THYROID

Volume 28, Number 2, 2018

ª Mary Ann Liebert, Inc.
DOI: 10.1089/thy.2017.0395

Alterations of the Gut Microbiota in Hashimoto’s

Thyroiditis Patients

Fuya Zhao,* Jing Feng,* Jun Li,* Lei Zhao, Yang Liu, Huinan Chen, Ye Jin, Biqiang Zhu, and Yunwei Wei

Background: Hashimoto’s thyroiditis (HT) is an organ-specific autoimmune disease in which both genetic pre-
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disposition and environmental factors serve as disease triggers. Many studies have indicated that alterations in the
gut microbiota are important environmental factors in the development of inflammatory and autoimmune diseases.
A comparative analysis was systematically performed of the gut microbiota in HT patients and healthy controls.
Methods: First, a cross-sectional study of 28 HT patients and 16 matched healthy controls was conducted. Fecal
samples were collected, and microbiota were analyzed using 16S ribosomal RNA gene sequencing. Second, an
independent cohort of 22 HT patients and 11 healthy controls was used to evaluate the diagnostic potential of the
selected biomarkers.
Results: Similar levels of bacterial richness and diversity were found in the gut microbiota of HT patients and
healthy controls ( p = 0.11). A detailed fecal microbiota Mann–Whitney U-test (Q value <0.05) revealed that the
abundance levels of Blautia, Roseburia, Ruminococcus_torques_group, Romboutsia, Dorea, Fusicatenibacter, and
Eubacterium_hallii_group genera were increased in HT patients, whereas the abundance levels of Fecalibacterium,
Bacteroides, Prevotella_9, and Lachnoclostridium genera were decreased. A correlation matrix based on the
Spearman correlation distance confirmed correlations among seven clinical parameters. Additionally, the linear
discriminant analysis effect size method showed significant differences in 27 genera between the two groups that
were strongly correlated with clinical parameters. The linear discriminant analysis value was used to select the first
10 species from the 27 different genera as biomarkers, achieving area under the curve values of 0.91 and 0.88 for
exploration and validation data, respectively.
Conclusions: Characterization of the gut microbiota in HT patients confirmed that HT patients have altered gut
microbiota and that gut microbiota are correlated with clinical parameters, suggesting that microbiome composition
data could be used for disease diagnosis. Further investigation is required to understand better the role of the gut
microbiota in the pathogenesis of HT.

Keywords: Hashimoto’s thyroiditis, gut microbiota, dysbiosis, clinical parameters, biomarker

Introduction Although the exact etiology remains unknown, HT is thought

to arise from interactions between genetic susceptibility

H ashimoto’s thyroiditis (HT), also known as chronic

lymphocytic thyroiditis, is a common organ-specific
autoimmune disorder characterized by the production of
autoantibodies against thyroid peroxidase (TPO) and thyro-
globulin (Tg) and infiltration of the thyroid gland by
inflammatory cells, which are often followed by hypothy-
roidism due to the destruction of thyroid follicles and even-
tual fibrous replacement of parenchymal tissue (1). The
clinical diagnosis of HT depends on both physical (typically
ultrasonography) and biochemical abnormalities and sero-
logical assessments of autoantibodies (TPOAb or TgAb) (2).
factors, epigenetic effects, and various environmental factors
(3). Studies of twins have provided epidemiological evidence
for a genetic susceptibility to HT. The concordance rate was
55% for monozygotic twins with HT, but it was 0% for di-
zygotic twins with HT (4). However, even in identical twins,
the concordance rate was only approximately 50%, empha-
sizing that other important factors, such as the environment,
including excessive iodine intake, selenium deficiency, He-
licobacter pylori infection, viral infection (hepatitis C virus,
human parvovirus B19, and enteroviruses), vitamin D defi-
ciency, hygiene, and new treatment modalities and chemical

Department of Oncological and Endoscopic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, People’s
Republic of China.
*These authors contributed equally to this work.

175
 176
agents, may have important roles in the pathogenesis of HT.
Additional unmodifiable predisposing factors include stress,
climate, age, and sex (5–7). The potential role of gut mi-
crobiota as an environmental factor that could alter the health
status of a host has recently drawn considerable attention.
Emerging evidence suggests a connection between the gut
microbiota and various autoimmune diseases, including
systemic lupus erythematosus (8), type 1 diabetes (T1D) (9),
rheumatoid arthritis (10), inflammatory bowel disease (IBD)
(11), psoriasis (12), multiple sclerosis (MS) (13), celiac
disease (14), Bechet’s disease (15), Graves’ disease, and
Graves’ orbitopathy (16–18).
However, few studies have addressed the link between gut
microbiota and HT, and these studies have only provided
indirect or weak evidence. First, a previous study indicated
that many interrelationships exist between the thyroid gland
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and the gastrointestinal tract. Similarities in embryology,
phylogeny, and function persist throughout adult life, even
after fragmentation of the primitive thyroglossal duct (19).
Therefore, it was hypothesized that thyroid disorders may be
associated with alterations of the gut microbiota. Second,
molecular mimicry mechanisms provide the most reasonable
explanation of the role of the gut microbiota in provoking
autoimmune disease, that is, the emergence of autoreactive
clones of T and B lymphocytes as a result of a cross-immune
response to homologous bacterial or viral antigens (20,21). It
was recently found that components of the cells of Bifido-
bacterium bifidum 791, Bifidobacterium adolescentis 94 BIM,
Bifidobacterium longum B379M, and Lactobacillus plantarum
B-01 selectively bind human antibodies (TPOAb and TgAb)
and compete with natural antigens (22). Third, some authors
have proposed that disruption of the mucosal barrier exposes
submucosal immune cells to bacterial and dietary antigens and
to self-antigens, leading to unfavorable immune activation or
failure of the tolerance reaction, with the consequent devel-
opment of autoimmune diseases (23,24). In accordance with
this hypothesis, morphological changes in gut epithelial cells,
increased intestinal permeability, and intraepithelial lympho-
cyte infiltration have been demonstrated in T1D patients and in
animal models (25,26). Interestingly, similar changes have
been detected in patients with HT (27), suggesting a patho-
genic role of a leaky gut barrier in the development of HT.
Finally, in nonobese diabetic mice with thyroiditis, polariza-
tion toward a prevalent Th1 or Th17 pathway has been re-
ported (28,29). Activation of Toll-like receptors in the
development of thyroiditis (30) and a protective inhibitory
effect of Treg cells (31) have also been reported in this model.
However, whether a similar mechanism (i.e., CD4+ Th path-
way imbalances) initiates autoimmune thyroiditis in humans
requires further investigation (32).
Therefore, it was hypothesized that HT patients have gut
microbial dysbiosis that influences HT development. To test
this hypothesis, the gut microbiota composition was analyzed
in euthyroid HT patients. The results showed that HT patients
have a distinct microbiota community profile from that of
healthy controls.
Materials and Methods
Ethics statement
The protocols used in this study were approved by the
Ethics Committee of the First Affiliated Hospital of Harbin
ZHAO ET AL.
Medical University (China). All subjects were informed of
the nature of the study and were asked to provide written
informed consent. The procedures were conducted in accor-
dance with approved guidelines.
Study cohort and recruitment of subjects
There were two study groups. First, a cross-sectional study
(exploration cohort) was conducted of 28 HT patients and 16
matched healthy controls. Second, an independent cohort
(validation cohort) composed of 22 HT patients and 11
matched healthy controls was used to evaluate the diagnostic
potential of selected biomarkers. Seventy-seven participants
were enrolled in the study from November 2016 to April
2017, including 50 HT patients recruited from the outpatient
departments of endocrinology at the First Affiliated Hospital
of Harbin Medical University and 27 healthy controls re-
cruited from the health screening center. The healthy vol-
unteers were matched with the HT groups in terms of age,
sex, and body mass index (BMI). All HT patients were di-
agnosed and underwent disease verification by an endocri-
nologist based on their histories, physical examination,
ultrasound, and biochemistry (2). The inclusion criteria for
HT patients were as follows: aged 18–65 years and the
presence of euthyroidism (normal free triiodothyronine
[fT3], free thyroxine [fT4], and thyrotropin [TSH] plasma
levels without hormonal therapy). The following exclusion
criteria were applied to all groups: pregnancy; lactation;
cigarette smoking; alcohol addiction; hypertension; diabetes
mellitus; lipid dysregulation; BMI >27 kg/m2; recent (<3
months prior) use of antibiotics, probiotics, prebiotics, sym-
biotics, hormonal medication, laxatives, proton pump inhib-
itors, insulin sensitizers, or Chinese herbal medicine; a
known history of disease with an autoimmune component,
such as MS, rheumatoid arthritis, irritable bowel syndrome
(IBS), or IBD; and a history of malignancy or any gastroin-
testinal tract surgery (e.g., gastrectomy, bariatric surgery,
colectomy, ileectomy, cholecystectomy, or appendectomy).
Sample collection
All subjects were examined in the morning after an over-
night fast (‡8 h). Peripheral blood (6 mL) was collected from
all subjects and stored in EDTA tubes at 4C for routine
blood, thyroid function, and thyroid autoantibody examina-
tions. In addition, all subjects were provided with Commode
Specimen Collection Kits for stool collection. Fecal samples
were collected by the patients using disposable sterile forceps
in the morning, when the patients had an empty stomach.
Basic information, such as acquisition time and patient name,
was recorded on the sample collection box. If the subjects
were unable to provide a stool sample during their stay at the
hospital outpatient clinic, they were instructed to collect the
sample at home in accordance with the above methods and to
send it to the laboratory in an ice pack within two hours. Upon
collection, each fecal sample was immediately divided into
aliquots, frozen on dry ice, and stored at -80C until DNA
extraction.
Thyroid function and thyroid autoantibodies tests
Serum TSH, fT4, and fT3 levels were measured by
chemiluminescent immunoassays (Abbott Diagnostics,
 HASHIMOTO’S THYROIDITIS AND GUT MICROBIOTA DYSBIOSIS
Tokyo, Japan). Reference ranges were defined as follows:
TSH 0.35–4.94 lIU/L; fT4 0.70–1.48 ng/dL; and fT3 1.71–
3.71 pg/mL. TgAb and TPOAb were measured using
chemiluminescent immunoassays (Beckman Coulter, Full-
erton, CA). Reference ranges were defined as follows:
TPOAb 0.00–5.61 IU/mL; TgAb 0.00–4.11 IU/mL.
gDNA extraction
DNA extraction was performed within one month after
sample collection. Bacterial DNA was extracted from the
fecal samples at Novogene Bioinformatics Technology Co.
Ltd. (Beijing, China) using a TIANGEN kit according to the
manufacturer’s recommendations. DNA concentration and
purity were monitored on 1% agarose gels. After determining
the concentration, DNA was diluted to 1 ng/lL using sterile
water. The extracted DNA was stored at -20C.
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Amplicon generation and purification
The bacterial genomic DNA was amplified with primers
341F (CCTAYGGGRBGCASCAG) and 806R (GGACTA
CNNGGGTATC-TAAT) specific to the V3–V4 hypervari-
able regions of the 16S rRNA gene (33). All polymerase
chain reactions (PCR) were carried out with Phusion High-
Fidelity PCR Master Mix (New England Biolabs, Ipswich,
MA). An equal volume of 1 · loading buffer (containing SYB
green) was mixed with the PCR products and subjected to
electrophoresis on 2% agarose gels for detection. Samples
with a bright, primary band between 400 and 450 bp were
chosen for further experiments. Products of the same sample
were combined and subjected to electrophoresis. DNA of the
correct size was purified using a Gel Extraction Kit (Qiagen,
Hilden, Germany) and quantified using a Qubit instrument
(Life Technologies, Carlsbad, CA).
Library preparation and sequencing
Sequencing libraries were generated using a TruSeq
DNA PCR-Free Sample Preparation Kit (Illumina, San
Diego, CA) following the manufacturer’s recommendations,
and index codes were added. Library quality was assessed
using a Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific,
Waltham, MA) and an Bioanalyzer 2100 system (Agilent,
Santa Clara, CA). Finally, the library was sequenced on an
Illumina HiSeq2500 platform, and 250-bp paired-end reads
were generated.
Paired-end reads assembly and quality control
Paired-end reads were assigned to samples based on their
unique barcode and were truncated by cleaving the barcode and
primer sequence. Paired-end reads were merged using FLASH
(v1.2.7), a rapid and highly accurate analysis tool designed to
merge paired-end reads when at least some of the reads overlap
the reads generated from the opposite end of the same DNA
fragment; the splicing sequences were termed raw tags.
Quality filtering of the raw tags was performed under
specific filtering conditions to obtain high-quality clean tags
according to the QIIME (v1.7.0) quality control process. The
tags were compared with a reference database (the Gold
database) using the UCHIME algorithm to detect chimaera
sequences, and the chimaera sequences were then removed.
The effective tags were finally obtained.
177
Operational taxonomic unit cluster and species
annotation
Sequence analysis was performed using Uparse software
(Uparse v7.0.1001). Sequences with ‡97% similarity were
assigned to the same operational taxonomic units (OTUs).
Representative sequences for each OTU were screened for
further annotation. For each representative sequence, the
SILVA128/16S database was used based on the RDP clas-
sifier (v2.2) algorithm to annotate taxonomic information. To
study the phylogenetic relationships between different OTUs
and differences in dominant species in different samples
(groups), multiple sequence alignment was conducted using
MUSCLE software (v3.8.31). OTU abundance information
was normalized using the sequence number corresponding to
the sample with the fewest sequences.
Statistical analyses
The analysis of clinical parameters, Firmicutes/Bacter-
oidetes (F/B) ratio, and Pearson correlation distance results
was performed using IBM SPSS Statistics for Windows
v19.0 (IBM Corp., Armonk, NY). Alpha diversity was ap-
plied to analyze the complexity of species diversity in each
sample based on six indexes: Observed-species, Chao1,
Shannon, Simpson, ACE, and Good’s coverage. These in-
dexes were calculated for the samples using QIIME (v1.7.0)
based on the rarefied OTU counts and were displayed using R
software (v2.15.3). A beta diversity analysis was used to
evaluate differences in the species complexity between
samples, and beta diversity-weighted UniFrac was calculated
using QIIME software (v1.7.0) based on the rarefied OTU
counts. A principal coordinate analysis (PCoA) was per-
formed to obtain the principal coordinates and to visualize
complex, multidimensional data. The results of the PCoA
were displayed using the WGCNA package, stats package,
and ggplot2 package in R software (v2.15.3). Differences
between the two groups were tested based on a distance
matrix using the nonparametric multivariate analysis test
(analysis of similarities [ANOSIM], used to examine whether
differences between groups were significantly greater than
differences within groups) included in R’s Vegan package.
The microbiota features differentiating the fecal micro-
biota were characterized using the linear discriminant anal-
ysis (LDA) effect size (LEfSe) method for biomarker
discovery, which emphasizes both statistical significance and
biological relevance (34). Based on a normalized relative
abundance matrix, LEfSe uses the Kruskal–Wallis rank-sum
test to detect features with significantly different abundance
levels between assigned taxa and performs an LDA to esti-
mate the effect size of each feature. An alpha significance
level of 0.05 and an effect size threshold of 3 were used for all
biomarkers discussed in this study. A differential abundance
analysis was performed using the Wilcoxon rank-sum test at
the phylum, family, and genus levels. Microbiome features of
healthy controls were compared to those of patients with HT
using Metastats based on the p-value and the false discovery
rate (Q-value) for non-normal distributions. Only taxa with
average abundance levels >1%, p-values <0.05, and Q-values
<0.05 were considered significant (35). Correlations between
variables were computed using the Spearman rank correla-
tion. Statistical analyses were performed using the SPSS
Data Analysis Program (v16.0; SPSS, Inc., Chicago, IL). To
 178 ZHAO ET AL.

Table 1. Clinical and Demographic Features of HT Patients and Healthy Controls

Exploration cohort Validation cohort
HT group Healthy group HT group Healthy group
Parameters (n = 28) (n = 16) p-Value (n = 22) (n = 11) p-Value
Sex (M/F) 3/25 2/14 1.000 3/19 2/9 1.000
Age (years), M – SD 44.29 – 12.25 44.63 – 10.33 0.100 45.82 – 10.70 43.73 – 10.95 0.567
BMI (kg/m2), M – SD 22.14 – 2.51 22.50 – 1.18 0.063 22.25 – 2.38 22.87 – 1.01 0.087
fT3 (pg/mL), M – SD 2.78 – 0.39 2.78 – 0.46 0.256 2.81 – 0.38 2.80 – 0.40 0.618
fT4 (ng/mL), M – SD 1.06 – 0.14 1.08 – 0.15 0.928 1.10 – 0.11 1.04 – 0.12 0.472
TSH (lIU/mL), M – SD 2.59 – 0.97 2.63 – 0.79 0.521 2.87 – 0.80 2.54 – 0.83 0.706
TPOAb (IU/mL), M – SD 460.23 – 341.38 0.81 – 0.36 0.001 530.69 – 339.20 0.83 – 0.32 0.001
TPOAb >5.61 IU/mL (P/N) 28/0 0/16 0.000 22/0 0/11 0.000
TgAb (IU/mL), M – SD 353.47 – 345.29 1.13 – 0.97 0.001 416.41 – 361.17 0.82 – 0.63 0.001
TgAb >4.11 IU/mL (P/N) 27/1 0/16 0.000 20/2 0/11 0.000
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HT, Hashimoto’s thyroiditis; M/F, male/female; TgAb, thyroglobulin antibody; TPOAb, thyroperoxidase antibody; fT3, free
triiodothyronine; fT4, free thyroxine; TSH, thyrotropin; BMI, body mass index; P/N, positive/negative ratio.

evaluate the discriminatory ability of the prediction model,
operating characteristic curves (receiving operational curve
[ROC]) were constructed, and area under curve (AUC) values
were calculated.
Results
Study population
All subjects (n = 77) were of Han nationality and were born
in northeastern China. The fecal samples of 50 HT patients and
27 healthy controls were sequenced and analyzed. Because of
traditional customs and the cold climate, typical meals include
foods that are rich in saturated fat and salt, such as pork fat,
blood sausage, and mutton. The demographics and clinical
parameters of the subjects are summarized in Table 1.
The gut microbiota of HT patients differs from that
of healthy controls
To identify whether HT was associated with a change in
microbiota diversity, the fecal samples of HT patients and
healthy controls were subjected to pyrosequencing and sta-
tistical analysis. A summary of the microbiota diversity is
shown in Table 2, and the detailed characteristics of each
sample are shown in Supplementary Table S1 (Supplementary
Data are available online at www.liebertpub.com/thy). A total
of 7,182,787 usable sequences were obtained from all samples
using the Illumina HiSeq2500 platform. Of these, 3,149,257
high-quality sequences were selected, yielding an average of
71,574 sequences per sample. A total of 784 OTUs were de-
lineated at a 97% similarity level. Good’s coverage for the two
groups was >99.5%, indicating a satisfactory sequencing depth
of the gut microbiota. Based on the OTU analysis results, the
rank-abundance curves for the bacterial communities of the
HT patients and the healthy controls exhibited similar patterns
(Fig. 1A). In the analysis of alpha diversity, the Chao1 index
considers only species richness. However, the Shannon di-
versity index considers both species richness and evenness.
The Wilcoxon rank-sum test results demonstrated that the gut
microbiota richness and diversity of the HT patients were
higher than those of the healthy controls, although the differ-
ences were not significant ( p > 0.05). Other estimators of
community diversity are shown in Table 2. Rarefaction curve
analysis estimates showed that the gut microbiota in HT pa-
tients exhibited species richness (observed OTU number)
similar to that of the healthy controls (Fig. 1B). There was also
a trend toward higher overall diversity (Shannon index) in HT
patients compared to healthy controls (Supplementary Fig.
S1). Additionally, a Venn diagram showing the overlapping
OTU data of the two groups was constructed to evaluate their
shared richness better. This analysis revealed that 660/783
OTUs accounting for the total richness were common to all
samples. Therefore, approximately 81 and 42 of the OTUs
were identified in the samples of HT patients and healthy
controls, respectively (Supplementary Fig. S2).
A taxon-dependent analysis using the Ribosomal Database
Project (RDP) classifier was conducted to describe the
composition of the fecal microbiota in HT patients and

Table 2. Comparison of Phylotype Coverage and Diversity Estimation of the 16S rRNA
Gene Libraries at 97% Similarity Based on the Pyrosequencing Analysis
Richness estimator Diversity index
No. of No. of Good’s ACE Chao1 Shannon Simpson
Group Samples reads OTUsa (%)b mean CI mean CI mean mean
HT 28 2,003,036 746 99.89% 434.98 [425.0769–464.4704] 437.04 [426.9908–464.3406] 3.9331 0.048749
Control 16 1,146,221 703 99.89% 413.04 [398.1339–443.9005] 412.88 [400.2511–439.3514] 3.8135 0.060105
a
The operational taxonomic units (OTUs) were defined at a 97% similarity level.
b
The coverage percentage (Good’s) was calculated using Good’s method, and richness estimators (ACE and Chao1) and diversity indices
(Shannon and Simpson) were calculated using the Mothur program.
CI, confidence interval.
 HASHIMOTO’S THYROIDITIS AND GUT MICROBIOTA DYSBIOSIS 179
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FIG. 1. Gut microbiota of Hashimoto’s thyroiditis (HT) patients differs from that of healthy controls. Rank-abundance
curves were used to explain species richness and evenness. The bacterial communities of the healthy controls and HT
patients exhibited similar patterns (A). Rarefaction curves were used to compare the species richness between HT
patients and healthy controls, and the microbiota richness of the HT patients was higher (B). The relative abundance of
fecal bacterial phyla and genera were clustered into two groups, and the microbiota compositions significantly differed.
In this analysis, only phyla and genera with relative abundances of >1% were included. All OTUs with lower abundances
were grouped as ‘‘other’’ (C and D). A principal coordinate analysis (PCoA) plot based on the Bray–Curtis distance
matrix. The first two coordinates are plotted with the percentage of variability explained indicated on the axis. Each point
represents a sample, and the colors represent different groups (E). Analysis of similarities (ANOSIM) indicated that
the difference between the HT group and the healthy control group was significant (R = 0.7154, p < 0.001) at the phylum
level (F). Color images available online at www.liebertpub.com/thy
 180 ZHAO ET AL.

healthy controls. Compared to healthy controls, the propor-
tions of Firmicutes and Actinobacteria were increased, and
the proportions of Bacteroidetes and Proteobacteria were
decreased in patients with HT (Fig. 1C). At the family and
genus levels, the microbiota composition of the HT patients
was also altered compared to that of the healthy controls
(Fig. 1D and Supplementary Fig. S3). To evaluate the extent
of similarity between the microbiota communities, beta-
diversity values were calculated using the weighted UniFrac
method, and a PCoA was performed. Despite significant inter-
individual variation, the gut microbiota of the HT patients and
the healthy controls were clearly separated using PCoA
(Fig. 1E). The HT patient samples also showed greater het-
erogeneity, with subsets resembling the healthy control sam-
ples. The analysis of group similarities (ANOSIM) indicated
that the differences between the HT patients and the healthy
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aceae were prevalent in the HT patient samples, whereas
members of Bacteroidaceae, Prevotellaceae, Streptococcaceae,
and Peptostreptococcaceae were enriched in the healthy con-
trol samples (Q-value <0.05; Fig. 2B). At the genus level, 11
genera differed dramatically between the HT patient samples
and the healthy control samples. The proportions of the Bac-
teroides, Fecalibacterium, Prevotella_9, and Lachnoclos-
tridium genera were decreased, whereas the proportions of the
Blautia, Ruminococcus_torques_group, Roseburia, Fusicate-
nibacter, Romboutsia, Dorea, and Eubacterium_hallii_group
genera were increased in the HT patient samples (Q-value
<0.05; Fig. 2B).
These data indicate that the HT patients had different
abundance levels of certain bacteria in the gut microbiota than
the healthy controls. Although bacterial diversity was not al-
tered dramatically, the aberrant compositions of the fecal mi-

controls were significant at the phylum and genus levels, with
R values of 0.7154 and 0.6653, respectively ( p < 0.001; Fig. 1F
and Supplementary Fig. S4). These results demonstrate that
the experimental grouping design was reliable.
The above results indicate that similar levels of bacterial
richness and diversity were found in the gut microbiota of HT
patients compared to those of healthy controls, whereas the
overall structures of the gut microbiota of the HT and control
groups were significantly different.
The abundance levels of certain bacteria are
associated with HT
To identify the specific bacterial taxa associated with HT,
the compositions of the fecal microbiota of HT patients and
healthy controls were compared using the LEfSe method. A
cladogram representing the structures of the fecal microbiota
and the predominant bacteria in the healthy controls and HT
patients, and the largest differences in the taxa between the two
communities were compared. In total, the LEfSe analysis re-
vealed 40 discriminative features (LDA >3, p < 0.05; Fig. 2A)
at the phylum (n = 3), family (n = 10), and genus (n = 27) levels.
Members of the Bacteroidetes bacterial taxa were enriched in
the healthy control samples, whereas members of the Firmi-
cutes and Synergistetes were enriched in the HT patient sam-
ples. Therefore, these taxa may be used as biomarkers to
discriminate HT patients. Changes in the composition of the
gut microbiota in the HT patient samples were also explored
using the Mann–Whitney U-test (a nonparametric test for two
independent groups) at different taxon levels, confining the
analyses to the taxa with average abundance levels >1%,
p-values <0.05, and false discovery rates (Q-values) <0.05.
Eighteen differentially abundant taxa were identified (Table 3).
At the phylum level, the proportion of Firmicutes was higher in
the HT patient samples than that in the healthy control samples,
whereas the proportion of Bacteroidetes was lower (Q-value
<0.05; Fig. 2B). At the family level, members of Lachnospir-
crobiota indicated gut dysbiosis in the patients with HT. The
F/B ratio was significantly higher in the HT patient samples
(9.534 – 3.207; p < 0.05; Fig. 2C) than that in the healthy
control samples (3.175 – 0.8209). A correlation matrix based
on Pearson correlation distance confirmed correlations be-
tween the F/B ratio and age and BMI, but no significant dif-
ferences ( p > 0.05) were detected in the correlations between
the healthy group or the HT group (Fig. 2D–G).
Clinical parameters correlated with the gut microbiota
The relationships between gut microbiota and host clinical
parameters were explored. A correlation matrix based on
Spearman correlation distance confirmed correlations among
seven clinical parameters, and 27 genera showed significant
differences between the two groups according to the LEfSe
method (Fig. 2A). The results revealed significant correlations
between different genera (not including Subdoligranulum,
Alloprevotella, and Lachnoclostridium) and HT-related clini-
cal diagnostic parameters, including TPOAb and TgAb
( p < 0.05). Eighteen genera were enriched in the HT patient
group and were positively correlated with TPOAb or TgAb,
while six genera were enriched in the healthy control group
and exhibited opposite correlations (Fig. 3A). Additionally,
Alloprevotella was positively correlated with fT4, while Fu-
sicatenibacter exhibited the opposite correlation. Romboutsia
was negatively correlated with TSH. However, other envi-
ronmental factors were not significantly correlated with the gut
microbiota, including fT3, age, and BMI ( p > 0.05).
Predictive model
The LEfSe analysis results revealed significant differences
(LDA >3; p < 0.05) between the two groups for 27 genera,
and the Spearman correlation test confirmed that 24 genera
were significantly correlated with TPOAb or TgAb. There-
fore, these differences in microbiota could serve as potential

‰
FIG. 2. Taxonomic cladogram obtained using linear discriminant analysis (LDA) effect size (LEfSe) analysis and Mann–
Whitney U-tests of the 16S sequences. LEfSe identified the taxa with the greatest differences in abundance between the HT patients
and the healthy controls. At the phylum, family, and genus level, the HT patient-enriched taxa are indicated by a positive LDA score
(blue), and healthy control-enriched taxa are indicated by a negative score (red). Only taxa meeting a significant LDA threshold
value of >3 are shown (A). Comparisons of the relative abundance at the bacterial phylum, family, and genus levels in HT patients
and healthy controls. *Adjusted Q-value <0.05; **adjusted Q-value <0.01; ***adjusted Q-value <0.001 (B). The Firmicutes/
Bacteroidetes (F/B) ratio in HT patients and healthy controls (C). #Adjusted p-value <0.05. Correlations of F/B ratio and age/body
mass index (BMI) in HT patients and healthy controls (D, E, F, and G). Color images available online at www.liebertpub.com/thy
 Downloaded by American Thyroid Association (ATA) from www.liebertpub.com at 03/14/18. For personal use only.

181
 182 ZHAO ET AL.

Table 3. Differential Taxa Abundances Between HT Patient and Healthy Control

Samples at Phylum, Family, and Genus Levels
Control Log2 fold HT Control
Species p-Value Q-value HT mean mean change prevalence prevalence
Phylum
Firmicutes 1.85E-06 1.38E-05 8.26E-01 6.91E-01 0.26 16 28
Bacteroidetes 4.70E-07 7.05E-06 9.85E-02 2.27E-01 –1.20 16 28
Family
Firmicutes; Lachnospiraceae 1.28E-06 5.82E-05 4.90E-01 3.30E-01 0.57 16 28
Bacteroidetes; Bacteroidaceae 1.67E-07 1.52E-05 6.13E-02 1.33E-01 –1.11 16 28
Bacteroidetes; Prevotellaceae 2.57E-05 5.85E-04 2.82E-02 7.77E-02 –1.46 16 28
Firmicutes; Peptostreptococcaceae 2.47E-03 3.54E-02 3.37E-02 1.22E-02 1.47 16 28
Firmicutes; Streptococcaceae 3.28E-03 4.39E-02 2.60E-02 1.50E-02 0.79 16 28
Genus
Firmicutes; Faecalibacterium 3.70E-03 2.45E-02 9.87E-02 1.51E-01 –0.61 16 28
Bacteroidetes; Bacteroides 1.67E-07 3.02E-05 6.13E-02 1.33E-01 –1.11 16 28
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Bacteroidetes; Prevotella_9 7.35E-05 2.05E-03 1.83E-02 6.01E-02 –1.72 16 28

Firmicutes; Blautia 1.19E-05 5.54E-04 9.77E-02 5.86E-02 0.74 16 27
Firmicutes; Roseburia 1.03E-02 4.25E-02 3.98E-02 3.12E-02 0.35 16 28
Firmicutes; Lachnoclostridium 1.28E-02 4.39E-02 2.41E-02 2.85E-02 –0.24 16 28
Firmicutes; Ruminococcus_torque_group 1.72E-03 1.50E-02 3.06E-02 2.00E-02 0.61 16 28
Firmicutes; Romboutsia 5.62E-03 4.12E-02 2.35E-02 1.46E-02 0.68 16 28
Firmicutes; Dorea 6.05E-03 5.84E-02 2.00E-02 1.38E-02 0.53 16 28
Firmicutes; Fusicatenibacter 1.63E-06 1.14E-04 1.86E-02 1.00E-02 0.89 16 28
Firmicutes; Eubacterium_hallii_group 2.16E-07 3.02E-05 2.58E-02 1.10E-02 1.24 16 28

novel biomarkers for noninvasive monitoring and diagnosis
of diseases. The performance of the model was assessed us-
ing ROC analysis. Finally, the first 10 species based on the
LDA value were selected from the 27 different genera as
biomarkers, achieving an AUC value of 0.91 (Fig. 3C). These
species included members of Bacteroides (LDA = 4.55),
Fecalibacterium (LDA = 4.34), Prevotella_9 (LDA = 4.31),
Blautia (LDA = 4.31), Eubacterium_hallii_group (LDA =
3.90), Ruminococcus_torques_group (LDA = 3.74), Strep-
tococcus (LDA = 3.67), Alloprevotella (LDA = 3.67), Rose-
buria (LDA = 3.63), and Fusicatenibacter (LDA = 3.62).
Identification of the most prevalent genera in the earlier
abundance analysis (Fig. 2B) indicated the robustness of both
analyses. A heat map was generated based on the abundance
levels of the first 10 species from the 27 different genera.
Hierarchical clustering (Euclidean distance, complete link-
age) shows that the two groups can be clearly separated
(Fig. 3B), and HT patient samples generally clustered to-
gether (Supplementary Fig. S5). Subsequently, the discrim-
inatory power of the model was evaluated using a validation
cohort of 22 HT patients and 11 healthy controls, and an AUC
value of 0.88 was achieved (Fig. 3D), confirming that the gut
microbiota–based classifier can distinguish HT patients from
controls.
Discussion
To date, no study has indicated a direct association be-
tween gut microbiota and HT. To bridge this gap, the 16S
rRNA sequencing technique was used to characterize gut
microbiota, and it was found that HT patients exhibit a gut
microbiota composition distinct from that of healthy controls.
A Spearman correlation analysis revealed correlations be-
tween altered gut microbiota and various clinical parameters.
Moreover, a prediction model was proposed based on the
LEfSe results, and high AUC values for both the exploration
and the validation cohort were achieved. These results
demonstrate that gut microbiota dysbiosis may play a role in
the development of HT. Notably, all HT subjects in this study
were euthyroid, whereas some previous studies revealed that
changes in thyroid hormone concentrations in hypothyroid-
ism or hyperthyroidism (16,17) may affect the composition
of gut microbiota. Regarding hypo- and hyperthyroidism, the
study was unable to reveal an intrinsic link between HT and
gut microbiota. Therefore, only euthyroid HT patients were
included in the study. However, in future studies, analyzing
gut microbiota at various thyroxine concentrations may
provide a better understanding of the changes in gut micro-
biota at different stages of the disease.
This cross-sectional study revealed that HT patients have
greater gut microbiota richness and diversity (a-diversity)
than healthy controls, although the differences did not differ
significantly. In previous studies, increased bacterial diver-
sity was also observed in hyperthyroid and hypothyroid pa-
tients and may be related to bacterial overgrowth in the
intestinal tract (17,36). The similarity of the gut microbiota
between HT patients and healthy controls was analyzed using
cluster analysis (PCoA). The bacterial structures were clus-
tered into two distinct groups, and the HT group displayed
relatively high homology, indicating common characteristics
among the HT patients. In this study, at the phylum level,
Firmicutes were more abundant in the HT patients, whereas
Bacteroidetes were less abundant. These findings are con-
sistent with those of a previous study that reported significant
increases in Firmicutes and decreases in Bacteroidetes spe-
cies in obese, IBS, and MS patients (13,37,38). To the best of
the authors’ knowledge, the Firmicutes/Bacteroidetes ratio is
considered representative of health status and may reflect
eubiosis of the gastrointestinal tract. In this study, the F/B
ratio was significantly increased in HT patients compared to
healthy controls (Fig. 2C), but this increase showed no cor-
relation with age or BMI (Fig. 2D–G). In addition, other
 HASHIMOTO’S THYROIDITIS AND GUT MICROBIOTA DYSBIOSIS 183
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FIG. 3. Spearman correlation analysis of environmental factors and the predictive model based on LEfSe analysis results. The
relationships among seven clinical factors and the relative abundance of top 27 altered genera (Fig. 2B) in HT patients and
healthy controls were estimated using Spearman correlation analysis. Heat maps showing correlations between clinical factors
and gut microbiota at the genus level. Color intensity represents magnitude of correlation. Red, positive correlations; green,
negative correlations. *Adjusted p-value <0.05; **adjusted p-value <0.01; ***adjusted p-value <0.001. TgAb, thyroglobulin
antibody; TPOAb, thyroperoxidase antibody; fT3, free triiodothyronine; fT4, free thyroxine; TSH, thyrotropin (A). Heat map
based on the abundance of the first 10 species from the 27 different genera. Hierarchical clustering (Euclidean distance, complete
linkage) shows that two groups can be clearly separated. The color of the spot corresponds to the Log10-transformed relative
abundance of each genera (B). Receiving operational curve (ROC) analysis was used to assess the predictive model performance
among exploration and validation cohorts (C and D). Area under the curve (AUC) = 0.91 for the exploration cohort (n = 44; red),
and AUC = 0.88 for the validation group (n = 33, blue). Color images available online at www.liebertpub.com/thy

studies have indicated that the intestinal flora of IBS patients
has a higher F/B ratio (36,38). Therefore, it is thought that an
increased F/B ratio corresponds to HT disease status.
At the genus level, it was found that the abundance levels of
many genera were decreased in HT patients according to the
LEfSe results (Fig. 2A). In previous studies, some species with
decreased abundance levels were found to play important roles
in maintaining human health. Similarly, other studies dem-
onstrated that Bacteroides may efficiently ferment fiber into
acetates and propionates (39) and that Phascolarctobacterium
can produce short-chain fatty acids, which are intestinal
epithelial-specific nutrients and energy components that pro-
tect the intestinal mucosal barrier and reduce inflammation
(40). Fecalibacterium may also produce butyrate, which is
essential for energy metabolism and the normal development
of colonic epithelial cells, therefore exhibiting a protective role
(41). Other studies have shown that Fecalbacterium praus-
nitzii supernatant, which contains a mixture of secreted prod-
ucts, has an anti-inflammatory effect (42) and may ameliorate
colitis in mice by regulating Th17 cell differentiation and in-
hibiting the excretion of relevant inflammatory cytokines (43).
A recent study indicated that F. prausnitzii levels were de-
creased in IBD patients (44). Prevotella and Oscillibacter are
also known to produce anti-inflammatory metabolites, which
subsequently reduce Th17 polarization and promote the dif-
ferentiation of anti-inflammatory Treg/Tr1 cells in the gut (45).
 184
Moreover, decreased Prevotella levels have been reported in
diseases such as MS, autism, and T1D (13,46,47). Alloprevo-
tella are obligate anaerobic, nonmotile, Gram-negative bacilli.
These strains are weakly to moderately saccharolytic and
produce acetic and succinic acids as the end products of fer-
mentation (48). Parabacteroides may reduce intestinal in-
flammation by inducing the anti-inflammatory cytokine
interleukin (IL)-10 and suppressing the secretion of the in-
flammatory cytokines IL-17, IL-6, and interferon-c (49). De-
creases in Bifidobacterium were also observed in the HT group,
although the difference was not significant ( p = 0.5338). Bi-
fidobacterium may beneficially affect a host by augmenting
its intestinal microbial population, possibly inhibiting path-
ogens. Decreased levels of Bifidobacterium may lead to poor
immune function (50). Therefore, in HT patients, decreased
levels of these species may lead to intestinal mucosal barrier
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destruction, resulting in the translocation of bacteria and their
products across the mucosal barrier and, consequently, the
activation of immune responses.
According to the LEfSe results, the abundance levels of
many genera were increased in HT patients (Fig. 2A).
Some species with increased abundance levels were shown
to be related to autoimmune or inflammatory diseases in
previous studies. Two Firmicutes, Blautia and Dorea, were
more abundant in HT patients than in healthy controls.
However, these species have been both positively (51) and
negatively (52) linked to inflammatory diseases, and mem-
bers of the genera Blautia and Dorea exhibited increased
abundance levels in MS and Parkinson’s disease (13,53).
Ruminococcus_torques is a mucin-degrading member of
the Clostridium_coccoides_group of Firmicutes in the human
intestinal microbiota (54) and has been associated with
Crohn’s disease (55). The Ruminococcus_gauvreauii_group is
a novel, strictly anaerobic, vancomycin-resistant, Gram-positive
coccus group, and acetic acid is the sole product of glucose
fermentation by these organisms (56). Coprococcus_catus,
Eubacterium_hallii, and Anaerostipes_caccae were shown to
use lactate as a substrate to produce propionate and butyrate
(57,58). Fusicatenibacter saccharivorans (FS), a member of
Clostridium cluster XIVa, is present at markedly lower levels
in active ulcerative colitis (UC) than in quiescent UC. La-
mina propria mononuclear cells (LPMCs) that were incu-
bated with FS produced higher amounts of IL-10. FS also
induced IL-10 production in human LPMCs isolated from
UC patients. These results suggest that human-derived FS
suppresses intestinal inflammation, probably through IL-10
induction, representing a possible future strategy of IBD bac-
teriotherapy to encourage these bacteria to colonize the intes-
tines of IBD patients (59). Phylogenetically, Subdoligranulum
is a member of Clostridium leptum and produces bacteriocins,
which are protein toxins that suppress the growth of similar or
closely related bacterial strains (60). Bacteriocins produced by
this genus may play a role in suppressing Bifidobacteria growth
during inulin fermentation.
Interestingly, the changes in gut microbial structure are
similar between IBS and HT patients. Several recent com-
prehensive studies of the microbiota in IBS patients have
reported increases in the relative abundance levels of Fir-
micutes, mainly Clostridium cluster XIVa (Blautia, Dorea,
Eubacterium_rectale_group, and Ruminococcaceae), Rumi-
nococcus_torques_group, Streptococcus, and Lachnospir-
aceae, and their abundance levels were positively correlated
ZHAO ET AL.
with bowel symptoms (38,61–63). The question of whether
the increased abundance levels of certain species can initiate
HT in humans requires further investigation.
A heat map was used to visualize the relationships between
species and clinical parameters, and the results show that
some gut microbiota were notably correlated with HT-related
clinical parameters (TPOAb, TgAb, fT4, and TSH), indi-
cating that the gut microbiota are closely related to HT
(Fig. 3A). These findings lay the foundation for research on
the interaction between the gut microbiota and a host relative
to the development of HT. A heat map based on the abun-
dance of the first 10 genera (biomarkers) from 27 different
genera was generated. Hierarchical clustering shows that the
two groups could be clearly separated (Fig. 3B) and that HT
patient samples generally cluster together (Supplementary
Fig. S5), with an AUC value of 0.91 (Fig. 3C). Subsequently,
good results were also obtained in the validation cohort
(Fig. 3D), thus confirming that the gut microbiota can be used
to identify patients accurately. Although this represents a new
method for the early diagnosis of HT, large samples are
needed to verify the accuracy of this method.
A key advantage of this study is that the 16S rRNA gene
sequencing technique was used to characterize the gut mi-
crobiota of HT patients. Environmental factors were exam-
ined using Spearman correlation analysis, and a novel
prediction model was proposed. However, this study has
several limitations that must be addressed in future studies.
First, this was a single-center, cross-sectional study involving
a small number of samples. Second, the study was unable to
observe variations among patients at different disease stages
of HT because all patients in the cohort were euthyroid (most
patients in the cohort were new-onset HT patients). Third,
although HT patients were matched for age, sex, and BMI in
the analysis, the results may be influenced by other con-
founding effects, such as stress and dietary factors. Fourth,
microbiota analyses must include studies of the ‘‘virome,’’
‘‘fungiome,’’ and so on. These factors warrant examination in
future studies. Finally, the study does not include animal
experiments or research regarding the possible mechanism
linking gut microbiota dysbiosis to the development of HT.
Therefore, further studies are needed with larger samples,
multicenter designs, animal experiments, and the use of in-
novative research techniques (metagenomics and metabo-
nomics) to explore the potential causal mechanisms between
gut microbiota and HT, such as intestinal barrier dysfunction,
microbiota translocation, and molecular mimicry in order to
facilitate future scientific studies and provide guidance for
the diagnosis, treatment, and prevention of HT.
Acknowledgments
This study was conducted at the First Affiliated Hospital of
Harbin Medical University. This study was supported by
Heilongjiang Provincial Science and Technology Depart-
ment Funds for Distinguished Young Scholar JC201416 and
the Translational Research Fund of Translational Medical
Centre between China and Russia CR201514. We thank all
the staff and participants for their important contributions.
Author Disclosure Statement
The authors declare no competing financial interests.
 HASHIMOTO’S THYROIDITIS AND GUT MICROBIOTA DYSBIOSIS
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Address correspondence to:
Yunwei Wei, PhD
Department of Oncological and Endoscopic Surgery
The First Affiliated Hospital of Harbin Medical University
No. 23, Youzheng Street
Nangang District
Harbin
Heilongjiang, 150001
People’s Republic of China
E-mail: hydwyw11@hotmail.com