Study cohort and demographics

To interrogate the impact of maternal gestational diet on the neonatal gut microbiome, we examined stool samples collected from neonates enrolled in a larger, prospective, population-based study that sought to characterize the early neonatal microbiome across multiple body sites (gut, skin, oral cavity, nares). Mother–infant dyads (n = 157) were sampled at the time of delivery, with a subset (n = 75) consented for longitudinal sampling at 4–6 weeks postpartum. As shown in Additional file 1: Table S1, the cohort was comprised of primarily Hispanic women (85.4 %), who delivered singleton pregnancies (95.5 %) at term (38.4 weeks). The rate of cesarean deliveries (33.8 %) and preterm birth (10.8 %) were similar to the US national incidence and were not enriched for [24, 25]. For the purposes of this study, we focused our efforts on only the neonatal stool samples collected at the time of delivery (first meconium) and at 4–6 weeks of age. All samples were subjected to standard 16S rRNA gene sequencing and analysis.

Assessment of maternal diet in pregnancy

In order to accurately assess maternal dietary intake during pregnancy, we employed the Dietary Screener Questionnaire (DSQ), which is a rapid dietary screener developed and validated by the National Health and Nutrition Examination Survey (NHANES) program [19, 20]. The DSQ is comprised of 26 questions that ask for the frequency of consumption in the past month of representative food and beverages. Based on the recorded responses, estimates of daily dietary intake of added sugars, fat, and fiber over the past month were determined, which, in our cohort, represented the maternal diet during the latter part of the third trimester.

Comparisons between the captured dietary intake data from the study cohort, the average intake of reproductive-age women in the US, and the recommended daily dietary intake are shown in Table 1. In aggregate, the cohort’s average consumption of added sugars (59.6 ± 42 g) and total intake of fat (33.1 ± 6.1 %) was at or above the Institute of Medicine’s recommended limit, which is largely reflective of the poor quality diet in America. Notably, the average intake of added sugars in our study cohort was significantly less than the reported national average (59.6 ± 42 g versus 68.8 g, p = 0.01). However, this difference is likely explained by the fact that pregnant women who develop gestational diabetes are placed on a diabetic diet that limits carbohydrate intake; we had a significant proportion of gestational diabetics (30 %) in our study cohort [26]. As expected, women with gestational diabetes consumed significantly less added sugars (p < 0.001) but not fiber or fat (Fig. 1a). To further evaluate if the captured dietary intake values were consistent with clinical expectations, we similarly examined if dietary intake correlated with gestational weight gain. Although identifiable risk factors for gestational weight gain are widely variable between studies and thus poorly understood, previous studies have shown no association between dietary composition and gestational weight gain [27, 28]. Consistent with the literature, intake of added sugars, fiber, and fat did not significantly differ with gestational weight gain subject strata (Fig. 1b). In sum, the dietary intake data captured by the DSQ were similar to the average for reproductive-aged women in the US and generally consistent with clinical expectations.

Table 1 Comparison of maternal dietary intake in pregnancy Full size table

Fig. 1 The maternal gestational diet is consistent with clinical expectations. Dietary intake of added sugars (g), fiber (g) and fat (percentage intake) during gestation compared a in mothers with or without gestational diabetes (GDM) or b in mothers with insufficient, normal, or excess gestational weight gain. Significance determined by Student’s t-test (**p = <0.01) Full size image

Impact of maternal fat intake during gestation on the neonatal microbiome

We previously demonstrated in a non-human primate model that, when compared with a control maternal diet (13 % fat), exposure to a maternal high-fat diet (36 % fat) during gestation and lactation persistently altered the offspring microbiome until up to one year of age [17]. To determine if this persistent effect of a maternal high-fat diet could be recapitulated in a human cohort, we modeled the conditions of our primate study by subdividing the study cohort into its extremes, defined as being one standard deviation greater or less than the cohort mean. Using this criterion, a high-fat maternal diet group (n = 13, 43.1 % fat intake) and maternal low-fat diet group (n = 13, 24.4 % fat intake) were identified for further analysis (Additional file 4: Figure S1). Notably, the average percentage dietary fat intake of the low-fat group fell within the recommended fat intake for the general population as indicated by the Institute of Medicine [29]; thus, for this analysis we considered this group as the control reference for the high-fat diet group comparisons and will refer to it as the “control group” from here on. As shown in Table 2, the percentage intake of fat significantly differed between the groups as expected (p < 0.001), while the intake of added sugar and fiber was not significantly different. Other potential confounders, such as pre-pregnancy BMI and mode of delivery, also did not significantly differ between the groups (all p > 0.05), though given the nested case-control design of this study, we may be underpowered to detect differences by these comparisons.

Table 2 Characteristics of groups segregated by extremes of maternal fat intake Full size table

To characterize and quantify the neonatal gut microbiome at the time of delivery, DNA from meconium samples was subjected to culture-independent 16S rRNA gene sequencing and analysis. With this approach, we identified 103 unique taxa classified down to at least the genus level that were represented in more than 10 % of all samples. To examine how maternal gestational diet correlated with the neonatal microbiome as a whole, we first projected the data as a heatmap, with each row representing the relative abundance of each taxa. Unsupervised hierarchical clustering on Euclidean distances revealed that a maternal high-fat diet in gestation varied in association with the neonatal microbiome (Fig. 2a). To further corroborate these findings, we next projected the data by principal coordinate analysis (PCoA) on unweighted UniFrac distances to reduce the dimensionality of the dataset to its components of greatest variation (principal coordinate (PC) axes 1 and 2). The neonatal microbiome again clustered significantly by virtue of maternal gestational diet (Fig. 2b; p = 0.04), which was best explained along the second PC axis (Fig. 2b, c). Notably, the variation of the neonatal microbiome along the second PC axis associated with maternal fat intake was not explained by maternal intake of fiber or added sugar (Fig. 3a; p > 0.05), nor was it confounded by other possible modifiers of the microbiome, including pre-pregnancy BMI, antibiotic usage, mode of delivery, gestational diabetes, or gestational weight gain (Fig. 3a, b; all p > 0.05).

Fig. 2 The neonatal gut microbiome at delivery varies according to maternal fat intake during pregnancy. a Heatmap showing unsupervised hierarchical clustering based on the relative abundance of each genera (columns) present in each meconium sample (rows). The maternal diet group (high-fat versus control) for each meconium sample and the phylum assignment for each genera are indicated by the horizontal and vertical colored bars, respectively. b Principal coordinate analysis of neonatal meconium on unweighted UniFrac distances, with the distribution of the samples along the second principal coordinate axis shown alongside on the right (***p < 0.001, Mann–Whitney U). c Linear regression between maternal gestational dietary fat intake and the second principal coordinate axis indicated by the solid black line with the 95 % confidence interval of the slope shown by the dashed lines Full size image

Fig. 3 Variation of the neonatal gut microbiome is not explained by other potential confounders. a Linear regression between the second principal coordinate axis and maternal intake of fiber, added sugars, maternal pre-pregnancy BMI, and gestational age at delivery. All regression lines were not significantly different from 0, indicating no correlation. b Principal coordinate analysis of neonatal meconium, stratified by intrapartum antibiotic use, gestational diabetes, mode of delivery, antepartum antibiotic use, and gestational weight gain Full size image

We next employed linear effect size (LEfSe) feature selection [23] to identify the specific taxa in the neonatal microbiome that were significantly associated with either a maternal high-fat or control diet. Seven taxa were identified as significant and are shown in Fig. 4 as a heatmap indicating the relative abundance of each taxa in each neonatal meconium sample. Hierarchical clustering of the taxa indicated by the dendrogram along the vertical axis demonstrates the specific signature associated with either dietary group. Notably, exposure to a maternal high-fat diet was significantly associated with an enrichment of Enterococcus and a relative depletion of Bacteroides, which is a known symbiont that aids in the maturation of mucosal immunity (Fig. 4) [30–32].

Fig. 4 Specific taxa in the neonatal meconium significantly associated with maternal gestational diet. Heatmap of taxa in the neonatal meconium identified by LEfSe feature selection that were significantly associated with either a maternal high-fat or maternal control diet during pregnancy Full size image

We next sought to determine if these changes in the neonatal gut microbiome associated with maternal gestational diet persisted beyond delivery. Similar to previous observations [8, 33], comparisons of the neonatal gut microbiome at delivery and at 6 weeks demonstrated significant differences at the phylum and OTU levels (Additional file 4: Figure S2), indicating broad rearrangements of the gut microbiome during this time period. When examining only the infant stool at 6 weeks, unsupervised hierarchical clustering again demonstrated significant clustering by maternal gestational diet (Fig. 5a), while clustering by unweighted PCoA trended toward significance (Additional file 4: Figure S3a; PERMANOVA p = 0.059). LEfSE analysis on the 6-week stool samples identified four taxa that were significantly associated with either the maternal high-fat or control diet group, though only Bacteroides was found to be significantly different at both delivery and 6 weeks (Additional file 4: Figure S3b). As in the neonatal meconium, the relative abundance of Bacteroides in the infant stool at 6 weeks of age was inversely correlated with the maternal fat intake during pregnancy (Fig. 5b; p = 0.02). Although the relative abundance of Enterococcus appeared subjectively higher in the infant stool exposed to a maternal high-fat diet in pregnancy, the correlation was not significant (Fig. 5c; p = 0.20). In addition, none of the other identified taxa at the time of delivery significantly correlated with maternal dietary intake (Additional file 4: Figure S4a; all p > 0.05). Breastfeeding practices have been shown to differentially impact the infant gut microbiome, with the greatest differences seen between those exclusively breast-fed or exclusively formula fed [33]. In our cohort, all infants received both breast milk and formula by the time they were sampled at 6 weeks of age, except for two in the high-fat diet group who were exclusively formula fed. When these two subjects were excluded from analysis, we still observed a significant correlation between the relative abundance of Bacteroides and maternal dietary fat intake during gestation (Additional file 4: Figure S4b; p = 0.04). Altogether, these findings indicate that a maternal high-fat gestational diet is significantly associated with specific alterations to the neonatal and infant gut microbiome, some of which persist to at least 6 weeks of age.