Study Subject Characteristics
In total 27 ICP patients and 31 control patients were enrolled in our study. Basic clinical information for subjects is summarized in Table 1 and Figure 1, in which the ages were equivalent between ICP and control patients. ICP patients were sampled at ~ 35.4 weeks while the control patients were sampled at ~39.4 weeks. Median values of bile, ALT, AST, Dbil, and Tbil were all significantly higher in ICP versus control patients (Figure 1, Table 1), Ibil median values for ICP patients were ~ two times higher than were the values in control patients (3.5 versus 1.7).
Table 1. Study subject characteristics.
|
ICP(n=27)
|
Control(n=31)
|
|
|
Median
|
IQR
|
Median
|
IQR
|
p-value
|
Age
|
28
|
7
|
29
|
6
|
0.5573
|
Sampling pregnancy weeks
|
35.4
|
3.5
|
39.4
|
2
|
1.97E-09
|
Bile (μmol/L)
|
26.6
|
57.1
|
8.1
|
3.3
|
2.56E-07
|
Total serum bilirubin (μmol/L)
|
15.3
|
12
|
6.5
|
2.9
|
7.18E-08
|
Indirect bilirubin (μmol/L)
|
3.5
|
4.3
|
1.7
|
5.2
|
0.08912
|
Direct bilirubin (μmol/L)
|
10.6
|
11
|
4.6
|
3.8
|
7.84E-08
|
Aspartate aminotransferase (U/L)
|
104
|
164
|
19
|
3
|
1.69E-06
|
Alanine aminotransferase (U/L)
|
168
|
285
|
17
|
9
|
1.28E-06
|
Characterizing Gut Microbiota in Women with ICP and controls
16S rRNA amplicon-based microbiome analysis was performed on stool samples from 27 ICP and 31 healthy control patients. 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 patient) were clustered into OTUs. There were no significant differences for the identified OTUs in comparisons between the two groups (median = 234 for ICP and 238 for control patients, Figure S1A). Alpha diversity analyses revealed that the number of OTUs in ICP patients was 9.8 % lower than were the resultant number of OTUs for control patients (p = 0.0165). Alpha rarefaction curves of numbers of observed OTUs for both patient groups showed a gradual leveling off by 60,000 sequences, while our sequences did not total greater than 400,000, suggesting there was adequate saturation at the selected rarefaction level (Figure S1B).
Collectively, the composition of gut microbiota of pregnant women was dominated by four major phyla including Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria, all of which occurred in more than 95 % of these samples. At the level of genus, 27 core genera, including Faecalibacterium, Streptococcus, Escherichia, existed in more than 95 % of the samples from pregnant women (Table S1).
The PCoA results based on overall bacterial community composition showed a marginally significant clustering in unweighted UniFrac distance matrices (Figure 2A, ANOSIM, p=0.0434), which only took the microbiota composition into account. In contrast, no significant clustering was observed in weighted UniFrac analysis (Figure 2B, ANOSIM, p=0.1578) in which more abundant OTUs were observed with an overall stronger effect.
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 for comparisons between ICP and control group patients (Figure S2, Table S2).
We next examined the correlation coefficient between OTUs and clinical parameters for both ICP and control patients. When we set up the estimates threshold above 0.4 or below -0.4, and p-value < = 0.001 (which means a moderate relationship), we observed that: 1) Lachnospiraceae, Weissella, Citrobacter, and Bacillus were positively associated with ALT; 2) Blautia_producta, and Clostridiales were positively associated with bile; 3) Dialister, Ruminococcus were negatively and Streptococcus_luteciae, Clostridiales were positively associated with Dbil; 4) Weissella, Bacillus, Streptococcus_luteciae were positively associated with AST; 5) Weissella, Streptococcus_luteciae, Clostridiales were positively and Oscillospira was negatively associated with Tbil (Table 2).
Table 2. Correlation coefficient between OTUs from all subjects and clinical parameters.
OTUs
|
Estimates
|
p-value
|
Occ
|
SampCount
|
Catalogue
|
Taxonomy
|
Otu1090
|
0.408477
|
0.001456
|
56
|
58
|
ALT
|
Lachnospiraceae
|
Otu319
|
0.43044
|
0.000745
|
18
|
58
|
ALT
|
Weissella
|
Otu411
|
0.434778
|
0.000649
|
36
|
58
|
ALT
|
Citrobacter
|
Otu711
|
0.406692
|
0.001535
|
16
|
58
|
ALT
|
Bacillus
|
Otu319
|
0.414393
|
0.001221
|
18
|
58
|
AST
|
Weissella
|
Otu711
|
0.441002
|
0.00053
|
16
|
58
|
AST
|
Bacillus
|
Otu737
|
0.44422
|
0.000477
|
25
|
58
|
AST
|
Streptococcus_luteciae
|
Otu147
|
0.419866
|
0.001034
|
58
|
58
|
bile
|
Blautia_producta
|
Otu973
|
0.426097
|
0.000853
|
29
|
58
|
bile
|
Clostridiales
|
Otu20
|
-0.41329
|
0.001262
|
27
|
58
|
Dbil
|
Dialister
|
Otu737
|
0.450102
|
0.000393
|
25
|
58
|
Dbil
|
Streptococcus_luteciae
|
Otu802
|
-0.41767
|
0.001106
|
6
|
58
|
Dbil
|
Ruminococcus
|
Otu973
|
0.464376
|
0.000241
|
29
|
58
|
Dbil
|
Clostridiales
|
Otu319
|
0.419613
|
0.001042
|
18
|
58
|
Tbil
|
Weissella
|
Otu411
|
0.405553
|
0.001587
|
36
|
58
|
Tbil
|
Citrobacter
|
Otu7
|
0.479112
|
0.000142
|
23
|
58
|
Tbil
|
Prevotella_stercorea
|
Otu718
|
-0.40842
|
0.001459
|
17
|
58
|
Tbil
|
Oscillospira
|
Otu737
|
0.412664
|
0.001286
|
25
|
58
|
Tbil
|
Streptococcus_luteciae
|
Otu973
|
0.430958
|
0.000732
|
29
|
58
|
Tbil
|
Clostridiales
|
At the genus level, the abundance of Butyricimonas was significantly lower in the group of ICP patients compared to patients making up the control group. However, Blautia, Citrobacter, Pseudomonas, Streptococcus and Weissella were significantly higher in ICP versus control patients. At the species level, Streptococcus lutecia was higher in ICP patients. At the class level, Bacilli and Gammaproteobacteria were higher in ICP patients. At the order level, Enterobacteriales, Lactobacillales, and Pseudomonadales were higher in ICP patients. At the family level, Enterobacteriaceae, Leuconostocaceae, Pseudomonadaceae, and Streptococcaceae were higher in ICP patients (Figure 3, Table 3). All these bacteria were considered to be rare (mean relative abundance < 5 %) in both the ICP control patient groups.
Table 3. Differential relative abundances of bacterial in six levels with FDR P-value <0.05.
Items
|
Mean(ICP)
|
SD(ICP)
|
Mean(Nor)
|
SD(Nor)
|
p.value
|
FDR
|
|
Bacilli
|
0.43725
|
0.590779
|
0.141213
|
0.182898
|
0.000676
|
0.015548
|
Class
|
Gammaproteobacteria
|
2.542838
|
5.428261
|
0.443897
|
0.691728
|
0.001971
|
0.022667
|
Enterobacteriales
|
2.186482
|
5.326455
|
0.386201
|
0.704164
|
0.002905
|
0.032923
|
Order
|
Lactobacillales
|
0.378908
|
0.577339
|
0.114305
|
0.178683
|
0.000462
|
0.008636
|
Pseudomonadales
|
0.003022
|
0.004634
|
0.000907
|
0.002216
|
0.000508
|
0.008636
|
Enterobacteriaceae
|
2.186482
|
5.326455
|
0.386201
|
0.704164
|
0.002905
|
0.04067
|
Family
|
Leuconostocaceae
|
0.009571
|
0.020579
|
0.00033
|
0.001309
|
0.000117
|
0.006011
|
Pseudomonadaceae
|
0.002195
|
0.003701
|
0.000363
|
0.000717
|
0.000322
|
0.006011
|
Streptococcaceae
|
0.340405
|
0.566579
|
0.077515
|
0.095561
|
0.000273
|
0.006011
|
Blautia
|
1.700667
|
1.161503
|
0.899852
|
0.689057
|
0.001317
|
0.026867
|
Genus
|
Butyricimonas
|
0.077094
|
0.112087
|
0.22485
|
0.30419
|
0.010086
|
0.098031
|
Citrobacter
|
0.041611
|
0.074845
|
0.002425
|
0.005323
|
0.00014
|
0.007293
|
Pseudomonas
|
0.002195
|
0.003701
|
0.000363
|
0.000717
|
0.000322
|
0.010948
|
Streptococcus
|
0.327829
|
0.566782
|
0.072758
|
0.094323
|
0.001317
|
0.026867
|
Weissella
|
0.009296
|
0.020585
|
0.000299
|
0.001305
|
0.000143
|
0.007293
|
Streptococcus luteciae
|
0.078866
|
0.155461
|
0.00461
|
0.017362
|
0.000431
|
0.029739
|
Species
|
In total, 51 metabolic pathways were identified by PICRUSt (Figure 4). There were no significant differences in examinations of the pathways for comparisons between ICP and control patients.
LEfSe identified eight significant (log10(LDA SCORE) > = 2 or log10(LDA SCORE) < = -2) OTUs (i.e., Streptococcus (genus), Lactobacillales (order), Streptococcaceae (family), Bacilli (class), Blautia (genus), Enterobacteriaceae (family), Enterobacteriales (order), and Gammaproteobacteria (class)) in ICP patients group and identified 12 significant OTUs (i.e., S24-7 (class), Betaproteobacteria (class), Burkholderiales (order), Sutterella (genus), Alcaligenaceae (family), Bacteroides ovatus (species), Odoribacteraceae (family), Odoribacter (genus), Butyricicoccus (genus), Bacteroides eggerthii (species), Bacteroides caccae (species), and Oscillospira (genus)) in controls (Figure 5).