European Journal of Cardiovascular Medicine

The establishment of the neonatal gut microbiome is a dynamic and critical process that begins at birth and has long-lasting implications for health and disease. This complex community of microorganisms influences key physiological functions such as immune system maturation, gastrointestinal barrier integrity, nutrient metabolism, and resistance to pathogenic colonization [1]. Disruptions in the early microbial ecosystem—commonly referred to as dysbiosis—have been linked to a spectrum of non-communicable diseases including allergies, obesity, autoimmune conditions, and neurodevelopmental disorders [2].

Among the earliest and most influential factors shaping the neonatal microbiome is the mode of delivery. Vaginal birth typically results in the vertical transmission of maternal vaginal and intestinal flora, facilitating the colonization of beneficial anaerobes such as Lactobacillus, Bifidobacterium, and Bacteroides species [3]. In contrast, cesarean section, especially when performed electively before the onset of labor, bypasses this natural exposure and favors colonization by skin-associated and environmental bacteria such as Staphylococcus and Clostridium [4]. These differences are not merely transient; emerging evidence suggests that the microbial disparities observed in cesarean-born neonates may persist for months or even years, with potential implications for immune and metabolic programming [5].

The increasing global prevalence of cesarean deliveries, particularly in urban and institutional settings, has raised concerns about its unintended biological consequences. While cesarean sections are often life-saving, their routine use without strict medical indications may inadvertently contribute to long-term health vulnerabilities in offspring [6]. Recognizing this, researchers have begun to explore interventions aimed at restoring microbial exposure, such as vaginal microbial seeding and probiotic supplementation [7]. However, the foundational step remains a precise understanding of how delivery mode alters the initial microbial landscape.

Despite advancements in microbiome research, most available data are derived from high-income countries with different sociocultural, dietary, and environmental contexts. There is a paucity of robust prospective data from developing regions such as South India, where microbial exposures may differ markedly. Moreover, the impact of cesarean delivery on microbiome development in such populations remains poorly characterized, limiting our ability to formulate context-specific recommendations [8].

This study was undertaken to evaluate the influence of delivery mode on the composition and diversity of the neonatal gut microbiome during the early postnatal period. By employing 16S rRNA sequencing to profile microbial communities in stool samples collected within 48 hours of birth, this research aims to provide empirical evidence on how vaginal versus cesarean birth modulates the neonatal microbial milieu. The study further seeks to inform strategies for microbiome restoration and optimize neonatal health outcomes, particularly in settings where cesarean delivery rates are rising.

Study Design and Setting

This was a hospital-based, prospective cohort study conducted in the Department of Obstetrics, Dhanalakshmi Srinivasan Medical College and Hospital, Siruvachur, over a 12-month period from August 2023 to July 2024. The primary objective was to compare neonatal gut microbiota composition based on the mode of delivery—vaginal versus elective cesarean section.

Participant Selection

A total of 160 healthy term neonates (≥37 weeks gestation) born to mothers aged 20–35 years were included in the study. Based on delivery mode, participants were categorized into two groups:

• Vaginal Delivery Group (n = 80): Neonates delivered through spontaneous vaginal delivery or assisted vaginal birth after labor onset.
• Cesarean Section Group (n = 80): Neonates delivered via elective cesarean section without labor or rupture of membranes.

Exclusion criteria were: maternal antibiotic use within one week prior to delivery, intrapartum or neonatal antibiotic administration, presence of congenital anomalies, NICU admission, and any evidence of neonatal sepsis or gastrointestinal pathology.

Data Collection

Maternal demographic details, delivery mode, intrapartum antibiotic exposure, gestational age, and neonatal anthropometrics were recorded using a standardized case record form. Stool samples were collected from neonates within the first 48 hours of life using sterile rectal swabs and stored in DNA/RNA Shield buffer at −80°C until processing.

Microbiome Analysis

Genomic DNA was extracted using the QIAamp DNA Stool Mini Kit. Bacterial 16S rRNA gene sequencing was performed targeting the V3–V4 hypervariable regions using the Illumina MiSeq platform. Raw sequences were quality-checked, trimmed, and clustered into operational taxonomic units (OTUs) at 97% similarity using QIIME 2. Taxonomic assignment was conducted using the SILVA reference database.

Outcome Measures

The primary outcomes were alpha diversity (Shannon and Simpson indices), beta diversity (Bray-Curtis dissimilarity), and relative abundance of dominant bacterial phyla and genera. Secondary outcomes included identification of distinct microbial signatures between delivery modes.

Statistical Analysis

Statistical analysis was performed using R (v4.2.0) and SPSS software. Continuous variables were reported as mean ± SD and compared using independent t-tests or Mann–Whitney U tests as appropriate. Categorical variables were analyzed using chi-square or Fisher’s exact test. Beta diversity differences were assessed via permutational multivariate analysis of variance (PERMANOVA). A p-value <0.05 was considered statistically significant.

Ethical Approval

Ethical clearance for the study was obtained from the Institutional Ethics Committee. Written informed consent was obtained from all participating mothers prior to enrollment.

Table 1: Baseline Demographic Characteristics of Mothers and Neonates

Table 2: Alpha Diversity Indices of Neonatal Gut Microbiota

Table 3: Relative Abundance of Dominant Bacterial Genera

Table 4: Identification of Unique Genera in Each Group

Fig 1: Distribution of bacteria by mode of delivery

The demographic comparison between neonates born via vaginal delivery and those delivered through cesarean section revealed no statistically significant differences in maternal age (27.8 ± 3.6 vs 28.1 ± 3.2; p = 0.542), gestational age (39.1 ± 0.8 vs 38.9 ± 0.9 weeks; p = 0.178), or birth weight (3050 ± 320 g vs 3120 ± 340 g; p = 0.209). This suggests comparability between groups and supports unbiased microbial comparison.

Alpha diversity indices demonstrated significantly greater microbial richness and evenness in the vaginal delivery group. The Shannon Index (3.8 ± 0.5) and Simpson Index (0.84 ± 0.07) were markedly higher compared to those in the cesarean section group (2.9 ± 0.4 and 0.72 ± 0.09, respectively), with p < 0.001 for both metrics. OTU richness also favored the vaginal group (112.4 vs 89.3; p < 0.001), indicating a more diverse gut ecosystem.

Relative abundance analysis showed that neonates born vaginally had significantly higher proportions of beneficial anaerobes such as Lactobacillus (21.6% vs 8.7%), Bifidobacterium (18.3% vs 9.1%), and Bacteroides (14.8% vs 5.4%). Conversely, Staphylococcus (4.3% vs 18.6%) and Clostridium (3.7% vs 13.9%) were significantly more dominant in the cesarean section group, suggesting skin and environmental microbial transmission.

Beta diversity analysis via PERMANOVA confirmed significant overall differences in microbial community structures between the two delivery modes (R² = 0.182, p = 0.002). Unique genera such as Prevotella were identified exclusively in the vaginal group, while Propionibacterium appeared predominantly in cesarean-born neonates

The early postnatal period is a critical window for microbial colonization, and the mode of delivery is one of the most influential determinants of the initial gut microbiota in neonates. This study aimed to evaluate how cesarean section versus vaginal birth affects the composition and diversity of neonatal gut microbiota using high-throughput 16S rRNA sequencing.

The rationale for this investigation lies in the growing body of evidence suggesting that alterations in early-life microbial exposure may predispose children to chronic diseases such as obesity, asthma, and immune dysregulation later in life [9]. With cesarean delivery rates increasing globally—including in South India—there is an urgent need to understand the microbial consequences of bypassing the maternal vaginal and fecal microbial transfer that typically occurs during natural childbirth [10].

Our findings confirm that neonates delivered vaginally harbor significantly more diverse and beneficial microbial communities than those born via elective cesarean section. Alpha diversity indices, including the Shannon and Simpson indices, were substantially higher in the vaginal group, reflecting greater microbial richness and evenness. Similar observations were reported by Dominguez-Bello et al., who demonstrated that vaginal delivery facilitates colonization by commensal genera such as Lactobacillus and Bacteroides, whereas cesarean delivery favors skin-associated taxa like Staphylococcus [11].

Importantly, cesarean-born neonates in our study exhibited higher proportions of potential opportunistic pathogens, including Clostridium, which has been implicated in early gastrointestinal disturbances and may contribute to inflammatory profiles in infancy [12]. The beta diversity analysis further supported the compositional distinction between the two groups, showing statistically significant clustering based on delivery mode.

From a clinical perspective, these findings underscore the importance of preserving natural childbirth where medically appropriate and offer a microbiological rationale against non-indicated cesarean deliveries. In cases where cesarean sections are unavoidable, emerging strategies such as vaginal microbial transfer and targeted probiotic interventions may help restore microbial balance, although these approaches remain under investigation [13].

This study highlights the significant influence of delivery mode on the initial composition and diversity of the neonatal gut microbiome. Neonates delivered vaginally exhibited greater microbial richness and a higher relative abundance of beneficial genera such as Lactobacillus, Bifidobacterium, and Bacteroides. In contrast, cesarean-born neonates showed reduced diversity and increased colonization by skin and environmental microbes, including Staphylococcus and Clostridium. These compositional differences may have implications for immune development and disease susceptibility in later life. Given the rising cesarean delivery rates, these findings underscore the need for judicious use of cesarean section and support the exploration of microbiome-restorative strategies. Early microbial modulation through delivery mode awareness could be a foundational step in neonatal and long-term preventive healthcare.

Acknowledgment

The authors would like to thank the staff and faculty of the hospital for their continuous support.

Conflicts of Interest

The authors declare no conflicts of interest related to this research.

1. Milani C, Duranti S, Bottacini F, Casey E, Turroni F, Mahony J, et al. The first microbial colonizers of the human gut: composition, activities, and health implications of the infant gut microbiota. Microbiol Mol Biol Rev. 2017;81(4):e00036-17.
2. Arrieta MC, Stiemsma LT, Amenyogbe N, Brown EM, Finlay B. The intestinal microbiome in early life: health and disease. Front Immunol. 2014;5:427.
3. Wampach L, Heintz-Buschart A, Hogan A, Muller EEL, Narayanasamy S, Laczny CC, et al. Colonization and succession within the human gut microbiome by archaea, bacteria, and microeukaryotes during the first year of life. Front Microbiol. 2017;8:738.
4. Shao Y, Forster SC, Tsaliki E, Vervier K, Strang A, Simpson N, et al. Stunted microbiota and opportunistic pathogen colonization in caesarean-section birth. Nature. 2019;574(7776):117–121.
5. Tamburini S, Shen N, Wu HC, Clemente JC. The microbiome in early life: implications for health outcomes. Nat Med. 2016;22(7):713–722.
6. Betrán AP, Ye J, Moller AB, Zhang J, Gülmezoglu AM, Torloni MR. The increasing trend in caesarean section rates: global, regional and national estimates. PLoS One. 2016;11(2):e0148343.
7. Korpela K, de Vos WM. Early life colonization of the human gut: microbes matter everywhere. Curr Opin Microbiol. 2018;44:70–78.
8. Das B, Goswami D, Singh D, Biswal D, Singh V, Vohra H, et al. Human gut microbiota and its association with health and diseases. J Microbiol Biotechnol. 2018;28(7):1031–1037.
9. Bäckhed F, Roswall J, Peng Y, Feng Q, Jia H, Kovatcheva-Datchary P, et al. Dynamics and stabilization of the human gut microbiome during the first year of life. Cell Host Microbe. 2015;17(5):690–703.
10. Blaser MJ, Dominguez-Bello MG. The human microbiome before birth. Cell Host Microbe. 2016;20(5):558–560.
11. Dominguez-Bello MG, Costello EK, Contreras M, Magris M, Hidalgo G, Fierer N, et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci U S A. 2010;107(26):11971–11975.
12. Penders J, Thijs C, Vink C, Stelma FF, Snijders B, Kummeling I, et al. Factors influencing the composition of the intestinal microbiota in early infancy. Pediatrics. 2006;118(2):511–521.
13. Cunnington AJ, Sim K, Deierl A, Kroll JS, Brannigan E. “Vaginal seeding” of infants born by caesarean section. BMJ. 2016;352:i227.