Rozprawy doktorskie na temat „Fetus – Diseases”

Thesis (Ph. D.)--Ohio State University, 2004.
Title from first page of PDF file. Document formatted into pages; contains xvi, 137 p.; also includes graphics (some col.). Includes bibliographical references (p. 127-137).

Orientadores: Edmur Franco Carelli, Donizeti Cesar Honorato
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: O diagnóstico pré-natal da mielomeningocele permite melhor planejamento de sua abordagem e, mais recentemente, um possível reparo intra-útero. Este estudo analisa a evolução neurocirúrgica de 98 crianças com mielomeningocele, tratadas no período pós-natal através de cirurgia tradicional, de janeiro de 1994 a dezembro de 2002, no centro de referência em medicina fetal da Universidade Estadual de Campinas (UNICAMP). Desta avaliação retrospectiva, foi elaborado um protocolo próprio para cirurgia fetal para prevenção de hidrocefalia, identificando-se os fetos que se beneficiariam com a correção intra-útero da mielomeningocele. O estudo revelou uma realidade caracterizada pelo prognóstico ruim e pelo alto índice de complicações decorrentes da mielomeningocele, principalmente no que se diz respeito à hidrocefalia. Com isso, no protocolo foram incluídos somente fetos com um tamanho ventricular menor que 14 mm no momento da cirurgia, fetos entre 20 e 25 semanas de gestação, fetos com defeitos situados abaixo de L3-L4, fetos com mielomeningocele como malformação isolada e ausência de anomalias cromossômicas, tendo como critérios de exclusão: a primiparidade, fetos com lesão abaixo de S1 e a incapacidade de entendimento das gestantes dos riscos materno-fetais. Apesar da correção intra-útero para prevenção de hidrocefalia ter uma aplicação bastante restrita em nosso meio, é uma nova opção de tratamento para as mães brasileiras, já que a legislação do país não prevê a interrupção médica da gravidez, quando complicada por fetos com mielomeningocele
Abstract: Prenatal diagnosis of myelomeningocele has permitted a better planning for optimum management of the disease. More recently, it has allowed for a possible intrauterine repair of the spinal defect. This study analyzed neurosurgical outcome of 98 children with myelomeningocele, postnatally treated with traditional surgery, from January 1994 to December 2002, in the Fetal Medicine referral center at the Universidade Estadual de Campinas (UNICAMP). From this retrospective evaluation, a suitable protocol for fetal surgery was developed for the prevention of hydrocephalus, identifying fetuses that would benefit from intrauterine repair of myelomeningocele. The study revealed a reality characterized by a poor prognosis and a high rate of complications due to myelomeningocele, particularly hydrocephalus. Thus, included in the protocol were only fetuses with ventricular size measuring less than 14 mm at the time of surgery; fetuses between 20 and 25 weeks of gestation; fetuses with defects located below L3-L4; fetuses with myelomeningocele as isolated malformation and absence of chromosomal abnormalities. Criteria of exclusion were: primiparity, fetuses with lesions below S1 and pregnant women¿s lack of understanding of the maternal-fetal risks. Intrauterine repair for the prevention of hydrocephalus has a very limited application in our setting. However, it is a new therapeutic option for Brazilian mothers, since in this country legislation is against medical termination of pregnancies affected with fetal myelomeningocele
Doutorado
Neurologia
Doutor em Ciências Médicas

Posterior urethral valves cause bladder outflow obstruction and damage to the developing fetal kidney. Posterior urethral valves affect 1 in 8000 new-born males. A third of these children develop end stage renal failure by adolescence, despite valve ablation in the early post-natal period, implying that majority of the damage to the kidneys occurs in utero. How does this damage occur, and should we intervene in utero? The answers to these questions require further research, and are the basis to this thesis. This thesis focused on the effect bladder outflow obstruction has on morphology and apoptosis in the fetal kidney in a fetal lamb model. It also looked at the effect of bladder outflow obstruction on fetal stress hormone levels. Bladder outflow obstruction was created surgically in fetal lambs at day 70 of gestation, and fetal kidneys were analysed at day 2, 5, 10, 20 and 30 after creation of obstruction. Controls undergoing sham surgery were used for comparison. Four aspects were investigated: - effects of bladder outflow obstruction on renal histology effects of bladder outflow obstruction on expression of pro-apoptosis gene Bax and anti-apoptosis gene Bcl-X - effects of bladder outflow obstruction on renal regional apoptosis effects of bladder outflow obstruction on serum fetal ACTH and cortisol levels. Bladder outflow obstruction resulted in sequential morphological change in the fetal kidney over time. By 2 days post-obstruction, cystic change was noted. In addition, patchy attenuation of the nephrogenic blastema was evident by 5 days post-obstruction, with more confluent blastemal attenuation as well as generalized renal architectural disorganization by 10 days post-obstruction. By 20 and 30 days post-obstruction, cystic renal dysplasia had developed. Bladder outflow obstruction resulted in an increase in the ratio of renal expression of pro-apoptosis gene Bax to anti-apoptosis gene Bcl-X. Regional apoptosis counts showed increased tubular apoptosis compared to controls at 2 days post-obstruction, and increased blastemal apoptosis compared to controls at 5 days post-obstruction. By 10 days post-obstruction, blastemal apoptosis counts were reduced compared to controls. There were no significant differences in fetal serum ACTH and cortisol levels between fetal lambs with bladder outflow obstruction and controls. In conclusion, the results of this thesis outline the spectrum of morphological change in the fetal kidney over 30 days of bladder outflow obstruction. They show that detectable changes in morphology occur within two days of bladder outflow obstruction. Likewise, detectable changes in gene expression occur within 2 days of bladder outflow obstruction. The increased ratio of expression of Bax to Bcl-X suggests a swing towards increased apoptosis in response to bladder outflow obstruction. Further research is required to ascertain if these changes are reversible. However, the early onset of these changes as shown in this thesis suggests that any fetal intervention to protect the fetal kidney from the effects of bladder outflow obstruction may need to be instituted very early in gestation

Previous epidemiological evidence from a number of studies supports the hypothesis that the risk of essential hypertension, coronary heart disease and non-insulin dependent diabetes is, in part, programmed by intrauterine nutritional status. An increasing number of human studies indicate that the developing kidney is particularly vulnerable to the adverse effects of fetal growth retarding influences. In animals growth retarding diets or other insults, which have an impact on the development of cardiovascular functions, also appear to impact upon nephron number. In this study, the feeding of a 9% casein diet to pregnant rats, a mild protein restriction, reduced nephron number in the offspring, which progressively declined with age compared to those exposed to an 1 8% control diet. At weaning low-protein exposed offspring had hypertension and evedence of renal insufficiency. On natural death, the kidneys from aged male rats exposed to both low-protein and control maternal diets had a higher incidence glornerulosclerosis and renal disruption than females. Supplementing the maternal 9% casein diet with 3% glycine, 1.5% urea and 3% alanine in the rat normalised nephron number in the offspring. Only the addition of glycinc in the maternal low- protein diet prevented the appearance of high blood pressure in the offspring. In this study it has been demonstrated that in humans, those of a low birth weight or ponderal index, a marker of fetal undernutrition, had evidence of increased glomerular permeability, but not elevated blood pressure at age 10. This association was not evident at age 12 or in a separate cohort of young adults. It is possible that hypertension and a reduced nephron reserve are not causally associated. The evidence from this thesis suggest that prenatal undernutrition may programme renal structure in later life, but that renal programming is not one of the primary mechanisms leading to hypertension

BACKGROUND: Chronic hypoxia due to placental insufficiency and prenatal undernutrition are probably the two major causes worldwide of an adverse intrauterine environment having an impact on neurodevelopment. Clinically, both situations manifest as an intrauterine growth restriction (IUGR), a situation defined as a significant reduction in fetal growth resulting in a birth weight below the 10th percentile. This situation is a well-recognized cause of neurobehavioral and cognitive impairments extending beyond childhood and early adulthood period. Although all these evidence, the structural ground of these functional impairments and the pathophysiological mechanisms are not fully characterized. An improvement in these two aspects would allow us to propose different therapeutic strategies aiming to ameliorate and even revert the long-lasting consequences of the IUGR. HYPOTHESIS: We hypothesized that IUGR produces subtle structural brain changes that underlie the long-term neurobehavioral and cognitive impairments. The severity of the neurodevelopmental consequences might be related to the severity of the prenatal insult (reduction in nutrients with or without a reduction in oxygen). High-resolution brain imaging along with specific histological techniques focused on neuronal connectivity could evidence these structural brain changes. Additionally, we hypothesized that an early postnatal stimulation might ameliorate the structural and functional impairments that persist at the long-term period after IUGR. METHODS: Two animal models of IUGR were used in this thesis: i. A cohort of pregnant rabbits was randomized to reproduce an undernutrition model based on maternal food reduction intake, ii. Another cohort of pregnant rabbits was randomized to the placental insufficiency model based on the surgical ligation of 40-50% of the uteroplacental vessels that irrigate each gestational sac. After the delivery in both models, IUGR and controls animals were followed up to the 70th postnatal days. At the 30th postnatal days, a subgroup of IUGR animals was randomized to an environmental enrichment strategy. In all the groups at the neonatal period, general motor skills, reflexes, and olfactory sensitivity were evaluated. Similarly, at the 70th postnatal days, anxiety, memory, and learning were evaluated. Afterward, animals were sacrificed and brains were fixed and diffusion MRI was then performed. In a subset of animals, changes at the microstructural level and differences in the number of fibers in two specific brain circuits (anxiety and memory circuits) were performed by using a Voxel-Based approach (VBA) and Tractography analysis, respectively. Moreover, brain networks were obtained and evaluated by means of a Connectomics. Finally, a subgroup of animals was also histologically evaluated by means of dendritic spine and perineural nets evaluation in the Hippocampus. RESULTS: IUGR animals showed poorer functional performance in both moments, especially in the model of placental insufficiency. At the long-term period, IUGR animals presented an altered brain network architecture, being again these differences more pronounced in the placental insufficiency model. Moreover, VBA analysis and Tractography analysis evidenced microstructural brain changes mostly affecting gray matter and a decreased in the number of fibers involved in the anxiety and memory circuits in the IUGR animals in comparison to controls. At the cellular level, IUGR animals presented abnormal neuronal connectivity with changes in the dendritic spine density and in the perineural nets. In contrast, stimulated IUGR animals presented a functional and structural improvement in comparison to non-stimulated IUGR animals over the long-term period. CONCLUSIONS: This thesis adds to the previous evidence new insights regarding the pathophysiological mechanisms underlying IUGR and gives strong evidence linking IUGR with altered brain connectivity as the basis for the neurological sequelae associated with IUGR. Additionally, it gives preliminary evidence suggesting that a strategy based on physical, sensory, cognitive as well as social stimulation applied during early postnatal life, where brain plasticity is higher, might ameliorate the neurodevelopmental consequences of IUGR.

BACKGROUND: Approximately, five million children have been born as a result of using assisted reproductive technologies (ART). Although the majority of ART children are born healthy; there are several reports of increased rate of pregnancy complications and worse perinatal outcomes in this population that may contribute to long-term health consequences according to the fetal programming hypothesis. Due to these techniques are relatively new, the effect of ART on later stages of development and adult susceptibility are uncertain. The main hypothesis of this thesis is that fetuses conceived by ART present worse perinatal outcomes together with primary cardiovascular remodeling and dysfunction as compared to those spontaneously conceived (SC), changes that persist postnatally and leads to increased cardiovascular risk in adulthood. METHODS: Cardiovascular morphology and functional assessment was performed in singleton and twin fetuses conceived by ART and spontaneously conceived; together with the presence of adverse perinatal outcomes. Finally, follow-up of these cohorts was made and cardiac and vascular function was assessed in childhood. RESULTS: ART fetuses showed: larger atria, shorter ventricles with lower sphericity index together with thicker myocardial walls. Systolic motion was decreased as measured by M-Mode and tissue Doppler; there were also sings of impaired relaxation, as demonstrated by a longer IRT and decreased deceleration time of E wave. All these changes were independent of the presence of been small for gestational age (SGA) due to these groups showed different cardiac phenotypes. ART children showed persistence of changes in cardiac morphology and function together with vascular remodeling (increased blood pressure and thicker carotid intima media). CONCLUSIONS: Adverse pregnancy outcomes seem to be present in infertile women, regardless of the use of ART. Singleton and twin ART fetuses present cardiovascular remodeling and subclinical dysfunction that persist postnatally in childhood. These changes are independent of the presence of SGA. These findings need to be take into account for further studies regarding higher cardiovascular risks in adulthood in this population.
INTRODUCCION: Aproximadamente, 5 millones de niños han nacido en el mundo gracias al uso de las tecnicas de reproduccion asistida (TRA). La mayoría de éstos niños son sanos al nacer; pero diversos estudios mencionan la presencia de peores resultados perinatales en esta población; los cuáles podrían tener consecuencias a largo plazo de acuerdo con la teoría de la programación fetal. Estas técnicas son relativamente nuevas, por lo que sus efectos en la vida adulta aún son desconocidos. La hipótesis principal de ésta tesis es que los fetos concebidos mediante TRA, presentan peores resultados perinatales que aquellos concebidos de manera espontánea (CE), junto con la presencia de cambios cardíacos morfológicos y funcionales que persisten de manera postnatal y que condicionaría un incremento del riesgo cardiovascular en la vida adulta. METODOS: Se llevó a cabo la evaluación cardiovascular morfológica y funcional de fetos únicos y gemelares concebidos por TRA comparados con fetos CE; junto con la presencia de resultados perinatales adversos. Se realiza un seguimiento y evaluación cardiovascular de dichas cohortes hasta la infancia. RESULTADOS: Los fetos TRA presentaron cambios al comparlos con los CE: aurículas más grandes, ventrículos más cortos junto con un menor índice de esfericidad así como paredes engrosadas. presentaron función sistólica disminuída de acuerdo con las mediciones de Doppler tisular y modo-M; también signos de disfunción diastólica demostrado por IRT alargados y disminución del tiempo de deceleración de la onda E. Todos estos cambios fueron independientes de la presencia de fetos pequeños para la edad gestacional (PEG), ya que dichos grupos presentaron fenotipos cardiacos diferentes. En la infancia, los niños TRA mostraron persistencia de dichos cambios cardíacos morfológicos y funcionales subclínicos, junto con remodelado vascular (presion arterial más alta y paredes engrosadas de la intima media de las carótidas). CONCLUSIONES: Las mujeres infértiles presentan resultados perinatales adversos independiente de el método de TRA usado. Los fetos concebidos mediante TRA presentan remodelado cardiovascular el cuál persiste postnatalmente en la infancia. Dichos cambios fueron independientes de la presencia de PEG. Estos hallazgos deben ser tomados en cuentra en futuros estudios del incremento de riesgo cardiovascular en la vida adulta de ésta población.

La prematuridad y el retraso de crecimiento intrauterino constituyen actualmente los problemas más importantes de la Medicina Fetal y de la Neonatología y son las causas más frecuentes de la morbilidad y mortalidad perinatal en los países desarrollados. OBJETIVO. Valorar la eficacia de un programa de intervención de apoyo prenatal (creado ex-novo) dirigido a madres gestantes de fetos Pequeños para la Edad Gestacional (PEG): detectar si este procedimiento mejora el desarrollo físico y neuroconductual del neonato, el estado emocional de la madre y el vínculo entre ambos. METODOLOGÍA. Estudio quasiexperimental tipo ensayo clínico controlado y sin asignación aleatoria de la intervención realizado en el área Materno-fetal de BCNatal (corporación del Servicio de Medicina Maternofetal del Hospital Clínic y el Hospital Sant Joan de Déu de Barcelona). El tamaño final de la muestra fue de 158 embarazadas, de las cuales 65 formaron parte del grupo intervención y 93 formaron parte del grupo control. RESULTADOS. Al finalizar el programa se observa que el feto y el neonato muestran una mayor ganancia de peso y mayor perímetro craneal en el grupo intervención. En cuanto a las capacidades y competencias del neonato, valoradas con la Escala de Brazelton, los del grupo intervención obtienen unos resultados discretamente superiores en casi todos los parámetros estudiados, destacando una mayor capacidad de habituación ante los estímulos auditivos. En relación a la embarazada, los resultados más relevantes al finalizar el programa son una disminución de la ansiedad (valorada con el cuestionario STAI) y una mayor vinculación afectiva materno-filial (valorada con la escala EVAP). CONCLUSIONES. Para las madres gestantes de fetos PEG, el hecho de haber participado en un programa de intervención de apoyo prenatal tiene un resultado beneficioso para ambos, madre e hijo, presentando menos ansiedad materna, mejores condiciones para establecer el vínculo así como una mejora en el desarrollo físico e indicios de mejores capacidades neuroconductuales en el neonato.
Prematurity and intrauterine growth restriction are currently the most important problems in Fetal Medicine and Neonatology and also are the most frequent causes of perinatal morbidity and mortality in developed countries.The Objectives were to evaluate the effectiveness of a prenatal support program (created ex-novo) aimed at pregnant mothers of small fetuses for Gestational Age (PEG): to detect if this procedure improves the physical and neurobehavioral development of the neonate, the emotional state of the mother and the bond between them. This was a quasiexperimental study of a controlled clinical trial and without random assignment of the intervention performed in the Maternal-fetal area of BCNatal (Hospital of the Maternal-Fetal Medicine Service of Hospital Clínic and Sant Joan de Déu Hospital in Barcelona). The final sample size was 158 pregnant women, of whom 65 were part of the intervention group and 93 were part of the control group. At the end of the program, it is observed that the fetus and the neonate show a greater weight gain and greater cranial perimeter in the intervention group. As for the abilities and competences of the newborn, evaluated with the Brazelton Scale, those in the intervention group obtained slightly better results in almost all the studied parameters, emphasizing a greater capacity of habituation before the auditory stimuli. In relation to the pregnant woman, the most relevant results at the end of the program are a reduction of anxiety (valued with the STAI questionnaire) and a greater maternal-filial affective attachment (valued with the EVAP scale). In conclusion, for pregnant mothers of PEG fetuses, having participated in a prenatal support intervention program has a beneficial outcome for both mother and child, with less maternal anxiety, better bonding conditions, and improved development physical and signs of better neurobehavioral abilities in the neonate.

University of Central Florida College of Arts and Sciences Thesis
Amniocentesis is one of the most widely used prenatal diagnostic techniques for congenital disorders. It was hypothesized that the spychological responses of mothers and fathers to amniocenthesis during high-rish pregnancies would be positively correlated on scales of Symptomatology (Anxiety, Depression, Anger, and Somatic Complaints) and Well-Being (Relaxed, Contented, Friendliness, and Somatic Well-Being). It was also hypothesized that Symptomatology would be negatively correlated with Well-Being. Nineteen couples, who were referred by their physicians, voluntarily participated in the study. Each partner completed the Symptom Questionnaire (Kellner, 1983), a self-rating scale of Symptomatology and Well-Being, in addition to the Pre-Amniocentesis and Post-Amniocentesis Questionnaires (original questionnaires developed for this study) at intervals prior to and following the procedure, while awaiting results. A Pearson product-moment correlation of the total scores revealed a positive correlation (p < 0.5) between the scores of fathers and mothers on the Symptomatology Scale, both pre- and post-amniocentesis (r = .47 and .47). In addition, there was a significant negative correlation (p < .05) between Symptomatolgy and Well-Being scores for both mothers (r = -.55 and -.60) and fathers (r = -.48 and -.74) at the pre- and post-amniocentesis periods, respectively. The hypothesis cannot be completely accepted because the positive correlation does not exist at the post-amniocentesis level. Mothers appear to experience more Symptomatology and less Well-Being than fathers at the post-amniocentesis level. The results are interpreted to suggest that fathers and mothers may both benefit from pre- and post-amniocentesis supportive intervention.
M.S.;
Masters
Arts and Sciences;
Clinical Psychology;
42 p.
vii, 42 leaves, bound : ill. ; 28 cm.

SUMMARY 1) Background Fetal growth restriction (FGR) is present in 5-10% of the pregnancies and is associated to high perinatal and long-term cardiovascular morbidity. Subclinical cardiac dysfunction has previously been described in severe and early FGR cases, but not in milder forms of late-FGR. The main aim of this thesis was to assess cardiac function by new echocardiographic techniques on myocardial imaging as Tissue Doppler Imaging (TDI), in early- and late-onset FGR cases. 2) Methods First, tissue Doppler was applied in a cohort of normally growth fetuses by TDI in order to describe its reproducibility and construct reference ranges for fetal annular peak velocities and myocardial performance index at 24-41 weeks of gestation. Secondly, cardiac function including conventional echocardiographic parameters and TDI was evaluated in a cohort of early-onset growth restricted fetuses with abnormal umbilical artery (UA) Doppler and, finally, in a cohort of late-onset small for gestational age (SGA) fetuses with normal UA Doppler. 3) Results Fetal TDI measurements demonstrated a good reproducibility. GA and estimated fetal weight (EFW) adjusted reference ranges for tissue Doppler indices at 24-41 weeks of gestation were provided. TDI demonstrated the presence of both systolic and diastolic cardiac dysfunction in early-onset FGR fetuses. Late-onset FGR fetuses with normal UA were also associated with cardiac dysfunction detected by TDI. 4) Conclusions Early- and late-onset growth restricted fetuses are associated with cardiac dysfunction. Subclinical cardiac dysfunction could be present from early stages of fetal deterioration and could be detected using TDI.

The convergence of compelling evidence that transmission of HIV from a pregnant woman living with HIV to her foetus can be significantly interrupted due to advances in antiretroviral and obstetrical interventions, and worrisome epidemiologic data documenting a rise in HIV infection among Canadian women, spurred the development in Canada and world wide of policies and programmes aimed at increasing the number of pregnant women who are tested for HIV. Responding to innovative therapy reducing perinatal HIV transmission risk by increasing the number of pregnant women who agree to test for HIV is clearly an important prevention objective. However, the process must be accomplished in a way that is of most benefit to the pregnant woman herself and in a way that does not compromise a pregnant woman's rights to the established Canadian principles of HIV counselling and testing.
Working with pregnant women in Ontario, the province with the highest level of HIV infection among Canadian women, this thesis articulates and interprets their experiences of prenatal HIV counselling and testing and details their perspectives on best practices. The pregnant women's evidence-based recommendations for the re-design of prenatal HIV testing programmes are provided. These unique data have important utility for federal and provincial policy makers as HIV counselling and testing policies and programmes that encompass and are grounded in pregnant womens' experiences and perspectives are likely to be maximally acceptable and thereby increase the number of pregnant women who can be apprised of prophylactic treatment to take care of their own health needs as well as those of their unborn children.
In order for pregnant women to increase control over their own health and that of their unborn children, there is clear value in all pregnant women being afforded the opportunity to know their HIV status. However, the voices of the women in this study suggest that the autonomy rights of pregnant women may well be at risk in a programme in which the current emphasis is on potential HIV infection of the foetus rather than on potential or actual infection of the pregnant woman.

Thesis (MNutr (Interdisciplinary Health Sciences. Human Nutrition))--University of Stellenbosch, 2006.
INTRODUCTION: The objective of the study was to determine the prevalence of certain known risk factors for intra-uterine growth restriction (IUGR) in women who gave premature birth to growth-restricted infants at a large regional hospital (Kalafong) in the Gauteng province of South Africa and to investigate the possible associations between the presence of various risk factors and the severity of growth restriction found in these infants. METHOD: The study was designed as cross-sectional, descriptive and observational. The subjects included singleton growth-restricted premature infants (n=80), without congenital abnormalities and their mothers (n=80). Anthropometric data [weight, height, mid-upper arm circumference (MUAC) and triceps skinfold thickness (TSF)] were collected from these mothers three to four days post-partum. Infant birth weights were recorded at birth, while the lengths and head circumferences were recorded within 2 days post-partum. Additional information, such as birth spacing, maternal age, smoking habits and alcohol use, was collected by personal interview and blood pressure data and HIV status was obtained from medical records. Data capturing and descriptive statistics were done using Microsoft Excel and comparative analytical statistics were performed with the Statistical Package for the Social Sciences (SPSS), version 12.0. RESULTS: The study demonstrated a high prevalence (69%) of infants born with a birth weight <3rd percentile. In the sample, 81% of the mothers were aged 17-34 years and most (93%) had their children 18 months or longer apart. Malnutrition prevalence was moderate. In 58% of the mothers the BMI was normal (18.5-24.9 kg/m2) and in 47% the upper arm muscle area (UAMA) was between the 10th-85th percentile. Grade III overweight occurred in 3% and TSF ≤5th percentile occurred in 35% of the mothers. About half (51%) of the mothers in the sample population had hypertension during the second trimester of pregnancy. Smoking and alcohol use during pregnancy was rare (1% and 6% respectively) and the prevalence of HIV infection in the mothers was 26%. The prevalence (16%) of Grade II overweight among the mothers of symmetric growth-restricted (SGR) infants was higher than among the mothers of asymmetric growth-restricted (AGR) infants (7%). Of the hypertensive mothers, 55% had infants with SGR compared to 45% with AGR (p=0.47). Although rare, smoking occurred only in mothers with AGR infants (3%). No significant differences were found between the smoking and non-smoking group (p=0.21). Although the use of alcohol was more prevalent at 6% in mothers with SGA infants and 7% in mothers with AGR infants, no significant associations were found (p=0.95). Although not significant (p=0.76), there was a higher prevalence of HIV infection in mothers with SGR infants at 29%, compared to 23% of mothers of AGR infants. CONCLUSION: Although further studies are needed before intervention strategies can be planned and implemented, the findings of this study suggest that apart from the usual factors (maternal age and nutritional status, smoking and alcohol use during pregnancy and birth spacing) that may influence intra-uterine growth, hypertension may contribute greatly to IUGR in this study population.

Orientador: Lourenço Sbragia Neto
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Gastrosquise é um defeito congênito da parede abdominal anterior no qual as alças intestinais ficam herniadas e em contato com o líquido amniótico (LA). Assim, a exposição ao LA resulta em várias disfunções intestinais pós-natal. Para reduzir o tempo de exposição ao LA em modelo animal, usou-se um hidrogel de N-isopropilacrilamida (NIPAAm) copolimerizado com ácido acrílico (Aac), que rapidamente intumesce na presença de LA. O hidrogel foi usado para cobrir as alças expostas até o fim da gestação. A gastrosquise foi induzida em fetos de ratas fêmeas da raça Sprague-Dawley através de um corte paramediano à direita do cordão umbilical para exposição parcial das alças com 18,5 dias de gestação. Os fetos foram separados em quatro grupos: controle (C), apenas gastrosquise (G), gastrosquise + cobertura das alças com adesivo de fibrina - Beriplast® (GA) e gastrosquise + cobertura das alças com adesivo de fibrina e aderido um pedaço de hidrogel seco (GAH). Os animais foram colhidos por cesárea com 21,5 dias de gravidez e o hidrogel foi cuidadosamente removido. Os fetos e as alças intestinais foram pesados e análise morfométrica foi realizada. Resultados mostraram que o hidrogel após intumescimento pesou 34X que seu peso seco; ele possui carga elétrica assim como a maioria das proteínas presentes no LA e sua retirada não provocou lesão à camada serosa do intestino exposto como visto na MEV. A comparação dos grupos C e GAH com os grupos G ou GA mostrou que o peso, o diâmetro, a espessura das camadas e da parede intestinais foi significativamente menor nos grupos C e GAH quando comparados aos grupos G e GA indicando processo inflamatório. Sendo assim, a aplicação do hidrogel aderido pelo adesivo de fibrina mostrou servir como uma efetiva proteção das alças herniadas, com uma redução significante da inflamação na gastrosquise.
Abstract: Gastroschisis is a congenital defect of the anterior abdominal wall which leads the fetal bowel to herniate into the amniotic cavity. There, exposition to amniotic fluid (AF), results in severe postnatal intestinal dysfunction. In order to reduce exposition time to AF in an animal model, has used a hydrogel of N-isopropylacrylamide (NIPAAm) copolymerized with acrylic acid (Aac), which undergoes rapid swelling in the amniotic fluid. The hydrogel was used to coat the bowel hernia until pregnance is completed. Gastroschisis was induced in the fetuses of female Sprague-Dawley rats by partial evisceration of the bowel through a right paramedian opening of the abdominal wall in day 18,5 of pregnancy. The fetuses were separated in four groups: control (C), gastroschisis alone (G), gastroschisis + coating of the bowel hernia with fibrin adhesive -Beriplast® (GA) and gastroschisis + coating of the bowel hernia with fibrin adhesive topped by a piece of adhered dry hydrogel (GAH). Animals were harvested by cesarean section at day 21.5 of pregnancy and the hydrogel was carefully removed. Fetuses and intestinal tract were weighed and morphometric analysis was performed. Results showed that the hydrogel weight was 34X heavier than its dry weight; its electric charge and also the AF charge were negative and there was no damage to serosa layer of the intestine exposed. Comparison of the C and GAH groups with G and GA showed that the bowel weight, diameter, the layers and wall thickness was significantly reduced in C and GAH compared to G and GA. Thus, application of the hydrogel bound onto the fibrin adhesive was shown to provide an effective protection of the herniated bowel, with a significant reduction of inflammation in gastroschisis.
Mestrado
Pesquisa Experimental
Mestre em Cirurgia

[Truncated abstract] The dramatic increase in the expression of allergic diseases such as asthma and allergy over the last 20-30 years has highlighted the urgent need to identify causative factors. It was hypothesised that direct immune interactions between mother and fetus contribute to the cytokine milieu of pregnancy, thus influencing immune maturation after birth. Further it was speculated that the cytokine responses produced as a result of maternalfetal interactions are Th-2 skewed in women allergic disease, which programmes their offspring towards developing an allergic phenotype after birth. To test this hypothesis a cohort of 169 pregnant women were recruited at 20 weeks gestation and defined as allergic or non-allergic based on both clinical history and skin prick test sensitisation. These women and their infants were followed up throughout pregnancy (20 weeks, 30 weeks, 36 weeks gestation and 6 weeks post-partum) and up to 2.5 years of age. Mixed lymphocyte reactions (MLR) were used to measure maternal cytokine (IL-6, IL-10, IL-13 and IFN-) and lymphoproliferative responses to fetal alloantigens at each pregnancy time-point. Human leukocyte antigen (HLA) typing of mothers and infants were performed to assess the effect of HLA mismatch on maternal MLR responses to their fetus. After delivery, mononuclear cells (MNC) were isolated from cord blood (CB) and stimulated with allergens, mitogen and toll-like receptor (TLR) ligands. .... As IL-6 also participates in adaptive immunity by promoting Th-2 differentiation it is proposed that the production of IL-6 as a results of maternal encounters with paternal antigens during pregnancy, contribute to the Th-2 skewed responses observed universally in most infants at birth. Associations between maternal-fetal interaction and clinical outcomes in infancy: Although clinical signs of allergy in infancy were not the main outcome measure of this thesis, there were interesting, yet complex relationships between the production of these maternal cytokines towards the fetus and allergic disease at infant follow-ups. Increased maternal IFN-¿ to fetal alloantigen was associated with asthma at 2.5 years and a trend towards recurrent wheeze at 12 months. In contrast decreased maternal IL-13 production was associated with IgE mediated food allergy at 12 months. Adjusting for maternal allergy and other potential confounders including infant gender, method of delivery, HLA mismatch, and paternal allergy did not account for these relationships. Further follow-ups of these infants are required to determine if these relationship last in to early childhood. In conclusion, the findings of this thesis provides further support for the hypothesis that immune responses at birth are programmed prenatally, and that this programming has implications later in life. Importantly, the placenta is the immunologically active interface between mother and fetus during pregnancy. Therefore it is emphasised that there is a crucial need for future research to focus on early immune programming at the placental level before the aetiological pathways of immune mediated diseases can be fully elucidated.

Barker's "fetal origin hypothesis" advocates that the diseases in later life originate through adaptations that fetus makes when it is undernourished. These adaptations, whether cardiovascular, metabolic, or endocrine, permanently change the structure, and functions of the body, and pave the road to chronic killer diseases in later life, such as; coronary heart diseases, related disorders, stroke, diabetes, and hypertension. The main objective of the study, both prospective, and retrospective, covering the subjects from 0 to 1 year, and 3 to 15 years, respectively, is to test the Barker "fetal origin hypothesis" that nutrition in early life does influence the disease pattern in later life. The research is specifically designed to study the relationship between infant body size, placental weight, blood pressure, and lipid profile in late infancy; and whether or not the relationship between high blood pressure, and low birthweight is initiated in uterus, or during the infancy. The prospective studies were carried out in Prince Salman Bin Abdulaziz Hospital in Riyadh, KSA. The sampling was performed systematically. Every fifth child born in the delivery room was selected in the obstetric ward. A total of 1026 neonates were included in the prospective studies. The retrospective studies were conducted in Deraya Primary Health Care Centre, and data were collected from the medical record department, which included 1505 subjects, aged 3 to 15 years. The babies with major congenital malformations were excluded both studies included questionnaires, anthropometric measurements, and critical evaluation of haematological and biochemical parameters. The data collected, both prospective and retrospective and retrospective studies, were categorised, analysed, and statistically interpreted by using the Statistical Package for Social Science SPSS/PC+V9.0. Normal distributions of data were confirmed by using the Kolmogorow-Smirnow Test. In all cases, significance was assumed at P < 0.05. The major findings do support Barker's epidemiological data, and evidences. Although, it is still somewhat too early, and premature to confirm these findings, due to the length of period covered, the data presented, both prospective and retrospective, do point out, and lead to the following major conclusions: Chronic diseases are being imprinted "Programmed" in feto-placental unit during pregnancy, and infancy, there is indeed a strong association between birthweight, especially, low birthweight, and placental weight, blood pressure, lipid metabolism in early infancy, and in childhood. Low birthweight, <2500gms is strongly associated with elevated systolic blood pressure, and low birthweight infants, if survived, are predisposed to inevitable disabilities of all kinds, and chronic diseases in later life.

Pre-eclampsia is a systemic disorder unique to human pregnancy and a leading cause of maternal-fetal morbidity and mortality worldwide. While its exact aetiology remains unknown, maternal immune changes in pre-eclampsia, including reduced regulatory T cells (Treg), support a breakdown in maternal-fetal immune tolerance. The current immune theory for pre-eclampsia is likely to be incomplete, however, as fetal immune function is not considered. Although obvious restrictions limit access to the human fetus, the thymus, a primary immune organ, can be readily observed by ultrasound at mid-gestation. Fetal thymus changes observed via ultrasound can therefore offer non-invasive insight into fetal immunity. Beyond its immediate consequences to maternal-fetal wellbeing, pre-eclampsia has been linked to childhood allergy. This suggests fetal programming of allergic disease, a concept also supported by studies linking newborn head size with allergy. A possible explanation for this link centres on altered neurotrophin levels, including BDNF. Neurotrophins are important mediators of brain growth and development, which are also dysregulated in allergic disease. Cord blood BDNF levels are also altered in pre-eclampsia. We therefore hypothesized that pre-eclampsia would be associated with altered fetal head growth. This thesis consists of five publications. Firstly, Chapters 3 and 4, observed a smaller fetal thymus in pre- eclampsia, with a smaller fetal thymus preceding clinical disease. The third study, Chapter 5, included a large prospective study which confirmed the findings of Chapter 4. Chapter 5 also included thymus histopathology, which revealed dramatic apoptosis of thymocytes in pre-eclampsia, especially affecting Treg cells. Adding to our theory of neurotrophin dysregulation as a possible factor in fetal programming, Chapter 6 demonstrated increased fetal head growth in pre-eclampsia. Finally, Chapter 7 found reduced fetal head size at mid-gestation to be associated with childhood allergy. In summary, this research has shown fetal thymus changes to precede clinical disease in pre-eclampsia, possibly reflecting an active role for fetal immunity in disease pathogenesis. Our findings also support the developmental programming of allergic disease.

Orientador: Egle Cristina Couto de Carvalho
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Contexto e objetivo: Algumas malformações congênitas têm origem vascular, e a trombose durante a organogênese já foi aventada como possível mecanismo para esta ocorrência. O objetivo deste estudo foi avaliar a associação entre trombofilia fetal e malformações de origem vascular. Tipo de estudo e local: Foi realizado um estudo caso-controle, desenvolvido no ambulatório de Medicina Fetal do CAISM UNICAMP, de 2005 a 2010. Métodos: Foram incluídos no estudo 100 fetos com malformações de sistema nervoso central (SNC), gastrosquise, limb body wall e redução de membros (casos), submetidos a cordocentese como rotina do serviço, cujos resultados de cariótipo foram normais. Como controles, foram incluídos 100 fetos sem malformações cujo sangue de cordão fora previamente doado para o Banco de Sangue de Cordão do HEMOCENTRO UNICAMP. A pesquisa das mutações Fator V de Leiden, G20210A-FII e C677T-MTHFR foi realizada no sangue fetal dos dois grupos, e os resultados foram comparados. A análise descritiva foi feita utilizando Qui-quadrado e Teste Exato de Fisher. Para avaliar a associaçãoo entre as variáveis, foram utilizados o teste de Wilcoxon e a regressão logística. Resultados: Foram incluídos 78 fetos com malformações de SNC, 14 com gastrosquise, 3 com redução de membros e 2 com limb body wall. As mutações fator V de Leiden e G20210A-FII não foram encontradas nos casos e nos controles. A mutação C677T-MTHFR foi encontrada na forma heterozigota (CT) em 24 casos (24,8%) e em 6 controles. A mutação homozigota (TT) foi encontrada em 7 casos (7,2%) e em 1 controle. Estas diferenças foram estatisticamente significativas (p<0,0001). Quando avaliados os fetos com malformações de SNC (Artigo 1), a mutação CT foi encontrada com frequência significativamente maior nos casos do que nos controles (OR 10.309 IC95% 3.344-32.258), e a mutação TT também mostrou diferença significativa (OR 12.346 IC95% 1.388-111.11). A avaliação dos 14 fetos com gastrosquise (Artigo 2) não mostrou diferenças significativas quanto à presença da mutação CT ou TT entre os casos e os controles. Conclusão: A presença da mutação C677T-MTHFR no sangue fetal mostrou associação com malformações de SNC, tanto na forma homozigota quanto heterozigota
Abstract: Context and objective: Some congenital malformations have vascular origin, and a thrombosis during organogenesis is a possible mechanism for them. The aim of this study was to evaluate the association between fetal thrombophilia and malformations of vascular origin. Study type and location: A case-control study was performed at the Fetal Medicine Outpatient Clinic of CAISM UNICAMP, from 2005 to 2010. Methods: Ninety-seven fetuses with central nervous system malformations, gastroschisis, limb body wall and limb reduction were included in the study (cases), after routine cordocentesis showed normal karyotype results. A hundred fetuses without malformations were included as controls. These fetuses' cord blood had been donated to the Cord Blood Bank of HEMOCENTRO UNICAMP. DNA was extracted from fetal cord blood to study the mutations Factor V Leiden, G20210A-FII and MTHFR-C677T in both groups. Descriptive analysis was realized using Chi-square and Fisher's Exact Test. Wilcoxon test and logistic regression were used to analise the associations among variables. Results: We found 78 fetuses with central nervous system malformations, 14 with gastroschisis, 3 with member reduction and 2 with limb body wall. The mutations Factor V Leiden and G20210A-FII were not detected in cases nor in controls. The mutation MTHFR-C677T was encountered in 24 cases (24.8%) and in 6 controls its heterozygous form (CT). The homozygous mutation (TT) was found in 7 cases (7.2%) and in one control. These differences were statistically significant (p <0.0001). When the fetuses with central nervous system malformations were evaluated separately (Article 1), the frequency of the CT mutation was significantly higher in cases than in controls (OR 10.309 95% CI 3.344-32.258), as did the TT mutation (OR 12.346 95% CI 1.388-111.11). The 14 fetuses with gastroschisis were also evaluated separately (Article 2), and the results showed no statistically significant differences between cases and controls when concerning to the presence of the mutation MTHFR-C677T. Conclusion: The presence of the mutation MTHFR-C677T in fetal blood was associated with central nervous system malformations, both in homozygous and heterozygous form
Doutorado
Saúde Materna e Perinatal
Doutora em Ciências da Saúde

Thesis (MTech (Radiography))--Cape Peninsula University of Technology, 2006
A prospective cohort study was done on Human Immunodeficiency Virus (HIV) infected and uninfected women attending Khayelitsha Midwifery Obstetric Unit (MOU) and Gugulethu MOU from June 2003 to December 2004, primarily to establish whether there is an association between HIV infection and Intra-uterine growth restriction (lUGR). B-Mode real time ultrasound imaging was used to monitor fetal growth from ±22 weeks to 36 weeks gestational age. Birth weight, gestational age at delivery, gender, placental weight, and maternal complications were also included. Maternal factors considered included age, weight parity, singleton versus multiple pregnancy, previous IUGR or preterm delivery, previous fetal abnormality, social habits viz. cigarette smoking, alcohol and drug use, and vascular disease viz. Diabetes, hypertension, renal disease, cardiac disease and collagen disease. A secondary objective was to establish whether the CD4 T-lymphocyte count possibly modulated the presence of IUGR. All HIV infected women were given antiretroviral therapy according to the standard Protocol of the Provincial Government of Western Cape (2002). The research questions were: • Does maternal HIV infection increase the risk of intrauterine growth restriction and associated preterm delivery? • Does the immune status of (CD4 T-lymphocyte count) of HIV infected pregnant women modulate fetal growth? The primary objective of this study was to establish whether there is an association between HIV infection and IUGR, and hence that HIV infection leads to an adverse perinatal outcome. Ultrasound was used as a diagnostic tool to establish normal or abnormal fetal growth patterns. Anecdotal reports from health workers in the obstetric field suggested that IUGR and preterm delivery may be associated with low birth weight infants in HIV infected pregnant women. However, preterm delivery is associated with various other factors including low socio-economic status (poor nutrition), cigarette smoking, drug and alcohol abuse, previous history of preterm delivery, over distention of the uterus (hydramnios, multiple gestation), premature rupture of membranes, cervical incompetence, vaginal infections (bacterial vaginosis) and maternal disease e.g. hypertension, heart disease (Lizzi, 1993: Symmonds, 1992; Odendaal et aI, 2002). HIV is now thought to be an added factor. Afier doing a systematic review and meta-analysis of 31 studies, Brocklehurst and French (1998) reported that there is an association (although not strong) between HIV infection and adverse perinatal outcome in developed countries; but in developing countries, there is an increased risk of infant death. By excluding or controlling for confounding variables that could affect fetal growth, this study aimed to determine whether there is a significant association between HIV and fetal growth by comparing fetal growth in HIV infected and uninfected women from midsecond trimester to the time of delivery. A secondary objective was to establish whether there is an association between the immune status (CD4 T-lymphocyte count) of the mother and IUGR. The immune status of the mother is probably one of the most important factors affecting the fetus and perinatal outcome. As the mother's viral load increases, her immune system is increasingly compromised, resulting in the occurrence of HIV-related diseases, and a concurrent increase in fetal complications. In this study a CD4 T-lymphocyte count was used to assess the level of immunodeficiency of all the HIV infected participants. Ideally the test should have been done each time the participant was scanned so that the CD4 T-lymphoc)1e count could be monitored simultaneously with the fetal growth parameters, however due to financial constraints and ethical considerations, one test was done on each HIV infected women. This study was based at two MOU's where different antiretroviral therapy (ARVT) regimens were used. The one MOU offered Zidovudine (ZDV) to mothers from 34 weeks gestation to the onset of labour, and the other MOU offered Nevirapine (NVP) as a single dose to the mother at the onset of labour and to the neonate within 72 hours of birth (Provincial Government Western Cape, 2002). This presented an opportunity to compare two groups of HIV infected women on different regimes. The intention was to establish whether ZDV had an adverse effect on fetal growth and resulted in low birth weight. However, 6 months after the study started a revised Prevention of Mother to Child Transmission (PMTCT) Protocol was implemented where women at both MOU's received the same ARVT i.e. ZDV and NVP. This objective was therefore abandoned due to a change in the PMTCT Protocol in the Western Cape. The study was based at two Midwife Obstetric Units (MOU) in the Western Cape where the prevalence of HIV in pregnant women is relatively high i.e. 20 - 24 % (Mother-to-child transmission Monitoring Team, 2001), viz. Gugulethu MOU and Khayelitsha MOU. A prospective cohort study was done with the intention of recruiting a sample of 400 pregnant women, 200 HIV infected and 200 uninfected. The actual sample size was 415. The study group was 194 HIV infected women and the control group was 221 uninfected women. Confounding variables such as cigarette smoking, alcohol and drug abuse. multiple gestation. grand multipara pregnancy, history of IUGR or preterm delivery. fetal abnormality detected at the time of the first scan in the current pregnancy, and maternal vascular disease - were excluded. Confounding variables such as maternal age, maternal weight and gestational age were controlled. Ultrasound imaging was used as a diagnostic tool to establish normal and abnormal fetal growth patterns. A B-mode real time ultrasound unit was used to confirm the gestation age and rule out any obvious fetal abnormalities at 20-24 weeks gestation. Fetal growth scans were done at 28 weeks, 32 weeks and 36 weeks gestation to compare fetal growth patterns in the study and control groups. Fetal biometry used to monitor fetal growth included biparietal diameter (BPD), head circumference (HC), femur length (FL), abdominal circumference (AC) and estimated fetal weight (EFW). Amniotic fluid index (AFI), placental thickness & placental grading were also included. The following variables were analyzed post delivery: • Gestation age at delivery: Normal term delivery is considered to be at 37 - 42 weeks and premature delivery is considered to be less than 37 weeks gestation. The HIV infected and uninfected groups were compared to assess if there \vas a significant difference in the number of preterm deliveries. • Birth weight: The HIV infected and uninfected groups were compared to assess if there was a significant difference in the number of infants with low birth weight. • Perinatal complications: The HIV infected and uninfected groups were compared to assess if there was a significant difference in the number of perinatal complications and to assess if there was an association between the immune status (CD4 T-lymphocyte count) of HIV infected women and perinatal complications. Appropriate ethical principles in medical research were applied. The participant's autonomy, rights and best interests were always considered a priority. Informed consent was obtained from all the participants. Strict confidentiality was adhered to regarding any data collected throughout the study. The Research Ethics Committees at Cape Peninsula University of Technology and University of Cape Town granted ethics approval for the study. Statistical analysis was performed using the statistical package SPSS 12.0.

Orientador: Renato Passini Junior
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Introdução: A legislação não permite a interrupção da gestação em casos de malformações fetais incompatíveis com a vida extra-uterina, cabendo ao Poder Judiciário decidir quando há uma solicitação deste tipo. Objetivos: Investigar a opinião de Magistrados e membros do Ministério Público sobre o abortamento nos casos de malformações fetais incompatíveis com a vida extra-uterina, especialmente em relação à anencefalia. Método: Análise parcial de dados obtidos em duas pesquisas realizadas pelo Centro de Pesquisas em Saúde Reprodutiva de Campinas (CEMICAMP), que objetivaram estudar a opinião destes profissionais acerca do aborto induzido. Foram obtidos dados de 1493 Magistrados e 2614 Promotores de Justiça. Foi constituído um banco de dados com as informações de interesse das pesquisas originais, analisado com auxílio do programa estatístico SAS versão 9.02, envolvendo análise bivariada e múltipla, por regressão logística. Resultados: Para 78,5% dos Magistrados e 82,6% dos membros do Ministério Público, a interrupção da gestação deveria ser permitida nos casos de qualquer malformação fetal incompatível com a vida extra-uterina. Em casos de diagnóstico de anencefalia, estes valores foram de 79,2% e 84,1%, respectivamente. Na análise multivariada, as variáveis associadas à opinião dos pesquisados foram a religiosidade, importância da religião e das concepções religiosas pessoais sobre as respostas dadas, experiência de gravidez indesejada que resultou em aborto, sexo, estado marital e o fato de possuir filhos. Conclusões: A grande maioria dos Magistrados e membros do Ministério Público foi favorável ao abortamento nas hipóteses estudadas, sendo as variáveis ligadas à religião as que mais influenciaram seu posicionamento
Abstract: Introduction: In Brazil abortion in cases of fetal malformation or anencephaly is prohibited by law. Pregnant women who want to perform an abortion in such cases must seek for a judicial order. Objectives: Evaluate the opinion of brazilian magistrates and Prosecutors about abortion in cases of fetal malformation incompatible with life and anencephaly. Methodology: It was a partial data analysis from data obtained in two researchs carried out by Centro de Pesquisas em Saúde Reprodutiva de Campinas (CEMICAMP ), to evaluate the opinion and conduct of these professionals about induced abortion. There were data from 1453 Magistrates and 2614 Prosecutors. It was made a data bank with data from the original studies, that was processed and analyzed using the statistical package SAS version 9.02. Results: For 78.5% of the Magistrates and 82.6% of the Prosecutors abortion should be permitted in cases of severe fetal malformation incompatible with life. In cases of anencephaly abortion should be permitted for 79.2% of the Magistrates and for 84.1% of the Prosecutors. Religiosity, influence of religion and personal religious convictions among responses, experience with unwanted pregnancy that ended in abortion, gender, marital status and the fact of having children had shown, in multivariable analysis, association with the opinion about abortion in the hypothesis studied. Conclusion: The great majority of Magistrates and Prosecutors had a favorable opinion about abortion in both hypothesis evaluated. Variables associated with religion had the strongest association with the opinion about abortion in cases of fetal malformation and anencephaly.Abstract: Introduction: In Brazil abortion in cases of fetal malformation or anencephaly is prohibited by law. Pregnant women who want to perform an abortion in such cases must seek for a judicial order. Objectives: Evaluate the opinion of brazilian magistrates and Prosecutors about abortion in cases of fetal malformation incompatible with life and anencephaly. Methodology: It was a partial data analysis from data obtained in two researchs carried out by Centro de Pesquisas em Saúde Reprodutiva de Campinas (CEMICAMP ), to evaluate the opinion and conduct of these professionals about induced abortion. There were data from 1453 Magistrates and 2614 Prosecutors. It was made a data bank with data from the original studies, that was processed and analyzed using the statistical package SAS version 9.02. Results: For 78.5% of the Magistrates and 82.6% of the Prosecutors abortion should be permitted in cases of severe fetal malformation incompatible with life. In cases of anencephaly abortion should be permitted for 79.2% of the Magistrates and for 84.1% of the Prosecutors. Religiosity, influence of religion and personal religious convictions among responses, experience with unwanted pregnancy that ended in abortion, gender, marital status and the fact of having children had shown, in multivariable analysis, association with the opinion about abortion in the hypothesis studied. Conclusion: The great majority of Magistrates and Prosecutors had a favorable opinion about abortion in both hypothesis evaluated. Variables associated with religion had the strongest association with the opinion about abortion in cases of fetal malformation and anencephaly
Mestrado
Ciencias Biomedicas
Mestre em Tocoginecologia

Las cardiopatías congénitas (CC) permanecen como una importante causa de morbimortalidad infantil, es conocido que existe relación entre su diagnóstico y alteracion del neurodesarrollo, los factores directamente implicados en este proceso aun no están identificados, reconocerlos genera un impacto positivo que permitiría una adecuada asesoria genética y establecer mayor oportunidad desde el período neonatal. Objetivo: Evaluar el flujo sanguíneo cerebral en una cohorte de fetos con CC y fetos sanos en gestantes con características semejantes, Identificando los patrones circulatorios que determinen un pronóstico neonatal adverso, en los fetos con CC aislada. Metodologia estudio prospectivo de cohorte de gestantes ingresadas al programa de anomalías cardíacas congénitas (CC) y gestantes con fetos sanos, de la Unidad de Medicina Fetal, Clínica Universitaria Bolivariana (Medellín – Colombia), durante el periodo 2010 – 2016. Mediante la medición del flujo sanguíneo cerebral a través de la herramienta 3D Power Doppler (3DPwD). Resultados: Se analizaron 108 pacientes, 52 casos - 56 controles. La validez interna de la herramienta se realizó en fetos normales. El coeficiente de correlación intraclase, fue de 0.89 95% (IC 0.7574 - 0.9474). Pearson ρ 0.8867 y factor de corrección de sesgos Cb (Precisión) 0.9980. Las características maternas basales fueron similares en ambos grupos, en características perinatales, hubo diferencia estadisticamente significativa, la edad gestacional al nacer, una media de 37.6 (SD 1,1) en CC vs 38,6 (S D1.2) en sanos, (p 0.001). El peso al nacer 2816.6 gr (SD 443.9) vs 3106.8 (SD 281.3) respectivamente, (p 0.001), la via del parto fue cesárea, 63.5 % vs 12,5 % de los sanos (p 0.001). A los 2 grupos se les realizó dos evaluaciones de los indices de flujos sanguíneo cerebral, 24-26 y 34-36 semanas: índice de vascularización (VI), índice de flujo (FI) y el índice de vascularización - flujo (VFI). Las variables tuvieron una distribución normal, se encontró una diferencia estadísticamente significativa al evaluar el IVF en el segundo y tercer trimestre entre los recien nacidos con CC y sanos, un valor IVF Medio 36.06 (DS 6.92) vs 32.14 (DS 6.49) respectivamente, (p 0.003) y al final IVF 41.62 (DS 6.6) vs 38.74 (DS 4.49), (p 0.032). El IF del segundo trimestre, 12.26 (DS 6.499) vs 9.38 (DS 4.4) respectivamente, (p 0.009). El Doppler pulsado, el apgar, el tono, el color y los reflejos mostraron diferencia entre los grupos. Conclusion Los fetos con CC presentan procesos en útero que llevan a mayor riesgo de cambios hemodinámicos desfavorables y alteraciones en el neurodesarrollo. Identificar estos factores de riesgo en la vida prenatal pemitiría mecanismos estratégicos que lleven a una adaptación neonatal en mejores condiciones y plantear un seguimiento estricto que incluya el screening neurológico desde muy temprana edad.
Congenital Cardiopathies (CC) remain as an important morbimortality cause in children, the relation between their diagnosis and an alteration of the neurodevelopement is known. Directly implicated factors in this process are still not identified, recognizing them generates a positive impact which would allow an adecuate genetic assessment and to stablish a more significant opportunity since the neonatal period. Objective: Evaluating Cerebral blood flow in a Cohort of fetuses with CC and healthy fetuses from pregnant women with similar characteristics, identifying circulatory patterns which determine an adverse neonatal prognosis in fetuses with isolated CC. Methodology: Cohort prospective study of pregnant women admitted to the Congenital Cardiac Anomalies program (CC) and pregnant women with healthy fetuses from the Fetal Medicine Unit, Clínica Universitaria Bolivariana (Medellín – Colombia) during the period 2010 – 2014 by measuring Cerebral blood flow through 3D Power Doppler (3DPwD) tool. Results: 108 patients were analyzed, 52 cases – 56 controls. The internal validity of the tool was carried out in normal fetuses. The intra class correlation coefficient was 0.89 95% (IC 0.7574 - 0.9474). Pearson ρ 0.8867 and a bias correction Factor Cb (Precision) 0.9980. Baseline maternal characteristics were similar in both groups; regarding perinatal characteristics, there was a statistically significant difference, gestational age at birth, median of 37.6 (SD 1,1) in CC vs 38,6 (S D1.2) in healthy, (p 0.001). weight at birth 2816.6 grs (SD 443.9) vs 3106.8 grs (SD 281.3), respectively, (p 0.001), the delivery route was cesarean, 63.5 % vs 12,5 % of the healthy (p 0.001). Both groups underwent two Cerebral blood flow index evaluations, 24-26 and 34-36 weeks: vascularization index (VI), flow index (FI) and vascularization – flow index (VFI). Variables had a normal distribution, a statistically significant difference was found when evaluating the VFI in the second and third trimester among newborns with CC and the healthy, a mean VFI value of 36.06 (DS 6.92) vs 32.14 (DS 6.49), respectively, (p 0.003) and in the end VFI 41.62 (DS 6.6) vs 38.74 (DS 4.49), (p 0.032). The FI of the second trimester, 12.26 (DS 6.499) vs 9.38 (DS 4.4) respectively, (p 0.009). Pulsed Doppler, the apgar, tone, color and reflexes showed difference between the groups. Conclusion: Fetuses with CC present in uterus processes which lead to a higher risk of hemodynamic unpromising changes and neurodevelopment alterations. Identifying these risk factors in the prenatal life will allow strategic mechanisms leading to a neonatal adaptation in better conditions and to set out a strict follow up which includes neurologic screening from an early age.

Minha experiência clínica atendendo gestantes com diagnóstico de cardiopatia fetal pôde propiciar a observação de alterações na condição emocional das pacientes que se mantinham em acompanhamento psicológico no hospital de Cardiologia, que passou a recebê-las desde o período pré-natal até o nascimento do bebê, para que estes fossem submetidos às intervenções cardíacas necessárias. Esta percepção motivou a pesquisa sobre a natureza destas repercussões emocionais uma vez que a gestação é considerada um período de transição e de crise para a mulher, no qual precisa se reorganizar emocionalmente devido às mudanças com a vinda do filho, bem como às expectativas e idealizações inerentes. O objetivo foi identificar as repercussões emocionais mais significativas a partir de aspectos da psicodinâmica das gestantes, visando contribuir para a assistência a esta população. O método utilizado foi o clínico qualitativo, tendo como instrumentos: a entrevista semidirigida e as técnicas projetivas, Desenho da Figura Humana (DFH) e Teste de Apercepção Temática (TAT). Foi realizada a análise de conteúdo, conforme Bardin, de uma sessão de atendimento psicológico, gravada, com o consentimento das participantes, e transcrita posteriormente. Os resultados confirmaram a presença de repercussões emocionais significativas, dentre elas: os sentimentos de impotência e de posse em relação ao bebê, sentimento de culpa pelo diagnóstico fetal, angústia de morte, desamparo, não aceitação do diagnóstico e medo do desconhecido. A partir da análise psicodinâmica, identificou-se também: as principais ansiedades das gestantes - conhecer o bebê, de separação e do parto; os mecanismos de defesa atuantes - negação, regressão, identificação, racionalização e idealização- e as principais formas de enfrentamento - confiança na equipe, acreditar que a barriga é a forma possível de proteção do bebê, controle emocional, busca de conhecimento sobre a cardiopatia, identificação com outros pais na mesma situação e oferecimento de ajuda a estes, e a fé. No DFH destacaram-se: a inclinação das figuras femininas e masculinas, que pode estar relacionado à tentativa de manter um equilíbrio corporal em virtude das modificações físicas ao final da gestação; a assimetria apresentada em metade dos desenhos, analisada como possível forma de expressar a percepção das anomalias corporais dos bebês, embora a cardiopatia não possa ser visualmente observada, pode se relacionar às fantasias sobre a aparência do bebê malformado. No TAT perceberam-se importantes conflitos como dependência x independência e maternidade x afiliação, com o uso de mecanismos de defesa tais como a regressão, racionalização e idealização. Concluiu-se que o diagnóstico de cardiopatia fetal traz intensas repercussões emocionais, relacionadas às fantasias de morte sobre o nascimento do filho, sendo atribuída ao parto uma representação simbólica ainda mais angustiante que nas gestações comuns ou não caracterizadas como de alto risco, pois parece ser vivenciada inicialmente como uma situação quase-certa de morte, o que torna importante que o acompanhamento psicológico seja oferecido nas instituições de saúde, por todo o ciclo gravídico-puerperal
My clinical experience serving pregnant women with diagnosis of fetus with heart disease made it possible to observe changes in the emotional condition of the patients who remained in counseling in the Cardiology Hospital, which has been admitting them from the prenatal period to childbirth, so that they were subjected to the necessary cardiac interventions. This realization led to research into the nature of these emotional repercussions since pregnancy is considered a period of transition and crisis to the woman, who needs emotional restructuring due to changes related to the child\'s coming, and the inherent expectations and idealizations. The objective was to identify the most significant emotional repercussions from the psychodynamic aspects of pregnant women, in order to contribute to their assistance. The research followed a clinical-qualitative method, with the following instruments: semi-structured interviews and projective techniques, the Human Figure Drawing Test (HFD) and Thematic Apperception Test (TAT). Content analysis was carried out according to Bardin during psychological counseling sessions, recorded with the consent of the participants, and later transcribed. The results confirmed the presence of significant emotional repercussions, such as: feelings of powerlessness and possession over the baby, guilt over fetal diagnosis, death anxiety, helplessness, denial and fear of the unknown. The psychodynamic analysis also identified: the main anxieties of pregnant women knowing the baby, separation and childbirth; the active defense mechanisms denial, regression, identification, rationalization and idealization; and the main coping mechanisms confidence in the team, belief in the bellys protection, emotional control, pursuit of knowledge about the disease, identification with other parents in similar situations and the ability to extend help to them, and faith. HFD highlighted: the angle of the female and male figures, which may be related to trying to maintain body balance because of physical changes at the end of pregnancy; the asymmetry identified in half of the drawings, analyzed as a possible way to express the perception of bodily abnormalities of babies which, even in the absence of visual observation of the abnormalities, can relate to fantasies about the appearance of a malformed baby. TAT showed important conflicts such as dependence vs. independence and motherhood vs. affiliation with the use of defense mechanisms such as regression, rationalization and idealization. The research concludes that the diagnosis of fetal heart disease causes intense emotional distress, related to death fantasies during childbirth with childbirth having a more distressing symbolic representation than during ordinary or low-risk pregnancies, since it seems to be early experienced as a situation of almost certain death, which makes it important that health institutions offer psychological counseling throughout the pregnancy and childbirth

Orientador: Lourenço Sbragia Neto
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A Hérnia Diafragmática Congênita (HDC) é um defeito da formação do músculo diafragma que incide em aproximadamente 1:2500 nascidos vivos e apresenta altos índices de mortalidade fetal e neonatal decorrentes da hipoplasia e da hipertensão pulmonares. Este defeito pode ser induzido experimentalmente em ratas grávidas administrando o herbicida nitrofen que causa HDC em 24% dos fetos. A análise microscópica do pulmão da HDC demonstra a presença de hipolasia pulmonar além de alveolização e vascularização alterada. Um dos fatores de crescimento envolvidos no desenvolvimento vascular é o VEGF (vascular endothelial growth factor) e seus receptores, no entanto ainda não se conhece como a expressão desta glicoproteína e de seus receptores varia ao longo do desenvolvimento pulmonar fetal nesta doença. Utilizando o modelo experimental de HDC induzido pelo nitrofen (2,4-dicloro-4'nitrodifenil éter) investigamos o grau de hipoplasia pulmonar e por meio de análise imunoistoquímica, comparamos a expressão dos receptores para o VEGF em três fases do desenvolvimento pulmonar, pseudoglandular, canalicular e sacular de fetos de ratos normais e com HDC. Dividimos o experimento em ratas da raça Sprague-Dawley em três grupos: controle externo (CE), exposto ao óleo de oliva (OO) e expostas ao nitrofen com e sem HDC. Estudamos quatro grupos de 20 fetos cada em cinco dias gestacionais (DG) diferentes 17,5, 18,5, 19,5, 20,5 e 21,5. As variáveis morfológicas estudadas foram: peso corporal (PC), peso pulmonar total (PPT), peso do pulmão esquerdo (PPE), relação PPT/PC, volume pulmonar total (VPT) e volume do pulmão esquerdo (VPE). As variáveis histométricas estudadas foram: parênquima pulmonar (Par), espaço aéreo (EA), densidade do parênquima (DAP) e volume do parênquima do pulmão esquerdo. A avaliação imumohistoquímica foi realizada por meio da contagem de pontos de receptor de VEGFR-1 e 2. Obtivemos 37 % (100/270) de HDC nas ratas expostas ao nitrofen, todas variáveis morfológicas e histométricas indicam diminuição dos resultados no grupo nitrofen com e sem HDC em relação aos demais, mas que se acentuam mais ainda no grupo HDC. Essas alterações são mais evidentes a partir dos DG 18,5 e 19,5. A imunomarcação para os receptores VEGFR-1 aumentou nos grupos nitrofen e foram progressivamente maiores no grupo nitrofen com HDC (p<0,005) que os fetos dos grupo CE e OO a partir do dia gestacional 17,5, fase pseudoglandular com pico máximo no dia gestacional 19,5. O mesmo ocorreu com os receptores de VEGFR-2 a partir do dia gestacional 17,5, fase pseudoglandular até o dia 21,5 fase sacular do desenvolvimento pulmonar. Concluímos que o modelo é valido e que os fetos expostos ao nitrofen com e sem HDC apresentam hipoplasia pulmonar primária sendo mais acentuada nos fetos portadores de HDC. O mesmo resultado ocorre com imunomarcação para os receptores de VEGFR-1 e 2 que foram maiores na HDC.
Abstract: The Congenital Diaphragmatic Hernia (CDH) is a defect in the embryogenesis of the diaphragm with an incidence of 1:2500 liveborns and high fetal and neonatal mortality due to pulmonary hypoplasia and hypertension. This defect can be experimentally induced in fetuses of pregnant rats by the administration of Nitrofen, an herbicide that causes CDH in 24% of the fetuses. The histology of lungs in CDH shows pulmonary hipoplasia and not only the alveolarization but also the vascularization are affected. These changes lead to a high neonatal mortality because of the thickening of the middle layer of the arterioles causing pulmonary hypertension. One of the factors involved in the growth of the arterioles is VEGF (vascular endothelial growth factor) and its receptors; however, it is not known how the expression of this glycoprotein and its receptors change during lung development in this disease. In Brazil, the experimental model has never been tested. So, we tested the model and verified the degree of pulmonary hipoplasia and, using imunohystochemistry, we compared the expression of the receptor of VEGF in three different stages of lung development, pseudoglandular, canalicular and saccular, of normal rat fetuses and fetuses with CDH. Female Sprague-Dawley rats were divided in three groups: external control (EC), exposed to olive oil (OO) and exposed to nitrofen (N). We studied four groups - EC, OO, N with CDH and N without CDH - with 20 fetuses in each five different gestational days (GD) 17,5, 18,5, 19,5, 20,5, 21,5. The morphologic variables studied were: body weight (BW), total lung weight (TLW), left lung weight (LLW), relationship TLW/BW, total lung volume (TLV) and left lung volume (LLV). The hystometric variables studied were: lung parenchyma (LP), air space (AS), left lung parenchyma density (PD) and left lung parenchyma volume (PV). The immunohystochemistry variables were: points positive and negative for the receptor for VEGF 1 and 2. We had 37% (100/270) of CDH frequency in the fetuses exposed to nitrofen. All the morphological and hystometrical variables show a reduction in the nitrofen group with and without CDH, which were more pronounced in the group of fetuses with CDH. These changes are more evident from the GD 18,5 and 19,5 on. The receptors VEGFR-1 e 2 are increased in the nitrofen groups with and without CDH, but this increase is higher in the fetuses with CDH. We conclude that the model is valid and that the fetuses exposed to nitrofen with and without CDH show primary pulmonary hypoplasia that is more pronounced in CDH, the same is also observed in the receptors of VEGFR-1 and 2.
Doutorado
Pesquisa Experimental
Doutor em Cirurgia

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro
O desenvolvimento da programação fetal é considerado um importante fator de risco para doenças não-transmissíveis da vida adulta, incluindo doença cardíaca coronariana. Com o objetivo de investigar a associação entre nutrição materna e o desenvolvimento das artérias coronárias (AC) em embriões de camundongos estadiados; embriões de camundongos C57BL/6 nos estádios de 16-23 foram retirados de mães alimentadas com dietas de proteína normal (NP) ou de baixa proteína (LP), e as AC foram estudadas. Embora os embriões LP possuam massa corporal menor, entretanto tinham taxas de crescimento cardíaco maior, quando comparados com os embriões NP. O Plexo subepicárdico foi observado no início do período pós-somítico (estádio 16) de embriões NP, enquanto que nos embriões LP apenas no estádio 17 (P <0,01), persistindo até o estádio 18 (P <0,01). As artérias coronárias foram detectadas inicialmente no estádio18 dos embriões NP, já nos embriões LP foram encontradas a partir do estádio 19 (P <0,01). Núcleos apoptóticos foram observados em torno do anel aórtico peritruncal no estádio 18 em embriões NP e LP. Células FLK1+ (Fetal Liver Kinase 1 = VEGFr2 = Vascular Endothelial Growth Factor Receptor 2) apresentaram uma distribuição homogênea nos embriões NP já no estádio 18, enquanto uma distribuição semelhante nos embriões LP foi visto apenas nos estádios 22 e 23. A restrição proteica materna em camundongos leva a um atraso no crescimento do coração no período embrionário modificando o desenvolvimento do plexo peritruncal subepicárdica e diminuindo a taxa de apoptose na região do futuro orifício coronariano.
Programming of fetal development is considered to be an important risk factor for non-communicable diseases of adulthood, including coronary heart disease (CHD). Aiming to investigate the association between maternal nutrition and the development of the coronary arteries (CA) in staged mice embryos, C57BL/6 mice embryos from stages 16 to 23 were taken from mothers fed a normal protein (NP) or low protein (LP) diet, and the CA were studied. Although the LP embryos had lower masses, they had faster heart growth rates when compared to the NP embryos. The subepicardial plexuses were observed earlier in the NP embryos (stage 20) than in the LP ones (stage 22) (P<0.01). Apoptotic nuclei were seen around the aortic peritruncal ring beginning at stage 18 in the NP and LP embryos. FLK1+ (fetal liver kinase 1 = VEGFr2 or vascular endothelial growth factor receptor 2) cells had a homogeneous distribution in the NP embryos as early as stage 18, whereas a similar distribution in the LP embryos was only seen at stages 22 and 23. Maternal protein restriction in mice leads to a delay in the growth of the heart in the embryonic period modifying the development of the subepicardial peritruncal plexus and the apoptosis in the future coronary orifice region.

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Fundação Oswaldo Cruz. Instituto Nacional de Saúde da Mulher, da Criança e do Adolescente Fernandes Figueira. Departamento de Ensino. Programa de Pós-Graduação em Saúde da Criança e da Mulher. Rio de Janeiro, RJ, Brasil.
Objetivo: Identificar fatores de risco associados à necessidade de exsanguíneotransfusão (EXT) em gestações acometidas por doença hemolítica perinatal (DHPN) e avaliar a influência da terapêutica aplicada. Métodos: Foi realizado estudo de coorte e analisados 124 casos referentes a nascimentos de crianças com DHPN no período de abril de 2006 a junho de 2009, cujo parto ocorreu no Instituto Fernandes Figueira. Dados da história materna, pré-natal, parto e recém-nascido foram avaliados quanto a sua relação com a necessidade de transfusão intrauterina (TIU) e com o desfecho grave da doença, definido por EXT. Resultados: Paridade (RR:1,22; IC:1,00-1,49), história de filhos com doença (RR:3,21; IC:1,27-7,99) e hidropisia fetal (RR:1,86; IC:1,11-3,11) são fatores de risco importantes para a necessidade de TIU. Realização de TIU (RR:1,82; IC:1,12-2,97), parto normal (RR:0,53; IC:0,30-0,93), icterícia (RR:2,31; IC: 1,46-3,68), nível de hematócrito ao nascimento (RR:0,95; IC:0,93-0,98), bilirrubina total máxima durante a internação (RR:1,23; IC:1,16-1,31), tempo de fototerapia (RR:0,90; IC:0,85-0,95) e tempo total de internação (RR:097; IC:0,95-0,99), são variáveis que estabelecem relação independente e estatisticamente significativa com o desfecho de gravidade. A necessidade de EXT após TIU difere entre fetos menos graves (RR: 1,29; IC:0,94-1,77) e mais graves (RR: 1,61; IC:1,11-2,34). Conclusão: A identificação de fatores de risco é possível e importante para o cuidado do recém-nascido no período neonatal precoce acometido por DHPN. O impacto da terapêutica no desfecho neonatal varia de acordo com a gravidade no momento do diagnóstico.
Objective : To identify the major risk factors related to exchange transfusion (EXT) in pregnancies afflicted with hemolytic disease of the fetus and newborn (HDFN) and to evaluate the influence of applied therapy . Methods: A cohort study of 124 infants born with HDFN between A pril 2006 and June 2009 at the Fernandes Figuei ra National Institute . Data on maternal history, prenatal care, delivery and neonatal paramet ers were subjected to analysis to de termine their relationship with the need for i ntrauterine transfusion (IUT) du ring prenatal care and severe disease outcome, represented by EXT. Results: Parity (RR:1,22; C I :1,00 - 1,49) , obstetric history related to HDFN (RR:3,21; C I :1,27 - 7,99) and hydrops fetalis (RR:1,86; C I :1,11 - 3,11) are important risk factors related with the need for IUT. The need for IUT (RR:1,82; C I :1,12 - 2,97), vaginal delivery (RR:0,53; C I :0,30 - 0,93), jaundice (RR:2,31; C I : 1,46 - 3,68), hematocrit level at birth (RR:0,95; C I :0,93 - 0,98), peak serum bilirubin leve ls during hospitalization (RR:1,23; C I :1,16 - 1,31), duration of phototherapy (RR:0,90; CI:0,85 - 0,95) and total duration of hospital stay (RR:097; C I :0,95 - 0,99 ) were the variables found to establish an independent and statistically significant relationship w ith severe outcome. The need for EXT after IUT was different between fetus with less severe disease (RR: 1,29; C I :0,94 - 1,77) and more severe disease (RR: 1,61; C I :1,11 - 2,34). Conclusion: To identify risk factors is possible and important in early neonatal assistance of newborn afflicted with HDFN. The impact of therapy on the neonatal outcome depends on severity of the disease at the moment of diagnosis.

Infectious disease continues to cause significant problems on reproductive efficiency in the cattle industry. The purpose of this project is to evaluate new testing strategies for Tritrichomonas foetus and Campylobacter fetus subsp. venerealis. This thesis describes the result of three studies that evaluated the use of real-time PCR for the identification of Tritrichomonas foetus and Campylobacter fetus subsp. venerealis in carrier bulls. The first study evaluated the specificity of a real-time PCR test for T. foetus in individual culture enriched samples, and the sensitivity of the assay for use in pooled samples of up to 25 bulls. Specificity estimates were 98.8% (95% CI 97-99.4) and 100% (95% CI 98.9-100) for culture and real-time PCR, respectively. The sensitivity of the real-time PCR assay for pooled preputial samples was: 96.8% (83.8-99.4) for pool ratios 1/3 and 1/5; 93.5% (79.3-98.2) for pool ratios 1/2, 1/15, 1/20 and 1/25; and 90.3% (75.1-96.6), and were not significantly different. However, 13 of the 217 pools tested were negative and 9 of these negative testing pools contained the same positive sample. The media in this positive sample showed evidence of contamination and could potentially explain the failure to detect T. foetus. The second study evaluated the sensitivity of a real-time PCR for the detection of T. foetus in individual and pooled direct preputial samples. Sensitivity of individual samples tested by culture, real-time PCR in direct and culture enriched samples were determined from 121 samples obtained from 9 infected bulls. Sensitivity estimates were: 95.0% (95% CI: 89.6% to 97.7%) for culture, 95.9% (95% CI: 90.7 to 98.2) for real-time PCR in cultured enriched samples, and 90.1% (95% CI: 83.5 to 94.2) for direct preputial samples and did not differ (P=0.12). Sensitivity estimates for direct pooled samples in groups of 5 or 10 were: 83.6% (95% CI: 75.6 to 89.4) and 77.3% (95% CI: 68.6-84.1), respectively and were not significantly different (P=0.08). The use of repeat sampling tested in pools by real-time PCR increased the sensitivity to 100% and 96% for 3 consecutive samples (pools of 5 or 10, respectively). The use of pooled direct preputial samples although sensitive, still requires the use of repeated sampling. The third study determined the sensitivity and specificity of a recently developed real-time PCR (qPCR) tests for Cfv. A total of 300 virgin bulls were tested by both culture and qPCR. Specificity estimates were 85% (95% CI: 80.5 to 88.6) for qPCR and 100% (95% CI: 98.7 to 100) for culture, and were significantly different (P<0.01). A total of 4 naturally infected bulls and 9 artificially infected bulls were sampled serially to obtain positive samples for a sensitivity analysis. Sensitivity estimates and 95% confidence intervals are as follows: qPCR (85.4%, 95% CI: 80.6-89.2); direct culture on blood agar (82.3%, 95% CI: 77.2-86.5), DFAT (72.1%, 95% CI: 66.2-77.4), direct culture on Skirrow agar (32.7%, 95% CI: 27.2-38.7), TEM and blood agar (30%, 95% CI: 23.4-37.5), and TEM and Skirrow agar (38.1%, 95% CI: 31-45.9). The sensitivity of the different tests evaluated varied significantly with different ambient temperatures (P<0.01). The sensitivity of the qPCR was significantly higher than any other test when temperatures exceeded 5°C. The use of repeated sampling at weekly intervals significantly improved the sensitivity of the qPCR. The real-time PCR assay for the detection of T. foetus in both individual and pooled samples appears to be highly sensitive and specific. Moreover, the possibility of using direct preputial samples provides a cost-effective diagnostic strategy. Real-time PCR in direct preputial samples for BGC diagnosis in bulls has good sensitivity and specificity. However, the use of repeated sampling maybe needed in order to maximize the ability to detect carrier bulls.

Another attempt was made to identify CpG-rich paralogues on chromosome 21 for dosage analysis. Methylation profiles of 14 paralogous CpG-rich clusters were screened by bisulfite genomic sequencing and/or combined bisulfite restriction analysis. One of the paralogue pairs showed similar differential methylation patterns, and three other CpG-rich clusters located on chromosome 21 were hypomethylated in the placentas and completely methylated in the maternal blood cells. Detection methods for these novel epigenetic markers were described and discussed, and potential applications were also suggested.
In the second part of this thesis, a technique called digital PCR was used for detecting and quantifying cell-free fetal DNA in maternal plasma. DNA templates are first diluted to a single molecule level and partitioned to separate compartments before subjecting to polymerase chain reaction amplification. Quantification is then made by counting the number of positive reactions directly. Such a technique has allowed the reliable detection of fetal DNA from a high background of maternal plasma DNA, and allows absolute quantification of fetal DNA without using a calibration curve. As a proof-of-principle project, a non-polymorphism-based approach called digital relative chromosome dosage (RCD) method was developed to detect chromosomal imbalance in trisomic cases. The implementation of digital PCR was further facilitated with the technology of integrated fluidics circuits (IFCs), by which nanolitre volumes of reaction mixtures could be manipulated in a high-throughput manner. Such a microfluidics digital PCR system was evaluated systematically and shown to be highly accurate, precise and sensitive compared to other existing detection platforms. The technology has been applied with the RCD approach for rapid detection of trisomy 21 from amniotic fluid samples and 100% accuracy was attained. With the development of new universal markers and robust detection platforms, it is envisioned that circulating fetal DNA in maternal plasma can be applied to an expanding range of clinical applications in the near future.
The first part of my thesis focused on the discovery of new epigenetic markers for fetal DNA detection. Methylation profiles of 7 selected CpG islands on chromosome 21 were revealed by bisulfite sequencing, in the hope of identifying regions with differential methylation patterns between placentas and maternal blood cells. Out of the 14 sub-regions of these CpG islands, five displayed significant difference between the two tissue type and were promising marker candidates.
The presence of circulating fetal DNA in maternal plasma provides a non-invasive source of fetal genetic materials for prenatal diagnosis. Reliance on Y-chromosomal sequences for detecting fetal-specific signals from the background of maternal plasma DNA, however, has restricted the applications to 50% of pregnancies. For a wider extent of diagnostic applications, sex- and polymorphism-independent fetal markers would be necessary. Recently, an epigenetic approach has been adopted to discriminate between the fetal and maternal DNA circulating in maternal plasma. Based on the differential DNA methylation status between the fetus and mother, universal fetal DNA markers have been developed and applied to detect fetal signals in maternal plasma. Identification of more of such markers is important for the development of the field.
Lun, Miu Fan.
Adviser: Yuk Ming Dennis Lo.
Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3292.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references (leaves 233-256).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

目前廣泛應用于胎兒醫學的唐氏綜合症篩查法，即結合早孕期胎兒頸項透明層的超聲檢查，及母體血清生化指標的綜合篩查法。頸項透明層是指在早孕期利用超聲檢測到的胎兒頸后的皮下積水，其作為預測胎兒異常的一項重要“軟指標，其臨床意義，尤其是與胎兒染色體異常及器官結構異常之間的關係，逐漸得到深入的認識，但其形成機制尚未明確。現在已知有一百餘種畸形及遺傳綜合征與胎兒頸項透明層增厚相關，但其染色體異常譜系，尤其是亞顯微的染色體異常仍有待明確。大部分頸項透明層增厚但核型正常的胎兒預後良好，但約3-10%的這部分胎兒會伴有畸形或出生后的神經智力發育缺陷。而傳統核型分析無法檢測到亞顯微的染色體異常，從而無法判斷這部分核型正常卻伴有缺陷的胎兒是否因為這類染色體異常而致病。
微陣列比較基因組雜交芯片作為檢測兒童發育遲緩者及器官結構異常原因的重要手段已廣泛應用于臨床。在染色體核型正常的胎兒中，若伴有器官結構異常的胎兒，5-12%被檢出與該畸形相關的微缺失及微重複；若僅伴有孕婦高齡或唐氏篩查高危，則微缺失及微重複檢出率約1%。
該課題旨在研究頸項透明層增厚但核型正常的胎兒中，染色體拷貝數變異發生的頻率及頻譜；評估微陣列比較基因組雜交芯片在協助臨床判斷胎兒預後中的作用。因此，我們開展該多中心隊列研究，通過納入449例頸項透明層厚度≧3.5 mm但正常核型胎兒的，檢測其染色體拷貝數變異，監測并記錄其圍產、產後及新生兒期情況。微陣列比較基因組雜交芯片總共檢出2.8%的異常拷貝數變異，其大小範圍為0.1 kb至18Mb。在伴有器官結構異常的胎兒組中，異常拷貝數變異檢出率達7.8%。對於頸項透明層厚度≧4.0 mm的胎兒，異常拷貝數變異檢出率可達7.3%。
對於頸項透明層增厚的胎兒，致病拷貝數變異暫未發現特定的頻譜。但，該研究中發現重複的致病拷貝數變異，如22號染色體長臂1區1帶的微重複或微缺失，2號染色體長臂2區2帶的微缺失。未在3號、7號、12號、13號、18號、20號、21號或Y染色體上發現與胎兒頸項透明層增厚相關的致病拷貝數變異。
頸項透明層增厚的胎兒79.3%預後良好；若經微陣列比較基因組雜交芯片未檢出致病拷貝數變異，則81.2%預後良好。如果僅頸項透明層增厚不伴有結構異常的胎兒，經微陣列比較基因組雜交芯片未檢出致病拷貝數變異，則93.5%預後良好。
綜上所述，微陣列比較基因組雜交芯片顯著提高了致病拷貝數變異的檢出率。可考慮將微陣列比較基因組雜交芯片作為頸項透明層厚度≧4.0 mm的胎兒染色體異常檢查的首要方法。對於僅頸項透明層增厚不伴有結構異常的胎兒，且經微陣列比較基因組雜交芯片未檢出致病拷貝數變異，絶大部分預後良好。對於頸項透明層增厚的胎兒，致病拷貝數變異暫未發現特定的頻譜，但發現重複出現的致病拷貝數變異。通過初步的基因本體分析及基因通路分析，神經嵴細胞的分化遷徙功能異常可作為今後研究頸項透明層增厚的病理生理機制的方向。
Measurement of nuchal translucency (NT) has been recognized as a sensitive marker for fetal chromosomal disorders for more than a decade, and is presently used as a routine first-trimester screening test. Although over 100 abnormalities and genetic syndromes have been reported to be associated with increased NT, these associations have not been fully explored and the relevant spectrum of associated submicroscopic chromosomal abnormalities has not been sufficiently investigated. The majority of euploid fetuses with increased NT have a good outcome, but around 3-10% of fetuses present with structural or neurodevelopmental abnormalities postnatally. A range of genetic syndromes has been reported, many of which are linked to submicroscopic chromosomal abnormalities that are typically missed by conventional karyotyping.
Microarray-based comparative genomic hybridization (arrayCGH) has been applied as the first-tier diagnostic tool for the evaluation of developmental delay and structural malformations in children. In fetuses with a normal karyotype, microarray analysis revealed clinically relevant deletions or duplications in 5-12% with a structural anomaly and in about 1% of those whose indications were advanced maternal age or positive screening results.
The objectives of this study were to delineate the frequency and spectrum of pathogenic chromosome copy number variants (CNVs) among fetuses with increased NT and normal karyotype; to evaluate the role of arrayCGH to predict the prognosis of the high NT fetuses; to explore the genotype-phenotype correlations of increased NT. Therefore, a multi-centre cohort of 449 fetuses with NT ≧3.5 mm and normal karyotype were further investigated by arrayCGH. Antenatal surveillance, pregnancy outcome and paediatric follow up were documented. ArrayCGH detected abnormal CNVs in 2.8% (14 of 449) of the fetuses with high NT; the size of CNVs ranged from 0.1 kb to 18Mb. Among fetuses with major congenital abnormalities the incidence of abnormal CNV reached 7.8% (4 of 51). By adjusting the NT to ≧4.0 mm as the referral indication, 7.3% (14 of 192) of the fetuses would have abnormal arrayCGH results. The spectrum of pathogenic CNVs found associated with increased NT was diverse. However, there were recurrent ones such as the deletions or duplications at chromosomal region 22q11, and deletions in ZEB2. There was no pathogenic CNV related with increased NT found in chromosomes 3, 7, 12, 13, 18, 20, 21, or Y. The total normal outcome rate of euploid fetuses with an increased NT was 79.3%; for fetuses with normal arrayCGH results 81.2% had a normal outcome. In fetuses with isolated increased NT, normal arrayCGH results predict a favorable prognosis of 93.5%.
In conclusion, arrayCGH significantly increased the diagnostic yield of pathogenic CNVs. In clinical practice arrayCGH may be considered as the first tier investigation in fetuses with an increased NT more than 4.0 mm. In cases with an isolated increased NT with normal arrayCGH results the pregnancy outcome is likely to be favorable. The spectrum of abnormal CNVs found by arrayCGH is diverse but there are recurrent cases such as del/dup 22q11 and del ZEB2. Our preliminary gene ontology and pathway analysis showed that gene pathways related to neural crest cells may be considered as a future study for physiopathologic mechanisms of NT.
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.
Detailed summary in vernacular field only.
Huang, Jin.
Thesis (Ph.D.) Chinese University of Hong Kong, 2014.
Includes bibliographical references (leaves 106-120).
Abstracts also in Chinese.

Prenatal diagnosis is an established obstetrics practice in many countries. Currently available methods to obtain fetal materials for a definitive diagnosis involve invasive procedures such as chorionic villus sampling and amniocentesis. Due to the invasive nature of the procedures, confirmatory testing is usually recommended only for pregnant women who are screened as being high risk of bearing a fetus with abnormalities. There has been an urge to develop safer alternatives in obtaining fetal genetic materials for prenatal assessment. Thus, the discovery of circulating fetal DNA in maternal plasma and serum has opened up new possibilities for noninvasive prenatal diagnosis.
The use of genetic markers such as Y chromosomal sequences from a male fetus or paternally-inherited polymorphic markers has been well-described in the literature. However, there is an obvious limitation on the use of these markers because they are gender- and/or polymorphism-dependent. This thesis focuses on the development of a universal fetal marker, namely SERPINB5 (encoding maspin), based on the intrinsic epigenetic differences between the placenta (the major contributor of fetal genetic materials in maternal plasma) and maternal blood cells (postulated to be the predominant source of cell-free DNA in the circulation). Analysis of the methylation profile of the SERPINB5 promoter in the placenta and maternal blood cells, and the development of methods and protocols to detect the differentially methylated SERPINB5 promoter molecules are described. The application of this epigenetic marker in prenatal assessment of the fetal chromosomal aneuploidy is illustrated by an epigenetic allelic ratio (EAR) approach. Basically, the ratio of a single-nucleotide polymorphism (SNP) on the placenta-derived SERPINB5 molecules is shown to be deviated in the maternal plasma from trisomic pregnancies when compared to the euploid ones. Results described in this thesis show the promising potential for the EAR approach for the noninvasive prenatal detection of fetal chromosomal aneuploidies. In addition, a novel approach for methylation analyses on the amplification and detection of restriction enzyme-digested DNA fragments will be discussed. This thesis has essentially described the evolution of a fetal epigenetic marker from basic science to potential clinical application.
Tong, Yu Kwan.
Adviser: Yuk-Ming Dennis Lo.
Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0953.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (leaves 149-172).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
School code: 1307.

Bibliography: leaves 192-212.
xv, 212 leaves ; 30 cm.
The present study investigates the relationships between fetal growth, as manifest in size and shape at birth, and later blood pressure and blood lipids, in an Australian cohort. Data on these outcomes for cohort members at age 8 years are available from a previous study. Birth details (body weight, placental weight, head circumference, chest circumference and length) are abstracted from hospital records. In addition, a follow up of cohort members is undertaken to collect new data pertaining to the two cardiovascular risk factors at 20 years of age. Socio-economic circumstances are characterised at birth, age 8 and age 20.
Thesis (Ph.D.)--University of Adelaide, Dept. of Public Health, 1997?

Lo, Yin Wai Wyatt.
"December 2010."
Thesis (M.D.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 186-206).
Abstracts in English and Chinese.
ABSTRACT --- p.II
摘要 --- p.VII
ACKNOWLEDGEMENTS --- p.X
PUBLICATIONS --- p.XI
TABLE OF CONTENTS --- p.XII
LIST OF TABLES --- p.XVIII
LIST OF FIGURES --- p.XXI
LIST OF ABBREVIATIONS --- p.XXIV
Chapter SECTION I: --- BACKGROUND --- p.1
Chapter CHAPTER 1: --- PRENATALTESTNG --- p.2
Chapter 1.1. --- THE AIM --- p.2
Chapter 1.2. --- INVASIVE PRENATAL DIAGNOSIS --- p.4
Chapter 1.3. --- NONINVASIVE PRENATAL SCREENING --- p.6
Chapter CHAPTER 2: --- NONINVASIVE PRENATAL DIAGNOSIS --- p.10
Chapter 2.1. --- CIRCULATING FETAL CELLS IN PRENATAL DIAGNOSIS --- p.10
Chapter 2.2. --- CIRCULATING FETAL NUCLEIC ACIDS IN PRENATAL DIAGNOSIS --- p.12
Chapter 2.3.1. --- Biology of circulating fetal DNA . --- p.14
Chapter 2.3.2. --- Clinical applications of circulating fetal DNA --- p.15
Chapter 2.3.2.1. --- Qualitative analysis of fetal DNA in maternal plasma --- p.16
Chapter 2.3.2.2. --- Quantitative analysis of fetal DNA in maternal plasma --- p.17
Chapter 2.4. --- CIRCULATING FETAL RNA IN MATERNAL PLASMA --- p.20
Chapter 2.4.1. --- Biology of circulating fetal RNA --- p.20
Chapter 2.4.2. --- Clinical applications of circulating fetal RNA --- p.22
Chapter 2.4.2.1. --- Quantitative analysis of fetal RNA in maternal plasma --- p.22
Chapter CHAPTER 3: --- TECHNICAL CHALLENGES IN ANALYZING CIRCULATING FETAL NUCLEIC ACIDS --- p.26
Chapter 3.1. --- INTRODUCTION --- p.26
Chapter 3.2. --- "PREANALYTICAL ISSUES IN MATERNAL PLASMA NUCLEIC ACID ANALYSE"";" --- p.28
Chapter 3.2.1. --- Low abundance of cell-free fetal nucleic acids in maternal plasma --- p.28
Chapter 3.2.2. --- High level of maternal background in maternal plasma --- p.30
Chapter 3.3. --- ANALYTICAL ISSUES IN MATERNAL PLASMA NUCLEIC ACID ANALYS --- p.IS
Chapter 3.3.1. --- Imprecise measurement of fetal nucleic acid quantity --- p.33
Chapter 3.3.2. --- Coexistence of fetal nucleic acids and maternal nucleic acid background in maternal plasma --- p.36
Chapter 3.4. --- AIMS OF THIS THESIS --- p.41
Chapter SECTION II: --- MATERIALS AND METHODS --- p.42
Chapter CHAPTER 4: --- QUANTITATIVE AND QUALITATIVE ANALYSIS OF NUCLEIC ACIDS --- p.43
Chapter 4.1. --- SAMPLE COLLECTION AND PROCESSING --- p.43
Chapter 4.1.1. --- Preparation of plasma and blood cells --- p.43
Chapter 4.1.2. --- Preparation of placental tissues --- p.44
Chapter 4.2. --- NUCLEIC ACID EXTRACTION --- p.45
Chapter 4.2.1. --- Extraction of total RNA --- p.45
Chapter 4.2.1.1. --- Plasma samples --- p.45
Chapter 4.2.1.2. --- Placental tissue samples --- p.46
Chapter 4.2.2. --- Extraction of genomic DNA --- p.48
Chapter 4.2.2.1. --- Plasma samples --- p.48
Chapter 4.2.2.2. --- Blood cell samples --- p.48
Chapter 4.3. --- CONVENTIONAL REAL-TIME PCR ANALYSIS OF NUCLEIC ACIDS --- p.50
Chapter 4.3.1. --- Principles of real-time polymerase chain reaction --- p.50
Chapter 4.3.2. --- Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) --- p.52
Chapter 4.3.3. --- Quantitative polymerase chain reaction (qPCR) --- p.53
Chapter 4.4. --- DIGITAL REAL-TIME PCR ANALYSIS OF NUCLEIC ACIDS --- p.55
Chapter 4.4.1. --- Principles of digital PCR (dPCR) --- p.55
Chapter 4.4.2. --- 384-reaction well dPCR v --- p.56
Chapter 4.4.3. --- Microfluidics dPCR --- p.57
Chapter 4.5. --- MATRIX-ASSISTED LASER DESORPTIONIONIZATION/TIME-OF-FLIGHT MASS SPECTROMETRY (MALDI-TOF MS) ANALYSIS OF NUCLEIC ACIDS --- p.59
Chapter 4.5.1. --- Principles of MALDI-TOF MS --- p.59
Chapter 4.5.2. --- DNA genotyping analysis by MassArray Homogenous MassExtend (hME) assay --- p.60
Chapter 4.6. --- CLONING AND DNA SEQUENCING --- p.63
Chapter SECTION III: --- IMPROVEMENTS ON MATERNAL PLASMA ANALYSIS OF CIRCULATING RNA --- p.65
Chapter CHAPTER 5: --- ENRICHMENT OF PLACENTA EXPRESSED MRNA MARKERS BY WHOLE TRANSCRIPTOME PREAMPLIFICATION --- p.66
Chapter 5.1. --- INTRODUCTION --- p.66
Chapter 5.2. --- MATERIALS AND METHODS --- p.69
Chapter 5.2.1. --- Study design --- p.69
Chapter 5.2.2. --- Subjects and sample collection --- p.71
Chapter 5.2.3. --- RNA extraction and sample dilution --- p.71
Chapter 5.2.4. --- Preamplification --- p.73
Chapter 5.2.5. --- qPCR analysis --- p.74
Chapter 5.3. --- RESULTS --- p.83
Chapter 5.3.1. --- Comparison of mRNA expression profiles in placental tissues with and without preamplification --- p.83
Chapter 5.3.1.1. --- Undiluted placental tissue RNA --- p.84
Chapter 5.3.1.2. --- Diluted placental tissue RNA --- p.88
Chapter 5.3.2. --- The effect of RNA input on the degree of amplification --- p.92
Chapter 5.3.2.1. --- Correlation between RNA input and RNA output --- p.94
Chapter 5.3.2.2. --- Correlation between RNA input and output/input ratio --- p.96
Chapter 5.3.3. --- Preamplification of maternal plasma RNA --- p.98
Chapter 5.3.3.1. --- Concentrations of placenta expressed mRNA in third trimester maternal plasma --- p.98
Chapter 5.3.3.2. --- Concentrations of placenta expressed mRNA in first trimester maternal plasma --- p.100
Chapter 5.4. --- DISCUSSION --- p.102
Chapter SECTION IV: --- IMPROVEMENTS ON MATERNAL PLASMA ANALYSIS OF CIRCULATING DNA --- p.105
Chapter CHAPTER 6: --- ACCURATE GENE DOSAGE ANALYSIS BY MULTIPLEX QPCR --- p.106
Chapter 6.1. --- INTRODUCTION --- p.106
Chapter 6.2. --- MATERIALS AND METHODS --- p.110
Chapter 6.2.1. --- Study design --- p.110
Chapter 6.2.2. --- Subjects and sample collection --- p.112
Chapter 6.2.3. --- DNA extraction and sample dilution --- p.113
Chapter 6.2.4. --- qPCR analysis --- p.113
Chapter 6.2.4.1. --- Monoplex assays --- p.114
Chapter 6.2.4.2. --- Multiplex assays --- p.114
Chapter 6.2.5. --- Microfluidics dPCR assay --- p.122
Chapter 6.2.6. --- Gene Dosage Comparison --- p.122
Chapter 6.2.6.1. --- In adult male samples --- p.123
Chapter 6.2.6.2. --- In maternal plasma samples --- p.123
Chapter 6.3. --- RESULTS --- p.125
Chapter 6.3.1. --- The influence of using the same and different sets of primers for amplifying different chromosomal loci --- p.125
Chapter 6.3.2. --- Effects of using monoplex and multiplex real-time PCR formulations --- p.130
Chapter 6.3.3. --- Effects of incorporating calibration curves for template quantification in conventional qPCR --- p.135
Chapter 6.4. --- DISCUSSION --- p.140
Chapter CHAPTER 7: --- DPCR DETECTION OF PATERNALLY INHERITED POINT MUTATIONS --- p.144
Chapter 7.1. --- INTRODUCTION --- p.144
Chapter 7.2. --- MATERIALS AND METHODS --- p.153
Chapter 7.2.1. --- Study design --- p.153
Chapter 7.2.2. --- Subjects and sample collection --- p.157
Chapter 7.2.3. --- DNA extraction and sample preparation --- p.158
Chapter 7.2.4. --- MassArray hME assays --- p.159
Chapter 7.2.5. --- dPCR assay --- p.159
Chapter 7.3. --- RESULTS --- p.161
Chapter 7.3.1. --- Validation of the digital HbE assay --- p.161
Chapter 7.3.2. --- Determination of the minimum fetal DNA amount required for digital PCR detection --- p.165
Chapter 7.3.3. --- Detection of paternally inherited fetal HbE mutation in maternal plasma --- p.172
Chapter 7.4. --- DISCUSSION --- p.175
Chapter SECTION V: --- CONCLUDING REMARKS --- p.180
Chapter CHAPTER 8: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.181
Chapter 8.1. --- IMPROVEMENTS ON QUANTITATIVE AND QUALITATIVE ANALYSIS OF FETAL NUCLEIC ACIDS IN MATERNAL PLASMA --- p.181
Chapter 8.2. --- PERSPECTIVES FOR FUTURE WORK --- p.184
REFERENCES --- p.186

Bibliography: leaves 231-266.
271 leaves : col. ill. ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Indicates that injections under the ureteric orifice can cure VUR and that the tissue response to the plastics becomes quiescent. Research into embolisation from solid implants from intravenous tubing and the possibility of antibody formation to implanted plastics is also included. A model for fetal VUR has been developed to clarify focus of the uncertainity about reflux disease.
Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1999

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

The clinical manifestations of hemolytic disease of the fetus and newborn mediated by anti-K, an antibody of the Kell blood group system, are distinguishable from the classical form of the disease. Affected fetuses have low numbers of circulating reticulocytes and antibody titers and bilirubin levels are not reliable predictors of anemia. These observations suggest that antibodies to Kell glycoprotein lead to anemia through suppression of erythropoiesis. This study established a liquid erythroid progenitor cell culture model in which to perform analyses on the mechanism of the suppressive growth effect of anti-Kell glycoprotein. Using this culture model, this study demonstrated the requirement for co-ligation of Kell glycoprotein by a bivalent antibody for growth suppression. The absence of markers of apoptosis in cell cultures treated with anti-Kell glycoprotein suggests that the mechanism of growth suppression is distinct from programmed cell death and necrosis. Furthermore, this growth suppression cannot be rescued by erythropoietin.

In its efforts to reduce maternal mortality and prevent Mother-to-Child Transmission of HIV, the government of Swaziland developed and implemented several programmes including a special antenatal care package for HIV-positive pregnant women in line with the WHO (2009) guidelines. Since the implementation of this latest special ANC package for HIV-positive women, little is known about how these services are experienced by the intended recipients. The purpose of this study was to explore and describe the actual experiences of HIV-positive women with the antenatal care services provided at a regional referral hospital in Swaziland, with the view of providing more insight into the quality of ANC services from the users' perspectives. A qualitative descriptive, exploratory design was used to address the above purpose. The researcher used purposive sampling to select the participants who met the inclusion criteria for the study. Semi-structured individual interviews were used and saturation was reached after 18 individual face-to-face interviews. Thematic content analysis was used to analyse the collected data. Forteen themes related to the participants experiences with the ANC services and seven related to measures for improvement emerged from data. In general HIV positive pregnant women expressed positive views towards ANC services they received at the target institution. The results give an indication on the quality of the focussed ANC package provided at the hospital and specific recommendations for improvement are outlined.
Health Studies
M. A. (Nursing Science)

Conotruncal anomalies are the leading causes of cyanotic congenital heart disease. We attempted to use live xPlane imaging of ductal arch view and in-plane view of IVS to screen the fetal conotruncal anomalies in 200 fetuses. There were 152 normal cases, 25 conotruncal anomalies and 23 other types of fetal CHDs were involved in this study. The visualization rate of the normal ductal arch view and in-plane view of IVS with live xPlane imaging was 100% (152/152), 100% (152/152) in normal cases, 8% (2/25), 12% (3/25) in conotruncal anomalies and 69.7% (16/23), 73.9% (17/23) in non-conotruncal CHDs, respectively. The visualization rate of abnormal ductal arch and in-plane view in conotruncal anomalies was much higher than that in non-conotruncal anomalies (P<0.001). Therefore, it may be a useful tool for the assessment and diagnosis of fetal conotruncal anomalies.
In conclusion, real-time 3DE is a novel and promising technique to perform the prenatal examination, both the fetal heart and other system. It represents the future of 3D ultrasound and will become a useful tool for prenatal screening and diagnosis.
This thesis summarized real-time 3D ultrasound in obstetric application. With the introduction of matrix transducer, 3D scanning the fetus in real time became available.
We attempted to use real-time 3D ultrasound in obstetrics outside the fetal heart. We evaluated the feasibility of using real-time 3D ultrasound to assist in obtaining a true midsagittal view in first trimester. Eight sonographers, including FMF-certified and non FMF-certified operators, were asked to perform ultrasound examinations on five patients and forty patients were examined in total. It showed that the deviation from true midsagittal view was reduced greatly with the guidance of live xPlane imaging. Real-time 3D ultrasound can improve the accuracy of acquisition of a defined sonographic plane, and reduce the difference in performance between operators who are formally certified or not.
We demonstrated a novel method to visualize the aortic and ductal arch with live xPlane imaging. The visualization rate is 100%. Ductal arch view can be visualized by placing the reference line through pulmonary artery and descending aorta and aortic arch view can be acquired by putting the reference line along the transverse view of aortic arch and descending aorta on the 3VT view with live xPlane imaging. Therefore, live xPlane imaging is an easy and feasible method for real-time imaging of the ductal and aortic arch.
We explored the feasibility to perform the fetal heart screening using real-time 3DE with live xPlane imaging. We developed and reported the methodology of acquiring and examining the screening planes of the fetal heart with live xPlane imaging. The procedure was simple and straight. When performing the fetal heart screening with live xPlane imaging, we just need display the apical four-chamber view and mid-sagittal view of fetal upper thorax and other thing could be done by moving the reference line. The overall detection of four cardiac screen planes can reach 100%.
We explored to evaluate the entire fetal IVS with both live xPlane imaging and live 3D imaging. We can successfully assess the entire IVS in most fetuses (153/154). We also compared the images acquired by real-time 3DE and STIC in this thesis. It showed that less motion artifact encounters with real-time 3DE and the image quality of real-time 3DE is similar to STIC volume acquired from the sagittal view (P>0.05) and superior to STIC volume from the four-chamber view (P<0.05). Therefore, real-time 3DE can be used to display the lateral view of the fetal IVS, and potentially may be a useful tool for the assessment and diagnosis of fetal VSDs.
Xiong, Yi.
Adviser: Tzekin Lau.
Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 116-138).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.