Saliva is a microenvironment for microbiota that acts as a bridge for the host and microbe to communicate with each other. Any disturbance of the microenvironment may contribute to the alteration of oral microbe and its relationship with host. Dramatic fluctuations of salivary hormone levels were observed in this study, which were similar to previous reports on pregnant and non-pregnant women. Figuero et al. measured the concentration of progesterone and estradiol in saliva with a similar longitudinal design (duration of pregnancy and 3-months postpartum), and found that hormone levels increased greatly during pregnancy, peaked at the third trimester and dropped after delivery[17]. In another study, Khosravisamani et al. evaluated the effect of the menstrual cycle on salivary progesterone and estradiol levels in non-pregnant women and found that values determined during ovulation (on days 12–14) and the pre-menstruation trimesters (on days 22–24) were significantly higher than at other days[18]. Therefore, in this study, the sampling time selected for non-pregnant women was days 17–21 within the menstrual cycle, and the results obtained were in agreement with previous findings.
The correlation between pregnancy and the oral bacteria has been profiled in many reports. On the one hand, accumulating evidence has indicated that oral bacteria play a significant role in intrauterine infections, which is a major cause of complications of pregnancy, including preterm birth and still birth[19, 20]. One the other hand, both clinical and laboratory studies have shown that oral diseases such as gingivitis and periodontitis are likely to worsen during pregnancy.The increase in sex hormone levels in saliva might be responsible for this, and the shift in dominant pathogens during pregnancy and its correlation with sex hormones has been demonstrated previously[20, 21].
It is worth noting that dramatic fluctuation of hormone levels might have a potential impact on the diversity of the salivary microbiome. This was evident in the results that the Shannon index of the salivary microbiome increased with the level of sex hormones. Both the highest concentration of salivary sex hormones and the richest Shannon index were found at the third trimester (t3, 33–37 weeks), and these values were reduced significantly after delivery. A higher Shannon index represents a higher diversity for the bacterial community. A similar trend in the Shannon index was obtained during pregnancy and after delivery, suggesting that the diversity of salivary microbial communities might be associated with the fluctuation of sex hormone levels detected in saliva. A possible explanation for the diversity between two groups is that the sex hormone levels promoted the colonization of different oral species[14]. The sex hormones could serve as a nutrient for some bacteria. Kornman et al. have demonstrated that pigmented Prevotella intermedia sensulato and Prevotella melaninogenica are able to substitute progesterone and estradiol for vitamin K, which is needed as an essential factor for their growth[21].
A co-occurrence bacterial community was observed and consisted of the core microbiome in this study. There is substantial evidence that the core microbiome is likely to be a group of bacteria that is stable over time and represents high relative abundance[22, 23]. Findings of the present study are consistent with this observation. From the level of the phylum to that of the genus, the core microbiome represents 99.46% and 95.09%, respectively, of relative abundance in pregnant women. No obvious composition differences could be found in the core microbiome at the phylum level between the two groups. However, more differentiation of low relative abundance taxa between pregnant and non-pregnant women were observed throughout the Lefse and LDA scores. As revealed by the Lefse and LDA scores, fourteen genera that were found to be significantly abundant in pregnant women represented a low relative abundance, and only three of six genera that were enriched in non-pregnant women were from the core microbiome. This interesting observation implies that it is the low abundance bacteria that mainly comprise the community of differential taxa between the pregnant and non-pregnant women. The importance of bacteria with low relative abundance has been widely reported in many fields, and might play an important role in the occurrence and process of diseases[24, 25]. Therefore, bacteria with low relative abundance should not be ignored.
To explore all the relevant bacteria that are associated with the fluctuation of salivary sex hormones during pregnancy, RDA analysis was applied in this study. Forty-three OTUs were observed to be related to the increase in the sex hormone levels, and the other 20 OTUs were negatively correlated with the hormones. This indicates that, among the 63 OTUs detected to be related to pregnancy by the bioinformatics strategy, more than two-thirds of the OTUs in saliva are enriched by the sex hormones. A possible explanation is that the increase in the sex hormones may influence directly the periodontal tissues[26, 27]. For instance, estradiol is reported to be a considerable factor in the proliferation, regeneration and differentiation of gingival epithelial cells and fibroblasts, and subsequently causes a reduced epithelial barrier to invasive bacteria[28]. Moreover, among the 63 hormone-related OTUs, 10 included Prevotella spp. The correlation of Prevotella spp. and pre`gnancy has been widely reported[14, 29]. Dareen Fteita and his colleague have reported that Prevotella nigrescens, Prevotella pallens and Prevotella aurantiaca, which are similar to Prevotella intermedia, have a higher relative abundance as estradiol levels increase during pregnancy[30].
Lactobacillus reuteri, Capnocytophaga gingivalis and Prevotella baroniae were detected by Pearson correlation coefficient to be relevant with the sex hormone levels in pregnant women. Lactobacillus also had higher LDA scores in pregnant group. It implies that Lactobacillus is significantly elevating in pregnant group and sex hormones might play a role in it. Lactobacillus reuteri is often used in the probiotic therapy to prevent and manage the oral inflammatory diseases through immunomodulation and down regulation of the inflammatory cascade. It was reported that Lactobacillus reuteri is useful in treating gingivitis in diabetic patients[31]. Capnocytophaga gingivalis was a periodontopathogenic bacteria. Emerging evidence detected the relationship between Capnocytophaga gingivalis and chronic periodontitis[32]. Santa Cruz I et al. also found that the presence of Capnocytophaga spp. were significantly associated with low-birth weight[33]. Therefore, it was suggested from this study that Lactobacillus reuteri and Capnocytophaga gingivalis might have antagonism effect. Prevotella baronia was found to be a relative of Prevotella intermedia[34]. Prevotella intermedia is a well-known periodontal pathogen and contributes a lot to the progress of pregnancy related gingivitis and periodontitis. Prevotella baronia is also reported to be found in primary endodontic infections and acute apical abscesses[35, 36]. However, direct evidence between sex hormones and Prevotella baronia need further investigations.
In this study, it is unable to avoid that small sample size and other potential factors such as diet habits, work, BMI and vitamin usage may have an impact on the results. Since it is an exploratory analysis, preliminary data were provided to suggest the correlation of salivary microbiome and salivary sex hormones, experimental procedures need to be done to explore the exact evidence between sex hormones and related microbes.