le développement intense du placenta et ses fonctions multiples requièrent des besoins en énergie importants. La régulation du métabolisme énergétique placentaire passe, en partie, par le contrôle de l’activité mitochondriale.La mitochondrie est l’organelle clé du métabolisme énergétique. Cependant, elle intervient dans de nombreuses autres fonctions cellulaires telles que l’apoptose et la biosynthèse des hormones stéroïdes. Des études récentes suggèrent que les mitochondries sont impliquées dans la différenciation cellulaire.Le récepteur apparenté aux récepteurs des oestrogènes-γ, (ERRγ), est un facteur de transcription qui joue un rôle crucial dans le contrôle du métabolisme énergétique. Des travaux préliminaires ont montré qu’ERR est fortement exprimé dans le placenta humain.Dans ce travail, nous nous sommes intéressés à l’implication d’ERRγ dans le développement placentaire.Dans une première partie, nous avons caractérisé l’expression d’ERRγ dans les trophoblastes de placentas humains de 1er et de 3ème trimestre. Nous avons montré que l’expression d’ERRγ i) augmente au cours de la grossesse et ii) est plus importante dans les cytotrophoblastes villeux en comparaison aux cytotrophoblastes extra-villeux.Dans une seconde partie, nous avons montré que la différenciation des cytotrophoblastes villeux est associée à des modifications du métabolisme énergétique et du contenu mitochondrial. De plus, nous avons clairement démontré qu’ERRγ contrôle positivement la différenciation des trophoblastes villeux en modulant les fonctions mitochondriales.Enfin, nous avons montré qu’ERRγ est moins exprimé dans les placentas issus de retards de croissance intra-utérins en comparaison à des placentas normaux. De plus, la diminution d’ERRγ est associée à une baisse du contenu mitochondrial placentaire.L’ensemble de ce travail a permis de mettre en évidence le rôle clé d’ERRγ et des mitochondries dans le développement du placenta humain. / Human placenta is a vital organ for pregnancy support and fetal development. The chorionic villi is the structural and functional unit of the placenta and is mainly constituted by trophoblastic cells. The trophoblast differentiate in villous endocrine and extra-villous invasive trophoblast. Placental development and its numerous functions require the availability of high energy. Placental energetic metabolism control is partially mediated by the regulation of mitochondrial activity.Mitochondria are key organelles of the energetic metabolism. However, mitochondria are involved in numerous other cellular functions such as apoptosis and steroid hormone biosynthesis. Moreover, recent studies suggest that mitochondria are involved in cell differentiation.Estrogen related receptor-γ (ERRγ) is a transcriptional factor implicated in the control of energetic metabolism. Preliminary studies showed that ERRγ is highly expressed in human placenta.In this work, we decided to study ERRγ implication in human placental development.In a first part, we characterized ERRγ expression in trophoblast from first and third trimester human placentas. We showed that ERRγ expression i) increased during pregnancy and ii) was higher in villous than extra-villous trophoblasts.In a second part, we showed that villous trophoblast differentiation was associated with modifications of energetic metabolism and mitochondrial content. Moreover, we clearly demonstrated that ERRγ positively controled villous differentiation by the modulation of mitochondrial functions.In a last part, we showed that ERRγ was less expressed in placentas from intra-uterine growth restriction as compared to non-pathological placentas. Moreover, this down-regulation was associated with a decrease of mitochondrial content.This work thus showed, for the first time, that ERRγ and mitochondria played a key role in placental development.

Pregnancy complications associated with placental development affect approximately one third of all human pregnancies. Genome health is essential for placental and fetal development, as DNA damage can lead to pregnancy loss and developmental defects. During this developmental phase rapid DNA replication provides an increased opportunity for genome and epigenome damage to occur[1]. Maternal nutrition is one of the principal environmental factors supporting the high rate of cell proliferation and differentiation. Folate functions in one-carbon metabolism and regulates DNA synthesis, DNA repair and gene expression[1]. Deficiencies or defects in gene-nutrient interactions associated with one-carbon metabolism can lead to inhibition of cell division, cell cycle delay and an excessive apoptotic or necrotic cell death rate [2], which may affect placentation. This study is the first to investigate the association between genomic damage biomarkers in late pregnancy complications associated with uteroplacental insufficiency (UPI) including preeclampsia and intrauterine growth restriction (IUGR). The results indicate that genome damage in the form of micronucleated cells in peripheral blood lymphocytes at 20 weeks gestation is significantly increased in women at risk of developing an adverse pregnancy outcome. The observed OR for the high micronuclei frequency may be the highest observed for any biomarker selected in relation to risk of pregnancy complications to date (15.6 – 33.0). In addition, reduced apoptosis was observed in association with increased micronuclei, suggesting that the cells may have escaped specific cell-cycle checkpoints allowing a cell with DNA damage to proceed through mitosis. This study demonstrated that an increase in plasma homocysteine concentration at 20 weeks gestation is associated prospectively with the subsequent development of UPI, indicating a causal relationship. The MTR 2756 GG genotype was significantly associated with increased plasma homocysteine concentration and UPI. Furthermore, the MTHFD1 1958 single nucleotide polymorphism was associated with increased risk for IUGR. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309296 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2007

<p>Glucose is the most important fetal nutrient and the production of this substrate increases in the pregnant woman. In the last trimester the increased insulin resistance directs energy substrates to the fetus. Fetal growth is sometimes disturbed, often without an obvious explanation.</p><p>After birth the newborn infant must produce its own glucose, primarily for the brain. Fatty acids from lipolysis are also important energy substrates. Hypoglycaemia can be a problem, occurring frequently in preterm infants and infants born small for gestational age (SGA). In addition, SGA infants are at risk of developing the metabolic syndrome in adulthood. Neonatal medication can influence energy metabolism. One such medication is theophylline, administered in preterm infants to prevent apnoea. </p><p>We investigated energy substrate production in women with normal and IUGR pregnancies, in preterm neonates, before and after theophylline treatment and in newborn SGA infants, using stable isotope-labelled compounds and gas chromatography-mass spectrometry. </p><p>We found that late pregnancy was associated with an almost twofold increase in the rate of lipolysis. This provides substrates for maternal energy metabolism, which may spare glucose for the fetus. Even though glucose production was comparable in the two groups of pregnant women, those with IUGR had a lower rate of lipolysis. A reduced supply of energy substrates could be one factor underlying IUGR. In spite of the insulin resistance of late pregnancy, insulin still had a regulatory role in energy substrate production in the women with normal pregnancies, but not in those with IUGR. </p><p>Although infants born preterm and/or SGA have limited energy stores, we demonstrated that they are capable of both lipolysis and glucose production. Theophylline had no adverse effects on energy substrate production. Data on insulin and IGFBP-1 in the SGA infants indicate that in such infants insulin sensitivity is increased peripherally but reduced in the liver.</p>

<p>The aim of this study was to examine the expression of Thrombomodulin, Tissue Factor, Tissue Factor Pathway Inhibitor and Endothelial Protein C Receptor in placenta throughout the three phases of the third trimester in the normal placenta and in IUGR placenta from full term.</p><p>Twenty-five normal placenta samples and twenty-five IUGR placenta samples were obtained and each sample was stained by immunohistochemistry using monoclonal antibodies. Each antibody was optimised for antigen retrieval method and for optimal dilution, before been applied to the test tissue.</p><p>The results showed that each of the antibodies mentioned was expressed in normal placenta and in IUGR placenta.</p><p>No significant difference could be established concerning the expression of each antibody mentioned between normal and IUGR placenta.</p>

Glucose is the most important fetal nutrient and the production of this substrate increases in the pregnant woman. In the last trimester the increased insulin resistance directs energy substrates to the fetus. Fetal growth is sometimes disturbed, often without an obvious explanation. After birth the newborn infant must produce its own glucose, primarily for the brain. Fatty acids from lipolysis are also important energy substrates. Hypoglycaemia can be a problem, occurring frequently in preterm infants and infants born small for gestational age (SGA). In addition, SGA infants are at risk of developing the metabolic syndrome in adulthood. Neonatal medication can influence energy metabolism. One such medication is theophylline, administered in preterm infants to prevent apnoea. We investigated energy substrate production in women with normal and IUGR pregnancies, in preterm neonates, before and after theophylline treatment and in newborn SGA infants, using stable isotope-labelled compounds and gas chromatography-mass spectrometry. We found that late pregnancy was associated with an almost twofold increase in the rate of lipolysis. This provides substrates for maternal energy metabolism, which may spare glucose for the fetus. Even though glucose production was comparable in the two groups of pregnant women, those with IUGR had a lower rate of lipolysis. A reduced supply of energy substrates could be one factor underlying IUGR. In spite of the insulin resistance of late pregnancy, insulin still had a regulatory role in energy substrate production in the women with normal pregnancies, but not in those with IUGR. Although infants born preterm and/or SGA have limited energy stores, we demonstrated that they are capable of both lipolysis and glucose production. Theophylline had no adverse effects on energy substrate production. Data on insulin and IGFBP-1 in the SGA infants indicate that in such infants insulin sensitivity is increased peripherally but reduced in the liver.

Placental development can be disturbed by various factors, such as mutation of specific genes or maternal diabetes. Our previous work on interspecies hybrid placental dysplasia (IHPD) and two additional models of placental hyperplasia, cloned mice and Esx1 mutants, showed that many genes are deregulated in placental dysplasia. Two of these candidate placentation genes, Cpe and Lhx3, were further studied. We performed in situ hybridization to determine their spatio-temporal expression in the placentas and placental phenotypes were analyzed in mutant mice. Our results showed that the placental phenotype in Cpe mutant mice mimics some IHPD phenotypes. Deregulated expression of Cpe and Cpd, a functionally equivalent gene, prior to the manifestation of the IHPD phenotype, indicated that Cpe and Cpd are potentially causative genes in IHPD. Lhx3 mutants lacked any placental phenotype. Deletion of Lhx3 and Lhx4 together caused an inconsistent placental phenotype which did not affect placental lipid transport function or expression of Lhx3/Lhx4 target genes. Down regulation of Lhx3/Lhx4 did not rescue the placental phenotype of AT24 mice and hence could be excluded as causative genes in IHPD. Analysis of placental development in diabetic mice showed that severe maternal diabetes resulted in fetal intrauterine growth restriction (IUGR) without any change in placental weight and lipid transport function. The diabetic placentas however exhibited abnormal morphology. Gene expression profiling identified some genes that might contribute to diabetic pathology. In another study, it was found that the heterochromatin protein CBX1 is required for normal placentation, as deletion of the gene caused consistent spongiotrophoblast and labyrinthine phenotypes. Gene expression profiling and spatio-temporal expression analysis showed that several genes with known function in placental development were deregulated in the Cbx1 null placenta.