| Objective: This study aims to clarify the relationship between oral microorganisms and pregnancy health and adverse pregnancy outcomes,to initially explore the pathways and mechanisms by which the main pathogenic bacteria of chronic apical periodontitis affect premature birth,and to provide certain guidance for the prevention and treatment of adverse pregnancy outcomes related to oral microorganisms.Methods: 1.High-throughput 16 S r RNA gene sequencing technology was used to compare the microbial community characteristics of supragingival plaque in 30 pregnant women and10 non-pregnant women,and identify specific microbial genera and strains that may be related to pregnancy health.2.Use high-throughput 16 S r RNA gene sequencing to compare the characteristics of the salivary microorganisms and placental microbial communities of 6 women in the spontaneous preterm birth group and 6 women in the term group,aiming to identify specific microorganisms that may be related to preterm birth.3.Explore the pathways through which the main pathogenic bacteria of chronic apical periodontitis affect the occurrence of spontaneous preterm birth from both animal experiments and cell experiments.We will establish a pregnant mouse model of chronic apical periodontitis through animal experiments to verify the correlation between chronic apical periodontitis and spontaneous preterm birth,and initially explore how Porphyromonas gingivalis and Fusobacterium nucleatum affect spontaneous preterm birth.occurrence;next,we will use Porphyromonas gingivalis and Fusobacterium nucleatum to intervene in umbilical vein epithelial cells through cell experiments to verify whether these two bacteria have the ability to enter the blood circulation by affecting vascular endothelial cells.4.Explore the impact of the main pathogenic bacteria of chronic apical periodontitis on placental trophoblast cells from both cell experiments and animal experiments.We will use Porphyromonas gingivalis and Fusobacterium nucleatum to intervene in placental trophoblast cells through cell experiments,observe the effects of these two bacteria on the proliferation and apoptosis of placental trophoblast cells,and evaluate the effects of bacteria on placental trophoblast cells.Adhesion and invasion ability and detect the expression of inflammatory factors and TLR4/MyD88/NF-κB pathway related proteins;then we established a pregnant mouse model of chronic apical periodontitis through animal experiments to verify the above cell experiment results.Results: 1.The alpha diversity analysis of the supragingival plaque microbial communities showed that the Chao 1 index of the third trimester group was significantly lower than that of the non-pregnant group,first trimester group,and second trimester group(P<0.05).The Simpson index of the second trimester group and third trimester group was significantly higher than that of the non-pregnant group(P < 0.05),and the Shannon index of the second trimester group was also significantly higher than that of the non-pregnant group(P < 0.05).Beta diversity analysis showed that the non-pregnancy group clustered together,and the first trimester,second trimester,and third trimester groups clustered together.The abundance of Spirochaetes,Bacteroidetes,Firmicutes,and Proteobacteria was the highest,accounting for 91.56%,91.07%,92.18%,and 89.64% of the total OTUs in the non-pregnant group,first trimester group,second trimester group,and third trimester group,respectively.Leptotrichia was the most abundant genus in the pregnancy groups,followed by Treponema,Prevotella,and Streptococcus,accounting for50.82%,51.85%,and 53.22% of the first trimester group,second trimester group,and third trimester group,respectively.Streptococcus was the most abundant genus in the non-pregnant group,followed by Treponema,Leptotrichia,and Neisseria,accounting for a total of 45.00% of the non-pregnant group OTUs.LEf Se analysis showed that the microbial species significantly different from the other groups in the non-pregnant group were mainly concentrated in the genus Neisseria,while those in the early pregnancy group were mainly concentrated in the genus Tannerella,and those in the mid-pregnancy and late pregnancy groups were mainly concentrated in the genus Leptotrichia.Metabolic functional pathways of the gingival microbial community were predicted using PICRUSt2,and principal component analysis(PCA)showed that the metabolic functional pathways of the non-pregnant group and early pregnancy group clustered together,while those of the mid-pregnancy group and late pregnancy group clustered together.LEf Se analysis showed that 11 metabolic functional pathways played a differentiating role among these four groups.2.Analysis of Alpha diversity of salivary microbial community showed that the Shannon index of the term group was higher than that of the preterm group.Beta diversity analysis showed that the salivary microbial communities of the term group and the preterm group formed different clusters.In the saliva samples of the term group,the phyla Bacteroidetes,Firmicutes,Proteobacteria,Patescibacteria,and Spirochaetes accounted for 29.07%,27.18%,16.67%,14.06%,and 7.21% of the total abundance,respectively.In the saliva samples of the preterm group,the phyla Bacteroidetes,Firmicutes,Proteobacteria,Patescibacteria,and Spirochaetes accounted for 33.13%,19.75%,29.68%,2.84%,and 9.13% of the total abundance,respectively.In the saliva samples of the term group,the genera Prevotella,Streptococcus,Neisseria,Porphyromonas,and Treponema accounted for 17.01%,14.71%,10.73%,6.74%,and3.77% of the total abundance,respectively.In the saliva samples of the preterm group,the genera Prevotella,Streptococcus,Neisseria,Porphyromonas,and Treponema accounted for 12.99%,10.09%,18.45%,13.34%,and 7.31% of the total abundance,respectively.LEf Se analysis found that the species significantly different from the other groups in the full-term group were mainly concentrated in the phylum Patescibacteria,while those in the preterm group were mainly concentrated in the phylum Firmicutes and the class Gammaproteobacteria.Alpha diversity analysis of the placental microbial community showed that the Shannon index of the term group was lower than that of the preterm group.Beta diversity analysis showed that the placental microbial communities of the term group and the premature group formed different clusters.In the placenta samples of the term group,the phyla Proteobacteria,Bacteroidetes,Firmicutes,Actinobacteria,and Spirochaetes accounted for 97.98%,0.70%,0.31%,0.03%,and 0.004% of the total abundance,respectively.In the placenta samples of the preterm group,the phyla Proteobacteria,Bacteroidetes,Firmicutes,Actinobacteria,and Spirochaetes accounted for95.79%,1.06%,2.15%,0.09%,and 0.05% of the total abundance,respectively.In the placenta samples of the term group,the genera Acinetobacter,Agrobacterium,Lactobacillus,Corynebacterium,Actinomyces,Pseudomonas,Porphyromonas,and Fusobacterium accounted for 26.17%,10.42%,10.03%,8.41%,8.20%,6.18%,0.002%,and 0.002% of the total abundance,respectively.In the placenta samples of the preterm group,the genera Acinetobacter,Agrobacterium,Lactobacillus,Corynebacterium,Actinomyces,Pseudomonas,Porphyromonas,and Fusobacterium accounted for 28.77%,2.61%,13.50%,12.78%,4.03%,4.53%,0.03%,and 0.04% of the total abundance,respectively.LEf Se analysis found that there were no significant differences in species between the term group and the other groups,while in the preterm group,the species significantly different from the other groups were mainly concentrated in the phylum Firmicutes and the class Bacilli.3.In animal experiments,it was found that compared with the control group,the pregnancy days of the Porphyromonas gingivalis periapical periodontitis group and the Fusobacterium nucleatum periapical periodontitis group were shortened,and the expression of serum TNF-α and IL-1β increased,with statistical significance(P<0.05);compared with the physiological saline injection group,the pregnancy days of the Porphyromonas gingivalis injection group and the Fusobacterium nucleatum injection group were shortened,and the expression of serum TNF-α and IL-1βincreased,with statistical significance(P<0.05).Compared with the control group,the bacterial gene copy numbers in the carotid artery bifurcation and placenta increased significantly in both the Porphyromonas gingivalis periapical periodontitis group and the Fusobacterium nucleatum periapical periodontitis group mice,with statistical significance(P<0.05).When compared with the physiological saline injection group,the bacterial gene copy numbers in the carotid artery bifurcation and placenta increased significantly in both the Porphyromonas gingivalis injection group and the Fusobacterium nucleatum injection group mice,with statistical significance(P<0.05).In cell experiments,when bacteria were co-cultured with Human Umbilical Vein Endothelial Cells,it was found that the proliferation activity and migration ability of umbilical vein endothelial cells decreased,with statistical significance(P<0.05).Bacteria were able to adhere to and invade umbilical vein endothelial cells.4.In cell experiments,when bacteria were co-cultured with HTR-8/SVneo,this study found that the proliferation activity of placental trophoblast cells decreased,with statistical significance(P<0.05).Bacteria were able to adhere to and invade placental trophoblast cells.Compared with the control group,the early and late apoptosis percentages of placental trophoblast cells in the Porphyromonas gingivalis and Fusobacterium nucleatum intervention groups increased,with statistical significance(P<0.05);the levels of TNF-α and IL-1β cytokines in the supernatant increased,with statistical significance(P<0.05);the expression levels of TNF-α and IL-1βcytokine-related genes in the cells increased,with statistical significance(P<0.05);MyD88 and TNF-α protein expression levels increased in the Porphyromonas gingivalis and Fusobacterium nucleatum intervention groups,with statistical significance(P<0.05).In animal experiments,it was found that the protein expression levels of TLR4,MyD88,NF-k B,and p-NF-k B in the placental samples of the bacterial infection group all increased,with statistical significance(P<0.05).Conclusions: In summary,this study confirms that the composition of the oral microbial community changes during pregnancy,and these changes are closely related to health during pregnancy.Oral microbes may be a risk factor for preterm birth.The main pathogenic bacteria of chronic apical periodontitis are likely to enter the placenta through blood circulation and cause premature birth.They may also increase the amount of inflammatory factors in blood vessels to cause a systemic inflammatory state and then lead to premature birth.The main pathogenic bacteria of chronic apical periodontitis are likely to increase the secretion of inflammatory factors by placental trophoblast cells through the TLR4/MyD88/NF-κB signaling pathway,thereby advancing the inflammatory activation process during delivery and leading to premature birth.The oral microbiome has great potential to serve as a predictive tool and therapeutic target for identifying high-risk pregnancies,providing new ideas for improving maternal and fetal health outcomes. |
PDF Full Text Request