Orientador: Lígia Maria S. S. Rugolo / Banca: Cleide Enoir Petean Trindade / Banca: José Carlos Peraçoli / Banca: Maria Fernanda Branco de Almeida / Banca: Lilian dos Santos Rodrigues Sadeck / Resumo: A rotura prematura de membranas pré-termo (RPM-PT) é uma das principais causas de morbimortalidade perinatal e fator de risco para infecção neonatal precoce. As citocinas pró-inflamatórias TNF-α, IL-8 e a antiiflamatória IL-10 são importantes mediadores da resposta imune, e na situação de risco infeccioso podem ser adjuvantes úteis no diagnóstico de infecção neonatal. Investigar se os níveis de TNF-α, IL-8 e IL-10 em sangue de cordão umbilical são marcadores de infecção precoce em prematuros advindos de gestações com RPM-PT. Estudo clínico, prospectivo e do tipo teste diagnóstico, realizado no Serviço de Obstetrícia e Neonatologia da Faculdade de Medicina de Botucatu, envolvendo prematuros de gestantes com rotura de membranas ≥ 12 horas. As citocinas TNF-α, IL-8 e IL-10 foram dosadas em sangue de cordão umbilical pelo método ELISA. Conforme a evolução clínica e laboratorial dos recém-nascidos foram constituídos dois grupos: Infectado e Não infectado, os quais foram comparados quanto às variáveis perinatais e neonatais por análise estatística univariada; com significância em 5%. A acurácia do teste diagnóstico foi obtida pela curva ROC, sendo calculados: sensibilidade, especificidade, valor preditivo positivo (VPP) e negativo (VPN). Foram estudados 55 prematuros: 27 infectados e 28 não infectados. O tempo de rotura não diferiu entre os grupos (54 x 29 hs; p=0,102) mas, no grupo infectado corioamnionite clínica e histológica foi mais frequente; a idade gestacional (31 x 33 semanas; p<0,001) e o peso de nascimento (1707 x 2109g; p=0,003) foram menores; a morbidade foi maior desde o nascimento com necessidade de assistência mais intensiva e o óbito ocorreu somente nesse grupo. As medianas de TNF-α (3.67 vs 1.76 pg/ml; p =0.084) e IL-10 (0 vs 4.10 pg/ml; p=0.291)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Preterm premature rupture of membranes (PPROM) is one of the major causes of perinatal morbidity and mortality, and an important risk factor for early-onset neonatal infection. The pro-inflammatory cytokines TNF-α, IL-8 and anti-inflammatory cytokine IL-10 are mediators of immune response and may be helpful as early indicator of neonatal infection in the presence of perinatal risk factor. To evaluate umbilical cord blood levels of TNF-α, IL-8 and IL-10 as markers for early-onset infections in premature infants from pregnancies complicated by PPROM. Clinical, prospective and diagnostic test study performed at Obstetrician and Neonatal Unit of Botucatu School of Medicine, enrolling premature infants from pregnant women with ≥ 12 hours of premature rupture of membranes. TNF-α, IL-8 and IL-10 cytokines were measured in umbilical cord blood by ELISA. According to clinical and laboratorial evaluation patients were classified into two groups: infected and noninfected. Perinatal and neonatal variables were studied. Comparisons between groups were performed by univariate statistical analysis; significance at p <0.05. The diagnostic test accuracy was obtained by ROC curve. Sensibility, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated by comparing infected with noninfected group. 55 premature infants were studied: 27 infected and 28 noninfected. There was no difference between the groups in the latency period (54 x 29 hours; p=0,102). In the infected group, clinical and histological chorioamnionitis were more frequent, gestational age (31 x 33 weeks; p<0,001) and birth weight(1707 x 2109g; p=0,003) were lower. Morbidity, mortality and resource use were higher among infected premature infants. The median levels of TNF-α (3.67 vs 1.76 pg/ml; p=0.084)... (Complete abstract click electronic access below) / Doutor

The vaginal microbial community is critical to a woman’s health and the health of her family. Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a shift of the vaginal microbiota from a Lactobacillus dominated community to a dense biofilm containing a complex mixture of organisms. Although BV is an important risk factor for poor reproductive health outcomes, the etiology of BV is poorly understood. Gardnerella vaginalis is a hallmark species of BV. Phylogenetic analysis of cpn60 universal target sequences from metagenomic studies of the vaginal microbiome and from G. vaginalis isolates resolved four subgroups within the species. This subdivision, supported by whole genome similarity comparisons, demonstrated that these subgroups might represent different species. Among a group of African women, only G. vaginalis subgroup B was significantly more abundant in women with BV relative to women with Nugent scores not consistent with BV. To characterize the subgroups further, several phenotypic and molecular factors of G. vaginalis subgroups were assessed. Proteomic profiles of isolates within each subgroup formed unambiguous clusters. Sialidase gene sequences were detected in all subgroups, however enzymatic activity was detected only in subgroup B. Two isolates of subgroup B isolates (N153 and N101) were incapable of growth in 7% CO2. Given the well-known relationship between an anaerobic microbiota and BV, anaerobic isolates of G. vaginalis are potentially important players in the vaginal microbial community. To determine genome content differences that could account for the phenotypic difference, whole genome sequences of four G. vaginalis subgroup B isolates representing facultative and anaerobic phenotypes were determined. Comparison of genomes led to the identification of genes predicted to encode proteins involved in cell wall biogenesis and protection from oxidative damage that might account for the observed phenotypes. The cpn60 universal target based methodology that improved resolution of the vaginal microbiota including G. vaginalis was applied in a prospective study of the vaginal microbiome of women with preterm premature rupture of membranes (PPROM). The objectives were to characterize the vaginal microbiota of women following PPROM, and to determine if microbiome composition at the time of rupture predicts latency duration and perinatal outcomes. Only 13/70 samples collected from 36 women were dominated by Lactobacillus spp., the expected profile for healthy women, while Megasphaera type 1 and Prevotella spp. were detected in all samples. Microbiome profiles at the time of membrane rupture did not cluster by gestational age at PPROM, or latency duration. Microbial profiles were unstable over the latency period, with dramatic shifts in composition between weekly samples, and an overall decrease in Lactobacillus abundance. Mollicutes were detected by PCR in 81% (29/36) of women, and these women had significantly lower gestational age at delivery and correspondingly lower birth weight infants than Mollicutes negative women. Taken together, the results presented in this thesis demonstrate the value of high resolution profiling of the vaginal microbiome using cpn60 UT sequences. The resolution of subgroups within G. vaginalis has potentially significant implications for women's health diagnostics, requiring a shift away from considering G. vaginalis as a single entity. The PPROM study provides foundational information that may lead to the identification of informative sequence patterns, providing clinicians with better tools for expectant management following PPROM.

Made available in DSpace on 2014-06-11T19:33:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-01Bitstream added on 2014-06-13T19:26:38Z : No. of bitstreams: 1 hashimoto_m_dr_botfm.pdf: 298656 bytes, checksum: 13b4b2d4d265946e9f647fb2dd27e0e4 (MD5) / A rotura prematura de membranas pré-termo (RPM-PT) é uma das principais causas de morbimortalidade perinatal e fator de risco para infecção neonatal precoce. As citocinas pró-inflamatórias TNF-α, IL-8 e a antiiflamatória IL-10 são importantes mediadores da resposta imune, e na situação de risco infeccioso podem ser adjuvantes úteis no diagnóstico de infecção neonatal. Investigar se os níveis de TNF-α, IL-8 e IL-10 em sangue de cordão umbilical são marcadores de infecção precoce em prematuros advindos de gestações com RPM-PT. Estudo clínico, prospectivo e do tipo teste diagnóstico, realizado no Serviço de Obstetrícia e Neonatologia da Faculdade de Medicina de Botucatu, envolvendo prematuros de gestantes com rotura de membranas ≥ 12 horas. As citocinas TNF-α, IL-8 e IL-10 foram dosadas em sangue de cordão umbilical pelo método ELISA. Conforme a evolução clínica e laboratorial dos recém-nascidos foram constituídos dois grupos: Infectado e Não infectado, os quais foram comparados quanto às variáveis perinatais e neonatais por análise estatística univariada; com significância em 5%. A acurácia do teste diagnóstico foi obtida pela curva ROC, sendo calculados: sensibilidade, especificidade, valor preditivo positivo (VPP) e negativo (VPN). Foram estudados 55 prematuros: 27 infectados e 28 não infectados. O tempo de rotura não diferiu entre os grupos (54 x 29 hs; p=0,102) mas, no grupo infectado corioamnionite clínica e histológica foi mais frequente; a idade gestacional (31 x 33 semanas; p<0,001) e o peso de nascimento (1707 x 2109g; p=0,003) foram menores; a morbidade foi maior desde o nascimento com necessidade de assistência mais intensiva e o óbito ocorreu somente nesse grupo. As medianas de TNF-α (3.67 vs 1.76 pg/ml; p =0.084) e IL-10 (0 vs 4.10 pg/ml; p=0.291)... / Preterm premature rupture of membranes (PPROM) is one of the major causes of perinatal morbidity and mortality, and an important risk factor for early-onset neonatal infection. The pro-inflammatory cytokines TNF-α, IL-8 and anti-inflammatory cytokine IL-10 are mediators of immune response and may be helpful as early indicator of neonatal infection in the presence of perinatal risk factor. To evaluate umbilical cord blood levels of TNF-α, IL-8 and IL-10 as markers for early-onset infections in premature infants from pregnancies complicated by PPROM. Clinical, prospective and diagnostic test study performed at Obstetrician and Neonatal Unit of Botucatu School of Medicine, enrolling premature infants from pregnant women with ≥ 12 hours of premature rupture of membranes. TNF-α, IL-8 and IL-10 cytokines were measured in umbilical cord blood by ELISA. According to clinical and laboratorial evaluation patients were classified into two groups: infected and noninfected. Perinatal and neonatal variables were studied. Comparisons between groups were performed by univariate statistical analysis; significance at p <0.05. The diagnostic test accuracy was obtained by ROC curve. Sensibility, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated by comparing infected with noninfected group. 55 premature infants were studied: 27 infected and 28 noninfected. There was no difference between the groups in the latency period (54 x 29 hours; p=0,102). In the infected group, clinical and histological chorioamnionitis were more frequent, gestational age (31 x 33 weeks; p<0,001) and birth weight(1707 x 2109g; p=0,003) were lower. Morbidity, mortality and resource use were higher among infected premature infants. The median levels of TNF-α (3.67 vs 1.76 pg/ml; p=0.084)... (Complete abstract click electronic access below)

Common, complex disorders are polygenic and multifactorial traits representing interactions between environmental, genetic and epigenetic risk factors. More often than not, contributions of these risk factors have been studied individually and this is especially true for complex reproductive traits where application of genomic technologies has been challenging and slow to progress. This thesis explores the potential of genetic and epigenetic components contributing to a better understanding of the biological pathways underlying disease risk in two specific female complex reproductive traits - polycystic ovary syndrome (PCOS) and preterm premature rupture of membranes (PPROM). The PCOS projects focus on characterization of a gene, DENND1A, whose association to PCOS has been established by Genome Wide Association Studies (GWAS) and is known to contribute to PCOS steroidogenic phenotype. In addition, differential microRNAs expression contributing to DENND1A expression regulation in PCOS theca cells was identified. The studies on PPROM utilize a Whole Exome Sequencing approach to identify rare variants in fetal genes contributing to extracellular matrix composition and synthesis contributing to PPROM risk. The results suggest that fetal contribution to PPROM is polygenic and is driven by a significant genetic burden of potentially damaging rare variants in genes contributing to fetal membrane strength and integrity. Tissue and location specific expression patterns of the Chromosome 21 miRNA cluster (miR-99a, miR-125b, let-7c) in fetal membranes from term pregnancies with spontaneous rupture were investigated. The results suggest that these miRNAs play potential roles in fetal membrane rupture and fetal membrane defects associated with T21.

Die nicht-invasive Analyse der fetalen Herzratenvariabilität mittels abdominaler Elektrokardiographie stellt eine neue Methode zur Beurteilung des fetalen Zustandes dar. Die Herzratenvariabilität gilt hier als ein sensitives Maß der autonomen Regulation. Bereits mit Beginn der zweiten Schwangerschaftshälfte ist es möglich, über das mütterliche Abdomen ein fetales Elektrokardiogramm abzuleiten und einer Herzratenvariabilitätsanalyse zuzuführen. Das Untersuchungskollektiv dieser Arbeit umfasste Frauen mit frühem vorzeitigem Blasensprung als Modell einer pathologischen Alteration des fetalen Zustandes sowie Frauen mit normalen Schwangerschaften zwischen der 20. und 28. Schwangerschaftswoche. Die technische Umsetzung und Analyse der fetalen Herzratenvariabilität erfolgte in Kooperation mit dem Institut für Biomedizinische Technik der TU Dresden. Insgesamt wurden 25 Datensätze der Auswertung zugeführt. Eine Reifung des autonomen Nervensystems des Feten mit ansteigendem Gestationsalter konnte mittels der Herzratenvariabilitätsanalyse gezeigt werden. Zur Risikostratifizierung der Feten bei frühem vorzeitigem Blasensprung konnten bei der Betrachtung der Herzratenvariabilitätsparameter keine signifikanten Unterschiede zum Normalkollektiv erhoben werden. Die Analyse der Parameter erfolgte hierbei unabhängig von den fetalen Verhaltenszuständen. Es konnte gezeigt werden, dass die nicht-invasive Analyse der Herzratenvariabilitätsanalyse methodisch und technisch in der Lage ist, den Fetalzustand und dessen Alterationen zu erfassen.

Die nicht-invasive Analyse der fetalen Herzratenvariabilität mittels abdominaler Elektrokardiographie stellt eine neue Methode zur Beurteilung des fetalen Zustandes dar. Die Herzratenvariabilität gilt hier als ein sensitives Maß der autonomen Regulation. Bereits mit Beginn der zweiten Schwangerschaftshälfte ist es möglich, über das mütterliche Abdomen ein fetales Elektrokardiogramm abzuleiten und einer Herzratenvariabilitätsanalyse zuzuführen. Das Untersuchungskollektiv dieser Arbeit umfasste Frauen mit frühem vorzeitigem Blasensprung als Modell einer pathologischen Alteration des fetalen Zustandes sowie Frauen mit normalen Schwangerschaften zwischen der 20. und 28. Schwangerschaftswoche. Die technische Umsetzung und Analyse der fetalen Herzratenvariabilität erfolgte in Kooperation mit dem Institut für Biomedizinische Technik der TU Dresden. Insgesamt wurden 25 Datensätze der Auswertung zugeführt. Eine Reifung des autonomen Nervensystems des Feten mit ansteigendem Gestationsalter konnte mittels der Herzratenvariabilitätsanalyse gezeigt werden. Zur Risikostratifizierung der Feten bei frühem vorzeitigem Blasensprung konnten bei der Betrachtung der Herzratenvariabilitätsparameter keine signifikanten Unterschiede zum Normalkollektiv erhoben werden. Die Analyse der Parameter erfolgte hierbei unabhängig von den fetalen Verhaltenszuständen. Es konnte gezeigt werden, dass die nicht-invasive Analyse der Herzratenvariabilitätsanalyse methodisch und technisch in der Lage ist, den Fetalzustand und dessen Alterationen zu erfassen.

Pregnancy-related complications such as gestational hypertension, preeclampsia, fetal growth restriction, gestational diabetes mellitus, spontaneous preterm birth, and preterm premature rupture of membranes may have severe consequences for both the mother and the child. The development of reliable early screening methods for pregnancy-related complications has therefore been a long-term goal of obstetrics. New possibilities for prenatal diagnostics have opened with the discovery of circulating microRNAs in maternal plasma. MicroRNAs are short, noncoding, 21 to 23 nucleotides long, single-strand RNAs whose main function is to regulate gene expression. During pregnancy, both common and unique miRNAs are expressed by the placenta, amongst them the miRNAs of the C19MC cluster. Several C19MC miRNAs have been shown to display a different expression profile associated with certain pregnancy-related complications. This thesis identifies the plasma exosomal profiles of six C19MC miRNAs (miR-516-5p, miR-517-5p, miR-518b, miR-520a-5p, miR-520h, and miR-525-5p) in patients in their first trimester of gestation who later developed pregnancy-related complications, and compares them with profiles in patients with normal pregnancies.