Ecological Shifts of Supragingival Microbiota in Association with Pregnancy

Abstract

Pregnancy is a physiological process with pronounced hormonal fluctuations in females, and relatively little is known regarding how pregnancy influences the ecological shifts of supragingival microbiota. In this study, supragingival plaques and salivary hormones were collected from 11 pregnant women during pregnancy (P1, ≤14 weeks; P2, 20-25 weeks; P3, 33-37 weeks) and the postpartum period (P4, 6 weeks after childbirth). Seven non-pregnant volunteers were sampled at the same time intervals. The microbial genetic repertoire was obtained by 16S rDNA sequencing. Our results indicated that the Shannon diversity in P3 was significantly higher than in the non-pregnant group. The principal coordinates analysis showed distinct clustering according to gestational status, and the partial least squares discriminant analysis identified 33 genera that may contribute to this difference. There were differentially distributed genera, among which Neisseria, Porphyromonas, and Treponema were over-represented in the pregnant group, while Streptococcus and Veillonella were more abundant in the non-pregnant group. In addition, 53 operational taxonomic units were observed to have positive correlations with sex hormones in a redundancy analysis, with Prevotella spp. and Treponema spp. being most abundant. The ecological events suggest that pregnancy has a role in shaping an at-risk-for-harm microbiota and provide a basis for etiological studies of pregnancy-associated oral dysbiosis.

Keywords: 16S rDNA sequencing; bacterial diversity; female steroid hormones; oral microbiota; pregnancy; supragingival plaque.

Figure 1

Alpha and beta diversity estimates. (A) Chao 1 and (B) Shannon index for pregnant and non-pregnant groups. The data were calculated with the Dunn's multiple comparisons test and are expressed as the median with the interquartile range. (C) Principal coordinates analysis (PCoA) plot. Each point represents the mean principal component score of individuals in a group, and the error bar represents the standard deviation. (“^**” 0.001 < P < 0.01).

Figure 2

Bacterial phylotypes whose abundances were changed with pregnancy, as identified by LEfSe. (A) A cladogram for taxonomic representation is shown in the panel. Red indicates enrichment in samples from pregnant women, and green indicates the taxa enriched in the samples from non-pregnant women. (B) A histogram of the linear discriminant analysis (LDA) scores was calculated for the selected taxa.

Figure 3

Co-occurrence network of the genera that are highly abundant in supragingival microbiota. The pregnant group is shown in (A), and the non-pregnant group is shown in (B). The size of the node indicates the mean relative abundance of the corresponding genus. The same color represents the genera belonging to the same phyla. The thickness of a connecting line corresponds to the coefficient values (p > 0.6), and the color of the line, namely, red or green, indicates a positive or negative correlation, respectively.

Figure 4

OTUs associated with sex hormones identified by two-step redundancy analysis (RDA). (A) The first biplot of the RDA is constrained by pregnancy as its explanatory variables. Responding OTUs that have 1.82% of the constrained variance of the supragingival microbiota are indicated by blue arrows. (B) The second biplot of the RDA shows the relationship between pregnancy-associated OTUs identified in (A) and sex hormones. Responding OTUs that have 3.1% of the constrained variance are indicated by blue arrows. (C) The heat map of the 64 key OTUs identified in the two-step RDA. The phylogenetic tree of the OTUs on the left is constructed in ARB based on distribution similarity. The OTUs shown in red indicate that they have higher relative abundances in the pregnant group than in the non-pregnant group, and the green OTUs are more abundant in non-pregnant group than in the pregnant group. Individuals at the bottom are arranged according to different trimesters, and accordingly, the black bars above indicate the log-transformed sex hormones levels. The color intensity of the spot represents the relative ratio of the OTUs in each sample. The taxonomic names of the OTUs are listed on the right.