Native amniotic fluid promotes barrier-associated maturation of the neonatal intestine under physiological conditions

Introduction

The intestinal barrier is a complex and dynamic system formed by a single layer of epithelial cells connected by tight junctions and supported by immune cells, commensal microbiota, and a mucus layer enriched with antimicrobial peptides and secretory immunoglobulin A (sIgA) (1). The integrity of this barrier can be compromised by infections, inflammatory cytokines, and dietary or environmental stressors, leading to increased intestinal permeability and disease (1,2). Barrier dysfunction has been strongly associated with inflammatory bowel disease (IBD) and neonatal necrotizing enterocolitis (NEC), both of which involve disruption of epithelial junctional complexes (3,4). Beyond disease settings, intestinal barrier development is also part of normal postnatal maturation, particularly in neonates whose epithelial defense system remains immature.

Tight junction proteins such as Claudins and Occludin play a central role in regulating paracellular permeability and maintaining epithelial integrity. Claudins are broadly classified into sealing types (e.g., Claudins-1, 3–5, 8) that strengthen intercellular adhesion and pore-forming types (e.g., Claudins-2, 10a/10b, 15–17) that create selective ion channels (5-7). The balance between these proteins determines the permeability properties of the intestinal epithelium. Occludin and the scaffold protein ZO-1 also contribute to junctional assembly, stability, and signaling, further influencing epithelial integrity (8). During early postnatal life, the intestinal epithelium undergoes dynamic maturation, with developmental factors driving region-specific changes in epithelial structure and function, which contribute to the establishment of an effective intestinal barrier (9-11).

Amniotic fluid (AF), which surrounds and nourishes the fetus, contains a wide array of bioactive components—including growth factors, cytokines, extracellular vesicles, and metabolites such as glutamine and tryptophan—that have been implicated in intestinal growth and repair (12-17). Previous studies have shown that AF-derived stem cells, conditioned media, or AF-associated factors can protect intestinal epithelium but these studies have mainly focused on inflammatory or injury settings (18-21). In contrast, the collective effect of native AF on neonatal intestinal barrier-associated maturation under physiological postnatal conditions remains unclear.

In this study, we investigated whether native AF supports neonatal intestinal barrier-associated maturation under physiological, non-injury conditions by assessing tight junction protein expression and epithelial barrier-related properties. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0598/rc).

Methods

Ethical approval and AF collection

This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University (approval No. LCKY2020-329). Written informed consent was obtained from 31 donors undergoing elective cesarean delivery. AF samples were collected at 34+4 to 39+5 weeks of gestation. Samples with visible contamination or clinical evidence of chorioamnionitis were excluded. After collection, AF samples were centrifuged to remove cellular debris, and the supernatants were pooled, aliquoted, and stored at −80 ℃ until use. Donor characteristics and selected biochemical features of the AF samples, including total protein, epidermal growth factor, heparin-binding epidermal growth factor-like growth factor, and hepatocyte growth factor, are summarized in Table S1. All procedures involving human samples were performed in accordance with relevant guidelines and regulations.

Cell culture

Caco-2 (human colonic epithelial) and IEC-6 (rat intestinal epithelial) cell lines were obtained from the Chinese Academy of Sciences. Cells were maintained in their recommended media [Caco-2 in minimum essential medium (MEM); IEC-6 in Dulbecco’s modified Eagle medium (DMEM); Thermo Fisher Scientific, USA] supplemented with 10 % fetal bovine serum at 37 ℃ in 5 % CO₂. Medium was replaced every 2 days.

Monolayer establishment

Cells in logarithmic growth were detached with 0.25% trypsin, centrifuged, counted, and seeded at 2×10⁵ cells per well in transwell inserts (Thermo Fisher Scientific, USA). After reaching confluence, monolayers were exposed for 24–72 h to control medium or medium containing 25% or 50% native AF. Native AF was diluted with the corresponding culture medium on a volume/volume basis. For Caco-2 cells, AF was diluted in MEM, whereas for IEC-6 cells, AF was diluted in DMEM. The 25% AF treatment contained 25% native AF and 75% culture medium, while the 50% AF treatment contained 50% native AF and 50% culture medium. No additional components were added during preparation.

Animal experiments

Newborn Sprague-Dawley rats aged 1–2 days, with an initial body weight of approximately 14 g, were purchased from Suzhou Zhaoyan Experimental Animal Co., Ltd. [Suzhou, China; license No. SCXK(Su)2018-0006; certificate No. 201908304]. Three litters were used, with each litter consisting of 13 newborn pups and one lactating dam. Animals were maintained under specific pathogen-free conditions in an approved animal facility [license No. SYXK(Zhe)2015-0008], with a temperature of 20–25 ℃ and relative humidity of 40–70%. Sterile ultrapure water was provided, and lactating dams were fed a maintenance diet supplied by Jiangsu Xietong Pharmaceutical Bioengineering Co., Ltd., according to the Chinese national standard GB14924.3–2010.

Newborn rats were randomly assigned to three groups: normal saline (NS) control, 25% AF, and 50% AF, with 13 pups allocated to each group. For oral gavage, native AF was diluted with NS on a volume/volume basis. The 25% AF solution contained 25% native AF and 75% NS, whereas the 50% AF solution contained 50% native AF and 50% NS. Control animals received NS alone. Each pup received 2 mL/day of the assigned solution from postnatal day 1 to day 14 or day 28. Fur color, body weight, activity, survival, hydration status, and stool output were monitored during the experiment. Neonatal rats were not fasted prior to anesthesia or tissue collection to prevent hypoglycemia. Body weight was recorded every 2 days from baseline to day 28. Body weight increased progressively in all groups during the experimental period, and no significant difference was observed between the AF-treated groups and the control group (Appendix 1). No obvious abnormalities in fur color, activity, hydration status, stool output, or survival were observed during routine monitoring.

Postnatal day 14 was selected to represent the pre-weaning lactation period, whereas postnatal day 28 was selected as a later post-weaning developmental time point for comparison.

At days 14 and 28, three rats from each group were euthanized, and terminal ileum and colon tissues were collected for subsequent analyses. For intestinal epithelial resistance and permeability assays, one rat from each group was used at each time point. The experimental unit was defined as the individual pup for body-weight analysis and as the individual intestinal tissue sample for ex vivo intestinal measurements. No animals were excluded from the final analysis. Sex was not used as an allocation factor, and sex-specific effects were not assessed.

All animal experiments were approved by the Animal Ethics Committee of Wenzhou Medical University (approval No. wydw2020-0909) and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Anesthesia and euthanasia

All terminal procedures were performed in accordance with the approved animal protocol and institutional guidelines for the care and use of laboratory animals. Neonatal rats were not fasted before anesthesia or tissue collection to reduce the risk of hypoglycemia. For terminal tissue collection, animals were anesthetized with isoflurane in oxygen using an induction chamber. The depth of anesthesia was monitored continuously and confirmed by loss of response to a mild paw pinch. No survival surgical procedures were performed, and animals were not allowed to recover from anesthesia.

After deep anesthesia was confirmed, animals were euthanized by cervical dislocation as a secondary physical method. Euthanasia was performed by trained personnel approved to conduct rodent anesthesia and euthanasia procedures. Death was confirmed by cessation of respiration and heartbeat and absence of reflex responses before tissue collection. Because all procedures were terminal and performed under deep anesthesia, no postoperative analgesia was required.

Western blot analysis

Proteins were extracted using Western and immunoprecipitation (IP) cell lysis buffer containing phenylmethylsulfonyl fluoride (PMSF) protease inhibitor, and protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit according to the manufacturer’s instructions. Equal amounts of protein (60 μg per lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA; cat. no. IPVH00010). Membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated overnight at 4 ℃ with primary antibodies against Claudin-1, Claudin-8, Occludin, and ZO-1 at a dilution of 1:1,000. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Lianke, China; cat. no. ab5465-100) was used as the loading control.

After washing with Tris-buffered saline with Tween 20 (TBST), membranes were incubated with Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Lianke, China; cat. no. GAM007) or HRP-conjugated goat anti-rabbit secondary antibody (Lianke, China; cat. no. GAR0072) at a dilution of 1:5,000 for 1 h at room temperature. Protein bands were visualized using an ECL Plus chemiluminescence kit and imaged with a ChemiDoc XRS+ system (Bio-Rad, USA). Band intensities were quantified using Image Lab software and normalized to GAPDH. Western blot experiments were performed in three independent experiments. Full-length uncropped blots and raw densitometric quantification data are provided in the Supplementary Information.

Measurement of transepithelial electrical resistance (TEER)

TEER was measured in intestinal epithelial cell monolayers using an epithelial voltohmmeter (WPI, USA), as described previously (22,23). Confluent monolayers were washed with phosphate-buffered saline and treated with control medium, 25% AF, or 50% AF. Readings were corrected for blank resistance, and TEER was calculated using the following formula:

TEER=(R1−R0)×A(Ω⋅cm2)[1]

where R₁ is the resistance of the sample insert, R₀ is the resistance of the blank insert, and A is the membrane surface area.

For ex vivo tissue TEER measurements, 2-cm ileal and colonic segments were mounted in an Ussing chamber system with six channels and a pore area of 0.031 cm². Tissues were perfused with oxygenated Krebs buffer (95% O₂/5% CO₂) at 37 ℃, and TEER was recorded for 120 min. Tissue TEER was interpreted as an ex vivo electrophysiological measurement of intestinal barrier-related properties, rather than a direct in vivo permeability assay.

Permeability assay

In vitro paracellular permeability was assessed in Caco-2 and IEC-6 cell monolayers using fluorescein isothiocyanate-conjugated inulin (FITC-inulin; Sigma-Aldrich), as described previously. FITC-inulin (1 mg/mL, 100 μL) was added to the apical chamber of each transwell insert. After incubation for 1 h at 37 ℃, basolateral samples were collected, and fluorescence intensity was measured at 485 nm excitation and 535 nm emission using a Tecan microplate fluorescence reader. Permeability was calculated using the following formula:

P=[(ΔC/ΔT×V)/(C0×A)]×100[2]

where ΔC/ΔT is the transfer rate, V is the basolateral chamber volume, C₀ is the initial apical FITC-inulin concentration, and A is the membrane surface area. This assay was performed only in epithelial monolayer models and was not used to assess direct in vivo intestinal permeability in neonatal rats.

Histology and morphometry

Ileal and colonic tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. Villus height, crypt depth, and mucosal thickness were quantified using Image-Pro Plus software (Media Cybernetics, USA). Histological assessment and morphometric quantification were performed by investigators blinded to the treatment groups.

Transmission electron microscopy (TEM)

Small intestinal tissue blocks (approximately 1 mm³) were fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, stained with uranyl acetate, dehydrated, and embedded in resin. Ultrathin sections (approximately 120 nm) were stained with lead citrate and examined using a TECNA-10 transmission electron microscope (Philips, Netherlands). Tight junctions, adherens junctions, and desmosomes were evaluated in randomly selected epithelial fields. Quantification was performed by investigators blinded to the treatment groups. For each animal, multiple representative fields were analyzed, and the individual tissue sample was considered the experimental unit. Structural observations were interpreted as assessments of junctional morphology and preservation rather than definitive evidence of functional barrier improvement.

Statistical analysis

Data are expressed as mean ± standard deviation (SD). The individual pup was used as the experimental unit for body-weight analysis, and the individual intestinal tissue sample was used as the experimental unit for ex vivo intestinal measurements. Comparisons among multiple groups were performed using one-way or two-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test when the overall ANOVA was significant. For repeated body-weight measurements, two-way ANOVA was used with treatment group and time as factors. When data did not meet assumptions of normality or homogeneity of variance, the Kruskal-Wallis test was applied for multiple-group comparisons. A two-tailed P value <0.05 was considered statistically significant. Exact n-values for each endpoint are reported in the corresponding figure legends and Results text.

Results

Cell monolayer models were established and verified by TEER

To assess the effect of AF on intestinal epithelial barrier properties, Caco-2 and IEC-6 cells were seeded in transwell inserts and cultured to form confluent monolayers. Cells proliferated primarily during the first 7 days after seeding, with extensive areas of cell fusion observed as density increased (Figure 1A-1D). TEER values rose rapidly after day 10 (P<0.001) and plateaued by day 15, indicating the formation of mature epithelial monolayers (Figure 1E,1F).

Figure 1 Establishment and verification of Caco-2 and IEC-6 cell monolayer models. (A,C) Early proliferation of Caco-2 and IEC-6 cells during the first 7 days after seeding in transwell inserts. (B,D) Confluent monolayers with extensive cell fusion as density increased. (E,F) TEER values of Caco-2 and IEC-6 monolayers measured at days 1, 4, 7, 10, 13, and 15. Unstained phase-contrast microscopy: (A,B) 100× magnification, (C,D) 400× magnification. Data are presented as mean ± SD from three independent experiments, each including three technical replicates per condition (n=9). Statistical significance: *, P<0.05; **, P<0.01; ***, P<0.001. SD, standard deviation; TEER, transepithelial electrical resistance.

AF increased the expression of tight junction proteins and TEER and decreased permeability in cell monolayers

Caco-2 and IEC-6 monolayers were treated with control medium, 25% AF, or 50% AF to examine the effect of AF on epithelial tight junctions. At baseline (control medium), Occludin expression was lower than Claudin-1 and Claudin-8. Treatment with 50% AF significantly increased the expression of all three tight junction proteins compared with control (P<0.05), while 25% AF caused a moderate, non-significant increase (Figure 2A-2D).

Figure 2 Effects of AF on tight junction protein expression, permeability, and TEER in Caco-2 and IEC-6 cell monolayer models. (A) Western blot analysis of tight junction proteins after 72 h treatment with medium alone, 25% AF, or 50% AF. (B-D) Quantification of Claudin-1, Claudin-8, and Occludin, respectively. (E) FITC-inulin permeability of Caco-2 and IEC-6 monolayers treated with 25% or 50% AF. (F,G) TEER measurements of Caco-2 (F) and IEC-6 (G) monolayers at 24, 48, and 72 h. Data are presented as mean ± SD from three independent experiments, each including three technical replicates per condition (n=9). Statistical significance: *P<0.05, **P<0.01, ***P<0.001 versus control; ^†P<0.05, ^††P<0.01, ^†††P<0.001 versus 25% AF; ^§P<0.05, ^§§P<0.01 versus 24 h. AF, amniotic fluid; FITC-inulin, fluorescein isothiocyanate-conjugated inulin; SD, standard deviation; TEER, transepithelial electrical resistance.

Barrier function was assessed using FITC-inulin permeability and TEER. Permeability was significantly reduced in both 25% and 50% AF-treated monolayers compared to control (P<0.001), with 50% AF producing the greatest reduction (P<0.01) (Figure 2E). TEER measurements at 24, 48, and 72 hours showed significant increases in both AF-treated groups relative to control (P<0.05), with 50% AF producing the highest values (Figure 2F,2G). TEER values also increased over time from 24 to 72 hours within each treatment group (P<0.05).

These findings demonstrate that AF enhanced tight junction protein expression, increased TEER, and reduced permeability within the tested concentration range, with the strongest response observed at 50% AF.

Effects of AF on ex vivo tissue TEER and tight-junction protein expression

To investigate whether AF influences intestinal barrier properties ex vivo, SD newborn rats on postnatal days 14 and 28 were treated with NS, 25%, or 50% AF. H&E staining and TEM showed normal villi, crypts, and mucosa in both ileum and colon, with no evidence of inflammation, erosion, or edema (Figure 3). Morphometric analysis revealed no significant differences in villus length, crypt depth, or mucosal thickness among groups (P>0.05), although measurements were slightly higher on day 28 compared to day 14 (Tables S2,S3). TEM analysis of junctions (tight junctions, intermediate junctions, and desmosomes) revealed no significant differences in junction numbers (P>0.05), indicating that AF did not disrupt overall junctional architecture (Figure 4; Tables S4,S5).

Figure 3 Effects of 25% or 50% AF on ileum and colon morphology in SD newborn rats on postnatal days 14 and 28, assessed by H&E staining. The villi and crypts of the ileum and colon appeared normal in all groups. Epithelial cells were orderly arranged, the lamina propria and goblet cell distribution were intact, and no inflammatory cell infiltration, erosion, ulcers, or edema were observed. Data represent n=10 rats per group. AF, amniotic fluid; H&E, hematoxylin and eosin; SD, standard deviation.

Figure 4 TEM images of ileum and colon from SD newborn rats on postnatal days 14 and 28 treated with NS, 25%, or 50% AF. Representative TEM images show the intestinal epithelial junctions. Squares indicate TJs, arrows indicate intermediate junctions, and circles indicate desmosomes. Quantification of junctional structures is summarized in Tables S2,S3. Data represent n=15 tissue samples per group. AF, amniotic fluid; NS, normal saline; SD, standard deviation; TEM, transmission electron microscopy; TJ, tight junction.

Ex vivo TEER measurements in ileum and colon samples revealed a time- and concentration-dependent effect (Figure 5A,5B). On day 14, 50% AF significantly increased TEER compared to control (P<0.01), whereas 25% AF had no significant effect. By day 28, TEER values were similar across all groups (P>0.05). These findings suggest that AF may accelerate early barrier maturation rather than provide a sustained or uniform enhancement.

Figure 5 Effects of 25% or 50% AF on ex vivo TEER and tight junction protein expression in the ileum and colon of SD newborn rats. (A) TEER of ileum tissues on postnatal days 14 and 28 after treatment with NS, 25%, or 50% AF. (B) TEER of colon tissues on postnatal days 14 and 28 under the same treatments. (C) Western blot analysis of ZO-1, Claudin-1, Claudin-8, and Occludin proteins in ileum tissues. (D-G) Quantification of ZO-1, Claudin-1, Claudin-8, and Occludin, respectively. Data are presented as mean ± SD from n=10 tissue samples per group. Statistical significance: *, P<0.05; **, P<0.01; ***, P<0.001. AF, amniotic fluid; NS, normal saline; SD, standard deviation; TEER, transepithelial electrical resistance.

Western blot analysis of tight junction proteins (ZO-1, Occludin, Claudin-1, Claudin-8) in ileum and colon tissues showed that 50% AF significantly upregulated all four proteins on day 14 compared with control and 25% AF (P<0.01) (Figures 5C-5G,6A-6E). On day 28, significant increases persisted for all proteins except Claudin-1 in the ileum, with 50% AF showing a stronger effect than 25% AF. Overall, AF treatment enhanced TEER and tight junction protein expression ex vivo, without detectable structural alterations, with effects most pronounced at day 14, consistent with accelerated early maturation of the neonatal intestinal barrier.

Figure 6 Effects of 25% or 50% AF on tight junction protein expression in the colon of SD newborn rats. (A) Western blot analysis of ZO-1, Claudin-1, Claudin-8, and Occludin proteins in colon tissues from rats treated with NS, 25%, or 50% AF. (B-E) Quantification of ZO-1, Claudin-1, Claudin-8, and Occludin, respectively. Data are presented as mean ± SD from n=10 tissue samples per group. Statistical significance for the indicated pairwise comparisons: **P<0.01; **P<0.001. AF, amniotic fluid; NS, normal saline; SD, standard deviation.

Discussion

AF contains multiple bioactive components, including growth factors and peptides, which have been implicated in neonatal intestinal development (16,21,24,25). In this study, AF exposure was associated with increased tight junction protein expression and higher TEER in both intestinal epithelial monolayers and neonatal rat tissues, while intestinal morphology remained unchanged. These results highlight molecular and ex vivo electrophysiological changes under physiological conditions, suggesting a role for AF in supporting intestinal barrier maturation during the neonatal period. Importantly, this study focuses on native AF under physiological, non-injury conditions and is distinct from prior research using AF-derived stem cells, conditioned media, or NEC/injury models (18,19,26), providing complementary insights into the physiological effects of AF on early intestinal barrier development.

Premature infants are defined as those born before 37 weeks of gestation (25). In 2010, approximately 11.1% of births worldwide were premature, with rates ranging from 5–13% in high-income countries and 12–18% in low-income regions, and the highest prevalence in Asia and Africa (27). AF contains multiple bioactive components and shares certain functional similarities with breast milk, which may contribute to intestinal development (24,26).

AF is a complex biological fluid containing multiple bioactive molecules, including growth factors, hormones, and peptides, whose concentrations vary during gestation (28,29). For example, Vrachnis et al. (28) reported that higher levels of fibroblast growth factor 21 (FGF21) and insulin in early second trimester AF were associated with greater reductions in fetal growth, highlighting the developmental relevance of AF composition. Other studies have similarly shown that the concentration of bioactive components in AF can differ between donors and gestational stages, potentially leading to variable biological effects (29). In the present study, we investigated the effect of AF on intestinal barrier properties at two concentrations (25% and 50%). Although 50% AF produced the strongest response among the tested concentrations, concentrations above 50% were not evaluated; therefore, a plateau, optimal concentration, or potential adverse effects at higher concentrations cannot be determined. Because ex vivo intestinal TEER was evaluated only at P14 and P28, the present study cannot define the complete temporal trajectory of AF-associated barrier changes before P14. Future studies incorporating additional earlier postnatal time points are needed to characterize these dynamic changes. Importantly, this study did not identify the specific active components responsible for these effects nor test the underlying signaling pathways, and therefore the molecular mediators remain to be elucidated in future work.

Tight junctions are intercellular junctions located at the apical region of epithelial cells that restrict the passage of molecules between cells and are a key component of the paracellular barrier (29). The tight junction complex includes Claudin family proteins (at least 27 subtypes) as well as scaffold proteins of the ZO family (ZO-1, ZO-2, ZO-3), occludin, and junctional adhesion molecules TJ (30,31). Alterations in tight junction protein expression can impair barrier function; for example, interleukin (IL)-1β and transforming growth factor (TGF)‑β have been shown to reduce occludin levels and weaken intestinal barrier integrity (32). Accordingly, modulation of tight junction proteins represents a potential mechanism influencing intestinal barrier properties, while recognizing that the present study does not directly test these signaling pathways or causal mechanisms.

Østergaard et al. (33) used newborn preterm pigs that received AF or no supplements orally to examine the effects of AF on intestinal structure and function. They found that, although crypt depth differed between the two groups in the proximal intestine, crypt depth in the distal intestine and villus height did not differ significantly. These observations were made under non-inflammatory conditions before birth. In contrast, Pan et al. (34) studied preterm pigs with chorioamnionitis and reported that villus height was reduced, while crypt depth remained unaffected; these alterations could be improved with timely enteral nutrition after birth. Additionally, Bohórquez et al. (35) investigated intestinal microstructure in embryonic chicken and turkey poults, showing that villus height steadily increased until reaching a plateau at day 8. Taken together, these findings suggest that intestinal structure develops normally in the absence of inflammation and that AF supplementation does not markedly alter intestinal morphology under physiological conditions.

The AF used in this study was collected from pregnant women undergoing cesarean delivery and processed by centrifugation to remove cellular debris, a method shown to preserve the biological activity of AF (36,37). Previous studies using similarly processed AF have primarily focused on experimental models of NEC or intestinal injury, reporting beneficial effects under inflammatory conditions (38,39). For example, Jain et al. (40) demonstrated that supplementation with AF reduced the incidence and severity of NEC in preterm rat models. More recently, AF has been increasingly recognized as an important but understudied contributor to gastrointestinal development, with its diverse bioactive components potentially influencing intestinal maturation during early life (21,41). In contrast, the present study examined the effects of native AF on intestinal barrier-related parameters in full-term neonatal rats under physiological, non-injury conditions. By clearly distinguishing our work from injury-focused studies, these findings complement existing NEC-related research by providing insight into how AF influences intestinal barrier properties under normal physiological conditions without implying therapeutic benefit.

This study has several limitations. First, all experiments were conducted under physiological conditions without the inclusion of an intestinal injury or inflammation model; therefore, the effects of AF on intestinal barrier function under pathological conditions cannot be inferred. Second, although TEER and tight junction protein expression were assessed, direct in vivo intestinal permeability was not evaluated using a functional tracer assay, such as oral FITC-dextran or sugar permeability testing, which limits conclusions regarding definitive in vivo barrier function. Third, Caco-2 and IEC-6 cells showed generally similar responses to AF treatment despite differences in species, intestinal segment, and cellular origin. However, these widely used cell lines may not fully recapitulate the developmental and functional characteristics of neonatal or preterm intestinal tissue, and the findings should not be directly extrapolated to the human preterm intestine. Possible species- or segment-specific differences require further investigation. Fourth, the prolonged separation of neonatal rats from their dams may influence gut permeability, host defense, stress, growth, and hydration, representing a potential confound in interpreting ex vivo barrier findings. Fifth, AF samples were pooled across a gestational range of 34+4 to 39+5 weeks, and batch-to-batch variability may contribute to experimental variability; while pooling ensured standardization and sufficient volume, it may mask biologically meaningful differences between individual donors. Sixth, this study did not identify the specific active components responsible for the observed effects, and mechanistic pathways underlying AF-induced changes were not investigated. Finally, the present study focused specifically on barrier-associated maturation through tight junction protein expression, TEER, permeability, intestinal morphology, and junctional ultrastructure. Immune maturation and oxidative homeostasis were not evaluated and should be investigated in future studies to provide a broader assessment of neonatal intestinal maturation. Future studies using neonatal intestinal organoid- or enteroid-derived epithelial monolayers would provide a more physiologically relevant system for validation and further mechanistic insight.

Conclusions

Native AF promotes early barrier-associated maturation of the neonatal intestine under physiological conditions, enhancing tight junction protein expression and TEER without altering intestinal structure. These findings add to current understanding of how AF influences neonatal intestinal barrier function, complementing previous studies using AF-derived stem cells, conditioned media, or pathological models. This work provides a foundation for future studies to identify the key mediators and optimal concentrations of AF to support intestinal development and neonatal gut health.

Acknowledgments

The authors thank the research personnel and the research volunteers involved with the project. During the preparation of this manuscript, the authors used AI-assisted tools to improve the readability and language of the text. After using these tools, the authors thoroughly reviewed and edited the content to ensure the accuracy, clarity, and integrity of the manuscript. The authors take full responsibility for the content of the publication.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0598/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0598/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0598/prf

Funding: This work was supported by the Health Commission of Zhejiang Province (No. 2018KY128) and the Wenzhou Science and Technology Bureau (No. Y2020075).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0598/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University (approval No. LCKY2020-329). Written informed consent was obtained from 31 donors undergoing elective cesarean delivery. All animal experiments were performed under a project license (No. wydw2020-0909) granted by the Animal Ethics Committee of Wenzhou Medical University, in compliance with NIH Guide for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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