IntroductionExtremely preterm infants commonly develop marked early changes in plasma glucose and sodium concentrations, but their clinical relevance remains unclear. We therefore aimed to characterize plasma glucose, sodium, and calculated osmolarity, and examine their associations with short-term neonatal outcomes.MethodsWe conducted a retrospective single-center cohort study of 42 extremely preterm infants (22+0 - 27+6 weeks’ gestation). Levels, variability, and temporal patterns in serial plasma glucose and sodium measurements, as well as calculated plasma osmolarity, were analyzed during the first postnatal week. Associations with neonatal outcomes were assessed in adjusted analyses.ResultsThe lowest glucose value and greater glucose variability during the first postnatal week were associated with duration of mechanical ventilation, bronchopulmonary dysplasia and the composite outcome of bronchopulmonary dysplasia or death. A larger decrease in glucose during the first 4 postnatal days was associated with necrotizing enterocolitis. Plasma sodium and calculated plasma osmolarity during the first postnatal week were associated with surgical ligation of patent ductus arteriosus but showed no consistent associations with the other outcomes studied.DiscussionThese exploratory findings suggest that associations with subsequent neonatal morbidity were more consistently observed for glucose-derived measures than for plasma sodium or calculated plasma osmolarity. Whether these associations are causal or reflect underlying physiological vulnerability requires further investigation.

OBJECTIVE:To investigate respiratory practices to prevent or treat evolving bronchopulmonary dysplasia in neonatal intensive care units (NICUs) across Europe. STUDY DESIGN:Between March and July 2024, a web-based survey was sent to European NICUs caring for infants born preterm with gestational age <28 weeks. RESULTS:We received replies from 447 of 721 (62%) NICUs across 24 European countries. Almost 16% of NICUs routinely intubate at birth, especially if the gestational age is <24 weeks. During transition most NICUs use continuous positive airway pressure ≥5 cmH2O and start with an FiO2 0.3. Volume-targeted ventilation is the primary ventilation mode in 60% of the NICUs. Permissive hypercapnia is a common practice. Higher SpO2 target limits have been adopted, although alarm settings vary across NICUs. Caffeine is routinely started (96%). Surfactant is used in all NICUs, mostly rescue (74%) via less invasive administration (81%). Prophylactic inhaled nitric oxide is not used. Treatment of patent ductus arteriosus varies; half of NICUs pharmacologically treat patent ductus arteriosus early, based on echocardiographic findings. Ureaplasma screening is done in 22% of NICUs. Most (97%) NICUs use postnatal corticosteroids, with dexamethasone being the preferred drug (65%) and starting 2-3 weeks after birth. Only 5% use corticosteroids prophylactically. After 2-3 weeks, diuretics are used frequently, inhaled corticosteroids/bronchodilators to a much lesser extent. CONCLUSIONS:This large survey shows considerable practice variation in preventing and treating evolving bronchopulmonary dysplasia across Europe, especially for interventions with limited evidence.

OBJECTIVES:To investigate fluid balance, sodium intake, and hyperglycemia during the first 2 weeks of life in infants born at 22-23 weeks of gestational age, and to examine their associations with hospital outcomes. STUDY DESIGN:Retrospective, single-center, cohort study of all infants surviving beyond 72 hours (22-23 weeks; January 2019-June 2024). Total fluid intake, weight, and urine output were collected during the first 14 days, and used to calculate daily insensible water loss. Daily intake of sodium, plasma sodium and glucose levels, and the occurrence of acute kidney injury (AKI) were recorded. Relationships with bronchopulmonary dysplasia, patent ductus arteriosus, necrotizing enterocolitis, and death were analyzed. RESULTS:Survival until discharge was 57% (39/69). The nadir weight loss was -15 ± 6% (mean ± SD) at median 3 (IQR 2-5) days. Average total fluid intake during the first week of life was 176 ± 25 mL/kg/day; peak mean urine output (119 ± 39 mL/kg/day) and insensible water loss (87 ± 54 mL/kg/day) occurred at days 2 and 5, respectively. The median daily sodium intake was 4.6 and 4.5 mmol/kg/day in the first and second weeks, respectively. Hypernatremia (plasma sodium >150 mmol/L) occurred in 42% and prolonged (>2 days) hyperglycemia (plasma glucose >10 mmol/L) was frequent (87%). AKI occurred in 28%. Mortality was independently associated with weight loss >15% (OR: 3.9, 95% CI: 1.4-11.1, P = .013), and AKI (OR: 3.5, 95% CI: 1.1-11.3, P = .034). CONCLUSIONS:In infants born at 22-23 weeks of gestation, a large loss of body weight and AKI are prevalent during the first 2 weeks of life and are associated with increased mortality. Preventing excessive weight loss might improve outcomes.

Background Our aim was to evaluate if increased survival and new ventilation strategies were accompanied by a changed incidence of bronchopulmonary dysplasia (BPD) in Sweden over a decade. Methods Data from two Swedish population-based studies of live-born infants with gestational age (GA) 22-26 weeks, born during 2004-2007 (n=702) and 2014-2016 (n=885), were compared for survival, any BPD, moderate BPD and severe BPD and the composite outcomes of any BPD or death and severe BPD or death at 36 weeks postmenstrual age (PMA). Ventilation strategies and interventions were analysed. Any BPD was defined as the use of supplemental oxygen or any respiratory support at 36 weeks PMA, moderate BPD as nasal cannula with <30% oxygen and severe BPD as >= 30% oxygen, continuous positive airway pressure (CPAP) or mechanical ventilation. Results Survival to 36 weeks PMA increased from 72% to 81% (p<0.001). Total days on mechanical ventilation increased from a median of 9 to 16 days (p<0.001). High-flow nasal cannula (HFNC) was introduced between the cohorts, and days of CPAP and HFNC increased from 44 to 50 days (p<0.001). Any BPD was unchanged at 65% versus 68%. Moderate BPD increased from 37% to 47% (p=0.003), while the incidence of severe BPD decreased from 28% to 23% (p<0.046). Severe BPD or death decreased from 48% to 37% (p<0.001), while any BPD or death remained unchanged at 74% versus 75%. Conclusion Even though an increased survival of infants born at 22-26 weeks GA was accompanied by an increased duration of invasive and non-invasive respiratory support, the incidence of any BPD remained unchanged, while severe BPD decreased in infants alive at 36 weeks PMA.

BACKGROUND:Invasive ventilation of infants born before 24 weeks of gestation is critical for survival and long-term respiratory outcomes, but currently there is a lack of evidence to guide respiratory management. We aimed to compare respiratory mechanics and gas exchange in ventilated extremely preterm infants born before and after 24 weeks of gestation. METHODS:Secondary analysis of two prospective observational cohort studies, comparing respiratory mechanics and indices of gas exchange in ventilated infants born at 22-24 weeks of gestation (N=14) compared to infants born at 25-27 weeks (N=37). The ventilation/perfusion ratio (VA/Q), intrapulmonary shunt, alveolar dead space (VDalv) and adjusted alveolar surface area (SA) were measured in infants born at the Neonatal Unit of King's College Hospital NHS Foundation Trust, London, UK. RESULTS:Compared to infants of 25-27 weeks, infants of 22-24 weeks had higher median (IQR) intrapulmonary shunt [18 (4 - 29) % vs 8 (2 - 12) %, p=0.044] and higher VDalv [0.9 (0.6 - 1.4) vs 0.6 (0.5 - 0.7) ml/kg, p=0.036], but did not differ in VA/Q. Compared to infants of 25-27 weeks, the infants of 22-24 weeks had a lower adjusted SA [509 (322- 687) vs 706 (564 - 800) cm2, p=0.044]. The infants in the two groups did not differ in any of the indices of respiratory mechanics. CONCLUSION:Ventilated infants born before 24 completed weeks of gestation exhibit abnormal gas exchange, with higher alveolar dead space and intrapulmonary shunt and a decreased alveolar surface area compared to extreme preterms born after 24 weeks of gestation.

Background:Unplanned extubation (UE) represents an unwanted adverse event in neonatal intensive care. Although skin-to-skin care (SSC) in intubated infants receiving mechanical ventilation (MV) is challenging, its impact on the incidence of UEs has not been reported. Purpose:To determine the incidence, infant characteristics, and short-term respiratory outcomes of UEs in a unit applying SSC as standard of care also during MV. Methods:Single-center retrospective cohort study including all infants receiving MV in a Swedish tertiary care center during 2021-2023. UE incidence was calculated per 100 days of MV related to time spent in conventional care (CC) and SSC, using automated chart review of electronic medical records. Pre-defined short-term respiratory outcomes were mode of respiratory support, ventilator settings and fraction of inspired oxygen (FiO2), at 30-120 min post-UE. Results:The UE incidence was 3.9 per 100 days of MV (3.0 in CC vs. 10.4 in SSC; p < 0.001). The UE incidence during SSC decreased from 14.5 in 2021, to 7.7 in 2023 (p = 0.07), whereas it remained the same during CC. After UE, 72% infants were reintubated within 120 min, and showed an increased mean FiO2 (0.37 vs. 0.43; p = 0.01). Conclusions:The number of UEs were high during SSC but decreased during the study period. Reintubation was not required in >25% of all UEs, regardless of type of care. Following UE, an increased need for supplemental oxygen was observed. Safe SSC in mechanically ventilated infants requires experienced staff and increased staff and parental risk awareness.

The early air distribution after surfactant instillation can have implications for the development of lung injury in extremely preterm newborns with respiratory distress syndrome (RDS). The objective of this study is to determine lung air distribution and focal heterogeneity after surfactant treatment of RDS, using computerized image analysis of chest radiographs (CXRs). This is a retrospective observational non-paired study where CXRs were assessed within 6 h after surfactant instillation in the surfactant-treated group [STG] (n = 52; median 24+4; range 22+0–27+5 weeks gestation) and at the same postnatal age in the pre-surfactant group [PSG] (n = 8; 25+3; 23+6–26+4). Digital image analysis was performed by ImageJ of lung intercostal segments 2–7 bilaterally: mean pixel intensity (MPI) determines focal lung density and correlates negatively with lung aeration; focal heterogeneity in pixel intensity (FHPI) defines focal differences in aeration and correlates positively with overexpansion/atelectasis. MPI was higher in the left lung in STG than in PSG (p = 0.008). MPI was higher in the apical compared to the basal regions bilaterally, in both STG (right, p = 0.024; left, p = 0.015) and PSG (right, p = 0.047; left, p < 0.001). FHPI was higher on the right compared to the left 7th intercostal segment in STG (p = 0.006). FHPI of the right 7th segment was higher in STG than in PSG (p < 0.001). Conclusions: In this retrospective observational study of CXRs in extremely preterm infants, the right lungs were more aerated than the left lungs after surfactant, whereas the basal regions were more aerated than the apical regions regardless of surfactant treatment. We hypothesize that increased focal heterogeneity in these more aerated regions after surfactant may reflect overexpansion/atelectasis, which may be secondary to an inhomogeneous surfactant distribution, an observation that needs to be further validated in future studies.

Collagen type VI (COL6) is an important component of the extracellular matrix (EM) and may have a major role in lung development and disease. Studies on COL6 expression during lung development are mainly based on animal models. The aim of the study was to define COL6 expression pattern in lung parenchyma in infants with different lung maturational stages.COL6 expression in 115 lung samples from deceased newborn infants (21-41 weeks’ gestational age; 0-228 days’ postnatal age) was studied by immunohistochemistry combined with digital image analysis.The distribution of COL6 expression was generally heterogeneous in the lung parenchyma of preterm and term infants. The size of the high-density and low-density areas appeared with logarithmic correlation and COL6 defined the basement membrane (BM) with a prominent expression around the air spaces in the canalicular stage during the first postnatal week. Infants at the alveolar stage showed linear correlation and a fine filamentous appearance during the first week of postnatal life, similarly to adults.COL6 is condensed to areas corresponding to the BM during the first postnatal week of the canalicular stage of lung development. After the first postnatal week COL6 expression changes to a microfibrillar appearance in the ECM, similar to the pattern that characterizes the later alveolar stage and adults. The localization of COL6 during the canalicular and saccular stages might have a higher impact on lung development than the amount of COL6.

BACKGROUND:Recommendations are limited regarding the placement of oral endotracheal tube (ETT), and umbilical arterial/venous catheter (UAC/UAC) in the tiniest extremely preterm infants. We aimed to determine optimal insertion depths, and assess the impact of a too deep ETT position on outcomes. METHODS:All infants born at 22-23 weeks gestation in 2019-2024 at Uppsala University Hospital, Sweden, were evaluated radiologically for accurate positions defined as: ETT (not right-sided/in main bronchus), UAC (T6-9 or L3-4), and UVC (right atrium/inferior vena cava junction). ETT position was further analyzed in relation to time to first extubation, respiratory severity score, duration of mechanical ventilation, bronchopulmonary dysplasia, and mortality. RESULTS:The cohort (n = 75; 22w n = 39; 23w n = 36) had a survival rate of 41 and 64%, respectively. The ETT was accurately placed in 75%, and lower birth weight was associated with a too deep tip position (p = 0.018). The optimal median (IQR) insertion depths were: ETT 5.5 (5.5-6.0); low UAC 6.0 (5.5-6.5); high UAC 9.6 (9.2-10.3), and UVC 5.5 (5.0-6.1) cm. ETT position was not associated with respiratory outcomes or mortality. CONCLUSION:The suggested insertion depths can be expected to result in accurate positioning of ETTs and umbilical lines in infants born at 22-23 weeks gestation. IMPACT:There is limited information to guide delivery room placement of endotracheal tube (ETT) and umbilical catheters (UC) in infants born at a gestational age (GA) of 22-23 weeks. An evaluation standardized insertion depths for ETT and UC, with use of x-ray based measurements of their positions, demonstrate the feasibility of using GA-based insertion depths. The suggested insertion depths can be expected to result in accurate ETT and UC tip positions in infants born at 22-23 weeks.

INTRODUCTION:Episodes of apnoea are common in extremely preterm infants (EPIs) and usually treated with caffeine and respiratory support. Understanding differences in apnoea definitions, monitoring practices, and use of respiratory stimulants is essential to improve future treatment. METHODS:Between March and July 2024, one lead consultant at European tertiary neonatal intensive care units (NICUs) was invited to complete a web-based survey on respiratory practices in EPIs. We sought information how they defined apnoea and monitored for it, and how they treated it with caffeine, doxapram, and non-invasive respiratory support. RESULTS:We received replies from 447/721 (62%) NICUs across 24 European countries. Most NICUs (74%) use both electrocardiogram electrodes and pulse oximetry for apnoea monitoring. All NICUs reported using caffeine citrate, with 102 centres (23%) starting it in the delivery room. The median loading, maintenance and maximum maintenance doses used are 20 mg/kg, 5 and 10 mg/kg/day, respectively. Caffeine is occasionally given twice daily in some NICUs (30%) and stopped at 34-35 weeks of postmenstrual age at most of them (74%). Doxapram is used at 111 (25%) NICUs, with geographical differences. Strategies for the use and escalation of non-invasive respiratory support in case of persistent apnoea are not clearly defined. Automatic closed-loop oxygen delivery is used at 25% of NICUs. CONCLUSION:Despite consistency in the dosing and weaning of caffeine, there is much variation in the management of apnoea in preterm infants across Europe. Future research should focus on timing and dosage of caffeine, the use of doxapram, and strategies for optimising non-invasive respiratory support.

Objective Bronchopulmonary dysplasia (BPD) remains the most common late morbidity for extremely premature infants. Care of infants with BPD requires a longitudinal approach from the neonatal intensive care unit to ambulatory care though interdisciplinary programs. Current approaches for the development of optimal programs vary among centers. Study Design We conducted a survey of 18 academic centers that are members of the BPD Collaborative, a consortium of institutions with an established interdisciplinary BPD program. We aimed to characterize the approach, composition, and current practices of the interdisciplinary teams in inpatient and outpatient domains. Results Variations exist among centers, including composition of the interdisciplinary team, whether the team is the primary or consult service, timing of the first team assessment of the patient, frequency and nature of rounds during the hospitalization, and the timing of ambulatory visits postdischarge. Conclusion Further studies to assess long-term outcomes are needed to optimize interdisciplinary care of infants with severe BPD. Key Points

Objective Defective Goblet cells have been proposed to be involved in necrotizing enterocolitis (NEC). The aim was to study the expression of the Goblet cell marker REG4 and its potential involvement in NEC in preterm infants with and without NEC. Study Design Seventy histologically intact intestinal biopsies were studied: 43 were collected during surgery due to NEC (NEC group: 26.5 ± 3.0 weeks' gestational age [wGA]), and 27 from individuals who underwent surgery due to other conditions (Control group; 36.1 ± 4.5 wGA). The tissue samples were immunohistochemically stained for REG4. REG4 expression was quantified with a semiautomated digital image analysis and with clinical data compared between the groups. Results REG4 expression was lower in the NEC group than in the Control group (p = 0.035). Low REG4 expression correlated to the risk of NEC (p = 0.023). In a multivariable logistic regression analysis including GA and REG4 expression for NEC risk, only GA (p < 0.001) and not REG4 expression (p = 0.206) was associated with NEC risk. Conclusion This study concludes that Goblet cell dysfunction may be involved in NEC development, as low expression of the Goblet cell marker REG4 was related to an increased NEC risk in preterm infants. Maturity could however not be excluded as a potential confounder for REG4 expression. Key Points

Background: Patent ductus arteriosus (PDA) in premature infants is associated with adverse clinical outcomes. Mode and timing of treatment are still controversial. Data are limited in the most extremely premature infants <26 weeks of gestational age (GA), where clinical problems are most significant and patients are most vulnerable. Aims: To investigate whether different approaches to surgical closure of PDA in two large Swedish centers has an impact on clinical outcomes including mortality in extremely preterm infants born <26 weeks GA.Study design: Retrospective, two-center, cohort study.Subjects: Infants born at 22+0-25+6 weeks GA between 2010-2016 at Uppsala University Children’s Hospital (UUCH; n=228) and Queen Silvia Children’s Hospital Gothenburg (QSCHG; n=220).Main outcome measures: survival, bronchopulmonary dysplasia (BPD), and retinopathy of prematurity (ROP). Results: Surgical closure of PDA was more common and performed earlier at QSCHG (50% vs 16%; median age 11 vs 44 days; p < 0.01). Survival was similar in both centres. There was a higher incidence of severe BPD and longer duration of mechanical ventilation at UUCH (p<0.01) There was a higher incidence of ROP, IVH and sepsis at QSCH (p<0.05, p<0.01 and p<0.01). A sub-group analysis matching all surgically treated infants at QSCHG with infants at UUCH with the same GA showed similar results as the total cohort.Conclusion: Earlier and higher rate of surgical PDA closure in this cohort of extremely preterms born <26 weeks GA did not impact mortality but was associated with lower rates of severe BPD and higher rates of severe ROP.

BACKGROUND:Patent ductus arteriosus (PDA) in premature infants is associated with adverse clinical outcomes. Mode and timing of treatment are still controversial. Data are limited in the most extremely premature infants <26 weeks of gestational age (GA), where clinical problems are most significant and patients are most vulnerable. AIMS:To investigate whether different approaches to surgical closure of PDA in two large Swedish centers has an impact on clinical outcomes including mortality in extremely preterm infants born <26 weeks GA. STUDY DESIGN:Retrospective, two-center, cohort study. SUBJECTS:Infants born at 22+0-25+6 weeks GA between 2010 and 2016 at Uppsala University Children's Hospital (UUCH; n = 228) and Queen Silvia Children's Hospital Gothenburg (QSCHG; n = 220). MAIN OUTCOME MEASURES:Survival, bronchopulmonary dysplasia (BPD), and retinopathy of prematurity (ROP). RESULTS:Surgical closure of PDA was more common and performed earlier at QSCHG (50 % vs 16 %; median age 11 vs 44 days; p < 0.01). Survival was similar in both centres. There was a higher incidence of severe BPD and longer duration of mechanical ventilation at UUCH (p < 0.01). There was a higher incidence of ROP, IVH and sepsis at QSCH (p < 0.05, p < 0.01 and p < 0.01). A sub-group analysis matching all surgically treated infants at QSCHG with infants at UUCH with the same GA showed similar results as the total cohort. CONCLUSION:Earlier and higher rate of surgical PDA closure in this cohort of extremely preterms born <26 weeks GA did not impact mortality but was associated with lower rates of severe BPD and higher rates of severe ROP.

Objective Regarding the use of lung ultrasound (LU) in neonatal intensive care units (NICUs) across Europe, to assess how widely it is used, for what indications and how its implementation might be improved. Design and intervention International online survey. Results Replies were received from 560 NICUs in 24 countries between January and May 2023. LU uptake varied considerably (20%-98% of NICUs) between countries. In 428 units (76%), LU was used for clinical indications, while 34 units (6%) only used it for research purposes. One-third of units had <2 years of experience, and only 71 units (13%) had >5 years of experience. LU was mainly performed by neonatologists. LU was most frequently used to diagnose respiratory diseases (68%), to evaluate an infant experiencing acute clinical deterioration (53%) and to guide surfactant treatment (39%). The main pathologies diagnosed by LU were pleural effusion, pneumothorax, transient tachypnoea of the newborn and respiratory distress syndrome. The main barriers for implementation were lack of experience with technical aspects and/or image interpretation. Most units indicated that specific courses and an international guideline on neonatal LU could promote uptake of this technique. Conclusions Although LU has been adopted in neonatal care in most European countries, the uptake is highly variable. The main indications are diagnosis of lung disease, evaluation of acute clinical deterioration and guidance of surfactant. Implementation may be improved by developing courses and publishing an international guideline.

Background Collagen type IV alpha 1 chain (COL4A1) in the basement membrane is an important component during lung development, as suggested from animal models where COL4A1 has been shown to regulate alveolarization and angiogenesis. Less is known about its role in human lung development. Our aim was to study COL4A1 expression in preterm infants with different lung maturational and clinical features. Methods COL4A1 expression in 115 lung samples from newborn infants (21-41 weeks’ gestational age; 0-228 days’ postnatal age [PNA]) was studied by immunohistochemistry combined with digital image analysis. Cluster analysis was performed to find subgroups according to immunohistologic and clinical data. Results Patients were automatically categorized into 4 Groups depending on their COL4A1 expression. Expression of COL4A1 was mainly extracellular in Group 1, low in Group 2, intracellular in Group 3, and both extra- and intracellular in Group 4. Intracellular/extracellular ratio of COL4A1 expression related to PNA showed a distinctive postnatal maturational pattern on days 1-7, where intracellular expression of COL4A1 was overrepresented in extremely preterm infants. Conclusions COL4A1 expression seems to be highly dynamic during the postnatal life due to a possible rapid remodeling of the basement membrane. Intracellular accumulation of COL4A1 in the lungs of extremely premature infants occurs more frequently between 1 and 7 postnatal days than during the first 24 hours. In view of the lung arrest described in extremely preterm infants, the pathological and/or developmental role of postnatally increased intracellular COL4A1 as marker for basement membrane turnover, needs to be further investigated.

Background This multicentre, international, retrospective study aimed to investigate whether respiratory system reactance (X-rs) assessed by respiratory oscillometry on day 7 of life is associated with respiratory outcomes in preterm infants below 32 weeks gestational age (GA). Methods Sinusoidal pressure oscillations (2-5 cmH(2)O peak-to-peak, 10 Hz) were superimposed on the positive end-expiratory pressure. We assessed the association of X rs z-score with the duration of respiratory support using linear regression and with bronchopulmonary dysplasia (BPD) using logistic regression. We used the likelihood ratio test to evaluate whether X-rs z-score adds significantly to clinical predictors, including GA, birthweight (BW) and the National Institute of Child Health and Human Development (NICHD) BPD prediction model. Results 137 infants (median (interquartile range) 28.43 (26.11-30.29) weeks GA) were included; 44 (32%) developed BPD. X-rs z-score was significantly associated with the duration of respiratory support (R-2=0.35). X rs z-score was significantly higher in infants who developed BPD (p<0.001); the optimal cutoff value was 2.6, associated with 77% sensitivity and 80% specificity. In univariable analysis, per z-score increase in X-rs, the odds ratio for BPD increased by 60% and the respiratory support by 8 days. In multivariable analysis, X-rs z-score added significantly to the NICHD model and to GA and BW z-score to predict respiratory support duration (p=0.016 and p=0.014, respectively) and BPD development (p=0.003 and p<0.001, respectively). Conclusion X(rs )z-score on the 7th day after birth improves the prediction of respiratory outcome in preterm infants.

Previous studies suggest that Paneth cells are involved in NEC development. Defensin alpha 6 (DEFA6) and guanylate cyclase activator 2A (GUCA2A) are selective protein markers of Paneth cells. The objective was to explore DEFA6 and GUCA2A expression in intestinal tissue samples from newborn infants with and without NEC. Tissue samples from histologically intact intestine were analyzed from 70 infants: 43 underwent bowel resection due to NEC and 27 controls were operated due to conditions such as intestinal atresia, dysmotility, aganglionosis, pseudo-obstruction or volvulus. Each tissue sample was immunohistochemically stained for DEFA6 and GUCA2A. Semi-automated digital image analysis was performed to determine protein expression. Clinical data and protein expressions were compared between the groups. DEFA6 expression was lower in the NEC group ( p = 0.006). Low DEFA6 correlated with risk of developing NEC in a logistic regression analysis, independently of gestational age and birth weight (OR 0.843 [CI 0.732–0.971]; p = 0.018). GUCA2A expression did not differ between the two groups. Conclusion : Lower expression of DEFA6 together with intact GUCA2A expression indicates that NEC patients have well-defined Paneth cells but diminished defensin activity. Our results suggest that DEFA6 could be used as a biomarker for NEC. What is Known: • Previous studies of defensin activity in NEC have been inconsistent, showing that defensin levels may be increased or diminished in NEC. GUCA2A has to our knowledge never been studied in NEC. What is New: • This study benchmarks two specific Paneth cell markers (DEFA6 and GUCA2A) and their activity in individuals with and without NEC. • The key finding is that the NEC group had a lower DEFA6 expression compared to the Controls, while the expression of GUCA2A did not differ between the groups.