Purpose: To investigate the factors affecting fecal calprotectin in preterm infants at the corrected age of 10-18 months.

Methods: This research employs a cross-sectional design. Infants with a gestational age of less than 37 weeks were recruited at a corrected age of 10 to 18 months (born between April 2023 and April 2024) from the Child Healthcare Department of Suzhou Hospital Affiliated to Nanjing Medical University between June 2024 to August 2024. The fecal calprotectin was measured and was classified into three grades: < 15μg/g, 15-60μg/g/g, and > 60μg/g. Relevant information was collected.

Results: Fecal calprotectin levels in preterm infants who received formula supplementation before 6 months of corrected age were lower than those in infants who received it after 6 months. In convalescent preterm infants with constipation, fecal calprotectin levels exhibit a mild elevation, typically below 60 μg/g. Probiotics demonstrate a more pronounced effect on improving fecal calprotectin grades rather than content.

Conclusions: Compared with non-exclusively convalescing preterm infants within the corrected age of 6 months, those who were exclusively breastfed showed increased intestinal inflammation mediated by neutrophils. Constipation moderately elevates the fecal calprotectin levels. Probiotics may serve as a protective factor against a lower degree of neutrophil-mediated intestinal inflammation. During early gastrointestinal development, long-term, low-grade, and safe inflammatory stimulation may be a positive factor for intestinal barrier development.

• Intestinal inflammation

• Premature

• Constipation

• Nutritional Support

• Probiotics

Liu Y, Ye K (2026) The Landscape of Influential Factors on Fecal Calprotectin in Convalescing Preterm Infants (Corrected Age of 10-18 Months). Pediatr Child Health 14(1): 1362.

CA: Corrected Age; FC: Fecal Calprotectin; NEC: Necrotizing Enterocolitis; NICU: Neonatal Intensive Care Unit; PTI: Preterm Infants.

Preterm infants (PTI) are neonates born at <37 weeks’ gestation. Recent advancements in neonatal intensive care unit (NICU) management have significantly improved their survival [1,2]. The NICU provides 24/7 intensive monitoring and care for high-risk neonates (e.g., PTI, low-birth-weight infants, those with life-threatening conditions). Owing to immature organ systems, PTI face increased risks of severe infections, growth/developmental delays, metabolic disorders, and post-feeding gastrointestinal issues (e.g., vomiting, bloating, diarrhea, gastric retention) compared to full-term infants [1-3]. The first postnatal year is the critical window for catch-up growth [4], and such gastrointestinal complications may impair PTI survival and developmental catch-up [3,4]. Neonatal necrotizing enterocolitis (NEC), an acute early-life intestinal disorder, is more prevalent in PTI. It arises from intestinal bacterial invasion, inducing inflammation that may progress to intestinal wall necrosis or perforation [5,6]. Severe NEC carries a poor prognosis and impairs PTI growth/development [5,6]; while medical progress has improved NEC survival—with mild cases (managed medically) having favorable outcomes [5]-the long-term impact of treated mild NEC on PTI intestinal development remains unclear. Fecal calprotectin (FC), a neutrophil-derived protein, is a validated marker of intestinal inflammation, used in diagnosing inflammatory bowel disease, infectious enteritis, and functional gastrointestinal disorders [7-9]. FC levels surge pre- and during NEC episodes and may remain elevated until NICU discharge [5,6], but data on long-term FC fluctuations in NEC-affected infants are scarce. Fecal consistency is a key determinant of FC detection, with diarrheal stools having higher FC concentrations than formed stools [8-10]. Building on prior work on perinatal factors influencing FC in PTI [11], this study explores how neonatal hospital-related stressors and feeding practices affect FC during the neonatal period, and extends to convalescent PTI (10–18 months corrected age [CA]) to assess long-term implications. Preterm infants’ corrected age (CA) is the “equivalent full-term age” calculated as chronological age minus prematurity weeks. Cow’s milk protein allergy is associated with elevated FC and gastrointestinal symptoms (e.g., bloating, bloody stools, diarrhea) [12,13], and is linked to allergic rhinitis, atopic eczema, and respiratory allergies [14,15]. However, no studies have explored the association between allergic diathesis (e.g., rhinitis, atopic eczema, respiratory allergies) and FC. Thus, this study aims to identify factors influencing FC in convalescent PTI (10–18 months CA; defined as chronological age minus prematurity weeks). For the first time, it examines the long-term impact of NEC on FC and explores the association between allergic diathesis and FC in this cohort. We hypothesize that convalescent PTI with a history of neonatal NEC or allergic predisposition will have higher FC levels than those without NEC.

Ethics Statement

The authors declare that this research adheres to the ethical principles outlined by the Ethics Committee, Suzhou Municipal Hospital (IRB approval No. K-2022 016-K02). All participants enrolled in this study provided written informed consent, which was obtained from their parents or legal guardians. All procedures involving human participants were conducted in accordance with the ethical principles outlined in the International Ethical Guidelines for Health-related Research Involving Humans issued by the Council for International Organizations of Medical Sciences (CIOMS) and the Declaration of Helsinki (1964), as revised in 2013.

Subjects

This study was designed as cross-sectional research. The recruitment of study participants was conducted from June 2024 to August 2024. Based on the number of subjects in relevant similar studies [10-13,16-17], a total of 133 PTI, with a gestational age under 37 weeks and at 10-18 months of CA, were randomly chosen at the Child Healthcare Department of Suzhou Hospital Affiliated to Nanjing Medical University. Two subjects with severe disabilities, malformations, chromosomal or genetic defects, three subjects whose mother had taken oral or intravenous antibiotics during breastfeeding, zero subjects whose mother had mastitis during breastfeeding, four subjects who had experienced diarrhea within one week before fecal sampling, and two of them received antibiotic treatment were excluded. Due to the absence of three fecal specimens, the final number analyzed was 109.

Investigation methods and content

During the recruitment period from June 2024 to August 2024, interviews and written questionnaires were administered, and medical record data were collected. Details, including date of birth, expected date of delivery, gestational age at birth (weeks), maternal gestational age (years), birth weight (gram), sex, days of hospitalization in the NICU at birth (days), and delivery type, were obtained from medical records. Compute the corrected age of PTI according to the expected date of delivery and express results in monthly increments. One week before stool sample collection, interviews and questionnaires were used to collect details about the timing of formula addition and cessation of breast milk, check for any history of neonatal NEC, rhinitis, eczema, and wheezing, antibiotic or probiotic use, and any gastrointestinal issues (diarrhea or constipation). Data collation and verification were conducted from September to October 2024.

Sampling and detection methods of FC

Fecal calprotectin measurements were performed between November 2024 and May 2025. Researchers collected around 5g of feces from each subject and placed it into a sample collection tube for preservation. After collection, the samples were kept at 2-8?. They were sent to the Clinical Laboratory of Suzhou Hospital, affiliated with Nanjing Medical University, within 12 hours for testing. We distributed 122 fecal collection tubes and retrieved 109 of them. The calprotectin concentration in the fecal samples was measured by using the Diagnostic Kit (Colloidal Gold) for Calprotectin (Xiamen Wiz Biotech Co., Ltd.-- Xiamen City, Fujian Province, 361026, P.R. China). The upper reference limit for healthy adults provided by the manufacturer is 60μg/g, and the lower detection limit of the kit is 15μg/g. During data analysis, the content less than 15μg/g was randomly assigned values ranging from 0 to 14μg/g using Microsoft Excel. The FC content of the subjects was classified into three grades: < 15μg/g, 15-60μg/g/g, and > 60μg/g.

Statistical Analysis

The data inspection and analysis were carried out between June and October 2025. SPSS 25.0 (https://www. ibm.com/products/spss-statistics, RRID: SCR_016479) was used for data analysis. For dichotomous variables, such as sex, neonatal conditions, time of formula introduction, probiotic use, constipation, and a history of eczema, rhinitis, or wheezing, frequencies and percentages are used for descriptive purposes. The Mann-Whitney U test was used to examine their associations with FC content. The Chi-square test was chosen to analyze their correlation to FC grades. Fisher’s exact test is recommended when the expected frequency in any cell of a contingency table is below five. The Spearman’s rank correlation analysis was used to investigate the relationships between gestational weeks, maternal age at pregnancy, and FC content and grades. We used the Mann-Whitney U test to examine their links to FC grades. Statistical significance was considered when P<0.05. Multiple linear regression was employed to analyze the relationship between relevant factors and the FC content. Ordinal Logistic regression was utilized to analyze the relationship between the factors and FC grades. The results are presented as regression coefficients (B), standard errors (SE), odds ratios (OR), and corresponding 95% confidence intervals (CI). An α level of 0.05 was set as the threshold for statistical significance.

Characteristics of the subjects

133 subjects born between 25 and 36 weeks of gestational age were recruited, and 109 were finally analyzed. For details, please refer to the flowchart (Figure 1). Females constituted 55% of the subjects (Table 1), and their mean birth weight was 1616.07 g (SD = 487.903). The mean birth weight of males was 1659.27 g (SD = 452.513). Other information about the subjects is as follows (Table 1).

FC content detection

109 fecal samples were collected for the quantitative determination of FC. The average concentration was 37.63 ± 37.796 µg/g, ranging from <15 to 213 µg/g.

Influencing factors of FC in convalescing PTI

No correlations were discovered between sex, history of NEC, timing of breastfeeding cessation, probiotic intake, history of eczema, rhinitis, or wheezing, gestational age, 

Figure 1 The flow diagram.

Table 1: The demographic and clinical features of the subjects (N=109).

Binary categorical variables	n (%)	Z	P
Sex		-1.203	0.229a
Male	60 (55.0)
Female	49 (45.0)
Neonatal NEC		-1.467	0.142a
Yes	4 (3.7)
No	105 (96.3)
Add formula within 6 months		-2.286	0.022*a
Yes	98 (89.9)
No	11 (10.1)
Stop breastfeeding within 6 months		-0.605	0.545a
Yes	67 (61.5)
No	41 (37.6)
Probiotic supplementation		-1.541	0.123a
Yes	22 (20.2)
No	87 (79.8)
Constipation		-2.254	0.024*a
Yes	13 (11.9)
No	96 (88.1)
History of eczema		-0.464	0.642a
Yes	24 (22.0)
No	85 (78.0)
History of rhinitis		-0.209	0.834a
Yes	8 (7.3)
No	101 (92.7)
History of wheezing		-0.435	0.664a
Yes	14 (12.8)
No	95 (87.2)
FC grades (μg/g)		-	-
< 15  (grade 1)	28 (25.7)
15 to 60 (grade 2)	70 (64.2)
> 60  (grade 3)	11 (10.1)
Continuous variable factors	M±SD	ρ	P
Maternal gestational age (y)	31.56±3.89	-0.016	0.866b
Gestational age (wks)	31.95±2.51	0.058	0.549b
Days of hospital stay at birth (d)	38.65±24.62	-0.134	0.164b

*P<0.05*

a Mann-Whitney U test.

b Spearman's rank correlation.

*NEC: necrotizing enterocolitis; FC: fecal calprotectin; CA: corrected age; NICU: Neonatal Intensive Care Unit;

109 subjects born between 25 and 36 weeks of gestational age were analyzed in this research. Females constituted 55% of the subjects, and their mean birth weight was 1616.07 g (SD = 487.903). The mean birth weight of males was 1659.27 g (SD = 452.513). The exclusively breastfed infants within the first six months of CA exhibit higher FC content compared to their non-exclusively breastfed counterparts. Individuals with constipation demonstrate elevated FC content relative to those without constipation.

Table A1: A comparison of the number of subjects with different timings of formula introduction for full-term infants and PTI.

	Full-term infants	PTI
≤ 6 months	41	98
>6 months	10	11

PTI: preterm infants; FC: fecal calprotectin.

Table A2: The varying impacts of the timing of formula introduction on the FC for full-term infants and PTI.

P value	Full-term infants	PTI
FC content	0.054a	0.022*a
FC grade	0.054b	0.114b

*P<0.05*.

a Mann-Whitney U test.

b Fisher’s exact tests.

PTI: preterm infants; FC: fecal calprotectin

No correlations were discovered between sex, history of NEC, timing of breastfeeding cessation, probiotic intake, history of eczema, rhinitis, wheezing, gestational age, maternal gestational age, number of days hospitalized in the NICU at birth and FC content.

maternal gestational age, number of days hospitalized in the NICU at birth, and FC content(P<0.05) (Table 1). Besides, no correlations were discovered between the above factors and FC grades (P<0.05) (Table 2).

Table 2: The relationship between potential correlated factors and FC grades.

Binary categorical variables	χ²	P	Fisher’s P
History of neonatal NEC	5.344	0.069a	0.101b
Formula addition time	4.850	0.088a	0.114b
Breastfeeding cessation time	2.197	0.333a	-
Probiotics supplementation	2.176	0.337a	-
Constipation	6.629	0.036*a	0.020*b
History of eczema	1.785	0.410a	-
History of rhinitis	1.029	0.598a	0.868b
History of wheezing	2.250	0.325a	0.458b
Continuous variables	ρ	P
Gestational age	0.091	0.346c
Maternal gestational age	0.033	0.735c
Days of hospital stay at birth	-0.142	0.141c

*P<0.05*

a Chi-square test.

b Fisher’s exact tests.

c Spearman's rank correlation.

*NEC: necrotizing enterocolitis; FC: fecal calprotectin; CA: corrected age; NICU: Neonatal Intensive Care Unit;

No correlations were discovered between sex, history of NEC, timing of breastfeeding cessation, probiotic intake, history of eczema, rhinitis, wheezing, gestational age, maternal gestational age, number of days hospitalized in the NICU at birth and FC grades.

The average content of FC in PTI who introduced formula milk powder within 6 months of CA was lower than that introduced after 6 months of CA (P = 0.022*) (Table 1). In another study of full-term infants conducted by our research team, such a difference was not observed (Table A1, Table A2). A significant difference was observed between the constipation and FC content (P = 0.024*) (Table 1) and grades (P = 0.020*) (Table 2). The FC content in the constipated group was relatively higher, but less than 60μg/g. The FC grade was also higher in the constipated group.

Multiple linear regression was employed to analyze the 

relationship between the above factors and the FC content. PTI with symptoms of rhinitis, eczema, or wheezing were considered to have an allergic constitution. Based on the above results, we developed two models. Model 1 included only control variables for which no correlations were detected: timing of breastfeeding cessation, allergic constitution, number of days hospitalized in NICU at birth, gestational age, maternal age, probiotic supplementation, and the presence of neonatal NEC. Model 2 was built upon Model 1 by introducing two key independent variables: the timing of formula introduction and the presence of constipation.

The model summary statistics indicated a significant improvement in explanatory power after the inclusion of the key independent variables. As shown in Model 1, the control variables were not statistically significant (F(7, 100) = 1.257, P = 0.279) and accounted for a negligible proportion of the variance in FC content (Adjusted R² = 0.017). In contrast, Model 2 was statistically significant (F(9, 98) = 2.562, P = 0.011*) and explained 11.6% of the variance (Adjusted R² = 0.116). The change in R² from Model 1 to Model 2 was 0.110, which was statistically significant (F Change (2, 98) = 6.631, P = 0.002**), indicating that the two additional variables collectively provided a significant incremental contribution to the model. The Durbin- Watson statistic of 1.918 suggested that the assumption of independent errors was met (Table 3).

Analysis of the regression coefficients in Model 2 revealed that both primary independent variables were significant predictors of FC content. Introduction of formula after 6 months was significantly associated with higher FC content (B = 27.613, P = 0.026*). The presence of constipation was a strong and significant positive predictor of FC content (B = 31.905, P = 0.006**) (Table 3).

None of the control variables included in Model 1 reached statistical significance in either model (all P> 0.05) (Table 3). Furthermore, collinearity statistics confirmed that multicollinearity was not a concern in the analysis, as all Variance Inflation Factor (VIF) values were well below the threshold of 10 (range: 1.047–3.479).

In summary, the results demonstrate that later introduction of formula (>6 months) and the presence of constipation are independent and significant factors associated with increased FC content in convalescing PTI, after controlling for relevant perinatal and clinical factors.

In order to enhance the sensitivity of the analysis, the influencing factors listed were also included in the ordinal logistic regression model to examine the relationship.

Table 3: Results of the multiple linear regression model.

FC content		B	t	P	Tolerance	VIF
Model 1	Intercept	0.956	0.01	0.992
	Breastfeeding cessation	8.359	1.082	0.282	0.934	1.071
	Allergic constitution	-11.044	-1.443	0.152	0.971	1.03
	Days of the NICU	0.019	0.068	0.946	0.298	3.357
	Gestational age	1.813	0.676	0.5	0.29	3.445
	Maternal age	-0.56	-0.585	0.56	0.955	1.048
	Probiotic use	-14.811	-1.547	0.125	0.914	1.094
	History of NEC	-16.853	-0.838	0.404	0.911	1.098
Model 2	Intercept	-42.243	-0.464	0.644
	Breastfeeding cessation	5.137	0.669	0.505	0.85	1.176
	Allergic constitution	-8.513	-1.163	0.248	0.955	1.047
	Days of the NICU	0.159	0.599	0.551	0.292	3.429
	Gestational age	2.537	0.993	0.323	0.287	3.479
	Maternal age	-0.282	-0.309	0.758	0.94	1.064
	Probiotic use	-14.747	-1.614	0.11	0.902	1.109
	History of NEC	-19.592	-1.015	0.312	0.889	1.125
	Time of formula adding	27.613	2.257	0.026*	0.862	1.161
	formula adding (Reference:≤6 Ma)	-	-	-	-	-
	Constipation	31.905	2.81	0.006**	0.927	1.079
	Constipation (Reference: No)	-	-	-	-	-

*P<0.05*, P<0.001**.

*Model 1: F=1.257, P=0.279, Adjusted R2=0.017; Model 2: F=6.631, P=0.011*,

Adjusted R2=0.116.

a Corrected age of 6 months.

*NEC: necrotizing enterocolitis; FC: fecal calprotectin; CA: corrected age; NICU: Neonatal Intensive Care Unit;

Introduction of formula after 6 months was significantly associated with higher FC content (B = 27.613, P = 0.026*). The presence of constipation was a strong and significant positive predictor of FC content (B = 31.905, P = 0.006**). The Variance Inflation Factor (VIF) values were well below the threshold of 10 (range: 1.047– 3.479).

In Model 1, the control variables were not statistically significant (F(7, 100) = 1.257, P = 0.279) and accounted for a negligible proportion of the variance in FC content (Adjusted R² = 0.017). In contrast, Model 2 was statistically significant (F(9, 98) = 2.562, P = 0.011*) and explained 11.6% of the variance (Adjusted R² = 0.116). The Durbin-Watson statistic of 1.918 suggested that the assumption of independent errors was met.

between each factor and the FC grades. The results showed that the history of NEC, the timing of formula addition, probiotic supplementation, and constipation, and the FC grades (P < 0.05) (Table 4).The FC grades of PTI without constipation tended to be lower (OR = 0.160, 95% CI: 0.039, 0.659) (P=0.011*). Starting formula milk powder supplementation earlier (≤6 months) resulted in lower FC grades than those who began later (>6 months) (OR = 0.162, 95% CI: 0.036, 0.732) (P=0.018*). Infants who did not receive probiotic supplementation had 3.165 times the risk of elevated FC grades compared to those who did (OR = 3.165, 95% CI: 1.102, 9.096) (P=0.032*). In comparison to PTI with NEC during the neonatal period, the FC grades of PTI without NEC were higher (OR = 18.915, 95% CI: 1.383, 259.105) (P = 0.028*). It should be noted that the 95%CI of OR for NEC history is extremely wide, revealing a result of considerable uncertainty (Table 4).

Table 4: Results of the Ordinal logistic regression model.

FC grades	B	SE	P	OR(95%CI)
Maternal age	0.035	0.055	0.521	1.036(0.931, 1.154)
Gestational age	-0.047	0.151	0.753	0.954(0.709, 1.281)
Days of the NICU	-0.011	0.016	0.485	0.989(0.959, 1.020)
Time of formula adding (≤6 M)	-1.82	0.77	0.018*	0.162(0.036, 0.732)
Time of formula adding(Reference:>6 M)	-	-	-	-
Breastfeeding cessation (≤6 M)	0.483	0.457	0.291	1.621(0.661, 3.971)
Breastfeeding cessation (Reference:>6 M)	-	-	-	-
Probiotic use: No	1.152	0.539	0.032*	3.165(1.102, 9.096)
Probiotic use (Reference: Yes)	-	-	-	-
Constipation: No	-1.831	0.722	0.011*	0.160(0.039, 0.659)
Constipation (Reference: Yes)	-	-	-	-
Allergic constitution: No	0.382	0.439	0.383	1.465(0.621, 3.462)
Allergic constitution (Reference: Yes)	-	-	-	-
History of NEC: No	2.94	1.334	0.028*	18.915(1.383, 259.105)

*P<0.05*

*NEC: necrotizing enterocolitis; FC: fecal calprotectin; CA: corrected age; NICU: Neonatal Intensive Care Unit;

The results showed the association between the history of NEC, the timing of formula addition, probiotic supplementation and constipation and the FC grades. In conclusion, constipation is a crucial risk factor for the elevation of FC, and introducing formula before 6 months of CA is a protective factor for the increase of FC. These have been confirmed in both regression models. Administering probiotics may be a protective factor for a lower degree of FC. There is insufficient evidence to establish a relationship between a history of NEC and FC.

The mean fecal calprotectin (FC) concentration in this study was lower than that reported in neonatal preterm infant (PTI) cohorts [16,17]. FC accumulates in fetal meconium (being highly degradation-resistant) and rises rapidly in the first postnatal week [11]; regional/ethnic differences or varied detection kits may also contribute to FC variability [9,11,18].

Constipation emerged as a risk factor for elevated FC content and grades. Few studies have explored this association, but a functional constipation study reported mean FC <50 μg/g consistent with our findings—indicating mild or subclinical neutrophil-mediated intestinal inflammation in constipation [18].

Contrary to our hypothesis, formula introduction after 6 months corrected age (CA) was linked to higher FC. Late formula initiators rely primarily on breast milk, which contains gut microbiota [19], and inflammatory factors [11], that induce intestinal immune responses, increasing 

basal FC-associated inflammation. Breast milk promotes PTI intestinal barrier maturation [20], so we hypothesize that low-intensity, sustained, safe inflammatory stimuli during early gastrointestinal development may benefit barrier function—low-grade neutrophil-mediated inflammation is not inherently detrimental.

While Mann-Whitney U, multiple linear regression, and Chi-square tests showed no correlation between probiotic use and FC, ordinal logistic regression revealed a significant association with reduced FC grades (not absolute values)—indicating probiotics exert grade- specific anti-inflammatory effects rather than linear reductions. This aligns with studies showing probiotics lowered FC in dermatitis-induced mice [21], and Bifidobacterium supplementation reduced FC in breastfed neonatal PTI [22].

Contrary to our hypothesis, PTI with a neonatal NEC history had lower FC grades at 10–18 months CA. However, only 4 NEC cases (vs. 105 non-NEC) were included, leading to a wide OR 95% CI (0.724, 259.105) and insufficient statistical power. While multivariable models identified NEC history as a FC grade predictor, this finding is preliminary and requires validation in large- scale prospective studies.

No significant associations were found between FC and NICU hospitalization duration or allergic constitution. While U.S. research linked neonatal NICU-related pain/ stress to early-life FC [11], our data suggest such intestinal stress responses diminish over time with environmental changes. Prior studies linked allergic enteritis/cow’s milk protein allergy to higher FC [23], but our cohort had mild, early-stage allergic symptoms (no overt food intolerance), explaining the lack of correlation. Future studies could stratify by allergy severity or include older children to explore this association.

In addition, the findings of that the FC grades of PTI with a history of neonatal NEC were lower than those of the non-NEC group at 10-18 months CA. This result was also contrary to our research hypothesis. It should be noted that only four subjects with a history of neonatal NEC, compared with 105 non-NEC PTI, were included in this re-search. Although the multivariable model recognized a history of neonatal NEC as a potent predictor of FC grades, this finding should be interpreted with prudence. The small sample size resulted in a wide 95% CI for OR (0.724, 259.105). This might lead to insufficient statistical power of the results. Consequently, the strength of the association between a history of neonatal NEC and FC grades requires verification in a prospective study with a larger sample size.

This study did not find a significant influence of the number of neonatal hospitalization days in NICU and allergic constitution on the FC content. A study from the United States indicated that the cumulative pain/stress endured by PTI during their NICU hospitalization might affect the FC content in early life [11]. Nevertheless, it did not focus on the long-term impact of that factor on FC. Our research indicated that the intestinal stress response from early life may diminish over time due to environmental changes.

Previous studies suggest that children with allergic enteritis and cow’s milk protein allergy tend to have higher intestinal inflammation markers (FC) [23]. In this research, the participants predominantly had mild eczema and rhinitis. They were young and did not display clear signs of food intolerance. Consequently, no correlation was detected between allergic constitution and FC. Stratification of subjects by allergy severity or inclusion of older children may be considered to further investigate the potential association.

Formula supplementation timing influences neutrophil- mediated intestinal inflammation in convalescent preterm infants. Constipation induces a mild elevation of fecal calprotectin (FC) that does not exceed the upper detection limit. Probiotics exert a stronger effect on improving FC categorical grades than on reducing its linear measured values. Evidence is insufficient to confirm an association between neonatal necrotizing enterocolitis (NEC) history and FC in convalescent preterm infants. No significant correlations were observed between FC (content or grades) and sex, gestational age, maternal age at pregnancy, NICU stay duration, breastfeeding cessation timing, or allergic diathesis in this cohort. This study also has limitations, including a small sample size of infants with NEC, lack of stratification by allergy severity, and cross-sectional FC data. Future research should use larger NEC samples, stratify by allergy severity, adopt a longitudinal design, and standardize FC detection.

This study was funded by the Jiangsu Commission of Health [Jiangsu Health Commission Office Maternal and Child Health Document No. 19 (2021)].

The author wishes to extend sincere gratitude to the participating infants and their parents, as well as the medical staff and workers in the Child Healthcare  Department of Suzhou Hospital Affiliated to Nanjing Medical University, and Suzhou Maternal and Child Healthcare Hospital. In particular, Dr Lu Xiaoting, Dr Li Beiquan, and Dr Pan Qiuping.Additionally, we extend our gratitude to the Clinical Laboratory of Suzhou Hospital Affiliated to Nanjing Medical University for their contributions to specimen testing, and especially to Prof. Chen Yan for inspecting the fecal specimens. Our gratitude also goes to Guo Yi for the help with specimen collection. We also thank Dr Chen Yiyuan, M.M., a public health expert, for her key contributions. She handled quality control and conducted a review of the statistical data.

 

1. Cao Y, Jiang S, Sun J, Hei M, Wang L, Zhang H, et al. Assessment of Neonatal Intensive Care Unit Practices, Morbidity, and Mortality Among Very Preterm Infants in China. JAMA network open. 2021; 4: e2118904.
2. Bai R, S. Jiang S, Guo J, Jiang S, Lee SK, Li Z, et al. Variation of Neonatal Outcomes and Care Practices for Preterm Infants <34 Weeks’ Gestation in Different Regions of China: A Cohort Study. Front Pediatrics. 2021; 9: 760646.
3. Sun H, Su X, Mao J, Du Q. Impact of pre-pregnancy weight on the risk of premature rupture of membranes in Chinese women Zhonghua fu chan ke za zhi. 2023; 49: 481-485.
4. Zeng J, Yu W, Gao X, Yu Y, Zhou Y, Pan X. Establishing reference values for age-related fecal calprotectin in healthy children aged 0-4 years: a systematic review and meta-analysis. Peer J. 2025; 13: e19572.
5. Bethell GS, Hall NJ. Recent advances in our understanding of NEC diagnosis, prognosis and surgical approach. Front Pediatr. 2023; 11: 1229850.
6. Cai X, Liebe HL, Golubkova A, Leiva T, Hunter CJ. A Review of the Diagnosis and Treatment of Necrotizing Enterocolitis. Curr Pediatr Rev. 2023; 19: 285-295.
7. Bryce C, Bucaj M. Fecal Calprotectin for the Evaluation of InflammatoryBowel Disease. Am Fam Physician. 2021; 104: 303-304.
8. D’Amico F, Rubin DT, Kotze PG, Magro F, Siegmund B, Kobayashi T, et al. International consensus on methodological issues in standardization of fecal calprotectin measurement in inflammatory bowel diseases. United European gastroenterology J. 2021; 9: 451-460.
9. Mari A, Baker FA, Mahamid M, Yacoob A, Sbeit W, Khoury T. Clinical utility of fecal calprotectin: potential applications beyond inflammatory bowel disease for the primary care physician. Ann Gastroenterol. 2019; 32: 425-430.
10. Velasco Rodríguez-Belvís M, Viada Bris JF, Plata Fernández C, García- Salido A, Asensio Antón J, Domínguez Ortega G, et al. Normal fecal calprotectin levels in healthy children are higher than in adults and decrease with age. Paediatr child health. 2020; 25: 286-292.
11. Xu W, Zhang Y, Zhao W, Chen J, Maas K, Hussain N, et al. Trends of fecal calprotectin levels and associations with early life experience in preterm infants. Interdisciplinary Nursing Res. 2022; 1: 36-42.
12. Anania C, Mondì F, Brindisi G, Spagnoli A, De Canditiis D, Gesmini A, et al. Fecal Calprotectin Determination in a Cohort of Children with Cow’s Milk Allergy. Nutrients. 2025; 17: 194.
13. Zhang ZH, Wang W, Zhang XH, J. Pan J, Chen X. Fecal Calprotectin in Self-Reported Milk Intolerance: Not Only Lactose Intolerance. Int Arch Allergy Immunol. 2023; 183: 1189-1197.
14. Hao L, Wang S, Ji W. Cow’s milk protein allergy: A comprehensive review of epidemiology, pathogenesis, clinical manifestations, diagnostics, and management strategies. Asia Pac J Clin Nutr. 2025; 34: 298-307.
15. Demirci B, Y?lmaz Topal O, Turgay Ya?mur I, Dibek M?s?rl?o?lu E. Development of respiratory allergic diseases according to cow’s milk protein allergy mechanisms. Postgrad Med. 2025; 137: 416-422.
16. Rougé C, Butel MJ, Piloquet H, Ferraris L, Legrand A, Vodovar M, et al. Fecal calprotectin excretion in preterm infants during the neonatal period. PloS one. 2010; 5: e11083.
17. Hannawayya R, Puentes R, Mirzadzare N, Cirone K, Amin H, Soraisham A, et al. Metabolomic Analysis and Visualization Engine for LC−MS Data. Pediatr Res. 2025; 98: 278-285.
18. Mahjoub FE, Zahedi N, Ashjai B, Ashtiani MT, Farahmand F, Monajemzadeh M, et al. Role of Fecal Calprotectin in Differentiating between Hirschsprung’s Disease and Functional Constipation. The Korean J Gastroenterol = Taehan Sohwagi Hakhoe chi. 2013; 62: 288-291.
19. Zhernakova A, Yassour M, Hall LJ, Collado MC. Unlocking the power of human milk and infant feeding: Understanding how nutrition and early microbiota interaction shapes health programming. Cell Host Microbe. 2025; 33: 820-835.
20. Roskes L, Chamzas A, Ma B, Medina AE, Gopalakrishnan M, Viscardi RM, et al. Early human milk feeding: Relationship to intestinal barrier maturation and postnatal growth. Pediatr Res. 2025; 97: 2065-2073.
21. Kim MJ, Kim JY, Kang M, Won MH, Hong SH, Her Y. Polydeoxyribonucleotides as Emerging Therapeutics for Skin Diseases: Clinical Applications, Pharmacological Effects, Molecular Mechanisms, and Potential Modes of Action. Int J Mol Sci. 2020 21:10.
22. Larke JA, Kuhn-Riordon K, Taft, DH, Sohn K, Iqbal S, Underwood MA, et al. Preterm Infant Fecal Microbiota and Metabolite Profiles Are Modulated in a Probiotic Specific Manner. J Pediatr Gastroenterol Nutr. 2022; 75: 535-542.
23. Czaja-Bulsa G, Bulsa K, ?okie? M, Drozd A. Adherence to Gluten-FreeDiet in Children with Celiac Disease Nutrients. 2024; 10: 1424.