In this study, we examined whether GDM influenced the offspring’s oral microbial colonization, and investigated the change of oral microbiota from birth to 1 month old in a well-controlled, carefully selected cohort of full-term infants without prenatal or postnatal antibiotic exposure. Although no significant differences were observed in alpha and beta diversity metrics between GDM and non-GDM groups, there were distinct differences in oral microbial community structures between D1 and D30 phases. We also found significant differences in the taxonomic composition of oral microbiota from phylum to genus levels between the two groups in both D1 and D30 phases. Metabolic pathways analysis showed that 5-aminoimidazole ribonucleotide biosynthesis and inosine-5'-phosphate biosynthesis were enriched in GDM subjects in the D30 phase. In addition, ecological network analysis revealed apparent differences between GDM and control groups, as well as in D1 and D30 phases, with the non-GDM group and D1 phase containing more high-degree nodes and microbial interactions compared with the GDM group and D30 phase, respectively.
It is well-established that maternal GDM has a long-term influence on the health of their children. However, the influences of GDM on the infant oral microbiota, which is closely related to oral and systemic health in offspring, are less documented. Recently, two studies from different countries have found a distinct oral microbial diversity in neonates born to mothers with GDM [10, 11]. In both studies, a significantly higher alpha-diversity, evaluated with Shannon’s index, in neonates born to mothers with GDM was observed. In the present study, Shannon’s index, Pielou’s index, and observed features value were applied to evaluate the diversity, evenness, and richness of the microbial community. There was no difference in Shannon’s diversity, Pielou’s evenness, or observed features value at any time points. As for beta-diversity visualized through PCoA, we also didn’t find a significant difference between the two groups, while He et al. [11] exhibited a significant separation between GDM and control groups. The inconsistency between our study and previous studies might partly be due to the different sequence processing methods. Mothur pipeline was used to handle and analyze the sequencing data in the study of He and colleagues [11], while in our study, QIIME2 software was applied, which is currently used more commonly. The different methodology may account for the inconsistent findings. More studies are needed in the future to confirm our findings.
To compare the overall oral microbiota composition, we further showed the abundance of taxa in the two groups and performed GLM analyses to identify taxonomic biomarkers that characterize the differences between offspring of mothers with and without GDM. Consistent with previous studies [10, 11], the dominant phyla of neonatal oral microbiota included Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes, accounting for approximately 90%, which was also similar to the composition of gut microbiota [23]. Moreover, significant differences were observed in the taxonomic composition of oral microbiota from phylum to genus level between the two groups, indicating that maternal GDM status would alter oral microbial composition from birth to infancy. Of note, we found that the GDM group exhibited less abundance of Veillonella (P < 0.001) in the D1 phase, and the difference persisted to the D30 phase (P < 0.05). Veillonella species within Firmicutes is one of the early colonizers of oral microbiota and is prevalent in oral microbiota [24]. Studies have shown that oral Veillonella was associated with oral diseases, such as dental caries and periodontal diseases [25,26,27]. In addition, Crusell et al. [28] reported that during pregnancy, gut Veillonella was depleted in women with GDM compared with the non-GDM group. Moreover, the gut Veillonella was also depleted in offspring born to mothers with GDM during both the neonatal period and infancy [8]. These studies, together with our findings, indicated that the genus Veillonella might be associated with GDM, and there might be some transmission paths from the maternal gut microbiota to neonatal oral and gut microflora. However, hitherto limited information is known about the mechanisms underlying the link between Veillonella and GDM.
Using metagenome function prediction, we found that 5-amnioimidazole ribonucleotide biosynthesis pathways were significantly enriched in GDM group in D30 phase. 5-aminoimidazole ribonucleotide (AIR) is a key intermediate of purine nucleotide and thiamine biosynthesis [29]. In addition, inosine-5'-phosphate biosynthesis pathway also showed the enrichment significance in saliva of infants born to GDM mothers. It's worth noting that the 5-aminoimidazole ribonucleotide biosynthesis pathway and inosine-5'-phosphate biosynthesis pathway make up the purine nucleotides de novo biosynthesis pathway, which plays an essential role in many cellular processes, including DNA replication, transcription, cellular signaling, and energy metabolism [30]. A recent study of neonatal gut microbiota also found that pathways related to carbohydrate and nucleotide metabolism were enriched in neonates born to GDM mothers [6]. Our study, together with previous studies [6, 11], indicated that maternal GDM might promote the succession of high-energy-providing microbiota with altered metabolism in their offspring. Consequently, maternal GDM could further mediate the development of macrosomia and childhood metabolic diseases such as obesity in later life.
GDM and non-GDM groups harbor distinct oral microbial ecological association networks, as well as the D1 and D30 phases. Ecological association networks showed more microbial correlations in the non-GDM group than the GDM group. We found that Gardnerella was an important genera in the non-GDM D1 group. It is known that Gardnerella was considered as one of the causes of bacterial vaginosis [31]. Previous studies also suggested that Gardnerella may change the host landscape in a way that makes other organisms more likely to colonize or cause disease [31]. However, the study of Lukic et al. [32] did not support a more prevalent vaginal infection by Gardnerella in diabetic women. In addition, some top genera observed in the GDM group, such as Morganella and Eikenella, were not found in the non-GDM group. The genus Desulfovibrio, which was previously found to be enriched in patients with GDM or type 2 diabetes [28, 33], also had more microbial interactions with other colonizers in the GDM group in our study. The difference in ecological networks between the GDM and non-GDM groups reflected maternal GDM might disrupt the ecology of oral microbiota in offspring. Meanwhile, the high-degree nodes and microbial interactions decreased dramatically in the D30 phase compared with the D1 phase, regardless of the GDM group or non-GDM group, indicating the oral microbiota has changed greatly over time. At birth, the neonatal oral microbiota was mainly derived from the maternal intrauterine environment, and influenced by vaginal or skin microbiota, while at 1 month of age, with the introduction of breast milk or formula, the infant oral microbiota changed dramatically [13, 34].
Strengths and limitations
One highlight of our study is the well-controlled, hypothesis-directed cohort study design. All subjects were full-term infants without prenatal or postnatal antibiotic exposure, and there were no significant differences in general characteristics between the GDM and non-GDM groups, eliminating the major confounders that typically interfered with early infant oral microbial colonization. Moreover, we not only investigated the influences of GDM on neonatal oral microbiota, but also followed up to 1 month old; therefore, for the first time, we explored the longitudinal changes in the oral microbiota composition from birth to infancy according to GDM status.
However, there were also certain limitations needed to be considered. Firstly, the sample size was limited. However, as mentioned above, the general characteristics were similar in the two groups, minimizing the influence of confounding factors on the results. Secondly, a causal relationship between maternal GDM and oral microbiota in offspring cannot be confirmed by the present study design. In addition, all participants in the current study were Han Chinese from one hospital. Given that the oral microbiota varies among different races, results of the present study cannot be directly transferred to other ethnicities. Further studies should be performed in other populations to validate and extend our findings.