INTRODUÇÃO: Gastrosquise é um defeito no fechamento da parede abdominal do feto que está relacionado a elevadas taxas de óbito intrauterino por mecanismos ainda desconhecidos. Em fetos normais, basicamente, todos os parâmetros da frequência cardíaca fetal (FCF) analisados na cardiotocografia computadorizada (CTGc) apresentam uma mudança significativa no decorrer da gestação. OBJETIVO: Descrever as características da FCF e o comportamento dos parâmetros avaliados pela CTGc anteparto, no período de 28 a 36 semanas de gestação. MÉTODOS: Estudo retrospectivo realizado na Clínica Obstétrica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, no período de janeiro de 2012 a junho de 2015, envolvendo pacientes com gestação única, feto vivo e portador de gastrosquise isolada que foram submetidas à avaliação antenatal pela CTGc (Sistema FetalCare). Os parâmetros avaliados foram: número de movimentos fetais por hora, frequência cardíaca fetal basal (FCF), desacelerações, acelerações, episódios de alta e baixa variação e variação de curto prazo. A análise não paramétrica para medidas repetidas (ANOVA não paramétrica) foi utilizada para análise comparativa dos parâmetros da CTGc em cada idade gestacional avaliada. RESULTADOS: O estudo envolveu 87 gestantes com média de 3,5 (1-9) avaliações cardiotocográficas por paciente. O número de avaliações cardiotocográficas em cada idade gestacional foi >= 20, exceto para a idade de 29 semanas (n = 16). Os principais parâmetros da FCF avaliados pela CTGc como a FCF basal e o STV não apresentaram mudança significativa. Apenas dois parâmetros da CTGc apresentaram mudança significativa no período avaliado: o número de exames com presença de episódios de baixa variação da FCF aumentou no decorrer da gestação (p = 0,019); e o número de acelerações acima de 15 batimentos por minuto aumentou no evoluir das idades gestacionais estudadas (p = 0,001). Nenhum dos outros parâmetros avaliados pela CTGc apresentou mudança significativa no decorrer do período avaliado: o número de movimentos fetais por hora (p = 0,244), a FCF basal (p = 0,606) e o STV (p = 0,145). CONCLUSÃO: O comportamento da FCF dos fetos com gastrosquise difere do padrão apresentado por fetos normais, já que a maioria dos parâmetros da FCF avaliados pela CTGc de fetos com gastrosquise não apresentou mudança significativa no período gestacional avaliado / INTRODUCTION: Fetal gastroschisis is an abdominal wall defect associated with high rates of intrauterine death of unknown mechanisms. In normal fetuses, basically all computerized cardiotocography (cCTG) parameters present a significant change across gestation. OBJECTIVE: To describe the antepartum cCTG parameters between 28 to 36 weeks gestation. METHODS: Retrospective study, accomplished in the Obstetrics Department of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo - Brazil between January 2012 and June 2015, involving singleton pregnancies, with alive fetus and isolated gastroschisis that underwent to cCTG (System 8002-Sonicaid) during the antenatal care. The cCTG parameters evaluated were: number of fetal movements per hour, baseline fetal heart rate (FHR) decelerations, accelerations, episodes of high and low variation and short-term variation. A non-parametric analysis for repeated measures (nonparametric ANOVA) was used for comparative analysis of the mean distribution of each cCTG parameters throughout the study period. RESULTS: The study involved 84 pregnant women with a mean of 3.5 (1 - 9) cCTG records per patient. The number of records in each gestational age was >= 20 except for the weeks 29 (n = 16). The cCTG parameters that presented significant change during the study period were: increase in the number of records with episodes of low variation (p = 0.019); and increase in the number of accelerations higher than 15 beats per minute (p = 0.001). None of the others analyzed parameters showed significant changes during the study period, the number of movements/hr (p = 0,244), basal FHR (p = 0,606) and the STV (p = 0,145). CONCLUSION: Only two of the cCTG parameters changed significantly during the study period. Therefore, it seems that the behavior patterns of cCTG parameters, in gastroschisis fetuses, are not similar to the normal fetuses

1997年，胎兒DNA被首次證實存在於母體血漿中。這一發現促進了無創產前診斷技術的發展。由於孕婦血漿中含大量來自母體的背景DNA，這給針對胎兒特異性DNA序列以外的產前診斷變得有挑戰性。近期開發的大規模平行測序技術把DNA定量精度提升到了一個空前的水平。本團隊已證實這一技術可應用於對胎兒染色體非整倍體和對胎兒全基因組的檢測。由於目前平行測序的費用仍相當昂貴，目標性測序技術可提高目標區域的數據比例從而降低測序成本。 / 在論文第一部分，本人論述了目標性測序在母體血漿DNA應用的可行性。本實驗採用雜交型富集技術對X染色體的外顯子進行富集。我們用平行測序比較了經由和未經富集處理的樣本。對比發現，經富集處理的樣本在目標區域的平均測序深度是未經富集處理樣本的213倍。目標區域的母體和胎兒DNA分子的富集程度相當。經富集處理後，目標區域的胎兒特異性等位基因的檢測率從3.5%提升至95.9%。 / 在論文第二部分，本人論述了目標性測序對胎兒21三體無創產前診斷的應用價值。我們對7，13，18和21號染色體上的單核苷酸多態性位點進行目標性測序。目標性測序數據顯示，在父源性21三體的樣本中，21號染色體上的胎兒特異性等位基因與共有性等位基因的比值上升約2倍。而在母源性21三體的樣本中，這一比值則下降約11%。本人採用電腦模擬實驗探討胎兒DNA濃度，有效等位基因數量和測序深度對檢測準確率的影響。 / 在論文第三部分，本人論述了目標性測序對胎兒單基因疾病無創產前診斷的應用。針對兩個需進行β地中海貧血產前診斷的家庭，我們對其β球蛋白基因進行目標性測序。我們用數字PCR技術推導了父母親β球蛋白基因區域的單倍型。經過相對性單倍型劑量分析，兩個胎兒的β地中海貧血遺傳狀況均得到了正確的推斷。其中一對夫婦位於致病區域的單倍型結構相近。 / 鑒於目標性測序技術可降低測序成本和提高目標序列的通量，其在血漿DNA的應用將有助於平行測序技術在無創性產前診斷、癌症診斷和移植監控等分子診斷學領域的發展。 / The presence of fetal DNA in the cell-free plasma of pregnant women was first reported in 1997. This discovery has facilitated the development of noninvasive prenatal diagnosis. The coexistence in maternal plasma of a minor population of fetal DNA among a major background of maternal DNA has posed challenges for extending noninvasive prenatal diagnostic applications that require analytical information beyond the detection of fetus-specific DNA sequences. The recent availability of massively parallel sequencing has enhanced the precision of DNA quantification to an unprecedented level. Our group has demonstrated the application of massively parallel sequencing in noninvasive prenatal diagnosis of chromosomal aneuploidies, as well as genome-wide fetal whole genome sequencing and mutational profiling. While the current costs of massively parallel sequencing are relatively expensive, targeted massively parallel sequencing may enhance the cost-effectiveness compared with the non-targeted approach because it increases the proportion of informative data from the regions-of-interest. / In the first part of this thesis, I have demonstrated the feasibility of targeted massively parallel sequencing in maternal plasma DNA. In this proof-of-principle study, hybridization-based target enrichment was used to enrich exons on chromosome X. Plasma DNA libraries with and without target enrichment were analyzed by massively parallel sequencing. For the targeted regions, the mean sequencing depth of the enriched samples was 213-fold higher than that of the non-enriched samples. Maternal and fetal DNA molecules were enriched to similar extents within the targeted regions. With target enrichment, the detection rate of fetus-specific alleles within the targeted regions increased from 3.5% to 95.9%. / In the second part of this thesis, I have demonstrated the potential application of targeted massively parallel sequencing of plasma DNA for noninvasive prenatal diagnosis of trisomy 21 using an allelic ratio approach. Targeted sequencing was used to enrich single nucleotide polymorphism loci on chr7, chr13, chr18 and chr21. The targeted sequencing data showed that the ratio between fetus-specific and shared alleles increased by approximately 2-fold on chr21 in a paternally-derived trisomy 21 case, and decreased by approximately 11% on chr21 for maternally-derived trisomy 21 cases. I have also used computer simulation to determine the impact of fractional fetal DNA concentration, number of informative alleles and sequencing depth on the detection accuracy. / In the third part of this thesis, I have demonstrated the feasibility of targeted massively parallel sequencing of maternal plasma DNA for noninvasive prenatal diagnosis of monogenic diseases. Targeted sequencing was used to enrich the β-globin gene region in two families undergoing prenatal diagnosis for β-thalassemia. Parental haplotypes of the β-globin gene region were deduced via digital polymerase chain reaction. Relative haplotype dosage analysis was performed successfully to determine the β-thalassemic status for the fetuses, including one family in which the parents had similar haplotype structures in the disease-causing region. / Because of its potential to save costs and increase throughput, targeted sequencing may catalyse the translation of massively parallel sequencing based molecular diagnostics into many fields, including noninvasive prenatal diagnosis, cancer diagnostics and transplantation monitoring. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liao, Jiawei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 147-155). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.vi / PUBLICATIONS --- p.vii / CONTRIBUTORS --- p.viii / TABLE OF CONTENTS --- p.ix / LIST OF TABLES --- p.xiii / LIST OF FIGURES --- p.xiv / LIST OF ABBREVIATIONS --- p.xvi / Chapter SECTION I : --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- Cell-free fetal DNA and targeted massively parallel sequencing --- p.2 / Chapter 1.1 --- Cell-free fetal DNA in maternal plasma --- p.2 / Chapter 1.2 --- NIPD by qualitative analysis --- p.3 / Chapter 1.2.1. --- Fetal sex assessment --- p.4 / Chapter 1.2.2. --- RHD genotyping --- p.5 / Chapter 1.2.3. --- Other implementations --- p.5 / Chapter 1.3 --- NIPD by quantitative analysis --- p.6 / Chapter 1.3.1. --- NIPD of chromosomal aneuploidies --- p.6 / Chapter 1.3.2. --- NIPD of autosomal recessive monogenic diseases --- p.8 / Chapter 1.4 --- Massively parallel sequencing of maternal plasma --- p.9 / Chapter 1.4.1. --- Massively parallel sequencing --- p.9 / Chapter 1.4.2. --- MPS-based NIPD of chromosomal aneuploidies --- p.11 / Chapter 1.4.3. --- MPS-based prenatal fetal whole-genome scanning --- p.15 / Chapter 1.5 --- Targeted massively parallel sequencing of maternal plasma --- p.19 / Chapter 1.5.1. --- Microdroplet-based PCR --- p.19 / Chapter 1.5.2. --- Molecular inversion probe --- p.20 / Chapter 1.5.3. --- On-array capture --- p.21 / Chapter 1.5.4. --- In-solution capture --- p.21 / Chapter 1.5.5. --- Implementation on plasma DNA --- p.22 / Chapter 1.6 --- Aims of this thesis --- p.29 / Chapter SECTION II : --- Feasibility of targeted MPS in maternal plasma DNA --- p.30 / Chapter CHAPTER 2: --- Targeted MPS of maternal plasma DNA permits efficient and unbiased detection of fetal alleles --- p.31 / Chapter 2.1 --- Introduction --- p.31 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- Study participants and sample collection --- p.34 / Chapter 2.2.2 --- Sample processing and DNA extraction --- p.34 / Chapter 2.2.3 --- DNA quantification --- p.36 / Chapter 2.2.4 --- Microarray genotyping --- p.39 / Chapter 2.2.5 --- Plasma DNA library preparation --- p.39 / Chapter 2.2.6 --- Plasma DNA library validation --- p.40 / Chapter 2.2.7 --- Target enrichment --- p.44 / Chapter 2.2.8 --- Massively parallel sequencing and alignment --- p.45 / Chapter 2.3 --- Results --- p.48 / Chapter 2.3.1 --- Efficiency of target enrichment --- p.48 / Chapter 2.3.2 --- Sequence coverage within the targeted region --- p.53 / Chapter 2.3.3 --- Fetus-specific allele detection --- p.59 / Chapter 2.3.4 --- Fractional fetal DNA concentrations before and after enrichment --- p.63 / Chapter 2.4 --- Discussion --- p.66 / Chapter SECTION III : --- NIPD of trisomy 21 by targeted MPS of maternal plasma DNA --- p.71 / Chapter CHAPTER 3: --- NIPD of fetal trisomy 21 by allelic ratio analysis using targeted MPS of maternal plasma DNA --- p.72 / Chapter 3.1 --- Introduction --- p.72 / Chapter 3.2 --- Methods --- p.74 / Chapter 3.2.1 --- Study participants and sample collection --- p.74 / Chapter 3.2.2 --- Sample processing and DNA extraction --- p.74 / Chapter 3.2.3 --- Targeted MPS of plasma DNA libraries --- p.74 / Chapter 3.2.4 --- F-S ratio calculation --- p.76 / Chapter 3.2.5 --- Microarray genotyping --- p.78 / Chapter 3.2.6 --- Computer simulation --- p.78 / Chapter 3.3 --- Results --- p.80 / Chapter 3.3.1 --- Efficiency of target enrichment --- p.80 / Chapter 3.3.2 --- Estimation of fractional fetal DNA concentrations --- p.83 / Chapter 3.3.3 --- F-S ratio calculation using non-targeted sequencing data --- p.83 / Chapter 3.3.4 --- F-S ratio calculation using targeted sequencing data --- p.85 / Chapter 3.3.5 --- Computer simulation --- p.85 / Chapter 3.4 --- Discussion --- p.90 / Chapter SECTION IV : --- NIPD of monogenic diseases by targeted MPS of maternal plasma DNA --- p.94 / Chapter CHAPTER 4: --- NIPD of monogenic diseases by targeted MPS of maternal plasma: application to Beta-thalassemia --- p.95 / Chapter 4.1 --- Introduction --- p.95 / Chapter 4.2 --- Methods --- p.98 / Chapter 4.2.1 --- Sample collection and DNA extraction --- p.98 / Chapter 4.2.2 --- Microarray-based genotyping --- p.100 / Chapter 4.2.3 --- Targeted MPS of plasma DNA libraries --- p.100 / Chapter 4.2.4 --- Genotyping by Sanger sequencing --- p.103 / Chapter 4.2.5 --- Haplotyping by digital PCR --- p.105 / Chapter 4.2.6 --- RHDO analysis --- p.105 / Chapter 4.3 --- Results --- p.110 / Chapter 4.3.1 --- Effectiveness of targeted sequencing --- p.110 / Chapter 4.3.2 --- Determination of fetal HBB genotype in the first family --- p.112 / Chapter 4.3.3 --- Determination of fetal HBB genotype in the second family --- p.113 / Chapter 4.4 --- Discussion --- p.115 / Chapter SECTION V : --- CONCLUDING REMARKS --- p.120 / Chapter CHAPTER 5: --- Conclusion and future perspectives --- p.121 / Chapter 5.1 --- Targeted MPS in plasma DNA --- p.121 / Chapter 5.2 --- Targeted MPS in NIPD of chromosomal aneuploidies --- p.124 / Chapter 5.3 --- Targeted MPS in NIPD of monogenic diseases --- p.126 / Chapter Appendix I: --- Primer names and sequences for genotyping and haplotyping of βeta-globin gene cluster region --- p.128 / Chapter Appendix II: --- Primers used in PCRs and sequencing for the parents in the first family --- p.132 / Chapter Appendix III: --- Primers used in PCRs and sequencing for the parents in the second family --- p.138 / Chapter Appendix IV: --- RHDO analysis on maternal plasma DNA in the first family --- p.140 / Chapter Appendix V: --- RHDO analysis on maternal plasma DNA in the second family --- p.145 / References --- p.147

Orientadores: Ricardo Barini, Lourenço Sbragia Neto / Tese (doutorado) - Universidade Estadual de Campinas. Faculdade de Ciencias Medicas / Made available in DSpace on 2018-11-27T12:29:22Z (GMT). No. of bitstreams: 1 Bueno_MarciaPereira_D.pdf: 2898828 bytes, checksum: 2c6dcaa4a20ec185bea4b47114c30a0e (MD5) Previous issue date: 2010 / Resumo: A restrição do crescimento intrauterino (RCIU) limita o desenvolvimento fetal adequado aumentando a morbidade e mortalidades perinatais. Os mecanismos fetais adaptativos na RCIU podem desencadear alterações endócrinas e metabólicas que explicariam a ocorrência de doenças na idade adulta. O objetivo do estudo foi avaliar na RCIU experimental pela ligadura da artéria uterina se existem alterações na morfometria e histologia do fígado, intesti no e rins e se existem diferenças na expressão dos receptores de insulina, IR-(3, IRS-1, IRS-2, IGF-IR(3 no grupo de fetos submetidos à RCIU. O presente experimento foi submetido ao Comitê de Ética e Experimentação Animal da Universidade Estadual de Campinas (CEEA-UNICAMP) e aprovado como projeto de pesquisa N° 1644-1. Para realização do estudo utilizamos fetos de ratas Sprague Dawley divididos em 3 grupos. Grupo I (RCIU) - 40 fetos submetidos à ligadura da artéria uterina unilateral com 18,5 dias de gestação, Grupo II (Controle-RCIU) - 40 fetos do corno oposto ao da ligadura da artéria uterina e Grupo III (Controle Externo) - 40 fetos sem procedimento cirúrgico ou alimentar. Os resultados mostraram no modelo experimental de RCIU uma diminuição do peso corporal (PC), hepático (PH) e intestinal (PI) (p<0,01) no grupo RCIU, as relações entre PH/PC, PI/PC, PR/PC foram mantidas, fetos RC IU tem diminuição das camadas submucosas e mucosas intestinais (p<0,05); diminuição da camada cortical renal e do número de glomérulos, com aumento do volume glomerular (p<0,05). Na RCIU encontramos menor expressão hepática do IR-(3, IRS-1 e IRS-2, menor expressão do IRS-2 no intestino e rins e maior expressão do IGF-IR(3 em todos os tecidos. O modelo experimental estudado causou uma RCIU simétrica com alterações morfométricas e do metabolismo da glicose que poderiam justificar no futuro um maior risco de doenças metabólicas / Abstract: Intrauterine growth restriction (IUGR) limits appropriate fetal development increasing morbidity and perinatal mortality. Adaptive mechanisms in fetal IUGR may leave to endocrine and metabolic alterations that could explain the occurrence of diseases in adulthood. The aim of this study was to evaluate whether experimental IUGR by uterine artery ligation causes changes in morphology and histology of the liver, intestines and kidneys. We also evaluated if there were differences in the expression of insulin receptors, IR-(3, IRS-1, IRS-2, IGF-IR(3 of fetuses subjected to IUGR. This experiment was submitted to the Ethics and Animal Experimentation of the Campinas State University (UNICAMP CEEA) and was approved as a research project No. 1644-1. The study used fetuses Sprague-Dawley rats divided into 3 groups. Group I (IUGR) - 40 fetuses who underwent uterine artery ligation sided with 18.5 days of pregnancy Group II (Control-IUGR) - 40 fetuses of the horn opposite to the uterine artery ligation, and Group III (External Control) - 40 fetuses without surgery or food The results showed the experimental model of IUGR, a reduction in body weight (BW), liver (PH) and intestine (PI) (p <0.01) in IUGR, the relationship between PH/PC, PI/PC, PR/PC have been retai ned, IUGR fetuses have reduced layers of the intestinal mucosa and submucosa (p<0,05), decreased renal cortical layer and the glomerular number and increased volume rate (p<0,05). In IUGR found lower hepatic expression of IR-(3, IRS-1 and IRS-2, reduced expression of IRS-2 in the intestine and kidney and increased expression of IGF-IR(3 in all tissues. The experimental model studied caused a symmetrical IUGR with histological changes and glucose metabolism that could justify a greater risk of metabolic diseasesin the future / Doutorado / Ciencias Biomedicas / Doutor em Tocoginecologia

BACKGROUND: Fetal growth is a complex process which depends on nutrient and oxygen availability and transport from the mother to the fetus across the placenta. This involves hormones and growth factors as well as maternal and fetal genes. The failure of the fetus to reach his or her full potential for growth is called Intrauterine Growth Restriction (IUGR) and implies risks for adverse short‐ and long‐ term outcomes. Imprinted genes are a specific subset of genes that display, in mammals and flowering plants, monoallelic expression depending on the parental origin of the allele. The regulation of imprinted expression depends on epigenetic mechanisms, a subset of heritable marks that have the ability to regulate DNA functions without altering its sequence. Imprinted genes tend to cluster in the genome due to coordinated regulation through Imprinting Control Centers, usually in the form of Differentially Methylated Regions between the paternally and maternally inherited alleles. Studies in both animals and humans as well as imprinting syndromes have uncovered a role for this group of genes in prenatal growth. Two imprinted genes (PLAGL1 and HYMAI) have been described in the 6q24 locus. Genetic and epigenetic defects in this region relate to the Transient Neonatal Diabetes Mellitus 1 phenotype, including severe growth restriction. We aimed to study the involvement of this region in non‐syndromic IUGR. PARTICIPANTS AND METHODS: One hundred placental samples from a cohort of healthy term singletons, fetal tissues from fifty‐four first trimester terminations and one hundred placental samples from healthy and complicated pregnancies of different gestational ages were used to analyze the role of the 6q24 region in normal fetal growth and IUGR, respectively. Relevant clinical data was obtained after informed consent. The methylation status of the 6q24 CpG islands was studied by array technology and bisulfite sequencing in normal term placenta and in first trimester fetal tissues. Methylation levels in the PLAGL1 DMR in healthy and IUGR placentas were compared by pyrosequencing. Allelic origin of expression was assessed by heterozygous DNA/cDNA SNP analysis. Levels of expression of imprinted transcripts were analyzed by qRT‐PCR. RESULTS: PLAGL1 P1, HYMAI and two newly described PLAGL1 isoforms (P3 and P4) were the only transcripts subjected to genomic imprinting in the investigated 6q24 region. Correspondingly, the CpG island associated to the P1 promoter was the only differentially methylated region. There was no correlation between PLAGL1 expression in the placenta and fetal size in uneventful pregnancies. In placentas from IUGR gestations, expression of HYMAI was significantly higher than in those from normally grown fetuses. Levels of expression of PLAGL1 were lower in IUGR and correlated positively and significantly with the presence of IUGR in placentas from girls, but not boys. These changes in expression were not mediated by Loss of Imprinting or abnormalities in the levels of methylation of the promoter‐associated DMR, but possibly by a change in regulatory posttranscriptional mechanisms, as suggested by the loss of correlation of PLAGL1 P1 and HYMAI expression in IUGR. CONCLUSIONS: Imprinted expression in the 6q24 region is limited to the PLAGL1/HYMAI locus, maybe due to demarcation of this region by CTCF boundaries. Intrauterine Growth Restriction is associated to abnormalities in expression of PLAGL1 and HYMAI in the placenta, which are not due to LOI or methylation changes.

Introdução- Os movimentos respiratórios têm influência na circulação fetal. Sua presença indica um sistema nervoso intacto, não deprimido, refletindo o bem-estar do concepto. Acredita-se que, em apnéia, a pressão exercida pelos órgãos intratorácicos no coração fetal, em particular os pulmões não expandidos, limita a distensibilidade ventricular. O padrão de fluxo das veias pulmonares, um parâmetro para avaliação Doppler-ecocardiográfica da função diastólica fetal, é determinado pelos eventos que ocorrem do lado esquerdo do coração, sendo influenciado pelas mudanças dinâmicas na pressão do átrio esquerdo criadas pela contração e pelo relaxamento do átrio e do ventrículo esquerdos. A impedância ao fluxo da veia pulmonar para o átrio esquerdo é representada pelo índice de pulsatilidade. Objetivo- Testar a hipótese de que o índice de pulsatilidade do fluxo venoso pulmonar fetal é menor na presença dos movimentos respiratórios fetais do que em apnéia. Métodos- Examinados 22 fetos normais de mães sem doença sistêmica, em apnéia (controles) e na presença de movimentos respiratórios fetais (casos). Os fetos foram examinados pela ecocardiografia pré-natal com Doppler e mapeamento de fluxo em cores. O índice de pulsatilidade da veia pulmonar foi obtido colocando-se a amostra volume do Doppler pulsado sobre a veia pulmonar superior direita ou inferior esquerda, e aplicando-se a fórmula velocidade máxima (sistólica ou diastólica)-velocidade pré-sistólica/velocidade média. Resultados- Os fetos apresentaram idade gestacional média de 28,9 ± 2,9 semanas. Na avaliação realizada nos fetos em apnéia as médias das velocidades sistólica, diastólica e pré-sistólica foram, respectivamente, 0,35 ± 0,08 m/s, 0,26 ± 0,07 m/s, 0,09 ± 0,03 m/s. Na avaliação realizada na presença de movimentos respiratórios fetais as médias das velocidades sistólica, diastólica e pré-sistólica foram, respectivamente, 0,33 ± 0,1 m/s, 0,28 ± 0,08 m/s, 0,11 ± 0,04 m/s. O índice de pulsatilidade da veia pulmonar médio, nos fetos em apnéia, foi de 1,25 ± 0,23 (1,69 a 0,82), e na presença de movimentos respiratórios fetais foi de 0,97 ± 0,2 (1,53 a 0,61). Conclusão- Demonstramos significante diminuição da impedância ao fluxo venoso pulmonar, representada pelo índice de pulsatilidade vascular, durante os movimentos respiratórios fetais, refletindo modificações da dinâmica atrial esquerda e da melhora complacência ventricular esquerda. / Introdution- Respiratory movements influence fetal circulation. Their presence indicates an intact, non-depressed nervous system, reflecting a good fetal clinical status. In apnea, the pressure of intrathoracic organs on the fetal heart, mainly the non-expanded lungs, limits ventricular distensibility. Flow pattern in pulmonary veins, a Doppler echocardiographic parameter in the assessment of fetal diastolic function, is determined by events occurring in the left heart and is influenced by dynamic changes in left atrial pressures created by left atrium and ventricle contraction and relaxation. Impedance to pulmonary venous flow to the left atrium is represented by the pulsatility index. Objective- To test the hypothesis that fetal pulmonary venous flow pulsatility index is lower during fetal respiratory movements than in apnea. Methods- Twenty-two normal fetuses of mothers without systemic disease were examined in apnea (controls) and in the presence of fetal respiratory movements (cases). Fetuses were examined by prenatal Doppler echocardiography with color flow mapping. The pulsatility index of the pulmonary vein was obtained placing the pulsed Doppler sample volume over the right upper or left lower pulmonary vein , and applying the formula [maximum velocity (systolic or diastolic)–pre-systolic velocity]/mean velocity. Results- Mean gestational age was 28.9 ± 2.9 weeks. During fetal apnea, mean systolic, diastolic and pre-systolic velocities were, respectively, 0.35 ± 0.08 m/s, 0.26 ± 0.07 m/s and 0.09 ± 0.03 m/s. In the presence of fetal respiratory movements, mean systolic, diastolic and pre-systolic velocities were, respectively, 0.33 ± 0.1 m/s, 0.28 ± 0.08 m/s and 0.11 ± 0.04 m/s. Pulsatility index pulmonary vein in apnea was 1.25 ± 0.23 (1.69 to 0.82), and during fetal respiratory movements it was 0.97 ± 0.2 (1.53 to 0.61). Conclusion- We showed a significant reduction in impedance of pulmonary venous flow, represented by pulmonary vein pulsatility index, during fetal respiratory movements, reflecting modifications of the left atrial dynamics and enhancement of left ventricular compliance.

Na Folha de Aprova??o consta o t?tulo: "An?lise das a??es dos Comit?s de Preven??o ao ?bito Infantil e Fetal em uma Regi?o de Sa?de do Vale do Jequitinhonha, Minas Gerais". / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2018-04-04T15:28:24Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) isabella_rodrigues_diamantino.pdf: 2271469 bytes, checksum: 6aa1269a10915ee8e6f7cc7e575910b3 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2018-04-09T18:53:54Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) isabella_rodrigues_diamantino.pdf: 2271469 bytes, checksum: 6aa1269a10915ee8e6f7cc7e575910b3 (MD5) / Made available in DSpace on 2018-04-09T18:53:54Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) isabella_rodrigues_diamantino.pdf: 2271469 bytes, checksum: 6aa1269a10915ee8e6f7cc7e575910b3 (MD5) Previous issue date: 2017 / Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG) / Funda??o Diamantinense de Apoio ao Ensino, Pesquisa e Extens?o (Fundaepe) / A Taxa de Mortalidade Infantil (TMI) ? considerada um dos mais sens?veis indicadores de sa?de utilizados pela sa?de p?blica para avaliar as condi??es de vida de uma popula??o em geral, sendo determinada, principalmente, pelas condi??es socioecon?micas de uma regi?o. A investiga??o de mortes infantis ? considerada uma importante estrat?gia para a redu??o desse fen?meno, tendo em vista o fato de propiciar mais visibilidade ? real situa??o dos munic?pios e possibilitar a implanta??o de medidas de preven??o e controle. O presente estudo foi desenvolvido no per?odo de 2013 a 2016, com o objetivo de analisar a atua??o dos Comit?s de Preven??o ao ?bito Infantil e Fetal na Regi?o de Sa?de de Ara?ua?, Minas Gerais. Trata-se de um estudo descritivo, realizado em 06 comit?s/equipes de investiga??o municipais que comp?em a regi?o, pertencente ? mesorregi?o do Jequitinhonha, Minas Gerais. A informa??o foi obtida a partir da utiliza??o de fontes secund?rias em base de dados nacionais, como o SIM e o Painel de Monitoramento da Mortalidade Infantil e Fetal. Os dados obtidos foram tabulados no Excel, pacote Officce (Microsoft?), e receberam tratamento para estat?stica descritiva. Como resultados observou-se a redu??o da mortalidade em crian?as menores de um ano na regi?o, com frequ?ncia de 15 casos em 2013 e 10 casos em 2016; e a eleva??o do n?mero de mortes fetais, com 14 ?bitos em 2013 e 16 ?bitos em 2016. Houve aumento de 57,2% nos ?bitos infantis evit?veis na regi?o, no per?odo analisado. De todos os seis munic?pios estudados, apenas um apresentou investiga??o de 100% dos ?bitos infantis e fetais em tempo oportuno. A organiza??o do sistema/servi?o de sa?de da regi?o foi respons?vel pela maioria dos problemas identificados ap?s a investiga??o dos ?bitos (34,8%), sendo a cobertura da aten??o prim?ria o principal problema relacionado ? organiza??o desses servi?os. As mortes por causas evit?veis destacaram-se por serem as mais comuns, de acordo com a classifica??o de evitabilidade, realizada pelos ?rg?os investigativos, citada 47 vezes durante os quatro anos. Dentre essas causas, nenhum ?bito reduz?vel por a??es de imunopreven??o foi registrado. Como causa de ?bitos fetais e perinatais, predominou-se a prematuridade, na Regi?o de Sa?de de Ara?ua?, Minas Gerais. Conclui-se que a inexist?ncia dos comit?s na maioria dos munic?pios analisados traz in?meras defici?ncias no processo de vigil?ncia do ?bito infantil e fetal na regi?o. Apesar da presen?a de profissionais designados para a investiga??o dos casos em cada localidade, a falta de uma equipe multiprofissional capacitada dificulta a an?lise, discuss?o dos casos e execu??o de medidas efetivas para a redu??o de novas ocorr?ncias. Foram identificadas falhas assistenciais nos cuidados com o pr?-natal e parto e um alto percentual de evitabilidade dos ?bitos. A implanta??o do Comit? de Preven??o ? Mortalidade Infantil e Fetal em toda a Regi?o de Sa?de de Ara?ua?, al?m do fortalecimento daqueles j? existentes, e o apoio cont?nuo de gestores municipais, estaduais e sociedade a estes ?rg?os, s?o fatores imprescind?veis para a melhoria dos cuidados prestados ?s gestantes e crian?as no territ?rio estudado. / Disserta??o (Mestrado Profissional) ? Programa de P?s-Gradua??o em Sa?de, Sociedade e Ambiente, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / The Child Mortality Rate (CMR) is considered to be one of the most sensitive health indicators used by Public Health services to assess the life conditions of the population in general, and is mainly determined by the socio-economic conditions of a region. The investigation of children death is regarded as an important strategy to reduce this phenomenon once it provides greater visibility to the cities real situation and enables the implementation of prevention and control measures. The current study was developed between 2013 and 2016, aiming to analyse the actions of the Children and Fetal Death Prevention Committees in Ara?ua?/MG health care territory. That is a descriptive study whose sample was composed for 06 municipal committees that comprehend Ara?ua? health care territory, belonging to the mesoregion of Jequitinhonha/ MG. The information was derived from secondary sources in national data bases such as the Mortality Information System (SIM) and the Children and Fetal Death Monitoring Panel. The survey data were organized on Microsoft Office Excel and were processed for descriptive statistics. The results observed were: The reduction of mortality among children under 1 year old in the region, being 15 cases in 2013 and 10 in 2016; and the increase of Fetal death rate, being 14 deaths in 2013 and 16 in 2016. There was an increase of 57,2% in preventable children death in the region in the period under review. Only one city out of the six studied presented in time a 100% investigation of children and fetal death. The health system organization of the region accounted for the majority of the problems identified after the death investigation ( 34,8%), being the primary - care coverage the main problem related to the organization of these services. The deaths for preventable causes excelled for being the most common, according to the avoidability rating carried out by investigative authorities, and they were mentioned 47 times during the 4-year period. Among these causes, it was not registered any death reducible for immunoprevention actions. As a cause of fetal and perinatal deaths prevailed prematurity, in Ara?ua? health care territory, Minas Gerais. The conclusion is that, the absence of committees in most studied cities brings several deficiencies in the process of monitoring child and fetal death in the region. Although there are professionals who perform the investigation of the cases in each locality, the lack of a capable multiprofessional team obstructs the analysis, discussion of cases and execution of effective measures to reduce new events. It has been identified care failures regarding prenatal and childbirth and a high percentage of death avoidability. The implementation of the Child and Fetal Death Prevention Committee in the whole Ara?ua? health care territory , the strengthening of those that already exist in conjunction with the permanent support from municipal, state managers and society to these bodies, are relevant factors to enhance the care provided to pregnant women and children in the studied area. / La Tasa de Mortalidad Infantil (TMI) es considerada uno de los m?s sensibles indicadores de salud utilizados por la salud p?blica para evaluar las condiciones de vida de una poblaci?n en general, siendo determinada, principalmente, por las condiciones socioecon?micas de una regi?n. La investigaci?n de muertes infantiles es considerada una importante estrategia para la reducci?n de ese fen?meno, teniendo en vista el hecho de propiciar m?s visibilidad a la real situaci?n de los municipios y posibilitar la implantaci?n de medidas de prevenci?n y control. El presente estudio fue desarrollado en el per?odo de 2013 a 2016, con el objetivo de analizar la actuaci?n de los Comit?s de Prevenci?n al ?bito Infantil y Fetal en la Regi?n de Salud de Ara?ua?, Minas Gerais. Se trata de un estudio descriptivo realizado en 06 comit?s / equipos de investigaci?n municipales que componen la regi?n, perteneciente a la mesorregi?n del Jequitinhonha, Minas Gerais. La informaci?n fue obtenida a partir de la utilizaci?n de fuentes secundarias en base de datos nacionales, como el Sistema de Informaci?n sobre Mortalidad (SIM) y el Panel de Monitoreo de la Mortalidad Infantil y Fetal. Los datos obtenidos fueron tabulados en Excel, paquete Officce (Microsoft?), y recibieron tratamiento para estad?stica descriptiva. Como resultados se observ? la reducci?n de la mortalidad en ni?os menores de un a?o en la regi?n, con frecuencia de 15 casos en 2013 y 10 casos en 2016; y la elevaci?n del n?mero de muertes fetales, con 14 muertes en 2013 y 16 muertes en 2016. Se observ? un aumento del 57,2% en las muertes infantiles evitables en la regi?n, en el per?odo analizado. De todos los seis municipios estudiados, s?lo uno present? investigaci?n del 100% de las muertes infantiles y fetales a tiempo oportuno. La organizaci?n del sistema / servicio de salud de la regi?n fue responsable de la mayor?a de los problemas identificados despu?s de la investigaci?n de los ?bitos (34,8%), siendo la cobertura de la atenci?n primaria el principal problema relacionado a la organizaci?n de esos servicios. Las muertes por causas evitables se destacaron por ser las m?s comunes, de acuerdo con la clasificaci?n de evitabilidad, realizada por los ?rganos investigativos, citada 47 veces durante los cuatro a?os. De entre estas causas, no se registr? ninguna muerte reducible por acciones de inmunoprevenci?n. Como causa de muertes fetales y perinatales, predomin? la prematuridad, en la Regi?n de Salud de Ara?ua?, Minas Gerais. Se concluye que la inexistencia de los comit?s en la mayor?a de los municipios analizados trae innumerables deficiencias en el proceso de vigilancia del ?bito infantil y fetal en la regi?n. A pesar de la presencia de profesionales designados para la investigaci?n de los casos en cada localidad, la falta de un equipo multiprofesional capacitado dificulta el an?lisis, discusi?n de los casos y ejecuci?n de medidas efectivas para la reducci?n de nuevas ocurrencias. Se identificaron fallas asistenciales en los cuidados con el prenatal y parto y un alto porcentaje de evitabilidad de las muertes. La implantaci?n del Comit? de Prevenci?n a la Mortalidad Infantil y Fetal en toda la Regi?n de Salud de Ara?ua?, adem?s del fortalecimiento de aquellos ya existentes, y el apoyo continuo de gestores municipales, estatales y sociedad a estos ?rganos, son factores imprescindibles para la mejora de los cuidados a las gestantes y ni?os en el territorio estudiado.

Introdução- Os movimentos respiratórios têm influência na circulação fetal. Sua presença indica um sistema nervoso intacto, não deprimido, refletindo o bem-estar do concepto. Acredita-se que, em apnéia, a pressão exercida pelos órgãos intratorácicos no coração fetal, em particular os pulmões não expandidos, limita a distensibilidade ventricular. O padrão de fluxo das veias pulmonares, um parâmetro para avaliação Doppler-ecocardiográfica da função diastólica fetal, é determinado pelos eventos que ocorrem do lado esquerdo do coração, sendo influenciado pelas mudanças dinâmicas na pressão do átrio esquerdo criadas pela contração e pelo relaxamento do átrio e do ventrículo esquerdos. A impedância ao fluxo da veia pulmonar para o átrio esquerdo é representada pelo índice de pulsatilidade. Objetivo- Testar a hipótese de que o índice de pulsatilidade do fluxo venoso pulmonar fetal é menor na presença dos movimentos respiratórios fetais do que em apnéia. Métodos- Examinados 22 fetos normais de mães sem doença sistêmica, em apnéia (controles) e na presença de movimentos respiratórios fetais (casos). Os fetos foram examinados pela ecocardiografia pré-natal com Doppler e mapeamento de fluxo em cores. O índice de pulsatilidade da veia pulmonar foi obtido colocando-se a amostra volume do Doppler pulsado sobre a veia pulmonar superior direita ou inferior esquerda, e aplicando-se a fórmula velocidade máxima (sistólica ou diastólica)-velocidade pré-sistólica/velocidade média. Resultados- Os fetos apresentaram idade gestacional média de 28,9 ± 2,9 semanas. Na avaliação realizada nos fetos em apnéia as médias das velocidades sistólica, diastólica e pré-sistólica foram, respectivamente, 0,35 ± 0,08 m/s, 0,26 ± 0,07 m/s, 0,09 ± 0,03 m/s. Na avaliação realizada na presença de movimentos respiratórios fetais as médias das velocidades sistólica, diastólica e pré-sistólica foram, respectivamente, 0,33 ± 0,1 m/s, 0,28 ± 0,08 m/s, 0,11 ± 0,04 m/s. O índice de pulsatilidade da veia pulmonar médio, nos fetos em apnéia, foi de 1,25 ± 0,23 (1,69 a 0,82), e na presença de movimentos respiratórios fetais foi de 0,97 ± 0,2 (1,53 a 0,61). Conclusão- Demonstramos significante diminuição da impedância ao fluxo venoso pulmonar, representada pelo índice de pulsatilidade vascular, durante os movimentos respiratórios fetais, refletindo modificações da dinâmica atrial esquerda e da melhora complacência ventricular esquerda. / Introdution- Respiratory movements influence fetal circulation. Their presence indicates an intact, non-depressed nervous system, reflecting a good fetal clinical status. In apnea, the pressure of intrathoracic organs on the fetal heart, mainly the non-expanded lungs, limits ventricular distensibility. Flow pattern in pulmonary veins, a Doppler echocardiographic parameter in the assessment of fetal diastolic function, is determined by events occurring in the left heart and is influenced by dynamic changes in left atrial pressures created by left atrium and ventricle contraction and relaxation. Impedance to pulmonary venous flow to the left atrium is represented by the pulsatility index. Objective- To test the hypothesis that fetal pulmonary venous flow pulsatility index is lower during fetal respiratory movements than in apnea. Methods- Twenty-two normal fetuses of mothers without systemic disease were examined in apnea (controls) and in the presence of fetal respiratory movements (cases). Fetuses were examined by prenatal Doppler echocardiography with color flow mapping. The pulsatility index of the pulmonary vein was obtained placing the pulsed Doppler sample volume over the right upper or left lower pulmonary vein , and applying the formula [maximum velocity (systolic or diastolic)–pre-systolic velocity]/mean velocity. Results- Mean gestational age was 28.9 ± 2.9 weeks. During fetal apnea, mean systolic, diastolic and pre-systolic velocities were, respectively, 0.35 ± 0.08 m/s, 0.26 ± 0.07 m/s and 0.09 ± 0.03 m/s. In the presence of fetal respiratory movements, mean systolic, diastolic and pre-systolic velocities were, respectively, 0.33 ± 0.1 m/s, 0.28 ± 0.08 m/s and 0.11 ± 0.04 m/s. Pulsatility index pulmonary vein in apnea was 1.25 ± 0.23 (1.69 to 0.82), and during fetal respiratory movements it was 0.97 ± 0.2 (1.53 to 0.61). Conclusion- We showed a significant reduction in impedance of pulmonary venous flow, represented by pulmonary vein pulsatility index, during fetal respiratory movements, reflecting modifications of the left atrial dynamics and enhancement of left ventricular compliance.

Introdução- Os movimentos respiratórios têm influência na circulação fetal. Sua presença indica um sistema nervoso intacto, não deprimido, refletindo o bem-estar do concepto. Acredita-se que, em apnéia, a pressão exercida pelos órgãos intratorácicos no coração fetal, em particular os pulmões não expandidos, limita a distensibilidade ventricular. O padrão de fluxo das veias pulmonares, um parâmetro para avaliação Doppler-ecocardiográfica da função diastólica fetal, é determinado pelos eventos que ocorrem do lado esquerdo do coração, sendo influenciado pelas mudanças dinâmicas na pressão do átrio esquerdo criadas pela contração e pelo relaxamento do átrio e do ventrículo esquerdos. A impedância ao fluxo da veia pulmonar para o átrio esquerdo é representada pelo índice de pulsatilidade. Objetivo- Testar a hipótese de que o índice de pulsatilidade do fluxo venoso pulmonar fetal é menor na presença dos movimentos respiratórios fetais do que em apnéia. Métodos- Examinados 22 fetos normais de mães sem doença sistêmica, em apnéia (controles) e na presença de movimentos respiratórios fetais (casos). Os fetos foram examinados pela ecocardiografia pré-natal com Doppler e mapeamento de fluxo em cores. O índice de pulsatilidade da veia pulmonar foi obtido colocando-se a amostra volume do Doppler pulsado sobre a veia pulmonar superior direita ou inferior esquerda, e aplicando-se a fórmula velocidade máxima (sistólica ou diastólica)-velocidade pré-sistólica/velocidade média. Resultados- Os fetos apresentaram idade gestacional média de 28,9 ± 2,9 semanas. Na avaliação realizada nos fetos em apnéia as médias das velocidades sistólica, diastólica e pré-sistólica foram, respectivamente, 0,35 ± 0,08 m/s, 0,26 ± 0,07 m/s, 0,09 ± 0,03 m/s. Na avaliação realizada na presença de movimentos respiratórios fetais as médias das velocidades sistólica, diastólica e pré-sistólica foram, respectivamente, 0,33 ± 0,1 m/s, 0,28 ± 0,08 m/s, 0,11 ± 0,04 m/s. O índice de pulsatilidade da veia pulmonar médio, nos fetos em apnéia, foi de 1,25 ± 0,23 (1,69 a 0,82), e na presença de movimentos respiratórios fetais foi de 0,97 ± 0,2 (1,53 a 0,61). Conclusão- Demonstramos significante diminuição da impedância ao fluxo venoso pulmonar, representada pelo índice de pulsatilidade vascular, durante os movimentos respiratórios fetais, refletindo modificações da dinâmica atrial esquerda e da melhora complacência ventricular esquerda. / Introdution- Respiratory movements influence fetal circulation. Their presence indicates an intact, non-depressed nervous system, reflecting a good fetal clinical status. In apnea, the pressure of intrathoracic organs on the fetal heart, mainly the non-expanded lungs, limits ventricular distensibility. Flow pattern in pulmonary veins, a Doppler echocardiographic parameter in the assessment of fetal diastolic function, is determined by events occurring in the left heart and is influenced by dynamic changes in left atrial pressures created by left atrium and ventricle contraction and relaxation. Impedance to pulmonary venous flow to the left atrium is represented by the pulsatility index. Objective- To test the hypothesis that fetal pulmonary venous flow pulsatility index is lower during fetal respiratory movements than in apnea. Methods- Twenty-two normal fetuses of mothers without systemic disease were examined in apnea (controls) and in the presence of fetal respiratory movements (cases). Fetuses were examined by prenatal Doppler echocardiography with color flow mapping. The pulsatility index of the pulmonary vein was obtained placing the pulsed Doppler sample volume over the right upper or left lower pulmonary vein , and applying the formula [maximum velocity (systolic or diastolic)–pre-systolic velocity]/mean velocity. Results- Mean gestational age was 28.9 ± 2.9 weeks. During fetal apnea, mean systolic, diastolic and pre-systolic velocities were, respectively, 0.35 ± 0.08 m/s, 0.26 ± 0.07 m/s and 0.09 ± 0.03 m/s. In the presence of fetal respiratory movements, mean systolic, diastolic and pre-systolic velocities were, respectively, 0.33 ± 0.1 m/s, 0.28 ± 0.08 m/s and 0.11 ± 0.04 m/s. Pulsatility index pulmonary vein in apnea was 1.25 ± 0.23 (1.69 to 0.82), and during fetal respiratory movements it was 0.97 ± 0.2 (1.53 to 0.61). Conclusion- We showed a significant reduction in impedance of pulmonary venous flow, represented by pulmonary vein pulsatility index, during fetal respiratory movements, reflecting modifications of the left atrial dynamics and enhancement of left ventricular compliance.

In the rat, before the establishment of the chorioallantoic placenta, the nutritional requirements of the post-implantation embryo, are met solely by the visceral yolk sac and therefore a study of its structure and functions is essential to a full understanding of early embryonic nutrition. A method has been developed for maintaining the rat visceral yolk sac in organ culture over a prolonged period, having first removed the embryo by microsurgery at 9.5 days or alternatively allowing it to die within its own amnion. The yolk sac continues to grow as a closed vesicle, and can reach a diameter of 2cm. The system has been called the 'giant' yolk sac. The 'giant' yolk sac and in vivo yolk sac have been compared using various criteria. A detailed morphological study was made, including a quantitative analysis of the vacuolar compartment. The endocytic capacity of both systems was studied using three different substrates; those used were 125I-polyvinylpyrrolidone (PVP), a non-degradable macromolecule, taken up in the fluid phase and accumulated within the yolk sac tissue, 125I-bovine serum albumin (BSA) taken up by adsorptive pinocytosis and digested within the lysosomes and 125I-IgG (and colloidal gold-IgG) taken up with great efficiency by specific receptor mediated endocytosis. Also a preliminary study of 14C-amino acid uptake was made. In many instances the 'giant' yolk sac functioned very similarly to the in vivo yolk sac and therefore seems an ideal model for studying transport across an epithelial sheet. It is particularly useful as its continuous epithelium separates the exocoelom from the external culture medium. The fluid maintained within the exocoelom of the 'giant' yolk sac should be an excellent source of processed histiotroph essential for embryonic nutrition during organogenesis. Experiments carried out indicate that some of the trophic factors necessary for growth are present in this fluid.

Glucocorticoids are steroid hormones that affect a variety of physiological and pathological processes both throughout development and in adult life. During mammalian fetal growth, the late gestation rise in fetal glucocorticoid levels is essential for the maturation of tissues and organs in preparation for birth. In humans, glucocorticoids are routinely administered to women threatened by a preterm labour to accelerate fetal lung maturation and prevent neonatal respiratory distress and mice lacking glucocorticoid receptor (GR-/- mice) die neonatally as they are unable to inflate their lungs due to severe pulmonary immaturity. Apart from their importance for proper lung maturation, the physiological role of glucocorticoids in the development of other organs and tissues is not well known. However, prenatal exposure to excess glucocorticoids was shown to elicit detrimental “programming” effects, raising the susceptibility to adult diseases such as hypertension, obesity and metabolic disturbances in both humans and animal models. I therefore used global and conditional GR knock out mouse models to investigate the role and importance of adequate glucocorticoid signalling in fetal heart development and maturation. I further confirmed the direct effects of glucocorticoids on the cardiomyocyte structure and function in an in vitro setting. GR-/- fetuses are under-represented in late gestation (>50% of the number of GR+/+ littermates) but are present in the expected mendelian ratio at E14.5. At E17.5, GR-/- fetuses show edema (increased fluid accumulation and body sodium content). Excess extracellular fluid accumulation could be a result of a congenital heart failure. During development, corticosterone levels sharply increase within the fetal hearts at E15.5-E16.5, coincident with nuclear translocation of GR. Consistent with activation of GR only after this time, the phenotypic consequences of GR deficiency can be seen after E16.5 and not before. At E17.5, hearts of GR-/- fetuses are smaller than in GR+/+ but display no structural abnormalities. Cardiac function however is severely impaired, with left ventricular systolic and diastolic performance inferior in GR-/- fetuses compared to their wild-type littermates. Microscopically, at E17.5, the structure of the cardiac muscle and individual cardiomyocytes are affected by the lack of GR. The normal outer muscle layer, with characteristic rod-shaped, aligned cardiomyocytes is not discernable in the GR-/- heart. Within the cardiomyocytes, myofibrils are short, undefined and randomly scattered within the cell. Lack of the maturational progression in the GR-/- hearts at E17.5 is evident in the pattern of gene expression. GR-/- fetuses do not display the normal gestational changes between E14.5 and E17.5 that are seen in control mice, including in genes involved in the maturation of cardiac structure (eg myosin heavy chain-α, MyHC-α), function (atrial natriuretic peptide, ANP), energy metabolism (eg hexokinase-1, PPARγ coactivator-1α, PGC-1α) and calcium handling (ryanodine receptor, RyR; sarcoplasmic reticulum Ca2+-ATPase, SERCA2a). However, there are no genotype or gestational alterations in mRNA encoding the mineralocorticoid receptor, which is also a receptor for glucocorticoids in the heart. The normal gestational changes in the levels of modified histone H3 associated with the promoters of some of the genes (MyHC-α, ANP, PGC-1α) are not seen in hearts of GR-/- fetuses. This cardiac phenotype was not secondary to adrenal catecholamine insufficiency reported in other GR-/- models, as peripheral tissue levels of adrenaline were not different between genotypes. In order to test the hypothesis that the effects of glucocorticoids on the heart are mediated via GR in cardiomyocytes and to further elucidate the direct effects of GR deficiency specifically within the heart, mice with conditional deletion of GR selectively in cardiac and vascular smooth muscle cells were generated ("SMGRKO" mice). These show ~65% reduction in cardiac GR mRNA and protein levels. Circulating levels of corticosterone do not differ between genotypes at E17.5. SMGRKO fetuses at E17.5 display a phenotype strikingly similar to that of global GR-/-, namely edema, impaired cardiac function, impaired cellular architecture within the ventricle and alterations in the gene expression, implying that the GR-deficient phenotype is largely due to the direct actions of GR within the heart and not secondary to effects on other systems (eg kidney or liver). In order to investigate the pathways by which GR stimulates cardiomyocyte maturation, an in vitro model of murine primary fetal (E15.5-E16.5) cardiomyocytes was developed. Cultures contain >98% of troponin Tpositive cells which beat spontaneously. Treatment of cardiomyocytes with either synthetic (dexamethasone) or physiological (corticosterone) glucocorticoid induces time- and dose-dependent changes in gene expression, consistent with glucocorticoid-dependent changes seen in vivo in the late gestation heart. The effects of glucocorticoids on gene expression were abolished by either siRNA mediated knock-down of GR or RU486 antagonism of GR, but were unaffected by a mineralocorticoid receptor (MR) antagonist. Moreover, cycloheximide pretreatment (to block protein synthesis) suggested PGC-1α as a direct genomic target of GR. RNAseq transcriptome analysis performed on cardiomyocytes treated with dexamethasone and cycloheximide for 2h identified >600 genes as possible rapid and direct glucocorticoid response targets. Among them are genes involved in energy metabolism, calcium handling and sarcomere assembly. Glucocorticoid treatment of fetal cardiomyocytes also induces striking structural changes – formation of stress troponin T-associated actin fibers and sarcomere assembly. Spontaneous contractile activity is improved by glucocorticoid treatment, with a decrease in both contraction and relaxation time (without a change in frequency) and an improvement in the relaxation kinetics. In summary, glucocorticoid signalling in cardiomyocytes is required for the functional, structural and transcriptional maturation of the fetal heart in late gestation in vivo. Glucocorticoid treatment of primary murine fetal cardiomyocytes replicated the contractile, transcriptional and structural changes seen in vivo and was dependent on GR. Thus, GR is essential in cardiomyocytes for the structural and biochemical changes that underlie the maturation of heart function around the time of birth and an inadequate glucocorticoid environment could potentially lead to detrimental and permanent changes in postnatal cardiac function. Since prenatal glucocorticoids are routinely used clinically, it is important to consider any possible effects they might have on the heart development and its function later in life.