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Page 4 of 19 van Beek et al. Microbiome Res Rep 2025;4:13 https://dx.doi.org/10.20517/mrr.2024.45

Table 1. Median (interquartile range) biomarker concentrations categorised by birth mode and age
5-6 months 11-12 months Total
Vaginal C-section Vaginal C-section Vaginal C-section
Number of samples 54 60 48 54 102 114
IAP mg/g 25.97 (6.20-58.85) 24.18 (16.32-32.73) 14.09 (8.28-23.19) 8.85 (3.14-16.07) 14.4 (3.9-32.7) 20.6 (10.1-29.7)
BPI ng/g 0.00 (0.00-0.00) 0.00 (0.00-0.00) 25.48595 (1.67-42.25) 0.00 (0.00-10.45) 0.00 (0.00-27.3) 0.00 (0.00-7.0)
HD5 pg/g 0.00 (0.00-142.21) 37.50 (0.00-230.32) 0.00 (0.00-34.46) 102.21 (52.49-160.04) 0.00 (0.00-73.2) 96.0 (0.00-184)
ECP pg/g 183.97 (0.00-1,037.84) 198.38 (0.00-558.94) 155.03 (0.00-331.03) 662.31 (488.187-772.91) 183 (0.00-684) 458 (173-672)
IgA μg/g 238.47 (54.26-609.15) 328.80 (227.96-452.58) 253.88 (123.26-1,031.91) 15.35 (11.40-18.67) 307.57 (208.23-497.60) 243 (80.6-905) 309 (208-491)
LTF μg/g 6.91 (2.53-12.25) 10.42 (6.49-16.08) 0.91 (0.34-2.69) 3.96 (1.90-6.68) 2.75 (0.89-9.11) 6.75 (3.75-12.40)
Alb ng/g 2.32 (0.33-20.63) 5.575 (2.74-25.19) 58.07 (7.31-198.96) 9.51 (6.39-17.34) 12.3 (1.91-72.1) 8.89 (4.50-22.6)
LCN2 ng/g 1.16 (0-5.47) 1.77 (0.00-17.51) 0.01 (0.00-22.70) 0.00 (0.00-1.24) 0.83 (0.00-8.95) 0.00 (0.00-6.55)
Cal ng/g 354.20 (88.01-1,602.02) 1,857.89 (1,199.08-2,707.68) 167.22 (0.00-465.38) 669.41 (266.36-1,958.44) 231 (21.0-842) 1,341 (601-2,542)
Tot.prot. mg/g 3.76 (0.31-13.79) 6.95 (0.00-17.16) 5.34 (0.87-8.37) 5.17 (0.46-11.8) 13.1 (6.16-17.6)
Muc2 ng/g 0.71 (0.30-2.42) 45.04 (22.53-75.84) 1.96 (0.20-7.41) 3.87 (1.75-7.99) 0.98 (0.24-5.81) 14.9 (4.00-51.5)
Lysozyme ng/g 0 (0-2.05) 0.00 (0.00-0.00) 0.00 (0.00-0.00) 0.9780727 (0-2.07) 0 (0-1.13) 0 (0-1.74)

Zonulin ng/g 0.0 (0.0-0.34) 0.7 (0.0-1.30) 1.5 (0.8-1.80) 0 (0-0) 0.7 (0-1.5) 0.0 (0-0.7)
Bristol 6 (6-6.00) 6 (6-6.00) 5 (5-5.00) 5 (5-5.75) 6.00 (5.00-6.00) 6.00 (5.00-6.00)
pH 6.065 (5.70-6.90) 7.240 (6.98-7.61) 7.220 (7.07-7.42) 6.600 (6.36-6.93) 6.95 (5.91-7.27) 6.97 (6.57-7.29)
Total bacteria (log 10) genomes/g 25.19 (23.98, 29.93) 28.02 (26.43, 29.93) 27.82 (24.75, 28.89) 25.24 (24.47, 26.80) 26.50 (24.40-29.41) 26.63 (25.45-28.40)

Tested biomarkers: Intestinal alkaline phosphatase (IAP), bactericidal/permeability-increasing protein (BPI), human alpha defensin 5 (HD5), eosinophil cationic protein (ECP), immunoglobulin A (IgA), lactoferrin
(LTF), albumin (Alb), lipocalin 2 (LCN2), calprotectin (Cal), mucin 2 (Muc2), Lysozyme and zonulin, total protein and total bacterial levels, pH, and Bristol score. All data points were included.

beating tube from the Ambion MagMAX Total Nucleic Acid Isolation Kit (Life Technologies). After three rounds of repeated bead-beating in a FastPrep®-96
TM
instrument (MP Biomedicals, Santa Ana, CA, United States) at a speed of 800 rpm for 60 s, the lysate was collected. A second round of bead beating was
conducted with 145 μL of fresh RBB buffer, repeating 3 times for 60 s each, to lyse the remaining intact cells. Pooled supernatant (250 μL) was used for DNA
extraction with a KingFisher Flex automated purification system (ThermoFisher Scientific) using a MagMAX Pathogen High Vol. DNA was quantified
TM
TM
using Quanti-iT Pico Green dsDNA Assay (Invitrogen) .
TM
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