Reliable data on causes of death (COD) in preterm infants are needed to assess perinatal care and current clinical guidelines. In this retrospective observational analysis of all deceased preterm infants born < 37 weeks’ gestational age (n = 278) at a Swedish tertiary neonatal intensive care unit, we compared preliminary COD from Medical Death Certificates with autopsy defined COD (2002–2018), and assessed changes in COD between two periods (period 1:2002–2009 vs. period 2:2011–2018; 2010 excluded due to centralized care and seasonal variation in COD). Autopsy was performed in 73% of all cases and was more than twice as high compared to national infant autopsy rates (33%). Autopsy revised or confirmed a suspected preliminary COD in 34.9% of the cases (23.6% and 11.3%, respectively). Necrotizing enterocolitis (NEC) as COD increased between Period 1 and 2 (5% vs. 26%). The autopsy rate did not change between the two study periods (75% vs. 71%). We conclude that autopsy determined the final COD in a third of cases, while the incidence of NEC as COD increased markedly during the study period. Since there is a high risk to determine COD incorrectly based on clinical findings in preterm infants, autopsy remains a valuable method to obtain reliable COD.

Background: Necrotizing enterocolitis (NEC) is a fatal disease where current diagnostic tools are insufficient for preventing NEC. Early predictive biomarkers could be beneficial in identifying infants at high risk of developing NEC.Objective: To explore early biomarkers for predicting NEC in extremely preterm infants (EPIs).Methods: Blood samples were collected on day 2 (median 1.7; range 1.5–2.0) from 40 EPI (median 25 gestational weeks; range 22–27): 11 developed NEC and 29 did not (controls). In each infant, 189 inflammatory, oncological, and vascular proteomic biomarkers were quantified through Proximity Extension Assay. Biomarker expression and clinical data were compared between the NEC group and Controls. Based on biomarker differences, controls were sorted automatically into three subgroups (1, 2, and 3) by a two-dimensional hierarchical clustering analysis.Results: None of the biomarkers differed in expression between all controls and the NEC group. Two biomarkers were higher in Control 1, and 16 biomarkers were lower in Control group 2 compared with the NEC group. No biomarker distinguished Control 3 from the NEC group. Perinatal data were similar in the whole population.Conclusions: Early postnatal comprehensive biomarkers do not identify EPIs at risk of developing NEC in our study. Future studies of predictors of NEC should include sequential analysis of comprehensive proteomic markers in large cohorts.

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.

Reliable data on causes of death (COD) in preterm infants is needed to assess perinatal care and current clinical guidelines. In this retrospective observational analysis of all deceased preterm infants born <37 weeks’ gestational age (n=278; 2012-2018) at a Swedish tertiary neonatal intensive care unit, we compared preliminary COD from Medical Death Certificates with autopsy defined COD (2002-2018), and assessed changes in COD between two periods (Period 1: 2002-2009 vs Period 2: 2011-2018). Autopsy was performed in 73% of all cases and was more than twice as high compared to national rates of infant autopsies in Sweden (33%). Autopsy defined COD differed from preliminary COD in 34.9% of the cases. Necrotizing enterocolitis (NEC) as COD increased between Period 1 and 2 (5% vs 26%), while intraventricular hemorrhage decreased (13% vs 6%). The autopsy rate did not change between the two study periods (75% vs 71%). We conclude that autopsy changed the preliminary COD in a third of cases, while the incidence of NEC as COD increased markedly during the study period. Since there is a high risk to determine COD incorrectly based solely on clinical findings in preterm infants, autopsy remains a valuable method to obtain reliable COD.