**Background:** The gut microbiota develops rapidly in the first 24 months of life and is critical for immune function, metabolism, and nutrition. Perturbations in early-life microbiota are linked to disease risk. Infants with congenital heart disease (CHD) often require surgery involving cardiopulmonary bypass (CPB) early in life, which may disrupt gut homeostasis and increase the risk of necrotising enterocolitis (NEC), a devastating intestinal condition with reported incidence of 3–9% in infants with CHD and all-cause mortality as high as 38% in cardiogenic NEC. The impact of CPB on the gut microbiome is poorly understood. A small case–control study (Salomon et al.) identified gut microbiota perturbations in children with CHD, with over-representation of proinflammatory bacteria exacerbated by CPB, but was limited by a short postoperative sampling window (24–48 hours) and did not assess intraoperative variables such as aortic cross-clamp time or CPB duration.
**Methods:** This is a single-site prospective cohort study at the National Centre for Paediatric Cardiac Surgery, Children’s Health Ireland (CHI) at Crumlin, Dublin. The target sample is 50 infants with CHD undergoing CPB. Inclusion criteria: full-term infants (>37 weeks gestation) with CHD scheduled for CPB, born in Ireland, with parental consent. Exclusion criteria: stillbirth, healthy infants without underlying illness, participation in another study, infants not undergoing CPB, gastrointestinal pathology or intestinal surgery (excluding gastrostomy tube). Stool samples are collected at seven time points: within 24 hours after birth (TP1), within 24 hours presurgery (TP2), 1 week postsurgery (TP3), 4 weeks postsurgery (TP4), 24 weeks postsurgery (TP5), 52 weeks (TP6), and 2 years of age (TP7). Urine samples are collected at 4–8 weeks postsurgery for metabolomic analysis. Shotgun metagenome sequencing (bioBakery suite: KneadData, MetaPhlAn2, HUMAnN3, StrainPhlAn) will be performed on stool samples. Urine metabolomic analysis will use LC-MS. Demographic, maternal, surgical (CPB duration, cross-clamp time), postoperative (PIM score, ventilation duration, inotrope score, PICU stay), feeding, and complication data (including NEC onset) will be recorded. Healthy age-matched controls (n=192) from the INFANTMET study (Cork University Maternity Hospital) will provide comparison data at 1, 4, 8, and 24 weeks and years 1, 2, and 4.
**Key Results:** This is a protocol paper; no results are reported. The study is ongoing: as of 5 February 2023, 84% of participants had been recruited, and laboratory analysis had been carried out on 25% of study samples. The sample size of 50 is justified by reference to Salomon et al. (17 cases, 12 controls), which was sufficiently powered to detect significant beta-diversity differences (F=5.6, p<0.001). Statistical analysis will include: Shapiro-Wilk normality testing; descriptive statistics (mean±SD for normal, median+IQR for non-normal); χ² tests for categorical variables; t-tests for normally distributed continuous variables; Mann-Whitney U for non-normal variables; subgroup analyses (NEC vs. no NEC, cyanotic vs. acyanotic, feeding mode); alpha diversity (Chao1, Shannon, Simpson; Wilcoxon rank-sum); beta diversity (Aitchison distance, PCA, PERMANOVA via adonis); multivariable associations (MaAsLin2); and sparse canonical correlation analysis for microbe-metabolite associations.
**Clinical Implications:** Understanding the gut microbiota dynamics in infants with CHD undergoing CPB may identify modifiable factors (e.g., mode of delivery, feeding type, antibiotic use) that influence microbiome recovery and clinical outcomes. The study’s extended follow-up (2 years) captures the critical developmental window of microbiome maturation and allows surveillance for NEC, repeat surgery, and mortality. Findings could inform perioperative care strategies, including probiotic administration, to promote a diverse gut microbiota and reduce the risk of NEC and other complications in this vulnerable population. The study is ethically approved (Clinical Research Ethics Committee of Children’s Health Ireland, GEN/826/20) and results will be disseminated to patients, families, and professional societies.