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Research Article
Effect of High-Fat Diet on the Intestinal Flora in Letrozole-
Induced Polycystic Ovary Syndrome Rats
Yan-Hua Zheng ,
1
Ying Xu ,
2
Hong-Xia Ma ,
3
Cheng-Jie Liang ,
4
and Tong Yang
5
1
Department of Traditional Chinese Medicine, e Second Affiliated Hospital of Guangzhou Medical University,
Guangzhou 510260, Guangdong, China
2
Department of Nutrition, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian, China
3
Department of Traditional Chinese Medicine, e First Affiliated Hospital of Guangzhou Medical University,
Guangzhou 510120, Guangdong, China
4
Animal Experiment Center, Guangzhou Medical University, Guangzhou 511436, Guangdong, China
5
Department of Pathology, e Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong,
China
Correspondence should be addressed to Yan-Hua Zheng; 332566964@qq.com
Received 18 November 2020; Revised 10 May 2021; Accepted 20 May 2021; Published 28 June 2021
Academic Editor: Sheng-Li Yang
Copyright ©2021 Yan-Hua Zheng et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Aim. e aim of this study was to explore whether letrozole and high-fat diets (HFD) can induce obese insulin-resistant polycystic
ovary syndrome (PCOS) with intestinal flora dysbiosis in a rat model. We compared the changes in the intestinal flora of letrozole-
induced rats fed with HFD or normal chow, to explore the effects of HFD and letrozole independently and synergistically on the
intestinal flora. Methods. Five-week-old female Sprague Dawley (SD) rats were divided into four groups: control (C) group fed
with regular diet; L1 group administered with letrozole and fed with regular diet; L2 group received letrozole and fed with HFD;
and HFD group fed with HFD. At the end of the experiment, ovarian morphology, hormones, metabolism, oxidative stress, and
inflammatory status of all rats were studied. 16S rDNA high-throughput sequencing was used to profile microbial communities,
and various multivariate analysis approaches were used to quantitate microbial composition, abundance, and diversity. Results.
Compared to the C group, the increased plasma fasting insulin and glucose, HOMA-IR, triglyceride, testosterone, and
malondialdehyde were significantly higher in the L2 group, while high-density lipoprotein cholesterol was significantly lower in
the L1 group and L2 group. e indices of Chao1 and the Abundance-based Coverage Estimator (ACE) (α-diversity) in the L2 and
HFD groups were significantly lower than that in the C group. Bray–Curtis dissimilarity based principal coordinate analysis
(PCoA) plots and analysis of similarities (ANOSIM) test showed obvious separations between the L2 group and C group, between
the HFD group and C group, and between the L2 and HFD groups. At the phylum level, Firmicutes and ratio of Firmicutes and
Bacteroidetes (F/B ratio) were increased in the L2 group; Bacteroidetes was decreased in the L2 and HFD groups. No significant
differences in bacterial abundance between the C group and L1 group were observed at the phylum level. Based on linear
discriminant analysis (LDA) effect size (LEfSe) analysis, the bacterial genera (the relative abundance >0.1%, LDA >3, p<0.05)
were selected as candidate bacterial signatures. ey showed that the abundance of Vibrio was significantly increased in the L1
group; Bacteroides and Phascolarctobacterium were enriched in the HFD group, and Bacteroides,Phascolarctobacterium,Blautia,
Parabacteroides,Akkermansia [Ruminococcus]_torques_group, and Anaerotruncus were enriched in the L2 group. Conclusion. e
effect of letrozole on intestinal flora was not significant as HFD. HFD could destroy the balance of intestinal flora and aggravate the
intestinal flora dysbiosis in PCOS. Letrozole-induced rats fed with HFD have many characteristics like human PCOS, including
some metabolic disorders and intestinal flora dysbiosis. e dysbiosis was characterized by an increased Firmicutes/Bacteroidetes
ratio, an expansion of Firmicutes, a contraction of Bacteroidetes, and the decreased microbial richness. Beta-diversity also showed
significant differences in intestinal microflora, compared with control rats.
Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2021, Article ID 6674965, 13 pages
https://doi.org/10.1155/2021/6674965
1. Background
PCOS is a common endocrine and metabolic syndrome
among women of reproductive age [1]. Alterations in
intestinal flora composition or “dysbiosis” have been
implicated in the pathological development of PCOS [2].
Testosterone (T) concentration may affect the composi-
tion of the intestinal microbial community, and several
studies have found that changes in the intestinal microbial
community in PCOS women are related to hyper-
androgenism and low α-diversity compared with the
control group [3, 4]. Intestinal flora may play a pathogenic
role in regulating energy balance and participate in the
development and process of obesity and metabolic dis-
eases [5]. Intestinal flora dysbiosis can interfere with
normal follicular development by triggering a chronic
inflammatory reaction and insulin resistance (IR), which
is closely linked to the occurrence and development of
PCOS [6]. e composition of the intestinal microflora is
affected by many environmental factors. Diet is consid-
ered to be one of the most important environmental
factors affecting the composition of the intestinal mi-
crobial community [7]. Diet-induced obesity is related to a
variety of metabolic and reproductive disorders, including
PCOS [8]. e heterogeneity of PCOS is frequently re-
flected in many animal models. erefore, if a rat model
can show not only the characteristics of ovarian and
metabolic syndrome but also the imbalance of intestinal
flora, it would be valuable for further study of new PCOS
therapy. Letrozole is a nonsteroidal aromatase inhibitor,
which can increase testosterone levels and reduce estrogen
levels by inhibiting the conversion of testosterone to es-
trogen [9]. According to the report, the letrozole-induced
model recapitulates many histological and biochemical
aspects consistent with human PCOS [10]. In the present
study, female Sprague Dawley (SD) rats were given oral
letrozole to establish a model of PCOS and fed with a
regular diet or HFD. We studied the reproduction,
metabolism, and intestinal flora community of these rats.
e findings of this study may also help us better un-
derstand the effects of HFD and letrozole on the intestinal
flora of PCOS.
2. Materials and Methods
2.1. Animals. At the beginning of the experiment, twenty-
one female specific pathogen-free (SPF) SD rats aged 5 weeks
came from the Experimental Animal Science Department of
Guangzhou University of Chinese Medicine, Guangzhou,
China (License number SCXK-2016-0168). is experiment
was approved by the Institutional Animal Care and Use
Committee of Guangzhou Medical University and was
conducted in strict accordance with the guidelines for
Ethical Review of the Welfare of Experimental Animals (GB/
T 35892-2018) in China. All rats were provided with humane
care in a temperature-controlled room with a 12 hr light/
dark cycle (lights on 07:00–19:00) and ad libitum access to
food and water in their cages (22°C–24°C and 60%
humidity).
2.2. Study Procedure. Rats were adaptively fed for one week
and then divided into four groups. e control group (n�5)
received an aqueous solution of 1% carboxymethyl cellulose
sodium (CMC) and consumed with normal chow (Research
Diets GB 14924.3-2010, energy%: 67% carbohydrate, 21%
protein, 12% fat, and total 3.45 kcal/g, provided by
Guangdong Medical Laboratory Animal Center). e PCOS
rat model in our study was established according to the
method of Kafali Het al. [10]. PCOS 1 group (L1, n�5) was
fed with regular diet and administered with letrozole (Target
Mol, American, 1 mg/kg) dissolved in solution CMC1% [10];
PCOS 2 group (L2, n�6) was fed with HFD (D12492, energy
%: 60% fat, 20% carbohydrates, and 20% protein, 5.24 kcal/g,
provided by Guangdong Medical Laboratory Animal Cen-
ter) and administered with letrozole (1 mg/kg) dissolved in
solution CMC 1%; HFD group (n�5) received an aqueous
solution of CMC 1% and consumed with HFD. All doses
were given orally via gavage, for 8 consecutive weeks, and
vaginal cytology analysis was done until the end.
2.3. Vaginal Smear. e stage of the estrus cycle was de-
termined by the main cell type in vaginal smears, which
started from 6 weeks of age to the end of the experiment
every day [11]. All rats were collected daily by using a
dropper filled with normal saline (0.9% NaCl).
2.4. Measurement of Hormone Profile and Biochemical
Indexes. e rats were anesthetized with 2% pentobarbital
sodium (100 μg/g of body weight). After the ovaries were
taken out, the chest was opened; about 4 ml of blood was
taken from the heart. e rats were sacrificed by overdose
pentobarbital sodium. Blood was withdrawn through orbital
sinus in a tube and separated by 10 min centrifugation (3,000
revolutions/min) at 4°C. Supernatant containing serum was
separated and stored immediately at −20°C until analyzed
for biochemical and hormonal analysis. Fasting blood glu-
cose (FBG) was analyzed by GOD-PAP. Testosterone (T),
superoxide dismutase (SOD), malondialdehyde (MDA),
interleukin-22(IL-22), fasting insulin (FINS), luteinizing
hormone (LH), follicle-stimulating hormone (FSH), lipo-
polysaccharide (LPS), Toll-like receptor 4 (TLR4), and tu-
mor necrosis factor-α(TNF-α) were determined using
enzyme-linked immunosorbent assay (ELISA) kit (Mlbio,
Shanghai, China). Low-density lipoprotein (LDL) choles-
terol, high-density lipoprotein (HDL) cholesterol, total
cholesterol (TC), and triglyceride (TG) levels were measured
using Chemistry Analyzer (UniCelDxC 600 Synchron,
Beckman Coulter, USA). IR was appraised with the ho-
meostasis model assessment of insulin resistance (HOMA-
IR) method. HOMA-IR was calculated using the following
formula: HOMA-IR �FBG (mmol/L) ∗FINS (mU/L)/22.5
[12].
2.5. Sample Collection. Fresh stool samples were extracted
from the colons of all rats, collected into 1.5 ml sterile EP
tubes, then frozen in liquid nitrogen quickly, and stored at
−80°C until further analysis. e right ovary of the rat was
2Evidence-Based Complementary and Alternative Medicine
fixed in 4% paraformaldehyde and embedded in paraffin.
5μm thick sections were prepared and stained with he-
matoxylin-eosin (HE) and histoanatomical changes were
observed and photographed under a light microscope (BX-
51, Olympus, Tokyo, Japan, at X40 magnification).
2.6. 16S rDNA Sequencing Data Analysis. e fecal micro-
biome for 21 fecal samples was collected from the rats in the
four groups. e 16S rDNA high-throughput sequencing
(V3-V4 region) was performed using an Illumina MiSeq
platform. After assembly, quality filtering, and the random
extraction of sequences at 97% similarity, the operational
taxonomic units (OTUs) for species classification were
obtained. e Chao1, ACE, Simpson, and Shannon indexes
were calculated to analyze α-diversity. We used Bray–Curtis
dissimilarity to analyze and compare the similarity of the gut
microbial communities (β-diversity). Analysis of similarities
(ANOSIM) test was used to check whether the differences
between groups were significantly greater than those within
groups. A principal coordinate analysis (PCoA) plot was
used to visualize whether the groups have significantly
different microbial communities. Linear discriminant
analysis effect size (LEfSe) analysis coupled with the
Kruskal–Wallis rank-sum test was performed to identify the
microbial differences among all groups. Note that while a
log-transformed LDA score of 2 was used as a threshold for
identification of significant taxa, the LDA >3.0 was set as the
threshold for selection of features.
2.7. Statistical Analyses. Most statistical evaluations were
performed with SPSS 21.0 for Windows (SPSS Inc., Chicago,
IL, United States). All data were presented as mean ±SEM.
One-way ANOVA was used to determine the significance,
and p<0.05 was considered significant. When the ANOVA
revealed significant differences among four groups, a post hoc
analysis was performed by a Tukey honest significant dif-
ference test. e Kruskal–Wallis test was used for not nor-
mally distributed values. α-diversity was analyzed using
Chao1, ACE, Shannon, and Simpson diversity indices. ese
indexes 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). β-Diversity analysis was used to evaluate
differences in the species complexity between samples, and
beta-diversity based on Bray–Curtis dissimilarity was cal-
culated using QIIME software (v1.7.0) based on the rarefied
OTU counts. e microbiota features differentiating the fecal
microbiota were characterized using the LEfSe method for
biomarker discovery, which uses the Kruskal–Wallis rank-
sum test to detect features with significantly different
abundance levels between assigned taxa and performs an
LDA to estimate the effect size of each feature.
3. Results
3.1. Reproductive and Metabolic Parameters. Body weight
was measured weekly. e weight of rats in the L1, L2, and
HFD groups increased more than that in the C group
(p<0.01) (Figure 1). As seen in Table 1, compared with the
C group, the increased plasma fasting insulin and glucose,
HOMA-IR, TG, T, and MDA were significantly higher in
the L2 group (p<0.01 or p<0.05), while HDL-C was lower
in the L1 group and L2 group (p<0.05). e level of LPS was
significantly higher in the HFD group than in the C group
(p<0.05). e reproductive function of the ovaries was
evaluated based on estrous cyclicity, follicle number, and
follicle morphology. Rats in the C and HFD groups showed
regular cycles of 4-5 days complete with the proestrus, es-
trus, metestrus, and diestrus stages. Ovaries from the C and
HFD groups exhibited follicles in various stages of devel-
opment, including some fresh corpora lutea. At the end of
the study, rats in the L1 and L2 groups had irregular cycles
and were in the diestrus stage which mainly showed leu-
kocytes. Hematoxylin-eosin (HE) staining was conducted to
evaluate the ovary structure. HE staining indicated that the
ovaries of the L1 and L2 group had a high incidence of
subcapsular ovarian cyst together with incomplete luteini-
zation and decreased number of corpora lutea (Figures 2(a)–
2(d)).
3.2. Diversity of the Intestinal Flora. OTU-level alpha-di-
versity metrics (ACE and Chao1) revealed significantly
lower diversity and richness in the L2 and HFD groups.
Compared with the C group, the ACE and Chao1 indices in
the L2 and HFD groups were significantly decreased
(p<0.05) (Figures 3(a)–3(d)). However, there were no
significant differences in the Shannon or Simpson index
between the groups. PCoA plot revealed distinct clustering
of C group that separated from both the L2 and HFD groups
(Figure 4). e significance of differences was confirmed by
the ANOSIM, C and L2 groups (R�1, p�0.003)
(Figure 5(b)), C and HFD group (R�0.98, p�0.008)
(Figure 5(c)), and L2 and HFD groups (R�0.885, p�0.004)
(Figure 5(d)), and R>0.5 implies that separation between
groups is good and intergroup variation is significantly
greater than intragroup variations.
3.3. e Composition of Intestinal Flora. We evaluate the
intestinal flora composition by comparing the relative
abundances at the phylum and genera levels. e 10 major
bacterial clades from the gut bacterial profiles of the groups
at phylum level are represented in Figure 6(a). e phyla
CL1L2HFD
∗
∗∗ ∗∗
0
Body weight (g)
450
400
350
300
250
200
150
100
50
Figure 1: e body weights were measured at the end of exper-
iment. ∗Compared with the C group, p<0.05; ∗ ∗ compared with
the C group, p<0.01.
Evidence-Based Complementary and Alternative Medicine 3
Firmicutes,Bacteroidetes,Proteobacteria,Verrucomicrobia,
and Actinobacteria dominate the intestinal flora community.
Compared with the C group, the relative abundance of
Firmicutes and ratio of Firmicutes and Bacteroidetes (F/B
ratio) were increased, and Bacteroidetes was decreased in the
L2 group (p<0.01). And the relative abundance of Bac-
teroidetes was also decreased significantly in the HFD group
(Figure 6(b)). Moreover (Figure 7(a) and 7(b)), compared
Table 1: Comparison of biochemical parameters among groups.
C L1 L2 HFD ANOVA Tukey HSD (adjusted for
multiple comparisons)
N�5N�6N�6N�5pValue P2 P3 P4
LH (mIU/ml) 5.30 ±0.81 5.51 ±0.78 5.38 ±0.65 6.06 ±0.73 0.372 0.966 0.894 0.331
FSH (mIU/ml) 7.36 ±1.13 7.00 ±0.98 6.91 ±0.72 7.71 ±1.18 0.558 0.945 0.876 0.942
T (pg/mL) 26.46 ±3.04 38.92 ±9.35 39.50 ±7.08 33.72 ±9.35 0.23 0.05 0.022 0.344
FINS (mU/L) 2.49 ±0.46 2.17 ±0.40 3.73 ±0.64 2.61 ±0.35 0 0.742 0.002 0.982
FBG (mmol/L) 5.46 ±0.83 5.5 ±0.1.37 8.33 ±1.03 6.84 ±1.75 0.003 1 0.006 0.329
HOMA-IR 0.59 ±0.07 0.54 ±0.19 1.34 ±0.42 0.79 ±0.25 0 0.991 0.001 0.655
HDL-C (mmol/L) 1.15 ±0.22 0.85 ±0.18 0.83 ±0.11 0.97 ±0.09 0.02 0.044 0.02 0.302
LDL-C (mmol/L) 0.41 ±0.13 0.43 ±0.10 0.42 ±0.02 0.38 ±0.04 0.843 0.992 0.997 0.952
TG (mmol/L) 0.46 ±0.08 0.46 ±0.10 0.72 ±0.15 0.53 ±0.12 0.004 1 0.008 0.799
TC (mmol/L) 1.25 ±0.41 1.44 ±0.38 1.64 ±0.20 1.44 ±0.19 0.247 0.771 0.187 0.753
TLR4 (ng/mL) 3.23 ±0.40 3.35 ±0.43 3.48 ±0.36 3.61 ±0.53 0.611 0.971 0.778 0.526
LPS (EU/L) 93.00 ±8.57 105.9 ±14.7 115.6 ±32.6 137.36 ±30.26 0.049 0.842 0.418 0.039
SCAF (pg/ml) 29.47 ±2.16 34.51 ±16.6 30.90 ±6.56 27.59 ±1.32 0.653 0.816 0.993 0.986
SOD (U/ml) 24.96 ±2.95 18.93 ±10.1 19.67 ±4.08 23.35 ±4.33 0.342 0.425 0.463 0.972
MDA (nmol/ml) 0.26 ±0.06 0.44 ±0.13 0.46 ±0.09 0.19 ±0.02 0 0.071 0.022 0.32
IL-22 (pg/ml) 3.81 ±0.57 4.09 ±0.29 4.21 ±0.49 3.52 ±0.50 0.109 0.794 0.502 0.754
TNF-α(pg/ml) 50.1 ±5.39 48.3 ±7.21 54.9 ±9.80 53.15 ±5.31 0.427 0.98 0.684 0.91
LH: luteinizing hormone; FSH: follicle-stimulating hormone; T: testosterone; FT: free testosterone; INS: fasting insulin; FBG: fasting blood glucose; HOMA-
IR: homeostasis model of assessment for insulin resistance index; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol;
TG: total triglyceride; TC: total cholesterol; TLR4: Toll-like receptor 4; LPS: lipopolysaccharide; SOD: superoxide dismutase; MDA: malondialdehyde; IL-22:
interleukin-22; TNF-α: tumor necrosis factor-α. Data are presented as mean ±standard deviation, analyzed by one-way analysis of variance followed by the
Tukey HSD test. P2: C group versus L1 group; P3: C group versus L2 group; P4: C group versus HFD group.
(a)
(c)
(b)
(d)
Figure 2: Photomicrographs of representative ovarian cross section from four groups: (a) C group, (b) L1 group, (c) L2 group, and (d) HFD
group. DF: developing follicles; CL: corpus luteum; CF: cystic follicles.
4Evidence-Based Complementary and Alternative Medicine
with the C group, the relative abundance of Verrucomicrobia
and Actinobacteria was enriched, and Tenericutes was de-
creased in the L2 group (p<0.05). e relative abundance of
Proteobacteria and Verrucomicrobia (p<0.05) was increased
in the HFD group, while Tenericutes and Cyanobacteria
(p<0.01) were decreased, compared with the C group. No
significant differences between the C group and L1 group
were observed at phylum level.
In this work, we used the LEfSe method to identify sig-
nificant, differentially abundant microbiome. At the genus
level, the results showed that four genera were distinctively
represented between the L1 group and C group, with two
(Peptococcus and Turicibacter) being abundant in the C
group, and two (Vibrio and Bifidobacterium) being abundant
in the L1 group (Figure 8). As seen in Figure 9, thirty-five
genera were obviously representative between the L2 group
and C group, with fourteen (Alloprevotella,Prevotella_9,
ruminantium_group, Bilophila,Prevotellaceae_Ga6A1_group,
CL1L2HFD
Chaol index
2,600
1,400
1,600
1,800
2,000
2,200
2,400
ns
ns
ns
ns
∗
∗
(a)
CL1L2HFD
ACE index
2,800
1,200
1,600
1,800
2,000
2,200
2,400
1,400
2,600
ns
ns
ns
ns
∗
∗
(b)
CL1L2HFD
Shannon index
9
5
6
7
8
ns
ns
ns
ns
ns
ns
(c)
CL1L2HFD
Simpson
1.15
0.90
1.00
1.05
1.10
0.95
ns
ns
ns
ns
ns
ns
(d)
Figure 3: Alpha-diversity analysis of the species distribution. (a) Chao1 index. (b) ACE index. (c) Shannon value. (d) Simpson value.
∗p<0.05, ns: not significant, p<0.05.
–0.4
–0.6
–0.4
–0.2
0.0
0.2
PCO2 (16.22%)
PCO1 (42.23%)
–0.2 0.0 0.2 0.4
C
L1
L2
HFD
Figure 4: Principal coordinate analysis (PCoA) plot of bacterial
community composition at the OTU level to evaluate the simi-
larities among the groups. Each dot represents the bacterial
community of the sample.
Evidence-Based Complementary and Alternative Medicine 5
Ruminococcaceae_NK4A214_group,Odoribacter,Catabacter,
Rikenella,Vibrio,pectinophllus_group,Anaerovorax,Rumi-
nococcaceae_UCG-007, and Papillibacter) being abundant in
the C group, and twenty-one (Bacteroides,Blautia,Akker-
mansia,Phascolarctobacterium,Parabacteroides,[Rumino-
coccus]_torques_group,Anaerotruncus,Allobaculum,
Faecalitalea,Streptococcus,Tyzzerella,Faecalibaculum,
Enterorhabdus,Bifidobacterium,Rothia,Lactococcus,Lach-
nospiraceae_FCS020_group,Holdemania, gauvreauii_group,
Lactonifactor, and Acetatifactor) being abundant in L2 group.
Fourteen genera differed dramatically between the HFD
group and C group, the proportions of the Alloprevotella,
Prevotella_9,Family_XIII_UCG_group,ruminantium_group,
Prevotellaceae_Ga6A1_group,Ruminococca-
ceae_NK4A214_group,Rikenella,Odoribacter, and Rumini-
clostridium_5 genera were decreased, whereas the
proportions of the Proteus,Lactonifactor,Holdemania,
Phascolarctobacterium, and Bacteroides were increased in the
HFD group samples (Figure 10). Subsequently, the genera
above with the average relative abundance >0.1% were ana-
lyzed by the Wilcoxon rank-sum test between the C, L1, L2,
and HFD groups (Figures 11(a)–11(c)). Compared with the C
group, the relative abundance of Romboutsia and Vibrio was
increased in the L1 group; Bacteroides,Blautia,Akkermansia,
Phascolarctobacterium,Parabacteroides,Clos-
tridium_sensu_sticto_1,[Ruminococcus]_torques_group,
Anaerotruncus, and Butyricimonas were enriched in the L2
group significantly, while the proportions of the Alloprevo-
tella, Prevotella_9, ruminantium_group, Pre-
votellaceae_Ga6A1_group, Ruminococca-
ceae_NK4A214_group, and Alistipes were decreased.
Bacteroides,Desulfovibrio,Phascolarctobacterium,
Between C L1
R = 0.216, P = 0.09
40
0
10
20
30
(a)
Between L2 C
R = 1, P = 0.003
40
0
10
20
30
50
(b)
Between HFD C
R = 0.98, P = 0.008
40
0
10
20
30
(c)
Between L2 HFD
R = 0.885, P = 0.004
40
0
10
20
30
50
(d)
Figure 5: Analysis of similarities (ANOSIM) plot showing dissimilarity between groups. (a) Between C and L1 groups. (b) Between L2 and C
groups. (c) Between HFD and C groups. (d) Between L2 and HFD groups. pvalue is a measure of the significance of the trend between
groups. R-value is a measure of the strength of the factors on the samples. R-value close to 1 indicates a high separation between groups.
6Evidence-Based Complementary and Alternative Medicine
Akkermansia,Parabacteroides, and Anaerotruncus were in-
creased in the HFD group; Alloprevotella,Prevotella_9,
Intestinimonas, and Ruminococcaceae_NK4A214_group were
decreased.
4. Discussion
PCOS is the most common endocrine disorder, with many
complications such as obesity and IR. e rats in the L1 and
L2 groups gained more weight than the controls and showed
C
Relative abundance (%)
L2 HFD
100
0
10
20
30
L1
90
80
70
60
50
40
Actinobacteria
Cyanobacteria
Proteobacteria
Firmicutes
Bacteroidetes
Verrucomicrobia
Unclassified
Others
Lentisphaerae
Saccharibacteria
Deferribacteres
Teneri cu te s
(a)
C
The Firmicutes/Bacteroid etes ratio
(F/B ratio)
L2 HFD
4
0
0.5
1
1.5
2
2.5
3
3.5
L1
(b)
Figure 6: Comparison of microbiota composition at the phylum level. (a) A bar plot about relative abundance (%) of bacterial taxa. (b) e
relative abundance of Bacteroidetes and Firmicutes, and the Firmicutes/Bacteroidetes ratio (F/B ratio). ∗ ∗ Compared with the C group,
p<0.01.
Verrucomicrobia
∗∗
∗∗
∗∗
Actinobacteria Tenericutes
Relative abundance (%)
5
4
3
2
1
0
C
L2
(a)
Proteobacteria Verrucomicrobia Cyanobacteria
Relative abundance (%)
25
20
15
10
5
0
Teneri cu te s
∗∗∗
∗
∗
C
HFD
(b)
Figure 7: Boxplot of comparing the relative abundance between groups at the phylum level. (a) Between C and L2 groups. (b) Between C
and HFD groups. ∗Compared with the C group, p<0.05; ∗ ∗ compared with the C group, p<0.01.
–4 –3 –2 –1 0
Vibr io
Turicibacter
Peptococcus
Bifidobacterium
LDA score (log 10)
12345
L1
C
Figure 8: LDA along with effect size measurements was applied to
present the enriched bacterial genera in the L1 group (red) and C
group (green).
Evidence-Based Complementary and Alternative Medicine 7
some reproductive phenotypes of PCOS, including hyper-
androgenism, anovulation (indicated by a lack of corpora
lutea in the ovaries), and the appearance of cystic ovarian
follicles. Combined with HFD, the metabolic disorder
seemed to aggravate, the fasting insulin and glucose,
HOMA-IR, and TG were significantly elevated, and HDL-C
was reduced in the L2 group, compared with the C group.
And the concentration of MDA was also raised in the L2
group. It is known that excessive intake of fat may affect the
intestinal flora, increase circulating LPS, trigger downstream
inflammatory events, and increase the risk of long-term low-
level systemic inflammation, obesity, and IR [13–18]. Diet is
considered to be one of the most critical environmental
factors for shaping intestinal flora structures. HFD can
influence the intestinal flora directly, and increase the cir-
culatory LPS [19]. In our study, the rats in the L2 and HFD
groups were fed with HFD, and the concentration of LPS in
the HFD group increased significantly, but there was no such
significant change in the L2 group. HFD feeding seemed to
interfere with the α- and β-diversity of the microbial
community more significantly than letrozole. OTU-level
α-diversity metrics (ACE and Chao1) revealed significantly
lower richness in the L2 and HFD groups. It has been proved
that individuals with low microbial richness are more prone
to obesity, IR, and dyslipidemia [20]. After correcting for age
and sex, OTU richness was negatively correlated with BMI
and TG, but positively correlated with HDL-C [21]. In line
with the fact that HDL-C was decreased in the L2 and HFD
groups, the fasting insulin and blood glucose, TG, and
HOMA-IR were elevated significantly in the L2 group.
Significant differences were found in β-diversity between the
L2 group and C group, between the HFD group and C group,
and L2 and HFD groups, but they were not found between
the C group and L1 group. e above results suggested that
the microbiota community in the HFD and L2 groups were
significantly different compared to the C group. And the
microbial environment was not changed significantly after
treating with letrozole alone but changed obviously after
feeding with HFD.
All predominant phyla, including Firmicutes,Bacter-
oidetes,Proteobacteria,Verrucomicrobia, and Actino-
bacteria, were largely consistent in different groups, and
different relative abundances could be observed. A decrease
of Bacteroidetes along with an increase of Firmicutes resulted
in an increased F/B ratio in the L2 group. An increased F/B
ratio has been widely considered a signature of gut dysbiosis
[22]. Gut microbial dysbiosis has been associated with in-
flammatory and metabolic disorders [23] and obesity [24].
e results showed that the rats in the L2 group had higher
body weight, fasting insulin, fasting blood glucose, and
HOMA-IR than in the C group. It was reported that Bac-
teroidetes-rich communities have a protective effect on
blood glucose level [25] and play a protective role in in-
testinal inflammation [26]. A reduction of Bacteroides is
related to some metabolic diseases, such as diabetes and
cardiac disease [27]. Increased Firmicutes was correlated
with obesity [28]. e relative abundance of Actinobacteria
was also enriched in the L2 group. e function of Acti-
nobacteria in gut microbiota was not thoroughly under-
stood. It was reported that Actinobacteria was increased in
human adults with type II diabetes [29]. In a survey about
thin and obese twins, a higher level of Actinobacteria in the
gut was found in obese subjects [30]. Intriguingly, different
from our study, Lindheim et al. found a reduced relative
abundance of bacteria from the Actinobacteria phylum in
L2
C
–6.0
Bacteroides
LDA score (log 10)
Blautia
Akkermansia
Phascolarctobacterium
Acetatifactor
Torques_group
Lactonifactor
Parabacteroides
Anaerotruncus
Allobaculium
Faecalitafea
Gauvreauii_group
Holdema nia
Streptococcus
Tyzzerella
Faecalibaculum
Rothia
Lactococcus
Enterorhobdus
Lachnospiraceae_FCS020_group
Bifidobacterium
Papillibacter
Ruminococcaceae_UCG_007
Anaerovorax
Pectinophilus_group
Vibr io
Rikenella
Catabacter
Odoribacter
Ruminococcaceae_NK4A214_group
Bilophila
Prevotellaceae_Ga6A1_group
Ruminantium_group
Prevotella_9
Alloprevotella
–4.8 –3.6 –2.4 –1.2 0.0 1.2 2.4 3.6 4.8 6.0
Figure 9: LDA along with effect size measurements was applied to
present the enriched bacterial genera in the L2 group (red) and C
group (green).
HFD
C
–6.0
Bacteroides
LDA score (log 10)
Phascolarctobacterium
Holdema nia
Lactonifactor
Proteus
Rikenella
Odoribacter
Ruminiclostridium_5
Ruminococcaceae_NK4A214_group
Prevotellaceae_Ga6A1_group
Ruminantium_group
Family_XIII_UCG_001
Prevotella_9
Alloprevotella
–4.8 –3.6 –2.4 –1.2 0.0 1.2 2.4 3.6 4.8 6.0
Figure 10: LDA along with effect size measurements was applied to
present the enriched bacterial genera in the HFD group (red) and C
group (green).
8Evidence-Based Complementary and Alternative Medicine
∗∗
∗
01
Relative abundance (%)
23
Vibr io
Romboutsia
C
L1
(a)
0510
Relative abundance (%)
15 20 25
Torques_group
∗∗
∗∗
∗∗
∗∗
∗∗
∗∗
∗∗
∗∗
∗
∗
∗∗
∗∗
∗∗
∗∗
∗∗
∗∗
Butyricimonas
Anaerotruncus
Alistipes
Ruminococcaceae_NK4A2 l 4_group
Prevotellaceae_Ga6A1_group
Ruminantium_group
Clostridium_sensu_stricto_l
Parabacteroides
Prevotella-9
Lachnospiraceae_NK4A136_group
Blautia
Akkermansia
Phascolarctobacterium
Alloprevotella
Bacteroides
C
L2
(b)
0510
Relative abundance (%)
15 20 25 30
Anaerotruncus
Ruminococcaceae_NK4A214_group
Intestinimonas
Parabacteroides
Prevotella_9
Akkermansia
Phascolarctobacterium
Desulfovibrio
Alloprevotella
Bacteroides ∗∗
∗
∗∗
∗∗
∗∗
∗∗
∗
∗
C
HFD
∗∗
∗
(c)
Figure 11: Comparisons of the relative abundances (%) of bacterial genera between groups. (a) Between C and L1 group. (b) Between C and
L2. (c) Between C and HFD group. ∗Compared with the C group, p<0.05; ∗ ∗ compared with the C group, p<0.01.
Evidence-Based Complementary and Alternative Medicine 9
PCOS patients [31]. e relative abundance of Verrucomi-
crobia was enriched, and Tenericutes was decreased in the L2
group and HFD group. Tenericutes phylum was found
enriched in healthy individuals compared with metabolic
syndrome patients [32]. In Europeans, PCOS was reported
to be related to the decrease of relative abundance of Ten-
ericutes [33]. And the decrease abundance of Tenericutes was
observed in intestinal dysbiosis of rats due to inflammatory
conditions [34], as well as the increase of Verrucomicrobia
[35]. Proteobacteria phylum, which includes a wide variety
of pathogens, was also more abundant in the HFD group.
e phylum Proteobacteria is the most unstable over time
among the main phyla in the intestinal flora [36]. e in-
creased prevalence of Proteobacteria reflects dysbiosis or an
unstable intestinal flora community structure [37]. ere-
fore, intake of HFD could increase the relative abundance of
Proteobacteria and interfere with the stability of the mi-
crobial community. Letrozole alone may not significantly
affect intestinal stability as HFD, but HFD combined with
letrozole have synergistic effects on altering the composition
and structure of intestinal flora.
At the genus level, we used the LEfSe method to compare
the intestinal flora compositions of the control group to the
other three groups and identify the specific bacterial taxa.
e larger the LDA score, the more significant the difference
between groups. Based on the LDA and Wilcoxon rank-sum
test, the bacterial genera (the relative abundance >0.1%,
LDA >3, p<0.05) were selected as candidate bacterial
signatures. Vibrio was enriched in the L1 group as a bio-
marker. Vibrio is known as an opportunistic bacterial
pathogen which might increase host susceptibility [38]. e
HFD group was characterized by a higher content of Bac-
teroides and Phascolarctobacterium. Intestinal microbial
communities are known to be affected by diet. Dietary habits
such as foods with saturated fats and animal protein can lead
to a high prevalence of Bacteroides [39, 40]. A higher
abundance of Bacteroides was observed in Japanese par-
ticipants who consumed a diet of animal origin in com-
parison to Indian adults who consumed a more plant-based
diet [41]. Bacteroides is also one of the major lineages of
bacteria and associated with gut inflammation [42, 43].
Bacteroides species are most commonly found in mixed
infections [44]. Moreover, increased levels of Bacteroides
were negatively correlated with energy intake and adiposity
[45]. Phascolarctobacterium can produce short-chain, which
is positively correlated with the metabolic status in the host
[46, 47]. It was also negatively correlated with many path-
ways, including environmental information processing and
metabolism [48]. Phascolarctobacterium is related to both
insulin sensitivity and secretion [49], and a higher abun-
dance of Phascolarctobacterium was observed in women
with metabolic syndrome [50].
Letrozole may have enhanced the effects of HFD on
intestinal flora imbalance. In addition to enrichment of
Bacteroides and Phascolarctobacterium, the relative abun-
dance of Blautia,Parabacteroides,[Ruminococcus]_tor-
ques_group,Akkermansia, and Anaerotruncus was presented
abundant in the L1 group. Blautia, which is considered to be
essential for a healthy status [51], may contribute to the
alleviation of inflammation, IR, and obesity by reducing the
intestinal endotoxins into the blood [52]. However, Blautia
has been found increased in disease groups in three out of four
cross-sectional studies for type 2 diabetes [53]. As a producer
of acetate, Blautia can drive the release of insulin and promote
metabolic syndromes, such as hypertriglyceridemia, fatty
liver, and IR [54]. e relative abundance level of Blautia was
positively correlated with bowel symptoms and increased in
patients with irritable bowel syndrome [55]. Blautia has been
shown to be associated with metabolites reflecting an un-
healthy metabolic state in individuals with a high BMI [56].
Many studies illustrated that Blautia can drive insulin release
and promote metabolic syndromes, such as hyper-
triglyceridemia, fatty liver disease, and IR [53, 57]. Patients
with type 2 diabetes and glucose intolerance had greater
numbers of Blautia [58]. Blautia was also positively correlated
with indicators of bodyweight (including waistline and body
mass index) and serum lipids (including LDL-C, TC, and TG)
[59]. Parabacteroides enrichment may alter gene expression in
pathways associated with metabolic function, neurodegen-
erative disease, and dopaminergic signaling [60]. Some
studies have reported that Parabacteroides is negatively
correlated with metabolic disorders [61, 62]. [Ruminococcus]
_torques_group may alter fat metabolism; low abundance of
Ruminococcus_torques_group is beneficial for the control of
body fat and promotes the effects of resistant starch on ab-
dominal adiposity [63]. Ruminococcus]_torques_group was
also reported to be associated with inflammatory bowel
disease [64], and more abundant in subjects consuming the
proinflammatory diets [65]. Anaerotruncus is a conditional
pathogenic bacterium and reported to be linked to hepatic
cirrhosis with Holdemania and Dorea and type 1 diabetes, but
not specific to IR [66]. In addition, in the mouse study, the
relative abundance of Anaerotruncus species is also related to
aging, age-related inflammation, and the increase of proin-
flammatory chemokines [67]. It is reported that Akkermansia
has both regulatory and inflammatory properties [68]. e
enrichment of Akkermansia has been found to be inversely
associated with obesity and diabetes mellitus [69]. Akker-
mansia has previously been reported to associate with im-
proved metabolic health, and the introduction of the
Akkermansia into the gut of diet-induced obese mice may
improve the host glucose homeostasis [70].
e results indicated that letrozole combined with HFD
apparently changed microbial diversity and composition,
which can influence the host metabolism mainly through
various mechanisms, including getting more energy from
the diet, disturbing metabolism, and immunologic function.
5. Conclusion
Letrozole has synergistic effects with HFD on intestinal flora
dysbiosis. e consumption of HFD might contribute to
accelerating the progression of oxidative stress status, ag-
gravating metabolic disorder in PCOS. e present findings
support the notion that the letrozole- and HFD-induced rat
model has many characteristics of human PCOS, including
some metabolic disorders and intestinal flora dysbiosis. e
rat model of PCOS may provide a useful tool for evaluating
10 Evidence-Based Complementary and Alternative Medicine
the efficacy and mechanism of new monotherapy or drug
combinations in treating PCOS.
6. Limitation
Because the biological samples of the microbial community
obtained in this study were limited, the effects of letrozole on
the intestinal microbial community may not be significant.
e search for intestinal microflora via stool carries specific
limitations. Stool may represent lower intestinal microflora,
but composition differs between upper and lower intestine
[72].
Abbreviations
PCOS: Polycystic ovary syndrome
SD: Sprague Dawley
HFD: High-fat diet
PCoA: Principal coordinate analysis
IR: Insulin resistance
SPF: Specific pathogen-free
OTUs: Operational taxonomic units
LH: Luteinizing hormone
FSH: Follicle-stimulating hormone
T: Testosterone
INS: Fasting insulin
FBG: Fasting blood glucose
HOMA-
IR:
Homeostasis model of assessment for insulin
resistance index
HDL-C: High-density lipoprotein cholesterol
LDL-C: Low-density lipoprotein cholesterol
TG: Total triglyceride
TC: Total cholesterol
TLR4: Toll-like receptor 4
LPS: Lipopolysaccharide
SCAF: Short-chain fatty acid
SOD: Superoxide dismutase
MDA: Malondialdehyde
IL-22: Interleukin-22
TNF-α: Tumor necrosis factor-α.
Data Availability
e datasets used and/or analyzed during the current study
are available from the corresponding author on reasonable
request.
Ethical Approval
is experiment was approved by the Institutional Animal
Care and Use Committee of Guangzhou Medical University
and was conducted in strict accordance with the guidelines
for Ethical Review of the Welfare of Experimental Animals
(GB/T 35892-2018) in China.
Conflicts of Interest
No conflicts of interest, financial or otherwise, are declared
by the authors.
Authors’ Contributions
YHZ, YX, and HXM conceived and designed the experi-
ments. CJL and TY performed the experiments. YHZ and
HXM analyzed the data. YHZ and YX wrote the manuscript.
Acknowledgments
is work was supported by research grants from the Na-
tional Natural Science Foundation of China (no. 81704107).
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