Long-term effect of caesarean section on the gut microbial taxonomical profile and metabolic function of children at pre-school age

To the Editor: In this study, we observed the long-term effect of caesarean section (CS) on the gut microbiome of pre-school age children, both on the microbial taxonomical profile and metabolic function. Interestingly, taxonomical differences due to CS were mostly found in children of younger age, but the microbial functional alterations were observed in children of older age. Gut microbiome is constantly evolving and adapting to the surrounding environments, especially during the early months of life when the microbial composition is undergoing turbulent changes.1 The gut microbiome of children tends to stabilise after 3 years of age,2 but continues to mature slowly thereafter.3 To date, only limited studies3, 4 have investigated the effect of CS on gut microbial profile of children beyond infancy (up to 12 months). These studies employed fluorescence in situ4 or 16S rRNA3 for microbial analysis, which were insufficient to explore microbial functions. We conducted this study with metagenomic sequencing method to examine the effect of CS on the gut microbiome of children of pre-school age. Between March and April 2017, 2199 children were recruited from 16 public kindergartens in Guangzhou, China. Faecal samples were collected and randomly selected (n = 1104) for gut microbial analysis. Among these children, 1034 with valid data regarding their parental, perinatal and birth characteristics derived from parent-administered questionnaires were included in this analysis. All participants provided written consents, and this study was approved by Guangzhou Women and Children's Medical Center ethic committee. Detailed methodologies regarding the study design, microbial analysis for faecal samples and statistical analysis are available in Supporting Information. Forty-three percent of the participants were delivered via CS (Table 1). CS-born children had higher weight Z-score at birth (.30 vs. .07, p < .001) than those delivered vaginally. Mothers who gave birth via CS were significantly elder at sampling (33 vs. 32 years, p < .001), had higher pre-pregnancy body mass index (BMI) (p < .001) than those who underwent vaginal delivery. Overall, for the entire population, the top five most abundant microbial phylum were Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Verrucomicrobia (Figure 1A), together representing nearly 96% of the total microbial composition. CS-born children scored significantly lower for richness (p = .019, FDR p = .076, FDR p = .110 for adjustment by age and sex; Figure 1B), but were similar to that of those born vaginally regarding other indices. The microbial beta-diversity of children born via different modes was significantly different after adjustment for age and sex (Figure 1C, Bray–Curtis p = .001, Jaccard p = .002). Significant enrichment of Clostridium spp. was observed in children born via CS as compared to those born vaginally (Figure 1E), and many of these are classified as opportunistic pathogens. Clostridium bolteae were found to be enriched in children of autism spectrum disorder (ASD) than in children without it.4 Clostridium ramosum enhances lipids uptake and hence promotes obesity as demonstrated in the in vitro study.6 In addition, several gene segments of C. bolteae, Clostridium clostridioforme and C. ramosum have been related to the antimicrobial resistance in various cultures.7 Altogether, these strains might possess adverse health implications on the host. Stratification by age groups revealed that the microbial beta-diversity was significantly different between children born via different modes of delivery in those of younger age only (p = .037 for age under 5, p = .012 for age 5, p = .744 for age above 5), although no differences for alpha-diversity in all age groups (Figure S1). Differences in microbial profile were mainly found at or below 5 years (age ranges were ≥60 and <72 months, <60 months, respectively; Figure S2). Notably, 51 downregulated pathways, mostly related to vitamin metabolism (35%), were found in CS born children aged above 5 (≥72 months), when compared to their vaginally born counterparts (Figure 2E,F; Table S1). It is possible that the children are exposed to more complexed environmental factors and food system over time, which then replaced mode of delivery and became the key determinants of microbial composition.2, 3 Previous meta-transcriptomic analysis has shown that the ability of gut microbiome to produce K and B group vitamins are at comparable levels across healthy population,8 and these microbiome-derived vitamins collectively contribute to 30% of our dietary requirement, lack of which might have adverse health consequences on the host. It is known that breastfeeding experience shapes the trajectory of gut microbial development,9 whether breast milk (BM) could ameliorate the adverse effect of CS on childhood gut microbiota is therefore of interest to explore. Over 60% CS-delivered children were fed on a predominantly BM diet within their first 6 months of life (Table S2). Infant formula (IF)-fed CS-born children scored lowest for gut microbial richness, as compared to either vaginally born or BM-fed CS-born children, although no other alpha-diversity indices were different (Table S3). On the whole population level, it seems that BM feeding ameliorated CS-induced changes in gut microbial metabolic functions. However, when breakdown by age groups (Tables S4–S6), for children born via same delivery mode, only minor differences in the gut microbial profile and metabolic activities were found to be due to early feeding practices. Our previous finding was largely driven by the younger age group, in whom the effect of BM is likely to be more substantial.9 This is the first study to examine the effect of CS on the gut microbiome of children at pre-school age using metagenomic sequencing. Recall bias was unavoidable due to information collected through questionnaire in a retrospective manner. In addition, information regarding medical history, the use of antibiotics and diet were not collected in this trial, both of which could substantially affect the gut microbiome of children. It must be acknowledged that our samples were derived from a single time point, limiting interpretation of changes or trajectory of gut microbiome of children over time. In addition, our interpretation regarding altered metabolic functions was based on metagenomic sequencing results only. Further study should combine the use of meta-transcriptomic data to confirm these observations. In conclusion, we found that CS-induced microbial change in pre-school age children is featured by enrichment of Clostridium spp. The altered microbial metabolic functions in older children are mostly related to vitamin metabolism, consequences of which remain to be explored. We would like to thank all families that have participated in this study, and the coordinating staff members on the grounds. We would also like to acknowledge BGI Shenzhen that has performed the microbial analysis work on our behalf. We are grateful to Prof. Jing-Yuan Fu and Dr. Hong-Wei Wang for their valuable feedbacks on our manuscript. The authors declare they have no conflicts of interest. Key Program of GuangDong Basic and Applied Basic Research Foundation, Grant Number: 2022B1515120080, 2020B1111170001; China Postdoctoral Science Foundation, Grant Number: 2022M710883; National Natural Science Foundation of China, Grant Number: 82173525, 82003471; GuangDong Basic and Applied Basic Research Foundation, Grant Number: 2021A1515110194; Guangzhou Science and Technology Project, Grant Number: 202201020656. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.