MicroRNA-320а as a novel biomarker at preclinical stage of necrotizing enterocolitis in term neonates with congenital heart defects

Table 1 Distribution of congenital heart defects in the study cohort, n (%)

Type of congenital heart defect	Total	No-NEC	NEC	Type of cardiac surgery
				No-NEC (n)	NEC (n)
Single-ventricle CHD
Hypoplastic left heart syndrome	2	1 (4)	1 (4)	Damus-Kaye-Stansel procedure (1)	Norwood procedure (1)1
Double-inlet left ventricle + Pulmonary artery/CoAo	2	1 (4)	1 (4)	MBTS placement (1)	Norwood procedure (1)
Unbalanced (right ventricle dominant) atrioventricular septal defect + CoAo	1	1 (4)	0	Modified Amato technique (1)	0
Total	5	3 (13)	2 (9)
Two-ventricle CHD
Transposition of the great arteries	7	5 (22)	2 (9)	ASO (4). ASO + Modified Amato technique (1)	ASO (1). MBTS placement (in left ventricular outflow tract obstruction) (1)2
Coarctation of the aorta	5	3 (13)	2 (9)	Modified Amato technique (2). End-to-end anastomosis (1)	Modified Amato technique (1). End-to-end anastomosis (1)
Pulmonary atresia/stenosis	4	2 (9)	2 (9)	mBTS placement (1). Antegrade palliation (1)	MBTS placement (2)
Aortic valve stenosis	2	1 (4)	1 (4)	Aortic commissurotomy (1)	Aortic commissurotomy (1)
Total	18	11 (48)	7 (30)

¹Mortality related to acute heart failure in congenital heart defects (CHD).

²Mortality related to acute heart failure in CHD and necrotizing enterocolitis.

ASO: Arterial switch operation; CHD: Congenital heart defects; CoAo: Coarctation of the aorta; MBTS: Modified Blalock-Taussig shunt; NEC: Necrotizing enterocolitis

Table 2 Characteristics of newborns with critical congenital heart defects enrolled in the study, n (%)

Newborns’ characteristic (n)		No-NEC (n = 14)	NEC (n = 9)	P value
					Sex
Female (6)	5 (36)	1 (11)	0.34
Male (17)	9 (64)	8 (89)
Gestational age (week) (23)		39 (39-40)	39 (38-39)	0.26
Birth weight (g) (23)		3345 (2960-3810)	3140 (3020-3580)	0.64
Mode of delivery	Unassisted vaginal delivery (15)	9 (64)	6 (67)	0.41
	Assisted vaginal delivery (1)	0	1 (11)
	Cesarean section (7)	5 (36)	2 (22)
Maternal factors	Chorioamnionitis (3)	3 (21)	0	0.22
	Prelabor rupture of membranes (2)	1 (7)	1 (11)
	Preeclampsia (0)	0	0
	Gestational diabetes (3)	3 (21)	0
Apgar score at 1 minute (23)		7 (7-8)	7 (7-7)	0.78
Apgar score at 5 minutes (23)		8 (8-8)	8 (8-8)	0.51
Intrauterine growth retardation (3)		1 (7)	2 (22)	0.54
Congenital heart defects	Cyanotic (15)	9 (64)	6 (67)	1.0
	Acyanotic (8)	5 (36)	3 (33)
Parameters before cardiac surgery
Feeding	Breast milk (7)	4 (29)	3 (33)	0.91
	Formula (7)	4 (29)	3 (33)
	Breast milk + Formula (9)	6 (43)	3 (33)
Feeding volume (mL/kg/day)		56.3 (28.0-80.8)	42.0 (34.1-72.7)	0.95
Superior mesenteric artery resistance index		0.99 (0.93-1.10)	0.95 (0.94-1.12)	0.79
Antibiotics at sample collection (Ampicillin + Sulbactam) (7)		3 (21)	4 (44)	1.0
Rashkind procedure/balloon valvuloplasty (6)		2 (14)	4 (44)	0.16
Prostaglandin E1 infusion (ng/kg/minute)		10 (10-20)	15 (10-25)	0.61
Inotropic therapy (3)		1 (7)	2 (22)	0.54
Parameters of cardiac surgery
Age at cardiac surgery (days)		7.5 (5.0-8.0)	4.0 (6.0-11.5)	0.72
Palliative surgery (10)		5 (36)	5 (56)	0.42
Duration of cardiac surgery (minute)		235.0 (212.5-347.5)	185.0 (161.0-353.0)	0.33
Aristotle score		7.9 (7.0-11.0)	7.0 (6.3-12.3)	0.50
Cardiopulmonary bypass (17)		10 (71)	7 (78)	1.0
Delayed sternal closure (11)		7 (50)	4 (44)	1.0
Parameters after cardiac surgery
Arterial hypotension on POD1 (13)		8 (57)	5 (56)	1.0
Heart rhythm disturbances on POD1 (9)		5 (36)	4 (44)	1.0
Maximum vasoactive-inotropic score during the first 24 hours after surgery		15.0 (12.6-31.3)	25.0 (7.5-38.8)	0.59
Duration of inotropic therapy (days)		4.0 (2.5-7.0)	10.0 (2.5-20.0)	0.17
Maximum arterial lactate concentration within 24 hours (mmol/L)		3.5 (2.7-5.1)	5.0 (2.7-9.6)	0.48
Arterial lactate concentration at 24 hours (mmol/L)		2.0 (1.8-2.6)	2.3 (1.5-6.5)	0.65
Start of enteral feeding on POD1		11 (79)	5 (56)	0.36
Start of enteral feeding (days)		1.0 (1.0-1.3)	1.0 (1.0-6.0)	0.14
Feeding volume on POD1 (mL/kg/day)		13.1 (6.91-16.0)	3.9 (0.0-10.8)	0.09
Feeding volume on POD3 (mL/kg/day)		33.9 (22.9-56.7)	1.0 (0.0-19.4)	0.004
NEC onset (days)		0	2.0 (1.0-5.0)	-
Intestinal wall thickening on POD1 by ultrasonography (9)		5 (36)	4 (44)	0.38
Pneumatosis intestinalis on POD1 by ultrasonography (4)		0	4 (44)	-
Neonatal intensive care unit duration (days)		6.5 (4.0-16.5)	14.0 (5.5-26.5)	0.17
Antibiotics at sample collection (20)		11 (cefoperazone + sulbactam: 5; cefuroxime: 2; cefotaxime: 1; ampicillin/sulbactam: 2; piperacillin/tazobactam: 1)	9 (cefoperazone + sulbactam: 3; cefuroxime: 1; ampicillin/sulbactam: 2; meropenem: 2; ertapenem: 1)	0.25

Data are n (%) or median (interquartile range). NEC: Necrotizing enterocolitis; POD: Postoperative day.

Table 3 Laboratory parameters of term newborns with critical congenital heart defects before and after cardiac surgery

Laboratory parameters		No-NEC (n = 14)	P value	NEC (n = 9)	P value	P value between groups
Hemoglobin (g/L)	Before	155.0 (141.0-175.5)	0.003	159.0 (128.5-180.0)	0.050	0.847
	After	129.5 (113.0-146.0)		120.0(109.0-143.0)		0.705
Erythrocytes (× 1012/L)	Before	4.68 (4.24-5.00)	0.110	4.64 (3.70-5.15)	0.208	0.787
	After	4.11 (3.74-4.64)		3.91 (3.55-4.87)		0.900
Platelets (× 109/L)	Before	345.5 (280.0-375.5)	0.583	291.0 (243.5-424.0)	0.674	0.758
	After	372.0 (227.0-501.0)		298.0 (232.0-421.0)		0.244
Leukocytes (× 109/L)	Before	13.65 (11.20-15.65)	0.724	11.45 (8.95-15.35)	0.327	0.375
	After	12.70 (11.70-15.00)		11.70 (10.40-19.40)		0.801
Band neutrophils (%)	Before	1.0 (1.0-1.5)	0.624	1.5 (1.0-2.0)	0.686	0.512
	After	1.0 (0.0-3.0)		1.0 (1.0-2.0)		0.395
Segmented neutrophils (%)	Before	40.0 (35.5-49.5)	0.023	42.5 (40.0-58.0)	0.779	0.217
	After	60.0 (52.0-65.0)		48.0 (44.0-58.0)		0.078
Eosinophils (%)	Before	5.0 (3.0-6.5)	0.103	4.5 (3.6-6.0)	0.447	0.969
	After	3.0 (1.0-5.0)		6.0 (0.0-10.0)		0.314
Lymphocytes (%)	Before	40.0 (36.0-44.5)	0.045	37.0 (25.5-40.5)	0.484	0.247
	After	20.5 (15.0-30.0)		25.0 (16.0-30.0)		0.825
C-reactive protein (mg/L)	Before	7.0 (4.0-10.5)	0.034	1.5 (0.8-5.0)	0.463	0.101
	After	8.2 (5.0-15.0)		6.1 (3.0-18.0)		0.550

Data are median (interquartile range). NEC: Necrotizing enterocolitis.

Table 4 Kyoto encyclopedia of genes and genomes and gene ontology pathways regulated by the differentially expressed microRNAs

MiRs	Term name	Term genes (n)	P value	False discovery rate	Target genes
Kyoto Encyclopedia of Genes and Genomes (pathways union)
MiR-155-5p miR-223-3p, miR-320a-3p	Forkhead box O signaling pathway	139	7.31e-10	1.66e-8	18 by miR-155-5p, 6 by miR-223-3p, 20 by miR-320a-3p
	Prostate cancer	101	5.77e-8	7.84e-7	15 by miR-155-5p, 4 by miR-223-3p, 15 by miR-320a-3p
MiR-155-5p, miR-320a-3p	Hippo signaling pathway	164	1.20e-10	4.07e-9	21 by miR-155-5p, 31 by miR-320a-3p
	Viral carcinogenesis	265	1.07e-10	4.07e-9	29 by miR-155-5p, 42 by miR-320a-3p
	Pathways in cancer	555	8.83e-9	1.50e-7	53 by miR-155-5p, 60 by miR-320a-3p
	Transcriptional misregulation in cancer	206	1.13e-7	1.28e-6	21 by miR-155-5p, 32 by miR-320a-3p
	Colorectal cancer	88	3.43e-7	3.33e-6	15 by miR-155-5p, 15 by miR-320a-3p
	Chronic myeloid leukemia	79	5.08e-6	2.88e-5	12 by miR-155-5p, 14 by miR-320a-3p
	Hepatitis B	177	8.83e-6	4.62e-5	22 by miR-155-5p, 21 by miR-320a-3p
	Protein processing in endoplasmic reticulum	194	2.04e-4	4.91e-4	20 by miR-155-5p, 22 by miR-320a-3p
MiR-223-3p, miR-320a-3p	Signaling pathways regulating pluripotency of stem cells	156	1.67e-6	1.26e-5	5 by miR-223-3p, 22 by miR-320a-3p
	Longevity regulating pathway	105	5.56e-5	1.89e-4	4 by miR-223-3p, 14 by miR-320a-3p
Gene Ontology (genes intersection)
MiR-155-5p, miR-223-3p, miR-320a-3p	Cytokine-mediated signaling pathway	319	2.93e-3	2.63e-2	2 by miR-155-5p, 2 by miR-223-3p, 2 by miR-320a-3p

MiRs: MicroRNAs.