In total, 27 ICP patients and 31 controls were enrolled in our study. Basic clinical information for subjects is summarized in Table 1 and Figure 1. Age brackets were similar and in very close range for both ICP and control groups. ICP patients and control subjects were sampled at ~ 35.0 and ~39.4 weeks respectively. Mean values of total bile acid, ALT, AST, Dbil, and Tbil were all significantly higher in ICP patients than in the control group (Figure 1, Table 1). Ibil mean values for ICP patients were ~relatively higher than the value in controls (4.7 ± 0.8 versus 3.2 ± 0.6).
Characterizing Gut Microbiota in Women with ICP and controls
16S rRNA amplicon-based microbiome analysis was performed on stool samples from 27 ICP patients and 31 healthy controls. After sequencing using the Miseq platform and preliminary data processing, clean reads were assembled into tags. Similar numbers of tags (on average 89265 ± 17127 tags for each ICP patient and 90964 ± 13947 tags for each control) were clustered into OTUs. A total of 875 OTUs were found from all samples. There was no significant difference in the identified OTUs between the two groups (median = 234 for ICP patients and 238 for controls, p = 0.3944, Figure S1A). Alpha diversity analyses revealed that the number of OTUs in ICP patients was 9.8 % lower than controls (p = 0.0165). Alpha rarefaction curves of numbers of observed OTUs for both patient groups showed a gradual leveling off by 60,000 sequences. The average number of reads for each sample was 287,000, which was much more than 60,000. (Figure S1B).
Collectively, the composition of gut microbiota of pregnant women was dominated by four major phyla: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria, all of which existed in more than 95 % of these samples. At the genus level, 27 core genera that included Faecalibacterium, Streptococcus and Escherichia existed in more than 95 % of the samples from pregnant women (Table S1).
To assess the structural similarities in the gut microbiota communities between the ICP patients and controls, a principle coordinates analysis (PCoA) was generated based on Bray-Curtis distance. The analysis is primarily based on the number of OTUs and not the specific taxonomic annotation. The PCoA results showed no separation of the ICP patients from the control group, indicating that the main composition of the gut microbiome of the ICP group was not significantly different from the control group (Figure 2, ANOSIM, p = 0.26).
The alpha-diversity between ICP patients and controls was also assessed. The five measures (Chao, ace, sobs, Shannon, simpson) were used to analyze the abundance and diversity of microbiota within samples. None of the five measures showed any significant differences between the two groups (Figure S2, Table S2).
The correlation coefficient between OTUs and clinical parameters was examined. The correlation between bacterial species (with prevalence ≥ 20%) and clinical parameters in ICP group was also assessed and no bacteria was identified or found. A subsequent slight change in conditions was made to aid observation of the correlated OTUs for clinical parameters in both ICP patients and controls. We observed the associations between specific microbiota and some liver parameters. Results showed that:
- Roseburia and Dorea were positively associated with ALT.
- SMB53, Roseburia, H. parainfluenzae, S. anginosus, L. rogosae and R. hominis showed some encouraging association with total bile acid
- O. coprococcus, H. parainfluenzae, B. pullicaecorum, R. mucilaginosa, R. lactaris, R. champanellensis were associated with Dbil.
- Associations between Paraprevotella, Dorea, Roseburia and B. product with AST, R. champanellensis and L. pacaense with Tbil and B. plebeius and L. pacaense with Ibil were observed as shown in Figure 3.
The Mann-Whitney U test (with an FDR correction) was then utilized in finding the differential relative abundances of bacteria at different taxonomy levels. As shown in Table 2, the abundance of Blautia, Citrobacter and Streptococcus at the genus level, was significantly higher in ICP patients than in controls. Similarly at the family level, Enterobacteriaceae, Leuconostocaceae and Streptococcaceae were higher in ICP patients. Bacilli and Gammaproteobacteria and Enterobacterials and Lactobacillales levels were higher at the class and order levels respectively. Streptococcus luteciae, at the species level was higher in ICP patients (Figure 4, Table 2). All these bacteria were considered to be rare (mean relative abundance < 5 %) in both the ICP patients and controls.
The functional profile of the gut microbiota from ICP patients and controls was also explored. The total OTUs were normalized by 16S rDNA copy number. The KEGG orthologs, Enzyme Classification and metagenomic functions were predicted from the KEGG pathways. A total of 638 KEGG Orthologs were identified significantly different between ICP patients and controls, all of which were less abundant in ICP patients (P<0.05; Table S3). A total of 138 pathways were identified significantly different between the two groups, whereas 136 pathways were significantly more abundant in controls (P<0.05; Table S4). Of these KEGG Orthologs, the greatest difference was observed among RNA polymerase primary sigma factor between the two groups (Figure 5 A). To the KEGG pathways, the greatest difference was observed among ketogluconate metabolism which was more represented in ICP patients than in controls (Figure 5 B).
The contribution of the taxa to the discrimination between ICP patients and controls was assessed by PLS-DA model and VIP score. PLS-DA score plot showed model discrimination between ICP patients and controls. A total of eight genera and seven species were recorded with VIP > 1.6. These were identified as key genera/species in ICP and control group. Of these, five were more abundant in ICP patients whereas the remaining 10 were more abundant in controls. Blautia genus (~2.6) and [C.] methylpentosum (~2.3) species recorded the highest VIP scores. (Figure 6).