Gut Microbiota Maturation and Neurological Injury in Extremely Preterm Infants

Gut Microbiota Maturation and Neurological Injury in Extremely Preterm Infants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Gut Microbiota Maturation and Neurological Injury in Extremely Preterm Infants Li-Ping Xu, Chun Xu, Xiaohong Fang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7858615/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: To examine the association between gut microbiota maturation and neurological injury in extremely preterm infants. Study Design: We prospectively studied 75 extremely preterm infants (28–<32 weeks, 1.0–<1.5 kg) and 20 term-born controls. Neurological outcomes were assessed by neuroimaging, amplitude-integrated EEG, and developmental evaluations up to 3 months corrected age. Stool samples collected on days 3, 10, and 20 were analyzed by 16S rDNA sequencing. Results: At day 3, preterm infants had reduced diversity versus term controls (P<0.01) and were enriched in facultative anaerobes. By day 20, neurologically normal infants showed increased colonization by Bifidobacterium and Akkermansia , whereas injury group infants retained facultative-dominated profiles. Beneficial taxa correlated positively with neurodevelopmental scores, while Enterococcus correlated negatively. Conclusions: Delayed microbial maturation is associated with neurological injury in extremely preterm infants and may serve as an early biomarker and target for intervention. Health sciences/Diseases/Neurological disorders/Paediatric neurological disorders Biological sciences/Neuroscience/Neurogenesis/Developmental neurogenesis extremely preterm infants gut microbiota neurological injury microbiome–gut–brain axis Bifidobacterium Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Neurological injury remains a major cause of morbidity and adverse neurodevelopmental outcomes among extremely preterm infants 1 . Despite advances in neonatal intensive care, survival improvements have not been accompanied by equivalent reductions in neurological impairment 2 . These complications contribute substantially to the burden of cerebral palsy, cognitive deficits, and long-term disability, underscoring the need for early biomarkers and novel preventive strategies. Recent evidence suggests that the gut microbiota plays a key role in early brain development via the microbiome–gut–brain axis 3 – 5 . Normal colonization involves a transition from facultative to strict anaerobes, with the establishment of Bifidobacterium, Bacteroidota, and Lachnospiraceae as critical milestones. Preterm birth disrupts this process, leading to delayed colonization by beneficial taxa and overrepresentation of facultative anaerobes or opportunistic pathogens 6 . These alterations may influence neurodevelopment through immune modulation, microbial metabolite production, and regulation of the hypothalamic–pituitary–adrenal axis 7 – 9 . Experimental models provide mechanistic insights. Dysbiosis during critical developmental windows impairs myelination, alters microglial activation, and disrupts synaptic plasticity 10 , 11 . Moreover, specific microbial metabolites, including short-chain fatty acids and tryptophan derivatives, regulate microglial maturation, blood–brain barrier integrity, and neurotransmitter pathways 12 – 16 . Disruption of these processes may contribute to the vulnerability of the preterm brain. Human data linking gut microbiota development and neurological injury remain limited. Most prior studies have been retrospective, lacked term-born controls, or focused primarily on sepsis and necrotizing enterocolitis rather than direct neurological outcomes 17 – 19 . Seki et al. demonstrated aberrant microbiota–immune–brain axis development in premature neonates with brain damage 2 , while Niemarkt et al. highlighted the role of the gut–brain axis in necrotizing enterocolitis-related brain injury 11 . Recent work also suggests that early microbial trajectories are closely associated with neurobehavioral development 18 , 19 . However, prospective studies integrating longitudinal microbial analyses with imaging and standardized neurodevelopmental assessments are scarce. In this study, we conducted a prospective cohort analysis of extremely preterm infants and healthy term-born controls. Serial stool samples were collected during the first three weeks of life and analyzed by 16S rDNA sequencing. Neurological outcomes were evaluated using cranial imaging, amplitude-integrated EEG, and standardized developmental assessments up to six months corrected age. We hypothesized that extremely preterm infants with neurological injury would show delayed transition from facultative to strict anaerobic colonization, with persistent enrichment of opportunistic taxa and reduced abundance of beneficial genera, compared with infants without injury and term controls. METHODS Study Design and Participants This prospective cohort study was conducted at the Neonatal Intensive Care Unit (NICU) of Zhangzhou Municipal Hospital (Fujian, China) between February 1, 2023, and January 31, 2024. Eligible participants were extremely preterm infants with a gestational age between 28 and < 32 weeks or a birth weight between 1.0 and < 1.5 kg, admitted to the NICU within 24 hours after birth. None had received antibiotics, probiotics, or prebiotics prior to enrollment. Infants were included only if their clinical condition improved sufficiently to allow hospital discharge and their caregivers agreed to follow-up until a corrected age of at least three months. Infants were excluded if they had congenital anomalies involving the nervous or gastrointestinal systems, neurological abnormalities attributable to genetic, metabolic, or viral causes, or if they were withdrawn from the study, transferred to another facility, or died before completing follow-up assessments. Additional exclusion criteria included severe intracranial pathology detected within the first three days of life, defined as intraventricular hemorrhage grade III or higher according to Papile’s classification, or periventricular leukomalacia. Intraventricular hemorrhage was graded as follows: grade I, confined to the germinal matrix; grade II, blood occupying ≤ 50% of the ventricular volume; grade III, blood occupying > 50% of the ventricular volume; and grade IV, hemorrhagic infarction involving the periventricular white matter 20 , 21 . A total of 75 extremely preterm infants met the inclusion criteria and were enrolled. For comparison, 20 healthy term-born infants (gestational age ≥ 37 weeks, birth weight ≥ 2.5 kg) matched for birth date and delivery mode were recruited as controls for gut microbiota assessment at postnatal day 3. Clinical Data Collection Demographic and clinical characteristics were extracted from medical records using a standardized form, including gestational age, birth weight, sex, delivery mode, Apgar scores at 5 and 10 minutes, head circumference at birth and discharge, feeding type (exclusive breast milk, mixed feeding, or exclusive formula), antibiotic use during hospitalization, major diagnoses, complications, and length of hospital stay. Data entry accuracy was verified independently by two researchers. Neurodevelopmental Assessment Neurological evaluations were performed during hospitalization and at follow-up. Cranial ultrasonography was conducted on postnatal days 3, 7, and 14, then every two weeks for infants without abnormalities or weekly for those with abnormal findings until discharge. Amplitude-integrated electroencephalography (aEEG) was performed at postnatal days 1–3, days 7–8, and at corrected ages of 32 and 36 weeks to assess background activity, sleep–wake cycling, and seizure patterns. Before discharge, cranial magnetic resonance imaging (MRI) was performed to detect structural brain injury. Post-discharge follow-up was performed at corrected ages of 40 weeks and 3 months. The Neonatal Behavioral Neurological Assessment (NBNA) was conducted at 40 weeks, and the General Movements Assessment (GMs) was conducted at 3 months. Infants with abnormal neuroimaging findings (cranial ultrasonography or MRI) or seizure activity detected by amplitude-integrated EEG during hospitalization, and who showed abnormal GMs at 3 months corrected age (defined as F−, reduced fidgety movements), were classified into the neurological injury group. In contrast, infants with consistently normal neuroimaging during hospitalization, no seizure activity on amplitude-integrated EEG, and normal GMs at 3 months (defined as NF, normal fidgety movements), were classified into the neurologically normal group. Cranial imaging abnormalities were defined as intraventricular hemorrhage or periventricular leukomalacia detected on any cranial ultrasound or MRI performed during hospitalization 1 , 22 . Fecal Sample Collection and DNA Extraction Fecal samples were collected from each infant at postnatal days 3, 10, and 20 (day 1 defined as the first 24 hours of life). Fresh stool was obtained from diapers using sterile swabs, placed into pre-labeled sterile 5 mL microcentrifuge tubes, and immediately stored at − 80°C until analysis. Genomic DNA was extracted from ~ 30–50 mg of stool using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, with an added bead-beating step for Gram-positive bacterial lysis. DNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). 16S rDNA Sequencing and Bioinformatic Analysis The V3–V4 hypervariable region of the 16S rRNA gene was amplified using primers 341F (5’-CCTACGGGNGGCWGCAG-3’) and 805R (5’-GACTACHVGGGTATCTAATCC-3’). PCR products were purified, quantified, and sequenced on the Illumina MiSeq platform (2 × 300 bp paired-end reads). Raw reads were processed with QIIME2 (version 2023.2) and denoised with DADA2 using a minimum quality score threshold of Q30. Chimeric sequences were removed, and taxonomy was assigned using the SILVA 138 database. Samples were rarefied to the minimum sequencing depth across all samples. Alpha diversity (Chao1, Shannon indices) and beta diversity (Bray–Curtis, weighted UniFrac) were calculated. Beta diversity differences were tested with PERMANOVA (999 permutations). Differential taxa were identified with LEfSe (LDA score > 2.0, adjusted P < 0.05). Statistical Analysis Continuous variables were tested for normality (Shapiro–Wilk test). Normally distributed variables are presented as mean ± standard deviation and compared using Student’s t-test or ANOVA. Non-normally distributed variables are expressed as median (interquartile range) and compared using the Wilcoxon rank-sum or Kruskal–Wallis tests. Categorical variables are presented as counts (%) and compared using chi-square or Fisher’s exact test. Microbiota differential abundance results were corrected for multiple testing using the Benjamini–Hochberg method. Analyses were performed in R (version 4.3.1) and SPSS Statistics (version 26.0; IBM Corp., USA), with P < 0.05 considered significant. RESULTS Participant Characteristics A total of 75 extremely preterm infants were enrolled, including 31 with neurological injury and 44 without injury, along with 20 healthy term-born controls. Baseline characteristics are summarized in Table 1 . Severe complications included late-onset sepsis, bronchopulmonary dysplasia grade ≥ III 13 , necrotizing enterocolitis stage ≥ II 11 , and retinopathy of prematurity requiring treatment 14 . Table 1 Clinical characteristics of the study population. *Values presented as mean ± SD, median (IQR), or frequency (%); significant differences indicated by * ( P < 0.05). Note: Severe complications include late-onset sepsis, bronchopulmonary dysplasia grade ≥ III, necrotizing enterocolitis stage ≥ II, and retinopathy of prematurity requiring treatment. Characteristic Normal group (n = 44) Injury group (n = 31) P -value Gestational age (weeks) 30.5 ± 1.2 30.3 ± 1.1 0.45 Birth weight (kg) 1.4 ± 0.2 1.3 ± 0.2 0.37 Head circumference (cm) 27.2 ± 1.0 26.9 ± 1.1 0.32 Male sex, n (%) 24 (54.5%) 17 (54.8%) 0.98 Cesarean delivery, n (%) 30 (68.2%) 22 (71.0%) 0.79 Antibiotic exposure, n (%) 36 (81.8%) 27 (87.1%) 0.53 Apgar score (5 min) 8 (7–9) 8 (7–9) 0.71 Apgar score (10 min) 9 (8–10) 9 (8–10) 0.68 Hospital stay (days) 46.2 ± 17.3 55.3 ± 17.7 0.03* Feeding type Breast milk, n (%) 24(54.5%) 12(38.7%) 0.29 Mixed, n (%) 6(13.6%) 8(25.8%) Formula, n (%) 14(31.8%) 11(35.5%) Incidence of severe complications (n%) 2(4.5%) 2(6.4%) 0.72 *Values presented as mean ± SD, median (IQR), or frequency (%); significant differences indicated by * ( P <0.05). Note: Severe complications include late-onset sepsis, bronchopulmonary dysplasia grade ≥III, necrotizing enterocolitis stage ≥II, and retinopathy of prematurity requiring treatment. The two preterm subgroups were comparable in gestational age, birth weight, sex, and delivery mode and the incidence of severe complications (P > 0.05 for all). Feeding type distribution (exclusive breast milk, mixed, exclusive formula) did not differ significantly between preterm subgroups. The neurological injury group had a significantly longer median hospital stay compared with the neurologically normal group (55.29 ± 17.72 days vs 46.20 ± 17.34 days, P = 0.03). Gut microbiota diversity At postnatal day 3, preterm infants exhibited a distinct gut microbiota profile compared with term-born controls (Fig. 1 ). Genus-level composition (Fig. 1 A) showed that facultative anaerobes—Enterococcus, Streptococcus, and Pseudomonas—dominated in preterm infants, whereas term controls had higher abundances of Bifidobacterium. Alpha diversity (Chao1 index) showed significant difference between groups (P <0.01; Fig. 1 B), and beta diversity (Bray–Curtis distance) revealed significant structural separation (Wilcoxon, P = 2.2 × 10⁻¹ 6 ; Fig. 1 C). Longitudinal microbiota composition Across postnatal days 3, 10, and 20, microbial diversity in preterm infants increased progressively (Fig. 2 ). Taxonomic succession analyses (Fig. 2 A) revealed dynamic changes from facultative to strict anaerobes. Beta-diversity trajectories (Fig. 2 B) demonstrated significant temporal shifts in microbial community composition (Kruskal–Wallis, P = 1.4 × 10⁻¹¹), indicating overall maturation of gut ecosystems during early life. Comparative composition between neurological-injury and normal groups At each time point, the genus-level compositions differed between the neurological-injury and neurologically normal groups (Fig. 3 ). Facultative anaerobes (Enterococcus, Streptococcus, Pseudomonas) remained predominant in the injury group throughout the first 3 weeks, whereas strict anaerobes (Bifidobacterium, Akkermansia, Veillonella) increased markedly in the normal group, reflecting faster microbial maturation. Effect-size comparison showed that Bifidobacterium abundance at day 10 was approximately 2.5-fold higher in the normal group (P < 0.01). Relative abundance of key taxa Bar-plot analyses (Fig. 4 ) summarized the relative abundances of dominant genera across groups. Strict anaerobes such as Bifidobacterium, Akkermansia, and Bacteroides were more abundant in the normal group, while facultative anaerobes including Enterococcus, Streptococcus, and Pseudomonas predominated in the injury group. Correlations with neurological outcomes LEfSe analysis (Fig. 5 ) identified genera that significantly discriminated between the two preterm subgroups (LDA > 2.0, adjusted P < 0.05). Bifidobacterium, Akkermansia, and Veillonella were enriched in the neurologically normal group, whereas Enterococcus, Streptococcus, and Alloprevotella were characteristic of the neurological-injury group. These findings confirm that delayed establishment of strict anaerobes and persistence of facultative taxa are closely associated with neurological injury in extremely preterm infants. Spearman correlation analyses demonstrated significant positive associations of abundances of Bifidobacterium (r = 0.42, P < 0.01) and Akkermansia (r = 0.30, P < 0.05) in day 10 and day 20 with NBNA scores at term-equivalent age. Conversely, Enterococcus abundance was negatively correlated with NBNA scores (r = -0.23, P < 0.05) (Table 2 ). These associations remained significant after adjustment for gestational age, feeding type, and antibiotic exposure. Table 2 Spearman correlations between gut microbiota taxa and NBNA scores at term-equivalent age D10 D20 Enterococcus Bifidobacterium Akkermansia Bifidobacterium Akkermansia r -0.23 0.42 0.2 0.26 0.30 P 0.04 <0.01 0.042 0.02 <0.01 DISCUSSION In this prospective cohort study, we demonstrated that extremely preterm infants with neurological injury exhibited delayed gut microbiota maturation compared with both neurologically normal preterm infants and term-born controls. Specifically, infants with neurological injury retained a microbiota dominated by facultative anaerobes such as Enterococcus , Streptococcus , and Pseudomonas , while normal preterm infants showed increasing colonization by strict anaerobes including Bifidobacterium and Akkermansia . Importantly, beneficial taxa were positively associated with neurodevelopmental scores, whereas facultative anaerobes correlated negatively. Comparison with previous studies Our results are consistent with previous reports showing altered microbial colonization in preterm infants, characterized by reduced diversity and delayed transition from facultative to strict anaerobes 10 , 15 , 23 . Seki et al. reported aberrant microbiota–immune–brain axis development in premature neonates with brain damage 2 . Other studies have indicated that dysbiosis may influence the risk of white matter injury and abnormal neurological development 16 . Together, these observations suggest that impaired microbial maturation is not only a marker of immaturity but also a potential contributor to brain vulnerability in preterm infants. Potential Mechanisms Several mechanisms may explain the observed associations between delayed microbiota maturation and neurological injury: Metabolite Deficiency – Bifidobacterium and Akkermansia are major producers of short-chain fatty acids (SCFAs), including acetate and propionate, which have been shown to promote blood–brain barrier integrity, modulate microglial maturation, and support myelination 24 , 25 . Reduced colonization by these taxa may limit SCFA availability during critical neurodevelopmental windows. Inflammatory Activation – Persistent colonization by Enterococcus and Pseudomonas may promote systemic inflammation via pathogen-associated molecular patterns (PAMPs) such as lipoteichoic acid and endotoxins, potentially exacerbating white matter injury 26 , 27 . Neurotransmitter Pathways – Dysbiosis may alter tryptophan metabolism and serotonin signaling, as well as vagal nerve-mediated pathways, all of which are implicated in cortical connectivity and motor development 28 – 30 . Clinical Implications Our results suggest that early microbial colonization patterns may serve as biomarkers of neurological risk in extremely preterm infants. Those who fail to transition from facultative to strict anaerobe dominance may warrant closer neurological monitoring. Furthermore, interventions aimed at accelerating microbiota maturation—such as human milk feeding, supplementation with Bifidobacterium, or targeted prebiotics—warrant further clinical evaluation 17 , 20 . Strengths and Limitations Strengths of this study include its prospective design, inclusion of a term-born control group, and integration of longitudinal microbiota analysis with multimodal neurological assessments. However, several limitations should be noted. The sample size was modest and derived from a single center, which may limit generalizability. 16S rDNA sequencing lacks strain-level and functional resolution, and future studies incorporating metagenomics and metabolomics are needed. Follow-up was limited to six months corrected age; long-term outcomes remain to be determined. In addition, infants in the neurological injury group had significantly longer hospital stays compared with the normal group. Although all stool samples were collected within the first 20 days of life and the incidence of severe complications outside the nervous system did not differ between groups, prolonged hospitalization could still act as a confounder. We therefore interpret the observed associations with caution, and further multicenter validation is required. Conclusion Extremely preterm infants with neurological injury exhibit delayed gut microbiota maturation, characterized by persistent facultative anaerobes and reduced beneficial strict anaerobes. These microbial signatures are associated with adverse neurological outcomes and may serve as early biomarkers and potential targets for intervention. Declarations Ethics Statement The study protocol was approved by the Ethics Committee of Zhangzhou Municipal Hospital (Approval No. 2022KYB179). Written informed consent was obtained from the parents or legal guardians of all participants prior to enrollment. All procedures were conducted in accordance with the Declaration of Helsinki and relevant national guidelines. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This work was supported by Fujian Provincial Natural Science Foundation (2023J011827). Data Availability Raw data is available upon reasonable request. Author Contributions Chun Xu: conceived and designed the study, collected clinical data and biological samples, performed microbiota sequencing and bioinformatic analyses, conducted statistical analyses and drafted the manuscript. Xiaohong Fang: collected clinical data and biological samples, performed microbiota sequencing and bioinformatic analyses. Lipin Xu: designed and supervised the study, revised the manuscript. All authors interpreted the data, critically revised the manuscript, and approved the final version for submission. References Ohuma, E. O. et al. National, regional, and global estimates of preterm birth in 2020, with trends from 2010: a systematic analysis. The Lancet 402 , 1261–1271 (2023). Seki, D. et al. Aberrant gut-microbiota-immune-brain axis development in premature neonates with brain damage. Cell Host Amp Microbe 29 , 1558-1572.e6 (2021). Back, S. A. Cerebral White and Gray Matter Injury in Newborns. Clin. Perinatol. 41 , 1–24 (2014). Franz, A. P. et al. 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Associations between dysbiosis gut microbiota and changes of neurotransmitters and short-chain fatty acids in valproic acid model rats. Front. Physiol. 14 , 1077821 (2023). Chen, Y., Xu, J. & Chen, Y. Regulation of Neurotransmitters by the Gut Microbiota and Effects on Cognition in Neurological Disorders. Nutrients 13 , 2099 (2021). Morris, G. et al. The Role of the Microbial Metabolites Including Tryptophan Catabolites and Short Chain Fatty Acids in the Pathophysiology of Immune-Inflammatory and Neuroimmune Disease. Mol. Neurobiol. 54 , 4432–4451 (2017). Bojović, K. et al. Gut Microbiota Dysbiosis Associated With Altered Production of Short Chain Fatty Acids in Children With Neurodevelopmental Disorders. Front. Cell. Infect. Microbiol. 10 , 223 (2020). Additional Declarations There is NO conflict of interest to disclose. 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05:38:01","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":230713,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7858615/v1/6fb132cffd2cfd7e9e6c9a27.png"},{"id":95502814,"identity":"7556dfd8-7344-4ce5-a2ea-cfe4ef95cb3e","added_by":"auto","created_at":"2025-11-10 05:38:01","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":78268,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7858615/v1/8222f0585d24110895e3e120.png"},{"id":95528726,"identity":"b6ad16f3-4873-4f04-95fa-94fd88a8e33f","added_by":"auto","created_at":"2025-11-10 10:16:26","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":155032,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7858615/v1/bf0d7543b5fa90d20dea84df.png"},{"id":95502809,"identity":"e26a004e-43cf-4daa-8a5f-723bacb8e1c9","added_by":"auto","created_at":"2025-11-10 05:38:01","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":78293,"visible":true,"origin":"","legend":"","description":"","filename":"2511800structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7858615/v1/3b43d7afb6b6e5631e124145.xml"},{"id":95502818,"identity":"8f4f0539-0970-4a4a-af94-65b7bba37c3a","added_by":"auto","created_at":"2025-11-10 05:38:01","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89775,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7858615/v1/a4e4279152dc9f78993b382d.html"},{"id":95502804,"identity":"8ab9dfd4-54d5-4ff3-8a61-dfdc2f804acd","added_by":"auto","created_at":"2025-11-10 05:38:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":655876,"visible":true,"origin":"","legend":"\u003cp\u003eDiversity (Chao1) and genus-level gut microbiota differences between extremely preterm and term infants at postnatal day 3. (A) Genus-level relative abundance profiles in extremely preterm (Pre) and term-born (Neo) infants. (B) Alpha diversity measured by the Chao1 index showing\u0026nbsp; significant difference between groups (P ＜0.01). (C) Beta diversity (Bray–Curtis distance) revealing distinct microbial community structures between groups (Wilcoxon, P = 2.2 × 10⁻¹\u003csup\u003e6\u003c/sup\u003e).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7858615/v1/8c58c4a0ab18a90f23dd9b6d.jpg"},{"id":95502798,"identity":"a9795445-d730-48f9-837f-209cadb16568","added_by":"auto","created_at":"2025-11-10 05:38:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":603845,"visible":true,"origin":"","legend":"\u003cp\u003eLongitudinal microbial diversity (β-diversity) in extremely preterm infants. (A) Temporal changes in genus-level relative abundance among six sampling groups (BasA–C, DagA–C). (B) Beta diversity (Bray–Curtis distance) demonstrating significant compositional shifts over time (Kruskal–Wallis, P = 1.4 × 10⁻¹¹).BasA–C: neurologically normal group at days 3, 10, and 20, respectively; DagA–C: neurological injury group at days 3, 10, and 20, respectively.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7858615/v1/e10466b2dce6e49f5f056205.jpg"},{"id":95502800,"identity":"09865a6a-0961-4b7f-a1ad-07177838b4d6","added_by":"auto","created_at":"2025-11-10 05:38:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1028400,"visible":true,"origin":"","legend":"\u003cp\u003eComparative genus-level microbiota composition between neurological injury and normal groups at postnatal days 3, 10, and 20. (A–C) Relative abundances of dominant genera at each time point comparing normal (BasA–C) and neurological injury (DagA–C) groups. Facultative anaerobes (Enterococcus, Streptococcus, Pseudomonas) predominated in the injury group, while strict anaerobes (Bifidobacterium, Akkermansia, Veillonella) were enriched in the normal group.BasA–C: neurologically normal group at days 3, 10, and 20, respectively; DagA–C: neurological injury group at days 3, 10, and 20, respectively.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7858615/v1/610dc9b558b6dab41c53106c.jpg"},{"id":95502802,"identity":"7884bd34-72e7-4991-8e01-636156b8950c","added_by":"auto","created_at":"2025-11-10 05:38:01","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":347142,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance rankings of differentially abundant genera. Bar plots illustrating genus-level relative abundances across all groups (BasA–C and DagA–C). Strict anaerobes (Bifidobacterium, Akkermansia, Bacteroides) were more abundant in the normal group, while facultative anaerobes (Enterococcus, Streptococcus, Pseudomonas) were dominant in the injury group.BasA–C: neurologically normal group at days 3, 10, and 20, respectively; DagA–C: neurological injury group at days 3, 10, and 20, respectively.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7858615/v1/b78f200f7da156a1dd46127b.jpg"},{"id":95528491,"identity":"70fc80d1-9883-4c02-8a94-d4cdff880f93","added_by":"auto","created_at":"2025-11-10 10:16:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":640056,"visible":true,"origin":"","legend":"\u003cp\u003eLEfSe analysis showing significantly enriched genera distinguishing neurological injury and normal groups (LDA \u0026gt; 2.0). (A–C) Genera significantly enriched in normal (BasA–C) and injury (DagA–C) groups. Bifidobacterium, Akkermansia, and Veillonella were characteristic of the normal group, whereas Enterococcus, Streptococcus, and Alloprevotella were enriched in the injury group.BasA–C: neurologically normal group at days 3, 10, and 20, respectively; DagA–C: neurological injury group at days 3, 10, and 20, respectively.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7858615/v1/0f96a8a3bb9cd288cfa422b5.jpg"},{"id":103049256,"identity":"46c6e7e4-1841-4ad1-8807-39a27b001874","added_by":"auto","created_at":"2026-02-20 07:39:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4092169,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7858615/v1/68b236c4-613b-4825-b0aa-7adeaac18622.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Gut Microbiota Maturation and Neurological Injury in Extremely Preterm Infants","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eNeurological injury remains a major cause of morbidity and adverse neurodevelopmental outcomes among extremely preterm infants\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Despite advances in neonatal intensive care, survival improvements have not been accompanied by equivalent reductions in neurological impairment\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. These complications contribute substantially to the burden of cerebral palsy, cognitive deficits, and long-term disability, underscoring the need for early biomarkers and novel preventive strategies.\u003c/p\u003e\u003cp\u003eRecent evidence suggests that the gut microbiota plays a key role in early brain development via the microbiome\u0026ndash;gut\u0026ndash;brain axis\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Normal colonization involves a transition from facultative to strict anaerobes, with the establishment of Bifidobacterium, Bacteroidota, and Lachnospiraceae as critical milestones. Preterm birth disrupts this process, leading to delayed colonization by beneficial taxa and overrepresentation of facultative anaerobes or opportunistic pathogens\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. These alterations may influence neurodevelopment through immune modulation, microbial metabolite production, and regulation of the hypothalamic\u0026ndash;pituitary\u0026ndash;adrenal axis\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Experimental models provide mechanistic insights. Dysbiosis during critical developmental windows impairs myelination, alters microglial activation, and disrupts synaptic plasticity\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Moreover, specific microbial metabolites, including short-chain fatty acids and tryptophan derivatives, regulate microglial maturation, blood\u0026ndash;brain barrier integrity, and neurotransmitter pathways\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Disruption of these processes may contribute to the vulnerability of the preterm brain.\u003c/p\u003e\u003cp\u003eHuman data linking gut microbiota development and neurological injury remain limited. Most prior studies have been retrospective, lacked term-born controls, or focused primarily on sepsis and necrotizing enterocolitis rather than direct neurological outcomes\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Seki et al. demonstrated aberrant microbiota\u0026ndash;immune\u0026ndash;brain axis development in premature neonates with brain damage\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, while Niemarkt et al. highlighted the role of the gut\u0026ndash;brain axis in necrotizing enterocolitis-related brain injury\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Recent work also suggests that early microbial trajectories are closely associated with neurobehavioral development\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, prospective studies integrating longitudinal microbial analyses with imaging and standardized neurodevelopmental assessments are scarce.\u003c/p\u003e\u003cp\u003eIn this study, we conducted a prospective cohort analysis of extremely preterm infants and healthy term-born controls. Serial stool samples were collected during the first three weeks of life and analyzed by 16S rDNA sequencing. Neurological outcomes were evaluated using cranial imaging, amplitude-integrated EEG, and standardized developmental assessments up to six months corrected age. We hypothesized that extremely preterm infants with neurological injury would show delayed transition from facultative to strict anaerobic colonization, with persistent enrichment of opportunistic taxa and reduced abundance of beneficial genera, compared with infants without injury and term controls.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy Design and Participants\u003c/h2\u003e\u003cp\u003e This prospective cohort study was conducted at the Neonatal Intensive Care Unit (NICU) of Zhangzhou Municipal Hospital (Fujian, China) between February 1, 2023, and January 31, 2024. Eligible participants were extremely preterm infants with a gestational age between 28 and \u0026lt;\u0026thinsp;32 weeks or a birth weight between 1.0 and \u0026lt;\u0026thinsp;1.5 kg, admitted to the NICU within 24 hours after birth. None had received antibiotics, probiotics, or prebiotics prior to enrollment. Infants were included only if their clinical condition improved sufficiently to allow hospital discharge and their caregivers agreed to follow-up until a corrected age of at least three months.\u003c/p\u003e\u003cp\u003eInfants were excluded if they had congenital anomalies involving the nervous or gastrointestinal systems, neurological abnormalities attributable to genetic, metabolic, or viral causes, or if they were withdrawn from the study, transferred to another facility, or died before completing follow-up assessments. Additional exclusion criteria included severe intracranial pathology detected within the first three days of life, defined as intraventricular hemorrhage grade III or higher according to Papile\u0026rsquo;s classification, or periventricular leukomalacia. Intraventricular hemorrhage was graded as follows: grade I, confined to the germinal matrix; grade II, blood occupying\u0026thinsp;\u0026le;\u0026thinsp;50% of the ventricular volume; grade III, blood occupying\u0026thinsp;\u0026gt;\u0026thinsp;50% of the ventricular volume; and grade IV, hemorrhagic infarction involving the periventricular white matter\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eA total of 75 extremely preterm infants met the inclusion criteria and were enrolled. For comparison, 20 healthy term-born infants (gestational age\u0026thinsp;\u0026ge;\u0026thinsp;37 weeks, birth weight\u0026thinsp;\u0026ge;\u0026thinsp;2.5 kg) matched for birth date and delivery mode were recruited as controls for gut microbiota assessment at postnatal day 3.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eClinical Data Collection\u003c/h3\u003e\n\u003cp\u003eDemographic and clinical characteristics were extracted from medical records using a standardized form, including gestational age, birth weight, sex, delivery mode, Apgar scores at 5 and 10 minutes, head circumference at birth and discharge, feeding type (exclusive breast milk, mixed feeding, or exclusive formula), antibiotic use during hospitalization, major diagnoses, complications, and length of hospital stay. Data entry accuracy was verified independently by two researchers.\u003c/p\u003e\n\u003ch3\u003eNeurodevelopmental Assessment\u003c/h3\u003e\n\u003cp\u003eNeurological evaluations were performed during hospitalization and at follow-up. Cranial ultrasonography was conducted on postnatal days 3, 7, and 14, then every two weeks for infants without abnormalities or weekly for those with abnormal findings until discharge. Amplitude-integrated electroencephalography (aEEG) was performed at postnatal days 1\u0026ndash;3, days 7\u0026ndash;8, and at corrected ages of 32 and 36 weeks to assess background activity, sleep\u0026ndash;wake cycling, and seizure patterns. Before discharge, cranial magnetic resonance imaging (MRI) was performed to detect structural brain injury.\u003c/p\u003e\u003cp\u003ePost-discharge follow-up was performed at corrected ages of 40 weeks and 3 months. The Neonatal Behavioral Neurological Assessment (NBNA) was conducted at 40 weeks, and the General Movements Assessment (GMs) was conducted at 3 months. Infants with abnormal neuroimaging findings (cranial ultrasonography or MRI) or seizure activity detected by amplitude-integrated EEG during hospitalization, and who showed abnormal GMs at 3 months corrected age (defined as F\u0026minus;, reduced fidgety movements), were classified into the neurological injury group. In contrast, infants with consistently normal neuroimaging during hospitalization, no seizure activity on amplitude-integrated EEG, and normal GMs at 3 months (defined as NF, normal fidgety movements), were classified into the neurologically normal group. Cranial imaging abnormalities were defined as intraventricular hemorrhage or periventricular leukomalacia detected on any cranial ultrasound or MRI performed during hospitalization\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eFecal Sample Collection and DNA Extraction\u003c/h3\u003e\n\u003cp\u003eFecal samples were collected from each infant at postnatal days 3, 10, and 20 (day 1 defined as the first 24 hours of life). Fresh stool was obtained from diapers using sterile swabs, placed into pre-labeled sterile 5 mL microcentrifuge tubes, and immediately stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis.\u003c/p\u003e\u003cp\u003eGenomic DNA was extracted from ~\u0026thinsp;30\u0026ndash;50 mg of stool using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer\u0026rsquo;s protocol, with an added bead-beating step for Gram-positive bacterial lysis. DNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003e16S rDNA Sequencing and Bioinformatic Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe V3\u0026ndash;V4 hypervariable region of the 16S rRNA gene was amplified using primers 341F (5\u0026rsquo;-CCTACGGGNGGCWGCAG-3\u0026rsquo;) and 805R (5\u0026rsquo;-GACTACHVGGGTATCTAATCC-3\u0026rsquo;). PCR products were purified, quantified, and sequenced on the Illumina MiSeq platform (2 \u0026times; 300 bp paired-end reads).\u003c/p\u003e\u003cp\u003eRaw reads were processed with QIIME2 (version 2023.2) and denoised with DADA2 using a minimum quality score threshold of Q30. Chimeric sequences were removed, and taxonomy was assigned using the SILVA 138 database. Samples were rarefied to the minimum sequencing depth across all samples. Alpha diversity (Chao1, Shannon indices) and beta diversity (Bray\u0026ndash;Curtis, weighted UniFrac) were calculated. Beta diversity differences were tested with PERMANOVA (999 permutations). Differential taxa were identified with LEfSe (LDA score\u0026thinsp;\u0026gt;\u0026thinsp;2.0, adjusted P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eContinuous variables were tested for normality (Shapiro\u0026ndash;Wilk test). Normally distributed variables are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and compared using Student\u0026rsquo;s t-test or ANOVA. Non-normally distributed variables are expressed as median (interquartile range) and compared using the Wilcoxon rank-sum or Kruskal\u0026ndash;Wallis tests. Categorical variables are presented as counts (%) and compared using chi-square or Fisher\u0026rsquo;s exact test. Microbiota differential abundance results were corrected for multiple testing using the Benjamini\u0026ndash;Hochberg method. Analyses were performed in R (version 4.3.1) and SPSS Statistics (version 26.0; IBM Corp., USA), with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eParticipant Characteristics\u003c/h2\u003e\n \u003cp\u003eA total of 75 extremely preterm infants were enrolled, including 31 with neurological injury and 44 without injury, along with 20 healthy term-born controls. Baseline characteristics are summarized in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Severe complications included late-onset sepsis, bronchopulmonary dysplasia grade\u0026thinsp;\u0026ge;\u0026thinsp;III\u003csup\u003e13\u003c/sup\u003e, necrotizing enterocolitis stage\u0026thinsp;\u0026ge;\u0026thinsp;II\u003csup\u003e11\u003c/sup\u003e, and retinopathy of prematurity requiring treatment\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eClinical characteristics of the study population.\u003c/strong\u003e *Values presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, median (IQR), or frequency (%); significant differences indicated by \u003cstrong\u003e*\u003c/strong\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Note: Severe complications include late-onset sepsis, bronchopulmonary dysplasia grade\u0026thinsp;\u0026ge;\u0026thinsp;III, necrotizing enterocolitis stage\u0026thinsp;\u0026ge;\u0026thinsp;II, and retinopathy of prematurity requiring treatment.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNormal group (n\u0026thinsp;=\u0026thinsp;44)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInjury group (n\u0026thinsp;=\u0026thinsp;31)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eGestational age (weeks)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eBirth weight (kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eHead circumference (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eMale sex, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24 (54.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17 (54.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCesarean delivery, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 (68.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22 (71.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eAntibiotic exposure, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36 (81.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27 (87.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eApgar score (5 min)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (7\u0026ndash;9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (7\u0026ndash;9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eApgar score (10 min)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (8\u0026ndash;10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (8\u0026ndash;10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eHospital stay (days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.2\u0026thinsp;\u0026plusmn;\u0026thinsp;17.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.3\u0026thinsp;\u0026plusmn;\u0026thinsp;17.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eFeeding type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBreast milk, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24(54.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12(38.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMixed, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6(13.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8(25.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFormula, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14(31.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11(35.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eIncidence of severe complications (n%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2(4.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2(6.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e*Values presented as mean \u0026plusmn; SD, median (IQR), or frequency (%); significant differences indicated by \u003cstrong\u003e*\u003c/strong\u003e (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). Note: Severe complications include late-onset sepsis, bronchopulmonary dysplasia grade \u0026ge;III, necrotizing enterocolitis stage \u0026ge;II, and retinopathy of prematurity requiring treatment.\u003c/p\u003e\n \u003cp\u003eThe two preterm subgroups were comparable in gestational age, birth weight, sex, and delivery mode and the incidence of severe complications (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05 for all). Feeding type distribution (exclusive breast milk, mixed, exclusive formula) did not differ significantly between preterm subgroups. The neurological injury group had a significantly longer median hospital stay compared with the neurologically normal group (55.29\u0026thinsp;\u0026plusmn;\u0026thinsp;17.72 days vs 46.20\u0026thinsp;\u0026plusmn;\u0026thinsp;17.34 days, P\u0026thinsp;=\u0026thinsp;0.03).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eGut microbiota diversity\u003c/h3\u003e\n\u003cp\u003eAt postnatal day 3, preterm infants exhibited a distinct gut microbiota profile compared with term-born controls (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Genus-level composition (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) showed that facultative anaerobes\u0026mdash;Enterococcus, Streptococcus, and Pseudomonas\u0026mdash;dominated in preterm infants, whereas term controls had higher abundances of Bifidobacterium. Alpha diversity (Chao1 index) showed significant difference between groups (P \u0026lt;0.01; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), and beta diversity (Bray\u0026ndash;Curtis distance) revealed significant structural separation (Wilcoxon, P\u0026thinsp;=\u0026thinsp;2.2 \u0026times; 10⁻\u0026sup1;\u003csup\u003e6\u003c/sup\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eLongitudinal microbiota composition\u003c/h2\u003e\n \u003cp\u003eAcross postnatal days 3, 10, and 20, microbial diversity in preterm infants increased progressively (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Taxonomic succession analyses (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) revealed dynamic changes from facultative to strict anaerobes. Beta-diversity trajectories (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB) demonstrated significant temporal shifts in microbial community composition (Kruskal\u0026ndash;Wallis, P\u0026thinsp;=\u0026thinsp;1.4 \u0026times; 10⁻\u0026sup1;\u0026sup1;), indicating overall maturation of gut ecosystems during early life.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eComparative composition between neurological-injury and normal groups\u003c/h2\u003e\n \u003cp\u003eAt each time point, the genus-level compositions differed between the neurological-injury and neurologically normal groups (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Facultative anaerobes (Enterococcus, Streptococcus, Pseudomonas) remained predominant in the injury group throughout the first 3 weeks, whereas strict anaerobes (Bifidobacterium, Akkermansia, Veillonella) increased markedly in the normal group, reflecting faster microbial maturation. Effect-size comparison showed that Bifidobacterium abundance at day 10 was approximately 2.5-fold higher in the normal group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eRelative abundance of key taxa\u003c/h2\u003e\n \u003cp\u003eBar-plot analyses (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) summarized the relative abundances of dominant genera across groups. Strict anaerobes such as Bifidobacterium, Akkermansia, and Bacteroides were more abundant in the normal group, while facultative anaerobes including Enterococcus, Streptococcus, and Pseudomonas predominated in the injury group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eCorrelations with neurological outcomes\u003c/h2\u003e\n \u003cp\u003eLEfSe analysis (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e) identified genera that significantly discriminated between the two preterm subgroups (LDA\u0026thinsp;\u0026gt;\u0026thinsp;2.0, adjusted P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Bifidobacterium, Akkermansia, and Veillonella were enriched in the neurologically normal group, whereas Enterococcus, Streptococcus, and Alloprevotella were characteristic of the neurological-injury group. These findings confirm that delayed establishment of strict anaerobes and persistence of facultative taxa are closely associated with neurological injury in extremely preterm infants.\u003c/p\u003e\n \u003cp\u003eSpearman correlation analyses demonstrated significant positive associations of abundances of Bifidobacterium (r\u0026thinsp;=\u0026thinsp;0.42, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and Akkermansia (r\u0026thinsp;=\u0026thinsp;0.30, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in day 10 and day 20 with NBNA scores at term-equivalent age. Conversely, Enterococcus abundance was negatively correlated with NBNA scores (r = -0.23, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). These associations remained significant after adjustment for gestational age, feeding type, and antibiotic exposure.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSpearman correlations between gut microbiota taxa and NBNA scores at term-equivalent age\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eD10\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eD20\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEnterococcus\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBifidobacterium\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAkkermansia\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBifidobacterium\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAkkermansia\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003er\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.042\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this prospective cohort study, we demonstrated that extremely preterm infants with neurological injury exhibited delayed gut microbiota maturation compared with both neurologically normal preterm infants and term-born controls. Specifically, infants with neurological injury retained a microbiota dominated by facultative anaerobes such as \u003cem\u003eEnterococcus\u003c/em\u003e, \u003cem\u003eStreptococcus\u003c/em\u003e, and \u003cem\u003ePseudomonas\u003c/em\u003e, while normal preterm infants showed increasing colonization by strict anaerobes including \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eAkkermansia\u003c/em\u003e. Importantly, beneficial taxa were positively associated with neurodevelopmental scores, whereas facultative anaerobes correlated negatively.\u003c/p\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eComparison with previous studies\u003c/h2\u003e\u003cp\u003eOur results are consistent with previous reports showing altered microbial colonization in preterm infants, characterized by reduced diversity and delayed transition from facultative to strict anaerobes\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Seki et al. reported aberrant microbiota\u0026ndash;immune\u0026ndash;brain axis development in premature neonates with brain damage\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Other studies have indicated that dysbiosis may influence the risk of white matter injury and abnormal neurological development\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Together, these observations suggest that impaired microbial maturation is not only a marker of immaturity but also a potential contributor to brain vulnerability in preterm infants.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003ePotential Mechanisms\u003c/h2\u003e\u003cp\u003eSeveral mechanisms may explain the observed associations between delayed microbiota maturation and neurological injury:\u003c/p\u003e\u003cp\u003e\u003cem\u003eMetabolite Deficiency\u003c/em\u003e \u0026ndash; \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eAkkermansia\u003c/em\u003e are major producers of short-chain fatty acids (SCFAs), including acetate and propionate, which have been shown to promote blood\u0026ndash;brain barrier integrity, modulate microglial maturation, and support myelination\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Reduced colonization by these taxa may limit SCFA availability during critical neurodevelopmental windows.\u003c/p\u003e\u003cp\u003e\u003cem\u003eInflammatory Activation\u003c/em\u003e \u0026ndash; Persistent colonization by \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e may promote systemic inflammation via pathogen-associated molecular patterns (PAMPs) such as lipoteichoic acid and endotoxins, potentially exacerbating white matter injury\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eNeurotransmitter Pathways\u003c/em\u003e \u0026ndash; Dysbiosis may alter tryptophan metabolism and serotonin signaling, as well as vagal nerve-mediated pathways, all of which are implicated in cortical connectivity and motor development\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eClinical Implications\u003c/h2\u003e\u003cp\u003eOur results suggest that early microbial colonization patterns may serve as biomarkers of neurological risk in extremely preterm infants. Those who fail to transition from facultative to strict anaerobe dominance may warrant closer neurological monitoring. Furthermore, interventions aimed at accelerating microbiota maturation\u0026mdash;such as human milk feeding, supplementation with Bifidobacterium, or targeted prebiotics\u0026mdash;warrant further clinical evaluation\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eStrengths and Limitations\u003c/h2\u003e\u003cp\u003eStrengths of this study include its prospective design, inclusion of a term-born control group, and integration of longitudinal microbiota analysis with multimodal neurological assessments. However, several limitations should be noted. The sample size was modest and derived from a single center, which may limit generalizability. 16S rDNA sequencing lacks strain-level and functional resolution, and future studies incorporating metagenomics and metabolomics are needed. Follow-up was limited to six months corrected age; long-term outcomes remain to be determined. In addition, infants in the neurological injury group had significantly longer hospital stays compared with the normal group. Although all stool samples were collected within the first 20 days of life and the incidence of severe complications outside the nervous system did not differ between groups, prolonged hospitalization could still act as a confounder. We therefore interpret the observed associations with caution, and further multicenter validation is required.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eExtremely preterm infants with neurological injury exhibit delayed gut microbiota maturation, characterized by persistent facultative anaerobes and reduced beneficial strict anaerobes. These microbial signatures are associated with adverse neurological outcomes and may serve as early biomarkers and potential targets for intervention.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the Ethics Committee of Zhangzhou Municipal Hospital (Approval No. 2022KYB179). Written informed consent was obtained from the parents or legal guardians of all participants prior to enrollment. All procedures were conducted in accordance with the Declaration of Helsinki and relevant national guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Fujian Provincial Natural Science Foundation (2023J011827).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; Raw data is available upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChun Xu: conceived and designed the study, collected clinical data and biological samples, performed microbiota sequencing and bioinformatic analyses, conducted statistical analyses and drafted the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eXiaohong Fang: collected clinical data and biological samples, performed microbiota sequencing and bioinformatic analyses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLipin Xu: designed and supervised the study, revised the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors interpreted the data, critically revised the manuscript, and approved the final version for submission.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eOhuma, E. 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F. \u003cem\u003eet al.\u003c/em\u003e International Classification of Retinopathy of Prematurity, Third Edition. \u003cem\u003eOphthalmology\u003c/em\u003e \u003cstrong\u003e128\u003c/strong\u003e, e51\u0026ndash;e68 (2021).\u003c/li\u003e\n \u003cli\u003eBresesti, I. \u003cem\u003eet al.\u003c/em\u003e The Microbiota-Gut Axis in Premature Infants: Physio-Pathological Implications. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 379 (2022).\u003c/li\u003e\n \u003cli\u003eLiu, L. \u003cem\u003eet al.\u003c/em\u003e Multi-omics analyses of gut microbiota via 16S rRNA gene sequencing, LC-MS/MS and diffusion tension imaging reveal aberrant microbiota-gut-brain axis in very low or extremely low birth weight infants with white matter injury. \u003cem\u003eBMC Microbiol.\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 387 (2023).\u003c/li\u003e\n \u003cli\u003eSodhi, C. P. \u003cem\u003eet al.\u003c/em\u003e Human milk oligosaccharides reduce necrotizing enterocolitis-induced neuroinflammation and cognitive impairment in mice. \u003cem\u003eAm. J. Physiol. Gastrointest. Liver Physiol.\u003c/em\u003e \u003cstrong\u003e325\u003c/strong\u003e, G23\u0026ndash;G41 (2023).\u003c/li\u003e\n \u003cli\u003eWang, Y. \u003cem\u003eet al.\u003c/em\u003e Pathogenesis from the microbial-gut-brain axis in white matter injury in preterm infants: A review. \u003cem\u003eFront. Integr. Neurosci.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1051689 (2023).\u003c/li\u003e\n \u003cli\u003eSun, Z., Xu, W., Cong, X., Li, G. \u0026amp; Chen, K. Log-contrast regression with functional compositional predictors: Linking preterm infants\u0026rsquo; gut microbiome trajectories to neurobehavioral outcome. \u003cem\u003eAnn. Appl. Stat.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, (2020).\u003c/li\u003e\n \u003cli\u003eInder, T. E., Volpe, J. J. \u0026amp; Anderson, P. J. Defining the Neurologic Consequences of Preterm Birth. \u003cem\u003eN. Engl. J. Med.\u003c/em\u003e \u003cstrong\u003e389\u003c/strong\u003e, 441\u0026ndash;453 (2023).\u003c/li\u003e\n \u003cli\u003eVolpe, J., Inder, T. \u0026amp; Darras, B. \u003cem\u003eVolpe\u0026rsquo;s Neurology of the Newborn\u003c/em\u003e. vol. 541 (Elsevier, 2018).\u003c/li\u003e\n \u003cli\u003eCandau, Marcolino Gomes, (30 May 1911\u0026ndash;25 Jan. 1983), Director-General Emeritus, World Health Organization, Geneva, since 1973. \u003cem\u003eWho Was Who\u003c/em\u003e https://doi.org/10.1093/ww/9780199540884.013.u162662 (2007) doi:10.1093/ww/9780199540884.013.u162662.\u003c/li\u003e\n \u003cli\u003eKorpela, K. \u003cem\u003eet al.\u003c/em\u003e Intestinal microbiota development and gestational age in preterm neonates. \u003cem\u003eSci. 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Sci.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 9577 (2023).\u003c/li\u003e\n \u003cli\u003eZhong, J.-G. \u003cem\u003eet al.\u003c/em\u003e Associations between dysbiosis gut microbiota and changes of neurotransmitters and short-chain fatty acids in valproic acid model rats. \u003cem\u003eFront. Physiol.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1077821 (2023).\u003c/li\u003e\n \u003cli\u003eChen, Y., Xu, J. \u0026amp; Chen, Y. Regulation of Neurotransmitters by the Gut Microbiota and Effects on Cognition in Neurological Disorders. \u003cem\u003eNutrients\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 2099 (2021).\u003c/li\u003e\n \u003cli\u003eMorris, G. \u003cem\u003eet al.\u003c/em\u003e The Role of the Microbial Metabolites Including Tryptophan Catabolites and Short Chain Fatty Acids in the Pathophysiology of Immune-Inflammatory and Neuroimmune Disease. \u003cem\u003eMol. Neurobiol.\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 4432\u0026ndash;4451 (2017).\u003c/li\u003e\n \u003cli\u003eBojović, K. \u003cem\u003eet al.\u003c/em\u003e Gut Microbiota Dysbiosis Associated With Altered Production of Short Chain Fatty Acids in Children With Neurodevelopmental Disorders. \u003cem\u003eFront. Cell. Infect. Microbiol.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 223 (2020).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"extremely preterm infants, gut microbiota, neurological injury, microbiome–gut–brain axis, Bifidobacterium","lastPublishedDoi":"10.21203/rs.3.rs-7858615/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7858615/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e To examine the association between gut microbiota maturation and neurological injury in extremely preterm infants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy Design:\u003c/strong\u003e We prospectively studied 75 extremely preterm infants (28–\u0026lt;32 weeks, 1.0–\u0026lt;1.5 kg) and 20 term-born controls. Neurological outcomes were assessed by neuroimaging, amplitude-integrated EEG, and developmental evaluations up to 3 months corrected age. Stool samples collected on days 3, 10, and 20 were analyzed by 16S rDNA sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e At day 3, preterm infants had reduced diversity versus term controls (P\u0026lt;0.01) and were enriched in facultative anaerobes. By day 20, neurologically normal infants showed increased colonization by \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eAkkermansia\u003c/em\u003e, whereas injury group infants retained facultative-dominated profiles. Beneficial taxa correlated positively with neurodevelopmental scores, while \u003cem\u003eEnterococcus\u003c/em\u003ecorrelated negatively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Delayed microbial maturation is associated with neurological injury in extremely preterm infants and may serve as an early biomarker and target for intervention.\u003c/p\u003e","manuscriptTitle":"Gut Microbiota Maturation and Neurological Injury in Extremely Preterm Infants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-10 05:37:56","doi":"10.21203/rs.3.rs-7858615/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"70b09cde-9f0b-45a8-9b99-0f008d1ce205","owner":[],"postedDate":"November 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56952007,"name":"Health sciences/Diseases/Neurological disorders/Paediatric neurological disorders"},{"id":56952008,"name":"Biological sciences/Neuroscience/Neurogenesis/Developmental neurogenesis"}],"tags":[],"updatedAt":"2026-02-16T10:45:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-10 05:37:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7858615","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7858615","identity":"rs-7858615","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}