In the last decades, using 16S rDNA sequences, there have been an increasing amount of research into GM, and it seems that most systemic diseases in the body are related to them [21].In our previous study, we found that there were differences in GM between irritable bowel syndrome patients and normal population in Nanchang, China [22]. In this study, we focused on the RSA patients and wanted to find the difference in GM between RSA patients and normal pregnant women in Nanchang, China. It is the first to analyze the differences in GM between RSA patients and normal pregnant women.
The structure of gut microbiota changes during pregnancy
The changes of GM in different stages of pregnancy have attracted much attention [23]. Studies from different regions have shown clear differences in the characteristics of GM between pregnant women and normal women. A US study reported that GM changed dramatically from first to third trimesters, and showed vast expansion of diversity between mothers, an overall increase in Proteobacteria and Actinobacteria [24]. A Sandi Arabia study indicated that bacterial diversity decreased in pregnant woman, whereas phylum Bacteroidetes declined significantly (p<0.05) in the first trimester, and a relatively high abundance of butyrate-producing bacteria (eg, Faecalibacterium spp. and Eubacterium spp.) in the gut of pregnant women [25]. A Japan research focused the differences in GM between early and late pregnancy, and they suggested that there were no obvious differences among four major phyla (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria) between early and late pregnancy, although the proportion of the TM7 phylum decreased in late pregnancy compared with that in early pregnancy [26]. A study in China reported that at genus level, Akkermansia, Bacteroides, Subdoligranulum, Oscillospira, Ruminococcacea UCG-004), and Alistipes showed higher abundance during pregnancy [27].
RSA patients have abnormal gut microbiota
There has been no report on the correlation between RSA and GM, but there are many reports focused on the correlation between abortion and vaginal or oral bacteria. A China study was aimed at analyzing the changes in gut microorganism of patients with positive immune antibody-associated recurrent abortion using the 16s rRNA gene sequencing microbiome assay, and found that Bacteroides had the highest relative abundance in the positive group, Bacteroides, Erysipelotrichaceae_UCG-003, Faecalibacterium, and Prevotella_9 had high relative abundance in the negative group[28]. First trimester miscarriage associated with reduced prevalence of Lactobacillus spp.-dominated vaginal microbiota classified using hierarchical clustering analysis [29]. A study of vaginal microbiota showed that at the genus level, the relative abundance of Fam_Finegoldia and Lac_Roseburia significantly differed in the embryonic miscarriage group [30]. In the present study, although there was no significant difference in the abundance of dominant bacteria (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria) between the two groups, but Actinobacteria and Proteobacteria tended to a higher in RSA patients at the phylum level. GM dysbiosis can cause insulin resistance (IR), which is closely linked to the occurrence of polycystic ovary syndrome (PCOS), and is associated with chronic inflammation, hormonal changes, follicular dysplasia, endometrial receptivity changes, and abortion or infertility [31].
Roseburia and Agathobacter may have a protective effect against RSA
In our study, one of the most striking result was a significant decrease in the abundance of genera Roseburia in the RSA group (P<0.05). In addition, Agathobacter was significantly increased in NR2 group, while Klebsiella (P<0.05) were significantly increased, Roseburia (P<0.05) and Prevotella.9 (P<0.05) were significantly decreased in RSA1 group. These results suggest that these bacteria play an important role in pregnancy, and the following studies can provide certain support for our research.
A Japan study in GM of infertile women showed that the abundance of the genera Roseburia and Phascolarctobacterium were decreased in patients with infertility [32]. A mice study indicated that the microbiota of conventional mice were significantly different at the end of pregnancy (day 18) as compared with pre-pregnancy (p < 0.05), and the abundance of Roseburia faecis was significantly different at day 18 compared with pre-pregnancy [33].
Moreover, autism spectrum disorder (ASD) children with sleep disorder exhibited declines in the abundance of Agathobacter, decreased levels of 3-hydroxybutyric acid and melatonin [34]. Jin M et al found that Prevotella.9 were significantly decreased in patients with positive immune antibody-associated recurrent abortion [28]. The enrichment of Faecalibacterium, Agathobacter and Roseburia were related to geriatric depression [35].
Gut mircobiota mechanism associated with RSA
Functional prediction analysis indicated that GM may play their role through membrane transport, carbohydrate metabolism, amino acid metabolism and other mechanisms.
Dietary composition:
The composition of the GM can be influenced by dietary composition. In our study, Roseburia (P<0.05) were significantly decreased in RSA group; furthermore, compared with RSA2 group, Roseburia (P<0.05) and Prevotella.9 (P<0.05) were significantly decreased in RSA1 group (patients with BMI>23.9 in RSA). The human gut Firmicute Roseburia intestinalis is a primary degrader of dietary β-mannans, gut Roseburia spp. metabolize dietary components that stimulate their proliferation and metabolic activities. They are part of commensal bacteria producing short-chain fatty acids, especially butyrate, affecting colonic motility, immunity maintenance and anti-inflammatory properties [36, 37]. In early pregnancy, the relative abundances of Roseburia and Lachnospiraceae increased in GM of vegetarian compared with omnivorous diet, and sub-analysis of GM showed an alterations in fermentation end products from a mixed acid fermentation towards more acetate/butyrate [38].
Immune maintenance and anti-inflammatory:
Roseburia is one of the most important microorganisms in human intestinal tract, and its main metabolite is butyric acid. Of note, the GMs that we found to differ significantly at the genus level, such as Roseburia, Prevotella.9, Agathobacter, were butyrate producing bacteriums. A ulcerative colitis (UC) study suggested that butyrate-producing species like Roseburia hominis, involved in the development of UC [39]. Several animal studies reported that abundance of Roseburia intestinalis (R.I), decreased significantly in patients with inflammatory bowel disease (IBD) and exerted an anti-inflammatory function in dextran sulfate sodium (DSS)‑induced colitis [40, 41, 42]. Another animal study on UC indicated that R.I flagellin plays an important role in the treatment of UC by inhibiting activation of the NLRP3 inflammasome and pyroptosis, [43]. In a murine model study suggested that Roseburia, a prominent gut-associated butyrate-producing bacterial genus, may provide a importion protection against atherosclerosis [44]. These studies suggest that R.I may be involved in the mechanism of immune maintenance and anti-inflammatory, which is related to butyrate.
Butyrate-producing bacteria may prevent RSA by regulating BMI
Previous evidence from animal studies suggests that butyrate-producing bacteria prevents high fat diet-induced obesity [45]. In the present study, the patients with BMI>23.9 in RSA have decreased Roseburia (P<0.05) and Prevotella.9 (P<0.05), both well-known butyrate-producing bacteria. Study suggested that chronic butyrate supplementation can prevent diet-induced obesity, hyperinsulinaemia, hypertriglyceridaemia and hepatic steatosis [46]. Abundances of butyrate-producing bacteria, such as R.I and Faecalibacterium prausnitzii, were lower in patients with type 2 diabetes (T2D). This result support that butyrate and other short-chain fatty acids are able to exert profound immunometabolic effects [47]. The abundance and butyrate-producing bacteria and butyrate production of overweight and obese women at 16 weeks gestation were significantly negatively correlated with blood pressure and plasminogen activator inhibitor-1 levels. These results suggest that increased butyrate production may help obese pregnant women maintain normal blood pressure. [48].