The study of intestinal microbiota changes on gestational metabolic syndrome

The study of intestinal microbiota changes on gestational metabolic syndrome

Shane Sheldon Mc Intyre1, Pan ShiLei

（南方医科大学珠江医院，广州， 510000）

Abstract

This study explored the association between intestinal microbiota and MS in pregnant women affected by. 40 pregnant women were included for analysis, and the intestinal bacterial community was compared based on the bar code 454 sequencing of the 16S rRNA gene amplified from fecal samples. We found that Erysipelotrichaceae and Ruminococcaceae were significantly more abundant in MS group.

Keyword: intestinal microbiota; metabolic syndrome; pregnancy; change.

Introduction

The prevalence of metabolic syndrome (MS) has been increasing worldwide, especially among the elderly, making it a serious public health problem (1). MS is a cluster of risk factors, including central obesity, insulin resistance, dyslipidemia and hypertension, which lead to adverse outcomes such as diabetes mellitus (DM) and cardiovascular disease (2). Among other pathogenic factors, human intestinal microflora plays an important role in the etiology of MS.

‘Dysbiosis’ aberrations in the composition of intestinal microorganisms associated with health status have been reported to be associated with MS. For example, some studies have reported a decrease in the proportion of Bacteroidetes in obese and overweight microflora, in some cases accompanied by an increase in the proportion of Firmicutes (3). In other characteristics, it was found that the proportion of Firmicutes and Clostridia members in DM subjects was lower than that in the control group, and the blood glucose level was positively correlated with the ratio of Bacteroidetes to Firmicutes (4).

Obesity increases the risk of gestational DM and preeclampsia, both of which are associated with an increased risk of cardiovascular disease in women later in life (5). However, so far, most studies have only evaluated inpidual metabolites during pregnancy: elevated triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) and reduced high density lipoprotein cholesterol (HDL-C) and gestational DM, preeclampsia was associated with an increased risk of spontaneous preterm birth (6, 7). In a small study, Greek women who were determined to have MS at the time of early pregnancy had a three-fold increase in the risk of premature delivery (8). However, it is unknown whether intestinal microbiota changes is associated gestational MS. is an urgent need to improve the understanding of the involvement of intestinal microflora in the development and etiology of obesity and metabolic diseases in order to guide us in improving the diagnosis, management and prevention of diseases (9).

Thus, the present study aimed to compare the intestinal microbiota communities between the pregnant women with or without MS and investigate the association between intestinal microbiota and gestational MS.

Method and Materials

Study Design

From September 2018 to February 2019, 100 pregnant women aged 25 to 40 years with or without disorder were included in the study. Participants were characterized as with MS (n=52) or without MS (n=48) according to ATP III criteria (10). Exclusion criteria for all participants included use of antibiotics during 3 months before enrollment. The study was approved by the ethics committee of our unit, and written informed consent was obtained from all study participants.

Anthropometric and biochemical measurements

Physical examination included the measurement of body mass index, systolic blood pressure (SBP) and diastolic blood pressure (DBP). Venous blood samples were collected immediately after fasting at night for different types of laboratory analysis, or cryopreserved at-80 ℃ for future analysis. The levels of total cholesterol, HDL-C, LDL-C, TG, fasting plasma glucose (FPG) and glycosylated hemoglobin (HbA1c) were measured.

Microbiota analyses

Stool samples were taken to the laboratory within 24 hours of collection stored in a household refrigerator of-20 °C before the study participants visited the clinic (11). The fecal samples are then stored at-80 °C until microbial DNA is separated by using the Maxwell ®16 tissue DNA purification kit and the Maxwell ®16 instrument plus additional lysozyme treatment (Promega, Madison,WI,USA). Universal primers 27F and 338R were used to amplify the V1-V3 hypervariable region of 16s rRNA gene by PCR, in which 338R primers carried unique sequence tags to bar code each sample. The reaction uses the FastStart high fidelity PCR system (Roche, Basel, Swiss) and the template DNA, with a total reaction volume of 25 μ l, and runs in three parts in the thermal cycler (Biometra, Göttingen, Germany) using the following cycle parameters: denaturation at 94 ℃ for 3 minutes. Then 30 cycles at 94 ℃ (denaturation) for 15 seconds, 45 s at 68 °C (annealing) and 60 s at 72 °C (elongation), and finally extended at 72 °C for 8 minutes. Using the 454 life science primer A (GATC Biotech AG,Constance,German), the purified amplifiers were collected and sequenced by 454FLX titanium pyrophosphate sequencing (Roche), using a protocol recommended by the manufacturer and modified by the center.

16S rRNA gene sequencing processing

Using PRINSEQ (12) to check the quality of the coking sequence and use FlowClus filtering and denoising coking markers. For mass filtration, readings shorter than 150 bases or longer than 1000 bases are discarded, and homopolymer runs longer than 6 bases and fuzzy sequences longer than 6 bases are excluded. A mass fraction of PHRED equal to or greater than 25 in a sliding window of 50 bases is considered to be the minimum allowable average and accepts a barcode correction and a mismatch between the two primers. Metaxa2 is used to process the denoising reading to extract and verify the 16s rRNA V1-V3 region. USEARCH v7 was used to copy, sort and cluster the extracted regions with 97% pairwise sequence identity. The chimera is removed by using the de-new method and the reference-based method as the features of the above tools. The RDP classifier training set N.15 is used as a reference database for chimerism. Using naive Bayesian RDP classifier (v.2.10) (13) to perform classification allocation to Greengenes database (v.201308), the minimum confidence is 0.6 (v.201308). The sequence data is available in the NCBI SRA database and is numbered SRP082486 and BioProject numbered PRJNA339677.

Statistical Analysis

Continuous variables with a normal distribution were reported as mean (±SD), skewed data as median (interquartile range). Categorical variables were presented as numbers (percentages). An unpaired t-test in normally distributed data or Mann-Whitney U-test in non-normally distributed data was applied for quantitative variable and chi-square for qualitative variables. Pearson analysis was used to investigate the association between Bacteroidaceae and BMI. Statistical analysis was performed with SPSS version 20 (SPSS, Inc, Chicago, IL) and values of P <0.05 were considered statistically significant.

Results

Clinical and nutritional characteristics of MS groups

Compared with pregnant women without MS, those with MS had significantly higher BMI, SBP, DBP, serum LDL-C, TG, FPG, levels with higher prevalence of hypertension and DM. Serum HDL-C levels were significantly lower in MS group than in no MS group (all

P<0.05). However, there was no significant difference of age between two groups (Table 1).

Table 1. Clinical characteristics in pregnant women with or without metabolic syndrome

Intestinal microbiota change in MS group

We then studied the abundance of intestinal microbiota between pregnant women with or without MS. As Figure 1 showed, in pregnant women diagnosed with MS, both Erysipelotrichaceae and Ruminococcaceae were significantly more abundant than those without MS. However, the abundance of Lachnospiraceae were less in MS group than in no MS group (all P<0.05).

Figure 1. Distribution of three intestinal microbiota between no MS and MS groups.

Correlation between intestinal microbiota change and BMI

We then test the association between the change of intestinal microbiota and BMI, a MS component. As shown in Figure 2, the abundance of

Bacteroidaceae was significantly with BMI values (r=0.800, P<0.001).

Figure 2. Bacteroidaceae abundance was negatively associated with BMI.

Discussion

In this present study, we found that the change of intestinal microbiota change played a role in the gestational MS. Specifically, the change of Erysipelotrichaceae and Ruminococcaceae was positively associated with MS, while the change of Lachnospiraceae was negatively associated with MS. Moreover, the change of Bacteroidaceae was negatively associated BMI, a MS component.

Microbial components found to be related to the anthropometric and clinical manifestations of MS suggest that they indicate or may contribute to certain aspects of MS. On the contrary, the members of the microbial flora, which are negatively correlated with these performances, suggest functions that are beneficial to metabolic health. In our study, Erysipelotrichaceae family were significantly correlated with the clinical indicators of MS, and found that the higher abundance of Erysipelotrichaceae was associated with MS, which was supported by other studies on inflammation, metabolic diseases and diet manipulation. This provides evidence for the close relationship between Erysipelotrichaceae and host metabolism, and points out that they may be used as microbial targets to combat metabolic disorders. It was shown that Erysipelotrichaceae was associated with host lipid metabolism (14) Consistently in our study, we also showed that Erysipelotrichaceae was significantly higher in pregnant women with MS.

As another important group in the Firmicutes gate, Lachnospiraceae seems to respond to the metabolic health of the host, as an increase in the abundance of this group can be seen in the MS group. Our data suggest that Lachnospiraceae is associated with metabolic disorders. The abundance of Lachnospiraceae, especially the abundance of Blautia, is associated with serum glucose and insulin levels, suggesting the role of host glucose metabolism. (15),

the Lachnospiraceae has also been demonstrated to be beneficial effect on the intestinal barrier (16). We found significant negative associations of Lachnospiraceae abundance in pregnant women with MS, suggesting its protective role in the incidence of MS.

The relatively high representation of bacterial members in the microbial flora indicates that metabolic disorders have a protective or balanced effect. There is an inverse relationship between the clinical manifestations of MS and the relative abundance of Bacteriodetes, which may point to a healthier metabolic state. In the major intestinal microflora, Bacteriodetes was often designated as a significant indicator of metabolic health and non-obese or normal body weight in rodent models and in human studies (17). In agreement with this, we showed that Bacteriodetes was negatively associated with BMI. Cani et al. found that Bacteriodetes-related bacteria, with a series of other microbiota groups, were strongly reduced in patients with obesity (18).

Firmicutes, which settles in the human intestinal tract, forms a highly dominant group with a wide range of phylogeny and functions (19). Therefore, in addition to metabolic disorders, Firmicutes bacteria also seem to be associated with metabolic health. Specifically, the ruminant family has an opposite relationship with the clinical markers of metabolic diseases. The latter is considered to be a powerful plant degradant, so it may play a protective role in metabolic diseases through a diet rich in plant ingredients (20). It is worth noting that Ruminococcaceae forms the most important family in the intestines of Hadza hunter-gatherers and has a foraging lifestyle (21).

There were two limitations to be highlighted. First, because of its cross-sectional design, the cause-effect relationship between intestinal microbiota change and gestational MS can not be identified. Second, the small sample size of this study might confound the generalized implication. Thus, further study with larger size is needed to verify our results.

Conclusion

The change of intestinal microbiota change was associated with the incidence of gestational MS. Exploring related confounding factors may eventually open up a new way to improve and maintain metabolic health by using human-microflora symbiosis.

Reference

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Sookoian S, Pirola CJ.Metabolic syndrome: from the genetics to the pathophysiology. Curr Hypertens Rep. 2011;13:149-157.

Turnbaugh PJ, Hamady M, Yatsunenko T, et al. A core gut microbiome in obese and lean twins. Nature. 2009;457:480-484.

1 Shane Sheldon Mc Intyre , 中文名：马系安，1983年出生，南方医科大学珠江医院研究生，研究生方向妇产科，E-mail：mcinshane@gmail.com,