Avaliação da microbiota de mulheres gestantes com e sem diabetes e de seus descendentes entre 2 e 6 meses de idade
Date
2021-10-26
Authors
Dualib, Patricia Medici [UNIFESP]
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Pititto, Bianca de Almeida [UNIFESP]
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Tese de doutorado
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Volume Title
Abstract
Objetivo: Analisar a composição da microbiota intestinal em cada trimestre da gestação e no puerpério de mulheres com e sem diabetes mellitus gestacional (DMG) pareadas por peso, bem como de seus bebês. Material e Métodos: O primeiro estudo foi uma revisão sistemática comparando a microbiota intestinal de mulheres com e sem DMG ao longo dos 3 trimestres da gestação. Após a revisão, fizemos um estudo que acompanhou 115 gestantes 18 anos, com IMC 25/kg/m2, com (n = 56) ou sem DMG (n = 59) admitidas nos ambulatórios de pré-natal da UNIFESP-EPM durante o primeiro/segundo ou terceiro trimestre de gestação durante os anos de 2018 a 2020. Destas, 84 mulheres, com (n = 40) ou sem diagnóstico de DMG (n = 44), foram reavaliadas no puerpério juntamente com seus respectivos bebês. Elas foram avaliadas com questionário padronizado, recordatório alimentar, exame clínico e coleta de amostras biológicas. Os dados clínicos e laboratoriais foram comparados entre os grupos com e sem DMG usando o teste t de Student e Mann Whitney ou teste qui-quadrado conforme fosse apropriado, com o auxílio do programa Statistical Package for the Social Sciences®, versão 16.0 (SPSS Incorporation, 2000). O perfil molecular da microbiota fecal foi obtido por meio do sequenciamento da região V4 do gene 16S rRNA (Illumina® MiSeq) e do pacote R utilizado para as análises. Foi calculada a alfa diversidade das amostras com e sem DMG nos diferentes momentos para as mães (gestação e puerpério) e os bebês. Para avaliar a beta diversidade, de acordo com diagnóstico de DMG, tipo de parto e aleitamento, foi usada a técnica de Principal Coordinate Analysis (PCoA), utilizando a distância de Jensen-Shannon e o teste de Permanova. Foi avaliada, também, a abundância relativa de bactérias, utilizando critérios rigorosos de p < 0,01 e descartando as bactérias que apresentavam abundância relativa de 0% em uma grande parte das participantes. As correlações entre as bactérias e parâmetros clínicos e laboratoriais foram testadas e consideradas estatisticamente significativa, se p < 0,01. Resultados: A revisão sistemática (Artigo 1) incluiu 23 estudos, envolvendo 3560 mulheres, que compararam a microbiota intestinal na gestação nos diferentes trimestres e foi visto que existe uma relação entre a microbiota intestinal e o DMG. Análises do estudo atual (Artigo 2) mostraram que mulheres com DMG (n=56) eram mais velhas [33.2 (6.2) vs. 28.1 (5.9) anos, p<0.01] e tinham um maior número de gestações do que as tolerantes normais (n=59). Apesar de apresentarem índice de massa corporal (IMC) semelhante [30.2 (3.9) vs. 29.2 (3.7), kg/m2, p = 0.39)], as mulheres com DMG apresentaram maior circunferência do pescoço no terceiro trimestre [36.5(2.4) vs. 34.6 (2.1) cm, p < 0.01). Em comparação com a ingestão de macronutrientes, o grupo não diabético consumiu mais carboidratos e menos proteína no primeiro e segundo trimestres gestacionais do que os GDM e menos proteína no terceiro trimestre. Não houve diferença na diversidade alfa e os grupos não diferiram em relação à estrutura geral da microbiota. Uma maior abundância de Bacteroides no grupo GDM foi encontrada. Foi observada uma correlação positiva entre as abundâncias de Christensenellaceae e Intestinobacter com a glicose plasmática pós-sobrecarga de 1 hora e uma correlação negativa entre Enterococcus e o nível de glicose plasmática de 2 horas. As abundâncias de Bifidobacterium e Peptococcus aumentaram no terceiro trimestre gestacional em ambos os grupos. No puerpério (Artigo 3), as mulheres com DMG anterior (n = 40) eram mais velhas [33.3 (5.9) vs. 28.6 (6.1) anos, p = 0,01], tiveram mais gestações [2.9 (1.7) vs. 1.9 (1.3), p = 0.04], níveis mais elevados de glicose plasmática pós sobrecarga de 2 horas [116.3 (38.5) vs. 98.5 (19.0) mg/dl, p = 0.02], HbA1c [5.6 (0.4) vs. 5.3 (0.3)%, p = 0.02] e colesterol LDL [126.6 (45.3) vs. 110.7 (27.0 ) mg/dl, p = 0.01] do que as mulheres com tolerância normal à glicose (n = 44) durante a gravidez. Em relação à dieta materna, apenas a ingestão total de fibras foi maior nas mulheres com DMG no período puerperal [11.9(9.1-14.5) vs 6.8 (3.9-13.9) g, p = 0.04]. As mulheres DMG amamentavam exclusivamente mais seus bebês com significância limítrofe [26 (60.5) vs. 19 (40.4) %, p = 0.06]. Além disso, esses bebês tiveram uma frequência maior de complicações neonatais, incluindo icterícia. As estruturas da microbiota intestinal das puérperas e seus bebês eram semelhantes. Estratificando de acordo com o tipo de parto, a abundância relativa do gênero Victivallis foi maior nas mulheres que tiveram parto normal. A exposição dos bebês ao aleitamento materno exclusivo foi associada a uma maior abundância de Bacteroides e Staphylococcus. O teste de abundância diferencial mostrou correlações com parâmetros clínicos e laboratoriais diversos. Conclusão: O estudo atual não observou diferença estrutural da microbiota intestinal ao longo da gestação ou no período pós-parto de mulheres e seus bebês com peso equivalente pré- e peri-gestacional (sobrepeso ou obesidade) de acordo com a presença ou não de DMG. No entanto, algumas abundâncias de gêneros mostraram associações com período da gestação e o metabolismo da glicose, o que pode motivar o aprofundamento do conhecimento sobre as alterações fisiológicas e fisiopatológicas da microbiota ao longo da gestação, contribuindo para implicações adicionais na prevenção e manejo do diabetes gestacional no futuro. No puerpério notamos maior abundância de Victivallis nas mães que tiveram parto normal, além da associação deste gênero com parâmetros metabólicos, que podem sugerir benefícios para o metabolismo energético destas mulheres. Em bebês amamentados caracterizamos a maior abundância de gêneros de bactérias que podem representar uma evolução ecológica da microbiota intestinal mais precoce, o que pode significar mais um benefício do aleitamento para bebês provenientes de gestação de risco, como a gestação em mulheres com obesidade e DMG. Porém estudos prospectivos são necessários para comprovar se, de fato, Bacteroides e Staphylococcus estarão associados a uma composição favorável da microbiota intestinal mais tarde na vida. Nossos resultados contribuem para a compreensão dos eventos precoces da vida na microbiota intestinal e seu possível papel no metabolismo futuro. O seguimento destas participantes poderá ajudar a esclarecer as hipóteses levantadas no presente estudo.
Objective: To analyze the composition of the intestinal microbiota in each trimester of pregnancy and in the puerperium of women with and without gestational diabetes mellitus (GDM) matched for weight, as well as their babies. Material and Methods: Our first study was a systematic review comparing the gut microbiota of women with and without GDM over the 3 trimesters of pregnancy. After the review, we performed a study that followed 115 pregnant women 18 years old, with BMI 25/kg/m2 , with (n = 56) or without GDM (n = 59) admitted to the prenatal clinics of UNIFESP-EPM during the first /second or third trimester of pregnancy from 2018 to 2020. Of these, 84 women, with (n = 40) or without (n = 44) diagnosis of GDM, were re-evaluated in the puerperium along with their respective babies. They were evaluated with a standardized questionnaire, dietary recall, clinical examination and collection of biological samples. Clinical and laboratory data were compared between groups with and without GDM using Student's t test and Mann Whitney test or chi-square test as appropriate, with the aid of the Statistical Package for Social Sciences®, version 16.0 (SPSS Incorporation, 2000). The molecular profile of the fecal microbiota was obtained by sequencing the V4 region of the 16S rRNA gene (Illumina® MiSeq) and the R package used for the analysis. The alpha diversity of samples with and without GDM was calculated at different times for mothers (pregnancy and puerperium) and babies. To assess beta diversity, according to GDM diagnosis, type of delivery and breastfeeding, the Principal Coordinate Analysis (PCoA) technique was used, using the Jensen-Shannon distance and the Permanova test. The relative abundance of bacteria was also evaluated, using strict criteria of p < 0.01, and discarding bacteria that had a relative abundance of 0% in a large part of the participants. Correlations between bacteria and clinical and laboratory parameters were tested and considered statistically significant if p < 0.01. Results: The systematic review (Article 1) included 14 studies, involving 1588 women, which compared the intestinal microbiota during pregnancy in different trimesters and it was seen that there is a relationship between the intestinal microbiota and GDM. Analyzes of the current study (Article 2) showed that women with GDM (n=56) were older [33.2 (6.2) vs. 28.1 (5.9) years, p < 0.01] and had a higher number of pregnancies than the normal tolerant ones (n = 59). Despite having similar body mass index (BMI) [30.2 (3.9) vs. 29.2 (3.7) kg/m2 , p = 0.39)], women with GDM had greater neck circumference in the third trimester [36.5(2.4) vs. 34.6 (2.1) cm, p < 0.01). Compared to macronutrient intake, the non-diabetic group consumed more carbohydrates and less protein in the first and second trimesters of pregnancy than the GDM and less protein in the third trimester. There was no difference in alpha diversity and the groups did not differ regarding the general structure of the microbiota. A greater abundance of Bacteroides in the GDM group was found. A positive correlation was observed between the abundances of Christensenellaceae and Intestinobacter with the 1-hour post-challenge plasma glucose and a negative correlation between Enterococcus and the 2-hour plasma glucose level. Bifidobacterium and Peptococcus abundances increased in the third trimester of pregnancy in both groups. In the puerperium (Article 3), women with previous GDM (n = 40) were older [33.3 (5.9) vs. 28.6 (6.1) years, p = 0.01], had more pregnancies [2.9 (1.7) vs. 1.9 (1.3), p = 0.04], higher plasma glucose levels after 2-hour overload [116.3 (38.5) vs. 98.5 (19.0) mg/dl, p = 0.02], HbA1c [5.6 (0.4) vs. 5.3 (0.3), p = 0.02] and LDL cholesterol [126.6 (45.3) vs. 110.7 (27.0), p = 0.01] than women with normal glucose tolerance(n=44) during pregnancy. Regarding the maternal diet, only total fiber intake was higher in women with GDM in the puerperal period [11.9(9.1-14.5) vs 6.8 (3.9-13.9) g, p = 0.04]. GDM women exclusively breastfed their babies more with borderline significance [26 (60.5) vs. 19 (40.4) %, p = 0.06]. In addition, these babies had a higher frequency of neonatal complications, including jaundice. The structures of the intestinal microbiota of postpartum women and their babies were similar. Stratified according to type of delivery, the relative abundance of the genus Victivallis was greater in women who had vaginal delivery. The exposure of infants to exclusive breastfeeding was associated with a greater abundance of Bacteroides and Staphylococcus. The differential abundance test showed correlations with different clinical and laboratory parameters. Conclusion: The current study did not observe structural differences in the intestinal microbiota during pregnancy or in the postpartum period of women and their babies with equivalent pre- and peri-gestational weight (overweight or obesity) according to the presence or absence of GDM. However, some genera abundances showed associations with the gestation period and glucose metabolism, which may motivate the deepening of knowledge about the physiological and pathophysiological changes in the microbiota throughout pregnancy, contributing to additional implications for the prevention and management of diabetes pregnancy in the future. In the puerperium, we noticed a greater abundance of Victivallis in mothers who had vaginal delivery, in addition to the association of this gender with metabolic parameters, which may suggest benefits for the energy metabolism of these women. In breastfed babies, we characterize the greater abundance of bacterial genera that may represent an earlier ecological evolution of the intestinal microbiota, which may mean another benefit of breastfeeding for babies from high-risk pregnancies, such as pregnancy in women with obesity and GDM. However, prospective studies are needed to prove whether, in fact, Bacteroides and Staphylococcus will be associated with a favorable composition of the intestinal microbiome later in life. Our results contribute to the understanding of early life events in the intestinal microbiota and their possible role in future metabolism. The follow-up of these participants may help to clarify the hypotheses raised in this study.
Objective: To analyze the composition of the intestinal microbiota in each trimester of pregnancy and in the puerperium of women with and without gestational diabetes mellitus (GDM) matched for weight, as well as their babies. Material and Methods: Our first study was a systematic review comparing the gut microbiota of women with and without GDM over the 3 trimesters of pregnancy. After the review, we performed a study that followed 115 pregnant women 18 years old, with BMI 25/kg/m2 , with (n = 56) or without GDM (n = 59) admitted to the prenatal clinics of UNIFESP-EPM during the first /second or third trimester of pregnancy from 2018 to 2020. Of these, 84 women, with (n = 40) or without (n = 44) diagnosis of GDM, were re-evaluated in the puerperium along with their respective babies. They were evaluated with a standardized questionnaire, dietary recall, clinical examination and collection of biological samples. Clinical and laboratory data were compared between groups with and without GDM using Student's t test and Mann Whitney test or chi-square test as appropriate, with the aid of the Statistical Package for Social Sciences®, version 16.0 (SPSS Incorporation, 2000). The molecular profile of the fecal microbiota was obtained by sequencing the V4 region of the 16S rRNA gene (Illumina® MiSeq) and the R package used for the analysis. The alpha diversity of samples with and without GDM was calculated at different times for mothers (pregnancy and puerperium) and babies. To assess beta diversity, according to GDM diagnosis, type of delivery and breastfeeding, the Principal Coordinate Analysis (PCoA) technique was used, using the Jensen-Shannon distance and the Permanova test. The relative abundance of bacteria was also evaluated, using strict criteria of p < 0.01, and discarding bacteria that had a relative abundance of 0% in a large part of the participants. Correlations between bacteria and clinical and laboratory parameters were tested and considered statistically significant if p < 0.01. Results: The systematic review (Article 1) included 14 studies, involving 1588 women, which compared the intestinal microbiota during pregnancy in different trimesters and it was seen that there is a relationship between the intestinal microbiota and GDM. Analyzes of the current study (Article 2) showed that women with GDM (n=56) were older [33.2 (6.2) vs. 28.1 (5.9) years, p < 0.01] and had a higher number of pregnancies than the normal tolerant ones (n = 59). Despite having similar body mass index (BMI) [30.2 (3.9) vs. 29.2 (3.7) kg/m2 , p = 0.39)], women with GDM had greater neck circumference in the third trimester [36.5(2.4) vs. 34.6 (2.1) cm, p < 0.01). Compared to macronutrient intake, the non-diabetic group consumed more carbohydrates and less protein in the first and second trimesters of pregnancy than the GDM and less protein in the third trimester. There was no difference in alpha diversity and the groups did not differ regarding the general structure of the microbiota. A greater abundance of Bacteroides in the GDM group was found. A positive correlation was observed between the abundances of Christensenellaceae and Intestinobacter with the 1-hour post-challenge plasma glucose and a negative correlation between Enterococcus and the 2-hour plasma glucose level. Bifidobacterium and Peptococcus abundances increased in the third trimester of pregnancy in both groups. In the puerperium (Article 3), women with previous GDM (n = 40) were older [33.3 (5.9) vs. 28.6 (6.1) years, p = 0.01], had more pregnancies [2.9 (1.7) vs. 1.9 (1.3), p = 0.04], higher plasma glucose levels after 2-hour overload [116.3 (38.5) vs. 98.5 (19.0) mg/dl, p = 0.02], HbA1c [5.6 (0.4) vs. 5.3 (0.3), p = 0.02] and LDL cholesterol [126.6 (45.3) vs. 110.7 (27.0), p = 0.01] than women with normal glucose tolerance(n=44) during pregnancy. Regarding the maternal diet, only total fiber intake was higher in women with GDM in the puerperal period [11.9(9.1-14.5) vs 6.8 (3.9-13.9) g, p = 0.04]. GDM women exclusively breastfed their babies more with borderline significance [26 (60.5) vs. 19 (40.4) %, p = 0.06]. In addition, these babies had a higher frequency of neonatal complications, including jaundice. The structures of the intestinal microbiota of postpartum women and their babies were similar. Stratified according to type of delivery, the relative abundance of the genus Victivallis was greater in women who had vaginal delivery. The exposure of infants to exclusive breastfeeding was associated with a greater abundance of Bacteroides and Staphylococcus. The differential abundance test showed correlations with different clinical and laboratory parameters. Conclusion: The current study did not observe structural differences in the intestinal microbiota during pregnancy or in the postpartum period of women and their babies with equivalent pre- and peri-gestational weight (overweight or obesity) according to the presence or absence of GDM. However, some genera abundances showed associations with the gestation period and glucose metabolism, which may motivate the deepening of knowledge about the physiological and pathophysiological changes in the microbiota throughout pregnancy, contributing to additional implications for the prevention and management of diabetes pregnancy in the future. In the puerperium, we noticed a greater abundance of Victivallis in mothers who had vaginal delivery, in addition to the association of this gender with metabolic parameters, which may suggest benefits for the energy metabolism of these women. In breastfed babies, we characterize the greater abundance of bacterial genera that may represent an earlier ecological evolution of the intestinal microbiota, which may mean another benefit of breastfeeding for babies from high-risk pregnancies, such as pregnancy in women with obesity and GDM. However, prospective studies are needed to prove whether, in fact, Bacteroides and Staphylococcus will be associated with a favorable composition of the intestinal microbiome later in life. Our results contribute to the understanding of early life events in the intestinal microbiota and their possible role in future metabolism. The follow-up of these participants may help to clarify the hypotheses raised in this study.
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DUALIB, Patricia Medici. Avaliação da microbiota de mulheres gestantes com e sem diabetes e de seus descendentes entre 2 e 6 meses de idade. São Paulo, 2021. 146 f. Tese (Doutorado em Endocrinologia e Metabologia) – Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2021.