Abstract not available

Dissertation--Marburg (Ad novi prorectoris inaugurationem). / Bibliographical foot-notes.

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

In five separate experiments, weight-adjusted doses of TEM, EMS, and ENU were injected intraperitoneally into twelve week-old female mice six hours after mating. On day seventeen of gestation, the females were sacrificed and their uterine contents were examined. The effect of each agent was determined by its ability to cause malformations and death to the developing embryos. All treatment groups showed statistically significant elevated levels of malformations in comparison to their corresponding control groups. The reproductive damage induced in these experiments cannot be singularly attributed to teratogenesis or mutagenesis but a combination of the two.

There has been an increase in rates of chromosomal abnormalities in newborns as a result of reproductive aging. For the past decades, a lot of effort has been placed on identifying pregnancies at risk of genetic defects. Conventional prenatal genetic diagnosis is achieved by invasive procedures that have been associated with an increased risk of pregnancy loss. This has led the researchers to explore the use of non-/minimally invasive techniques for prenatal diagnosis. Trophoblasts are known to be shed from regressing chorionic villi into the lower uterine pole of pregnant women during the first trimester. These cells are trapped within cervical mucus, which can be retrieved with a cytobrush. By using human leukocyte antigen G (HLA-G) and cytokeratin-7 (CK7) as trophoblast markers, this study aims to investigate the possibility of isolating individual fetal trophoblast from transcervical samples for genetic diagnosis. 195 healthy pregnant women requesting for legal termination of pregnancy (TOP) were recruited in this study. Transcervical cells were collected from them with the use of a cytobrush before TOP. HLA-G+ or CK7+ cells were then isolated by a combination of mucolytic action, fluorescent immunohistochemistry, and micromanipulation. The origin of these cells was subsequently investigated by either fluorescent in situ hybridization (FISH) or allelic profiling by quantitative fluorescent polymerase chain reaction (QF-PCR) based on chromosome 16, chromosome X, amelogenin gene and sex determining region Y (SRY) gene. This study first demonstrated the presence of fetal cells in transcervical samples based on the detection of chromosome Y signal by ordinary PCR. Cells expressing HLA-G and CK7 were also identified among transcervical cells. Immunopositive cells were isolated by micromanipulation under fluorescent microscopy. One isolated cell expressing CK7 was shown to inherit paternal allele at a locus on chromosome 16, suggesting the possible fetal origin of this cell. However, this study was still hampered by a number of technical factors. Further optimization of the protocol is required before transcervical trophoblasts can be retrieved in a reliable manner. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Thesis--Copenhagen. / Summary in Danish. Bibliography: p. 219-235.

Thesis--Copenhagen. / Summary in Danish. Bibliography: p. 219-235.

Lam, Yuk Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 142-157). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / CONTRIBUTORS --- p.viii / PUBLICATIONS --- p.ix / LIST OF TABLES --- p.x / LIST OF FIGURES --- p.xi / LIST OF ABBREVIATIONS --- p.xiii / TABLE OF CONTENTS --- p.xiv / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- PRENATAL DIAGNOSIS OF FETAL ANEUPLOIDIES --- p.2 / Chapter 1.1 --- The need for prenatal screening and diagnosis --- p.2 / Chapter 1.2 --- Patau Syndrome (Trisomy 13) --- p.2 / Chapter 1.3 --- Current methods for fetal aneuploidy detection --- p.4 / Chapter 1.3.1 --- Routine prenatal screening tests --- p.4 / Chapter 1.3.2 --- Definitive prenatal diagnosis by invasive procedures --- p.7 / Chapter 1.4 --- New approach for noninvasive prenatal diagnosis --- p.11 / Chapter 1.4.1 --- Circulating fetal cells --- p.11 / Chapter 1.4.2 --- Cell-free fetal nucleic acids in maternal circulation --- p.12 / Chapter 1.4.3 --- Diagnostic applications of cell-free fetal nucleic acids in maternal plasma --- p.12 / Chapter CHAPTER 2: --- DEVELOPMENT OF FETAL EPIGENETIC MARKERS IN MATERNAL PLASMA --- p.17 / Chapter 2.1 --- Limitations of fetal DNA markers --- p.17 / Chapter 2.2 --- DNA methylation is an actively-researched area under the field of epigenetics --- p.18 / Chapter 2.3 --- Genome-scale DNA methylation analysis brings new insight into epigenetics --- p.20 / Chapter 2.4 --- The first demonstration of using an epigenetic method for detecting maternally-inherited fetal DNA in maternal plasma --- p.22 / Chapter 2.5 --- The first universal marker for fetal DNA in maternal plasma --- p.24 / Chapter 2.6 --- Discovery of more fetal epigenetic markers --- p.25 / Chapter 2.6.1 --- Methylated fetal epigenetic markers are more desirable --- p.25 / Chapter 2.6.2 --- Discovery of hypermethylated fetal epigenetic markers by studying tumor suppressor genes --- p.26 / Chapter 2.6.3 --- Discovery of hypermethylated fetal epigenetic markers on chromosome 21 --- p.28 / Chapter 2.7 --- Noninvasive detection of fetal aneuploidies using fetal epigenetic markers --- p.29 / Chapter 2.7.1 --- Noninvasive detection of fetal trisomy 18 by the epigenetic allelic ratio (EAR) approach --- p.29 / Chapter 2.7.2 --- Noninvasive detection of fetal trisomy 21 by the epigenetic-genetic (EGG) approach --- p.30 / Chapter 2.8 --- Aim of thesis --- p.32 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.34 / Chapter CHAPTER 3: --- METHODS FOR QUANTITATIVE ANALYSIS OF DNA METHYLATION --- p.35 / Chapter 3.1 --- Subject recruitment and sample collection --- p.35 / Chapter 3.2 --- Sample processing --- p.38 / Chapter 3.3 --- DNA extraction --- p.38 / Chapter 3.3.1 --- Placental tissues --- p.38 / Chapter 3.3.2 --- Maternal blood cells --- p.39 / Chapter 3.3.3 --- Maternal plasma --- p.40 / Chapter 3.4 --- Methylated DNA immunoprecipitation and tiling array analysis (MeDIP-chip) --- p.41 / Chapter 3.4.1 --- Principles --- p.41 / Chapter 3.4.2 --- DNA sample and array processing --- p.43 / Chapter 3.4.2.1 --- DNA preparation and target hybridization --- p.43 / Chapter 3.4.2.2 --- Data analysis --- p.44 / Chapter 3.5 --- DNA methylation analysis on randomly-chosen regions on chromosome / Chapter 3.6 --- Bisulfite conversion --- p.46 / Chapter 3.6.1 --- Principles of bisulfite conversion --- p.46 / Chapter 3.6.2 --- Procedures of bisulfite conversion --- p.46 / Chapter 3.7 --- Quantitative analysis of DNA methylation --- p.47 / Chapter 3.7.1 --- Bisulfite PCR and genomic sequencing --- p.47 / Chapter 3.7.1.1 --- Primer design for bisulfite polymerase chain reaction (PCR) --- p.47 / Chapter 3.7.1.2 --- Bisulfite PCR --- p.49 / Chapter 3.7.1.3 --- Cloning --- p.50 / Chapter 3.7.1.4 --- Bisulfite genomic sequencing --- p.52 / Chapter 3.7.1.5 --- Data acquisition and interpretation --- p.53 / Chapter 3.7.2 --- EpiTYPER，a mass-spectrometry-based method --- p.54 / Chapter 3.7.2.1 --- Principles of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) --- p.54 / Chapter 3.7.2.2 --- Primer design of the EpiTYPER assay --- p.55 / Chapter 3.7.2.3 --- The EpiTYPER assay and its principle --- p.56 / Chapter 3.8 --- Methylation-sensitive restriction enzyme (MSRE)-mediated real-time quantitative PCR (qPCR) --- p.61 / Chapter 3.9 --- Digital PCR --- p.66 / Chapter 3.9.1 --- Principles of digital PCR --- p.66 / Chapter 3.9.2 --- Poisson distribution --- p.68 / Chapter 3.10 --- Statistical analyses --- p.69 / Chapter SECTION III: --- SYSTEMATIC IDENTIFICATION OF A FETAL DNA METHYLATION MARKER ON CHROMOSOME 13 FOR DETECTION OF FETAL TRISOMY 13 --- p.70 / Chapter CHAPTER 4: --- SYSTEMATIC IDENTIFICATION OF POTENTIAL FETAL EPIGENETIC MARKERS BY MEDIP-CHIP ANALYSIS --- p.71 / Chapter 4.1 --- Systematic discovery of fetal epigenetic markers on chromosome 13 by MeDIP-chip analysis --- p.71 / Chapter 4.2 --- Experimental design --- p.73 / Chapter 4.3 --- Results --- p.76 / Chapter 4.3.1 --- Identification of differentially methylated DNA regions by MeDIP-chip or non-MeDIP-chip approaches followed by EpiTYPER analysis --- p.76 / Chapter 4.3.2 --- Confirmation of differential methylation patterns and exclusion of regions with high inter-individual variations by EpiTYPER analysis --- p.82 / Chapter 4.3.3 --- Confirmation of differential DNA methylation patterns with higher resolution by bisulfite sequencing --- p.85 / Chapter 4.4 --- Discussion --- p.95 / Chapter CHAPTER 5: --- THE APPLICATION OF FETAL EPIGENETIC MARKER ON CHROMSOME 13 FOR DETECTION OF FETAL TRISOMY 13 --- p.98 / Chapter 5.1 --- Identification of a fetal epigenetic marker on chromosome 13 for the detection of fetal trisomy 13 by the epigenetic-genetic (EGG) chromosome dosage approach --- p.98 / Chapter 5.2 --- Experimental design --- p.101 / Chapter 5.3 --- Results --- p.105 / Chapter 5.3.1 --- Optimization of the digestion protocol --- p.105 / Chapter 5.3.2 --- Detection of digestion-resistant EFNB2-3'UTR moleculesin maternal plasma --- p.109 / Chapter 5.3.3 --- Evaluation of the fetal specificity of digestion-resistant EFNB2´ؤ3 'UTR DNA molecules in maternal plasma --- p.111 / Chapter 5.3.4 --- Comparison of EFNB2-3'UTR methylation profiles between the euploid and trisomy 13 placental tissue samples --- p.115 / Chapter 5.3.5 --- Chromosome dosage analysis by the EGG analysis using placental tissue samples --- p.118 / Chapter 5.4 --- Discussion --- p.122 / Chapter SECTION IV: --- CONCLUDING REMARKS --- p.125 / Chapter CHAPTER 6: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.126 / Chapter 6.1 --- Development of fetal epigenetic markers for noninvasive prenatal diagnosis --- p.126 / Chapter 6.2 --- Systematic identification of fetal epigenetic markers on chromosome13 --- p.127 / Chapter 6.3 --- Detection of fetal trisomy 13 by the epigenetic-genetic (EGG) relative chromosome dosage analysis --- p.129 / Chapter 6.4 --- Future perspectives --- p.132 / Appendix I --- p.134 / Appendix II --- p.136 / REFERENCES --- p.142

Orientador: Lourenço Sbragia Neto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T01:31:04Z (GMT). No. of bitstreams: 1 Schmidt_AugustoFredericoSantos_D.pdf: 2455947 bytes, checksum: 2419510f01584a15bbc44f3555be5714 (MD5) Previous issue date: 2010 / Resumo: A hérnia diafragmática congênita (HDC) é uma doença grave com alta mortalidade devido à hipoplasia e hipertensão pulmonar. A via do ácido retinóico tem sido implicada na patogênese da HDC e pode ser uma alternativa de intervenção na promoção da alveolarização e vascularização pulmonar. O fator de crescimento vascular do endotélio (VEGF - Vascular Endothelial Growth Factor) e seus receptores VEGFR1 e VEGFR2 têm importante função no crescimento e na vascularização pulmonar, e, possivelmente, na patogênese da HDC. No entanto, não se conhece como o tratamento pré-natal com ácido retinóico pode afetar a vascularização pulmonar e seus fatores de crescimento. O objetivo deste estudo foi analisar o efeito do tratamento pré-natal com ácido retinóico na vascularização pulmonar e na expressão pulmonar de VEGF e seus receptores VEGFR1 e VEGFR2 em fetos de rato com HDC induzida pelo nitrofen (2,4-dicloro-4'nitrodifenil éter). Fetos de ratas Sprague-Dawley prenhes foram divididos em oito grupos: 1) controle externo, 2) placebo óleo nitrofen; 3) placebo óleo ácido retinóico, 4) tratados com ácido retinóico, 5) expostos ao nitrofen sem HDC, 6) expostos ao nitrofen com HDC, 7) expostos ao nitrofen sem HDC e tratados com ácido retinóico, 8) expostos ao nitrofen com HDC e tratados com ácido retinóico. Nitrofen foi administrado por via oral (gavagem) com 9,5 dias de gestação. Ácido retinóico foi administrado por via intraperitoneal com 18,5, 19,5 e 20,5 dias de gestação na dose de 5 mg/kg/dia, a coleta fetal foi realizada com 21,5 dias (termo=22 dias). Cada grupo fetal foi composto por 25 fetos. As variáveis morfológicas estudadas foram: peso corporal (PC), peso pulmonar total (PPT), peso do pulmão esquerdo (PPE) e a relação peso pulmonar total / peso corporal (PPT/PC). A morfologia pulmonar foi estudada pela mensuração da média linear de interceptação (MLI) e seus componentes diâmetro interno dos espaços aéreos (DEA) e a relação de comprimento de transecção do parênquima / espaço aéreo (MCTP). A morfologia vascular foi estudada pela mensuração do diâmetro externo (DE), diâmetro interno (DI) e espessura proporcional da camada muscular média (ECM) de arteríolas pulmonares de resistência. A expressão de VEGF e dos receptores VEGFR1 e VEGFR2 foi analisada por meio de imunoistoquímica e western blotting. Os dados morfológicos e morfométricos foram analisados pelo teste ANOVA com pós-teste de Tukey-Kramer, a avaliação semiquantitativa da imunoistoquímica foi analisada pelo teste de Kruskal-Wallis com pós-teste de Dunn, sendo considerados significativos valores de p<0,05 para ambos os testes. A freqüência de HDC observada entre os fetos expostos ao nitrofen foi de 40%. As variáveis morfológicas apresentaram diminuição significativa nos grupos expostos ao nitrofen, especialmente nos fetos com HDC (p<0,05). O tratamento com ácido retinóico não alterou as variáveis morfométricas pulmonares. Fetos com HDC apresentaram aumento da ECM, enquanto o tratamento com ácido retinóico reduziu a ECM nos fetos com HDC (p<0.001). A presença de HDC levou à diminuição da expressão de VEGF, VEGFR1 e VEGFR2, enquanto o tratamento com ácido retinóico recuperou a expressão de VEGF e VEGFR1. A alteração sinalização da via do VEGF na HDC pode estar associada à patogênese da hipoplasia e da hipertensão pulmonar. O tratamento pré-natal com ácido retinóico pode fornecer vias para tratamento da hipertensão pulmonar na HDC por meio da redução da ECM das arteríolas pulmonares e recuperação do VEGF e seus receptores / Abstract: Congenital diaphragmatic hernia (CDH) is a life-threatening disease with high mortality due to the pulmonary hypertension and hypoplasia. Vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 play a major role in lung vascularization, growth, and possibly in the pathogenesis of CDH. However it is not know how prenatal treatment with retinoic acid can affect lung vascularization by acting on the VEGF signaling. The purpose of this study was to analyze the effect of antenatal retinoic acid treatment on the expression of pulmonary VEGF and its receptors VEGFR-1 and VEGFR-2 in rat fetuses with CDH induced by nitrofen. Fetuses from pregnant Sprague-Dawley rats (term=22 days) were divided in eight groups: 1) external control, 2) placebo oil nitrofen, 3) placebo oil retinoic acid, 4) treated with retinoic acid, 5) exposed to nitrofen without CDH, 6) exposed to nitrofen with CDH, 7) exposed to nitrofen without CDH and treated with retinoic acid, 8) exposed to nitrofen with CDH and treated with retinoic acid. Nitrofen (2,4- dichloro-4'-nitrodiphenyl ether) was administered by gavage (100 mg) at 9,5 days of gestation. Retinoic acid was administered intraperitoneally on days 18.5, 19.5 and 20.5 of gestation (5 mg/kg), harvest was performed at 21.5 days (term =22 days). Each fetal subgroup was composed of 25 fetuses. The morphologic variables studied were: body weight, total lung weight, left lung weight, and total lung weight to body weight ratio. Pulmonary morphometry was studied by measuring the mean linear intercept and its components: internal diameter of airspaces and mean transection length / airspace. Vascular morphometry was studied by measuring the external diameter, internal diameter and proportionate thickness of the medial muscular layer of pulmonary resistance arterioles. Immunohistochemistry and Western blotting analysis were used to assess VEGF, VEGFR-1 and VEGFR-2 expression. Data was analyzed using ANOVA with Tukey's post-test and immunohistochemistry was studied semiquantitatively using Kruskal-Wallis test with Dunn's post-test. The frequency of CDH was 40%. The morphological variables showed reduction in the nitrofen group with and without CDH, which were more pronounced in the latter (p<0.05). Retinoic acid did not affect fetal morphology. There was no difference in pulmonary morphometry among groups. Fetuses with CDH had increased proportionate thickness of the medial muscular layer of pulmonary arterioles, while treatment with retinoic acid reduced this variable in fetuses with CDH (p<0.001). Fetuses with CDH had reduced VEGF, VEGFR1 and VEGFR2 expression, while retinoic acid treatment restored expression VEGF and VEGFR1. VEGF signaling disruption may be associated with pulmonary hypertension in CDH. Retinoic acid may provide a pathway for acting on pulmonary hypertension by reducing medial thickness of pulmonary arterioles and restoring expression of VEGF and its receptors / Doutorado / Cirurgia / Doutor em Ciências da Cirurgia

Este trabalho trata da interrupção da gestação, em casos de diagnóstico de malformação fetal letal e os processos psíquicos dela decorrentes. São feitas algumas considerações sobre os aspectos históricos e políticos da reprodução e da sexualidade, explorando, em seguida, aspectos relativos ao contexto cultural do aborto; o debate sobre o início da vida humana; questões da bioética e da legislação. O trabalho explora, ainda, questões sobre a legislação brasileira, Medicina Fetal e os processos psíquicos desencadeados a partir do diagnóstico de anomalia fetal letal. Foi desenvolvida uma pesquisa de campo, na Divisão de Clínica Obstétrica do Hospital das Clínicas da FMUSP, para aprofundar as questões teóricas discutidas. No período de agosto de 1998 a dezembro de 2003, foram realizadas entrevistas abertas com 249 mulheres, após terem recebido o diagnóstico de malformação fetal letal e entrevista semidirigida com trinta e cinco destas pacientes após a interrupção da gravidez. Este trabalho tem como objetivos específicos: identificar os processos psíquicos desencadeados nas mulheres, após o diagnóstico de malformação fetal letal; no processo de decisão pela interrupção judicial da gravidez; após a interrupção da gravidez; e identificar, na opinião das mulheres que receberam o diagnóstico de malformação fetal letal e que realizaram a interrupção da gestação, qual o papel da consulta psicológica nesse processo. A análise dos dados se deu de forma quantitativa e qualitativa. Os resultados obtidos versam tanto sobre o momento do diagnóstico como experiência que propicia um caos temporário com perda do raciocínio lógico, não permitindo reflexões imediatas. Demonstra as angústias vivenciadas no processo de decisão pela interrupção ou manutenção da gravidez, apresentando o processo de reflexão como de fundamental importância para decisão consciente e para posterior satisfação com a decisão tomada. O acompanhamento psicológico foi destacado como de fundamental importância para elaborar a situação vivida. Conclui que o diagnóstico de malformação fetal letal ativa mecanismos de defesa para manutenção do equilíbrio psíquico. O processo de decisão pela interrupção da gravidez deve ser acompanhado por um psicólogo para que ocorra revisão dos valores morais e culturais permitindo uma decisão adequada que visa minimizar o sofrimento vivido. / This research is about the termination of pregnancy in situations where lethal fetal malformation has been diagnosed, and the psychic process that the patient goes through in these cases. The study was done with some consideration for the historical and political process of reproduction and sexuality, exploring aspects about the cultural context of abortion, the beginning of human life, issues about bioethics, and specific Brazilian laws on abortion. It discusses the point of view of the Catholic Church on the termination of pregnancy. This research also explores questions about Brazilian laws, fetal medicine, and the psychic processes triggered after the diagnosis of fetal anomaly. This study was performed at the Hospital das Clínicas da Faculdade de Medicina da Universidade de Sao Paulo, in the Department of Obstetrics Between august, 1998 and December, 2003 open interviews was performed with 249 women after they have been diagnosed with lethal malformation of the fetus, and semi-direct interviews with 35 women after their pregnancy had been terminated. The objective of this research was not only to identify the psychic process women undergo after the diagnosis of lethal fetal malformation, during the decision-making process for the judicial intervention in the pregnancy, and after the termination itself; but also to know their opinion about the function of the psychological consult in this process. The data analysis was quantitative and qualitative. The results show that the moment of the diagnosis is an experience that creates a temporary chaos that deprives logical reasoning, and this situation does not allow an immediate decision. It shows the distress experienced in the decision-making process, showing that a reflective process is essential to the conscious decision and to being satisfied with the decision once it has been made. The psychological follow-up was determined to be of essential importance to understanding this situation. The study concludes that the diagnosis of lethal malformation of the fetus triggers a defense mechanism to maintain the psychic equilibrium. A psychologist must follow the process of the decision through to the termination of the pregnancy in order to provide a moral and cultural reflection leading to the correct decision and minimizing the emotional distress for the patient.