257
Journal of Neonatal-Perinatal Medicine 6 (2013) 257–262 DOI 10.3233/NPM-1370813 IOS Press
Original Research
Cord blood epidermal growth factor as a possible predictor of necrotizing enterocolitis in very low birth weight infants W.A. Wahab Mohameda,b,∗ and A.M. Aseeric a Department
of Pediatrics, Faculty of Medicine, Minia University, Minia, Egypt of Pediatrics, College of Medicine, King Khalid University, Abha, Saudi Arabia c Department of Biochemistry, Khamis Civil Hospital, Khamis Mushayt, Saudi Arabia b Department
Received 15 February 2013 Revised 06 May 2013 Accepted 13 June 2013
Abstract. OBJECTIVE: To evaluate epidermal growth factor (EGF) levels in cord blood as a possible marker for predicting the subsequent development of necrotizing enterocolitis (NEC) in very low birth weight (VLBW) infants. PATIENTS AND METHODS: A total of 241 VLBW infants (38 infants developed NEC and 203 had no NEC) were enrolled. EGF concentrations were measured by enzyme-linked immunosorbent assay. RESULTS: VLBW infants who subsequently developed NEC had significantly lower cord serum concentrations of EGF than those who did not (p < 0.0001). Moreover, cord blood concentrations of EGF were significantly lower in infants with stage III than those with stage II or I NEC (p < 0.001). Logistic regression analysis demonstrated that low cord blood EGF was independently associated with the subsequent risk of NEC (OR = 0.978 [95% CI: 0.959–0.997]; p = 0.02). CONCLUSION: Low cord blood EGF concentrations may predict the subsequent development of NEC in VLBW infants. Keywords: Epidermal growth factor, necrotizing enterocolitis, very low birth weight infants
1. Introduction Despite modern medical advances, necrotizing enterocolitis (NEC) remains a significant cause of morbidity and mortality in neonatal intensive care units (NICUs). It is more common in very low birth weight (VLBW) infants, with highly variable incidence affecting 2.6% to 28% of these infants [1, 2]. Mortality rate ranges from 20% to 40%, and survivors after either ∗ Corresponding
author: Dr. Walid Abdel Wahab Mohamed, Department of Pediatrics, College of Medicine, King Khalid University, Abha P.O. Box 941, Saudi Arabia. Tel.: +966 502507894; Fax: +966 72418194; E-mail: [email protected].
medical or surgical therapy can present with failure to thrive, feeding abnormalities, diarrhea, bowel obstruction or neurodevelopmental delay [3–5]. In spite of extensive epidemiological, clinical, and basic research, the pathogenesis of NEC remains unknown, and subsequently efforts to prevent the disease remain ineffective. Several potential preventive strategies have aimed at induction of gastrointestinal maturation in premature infants, however, none of them have led to consistently positive results [6–9]. Identification of a predictive marker would provide the opportunity for both early prevention and possibly a treatment specifically directed at one or more
1934-5798/13/$27.50 © 2013 – IOS Press and the authors. All rights reserved
258
W.A. Wahab Mohamed and A.M. Aseeri / Epidermal growth factor and NEC
steps in the pathophysiologic cascade leading to NEC. The search for useful biomarkers based on cytokines or other mediators continues but none have shown sufficient sensitivity and specificity for clinical use [10]. In recent years, studies have focused on the role of the inflammatory cascade and its impact on the disease process, and investigators are evaluating strategies to attenuate inflammatory signaling that might prevent and/or ameliorate neonatal NEC [11]. Epidermal growth factor (EGF), is a potent peptide that stimulates cell growth, proliferation and differentiation. EGF was first purified from the mouse submandibular gland, but since then found in many human tissues and fluids including submandibular and parotid glands, human platelets, macrophages, urine, saliva, and plasma [12]. EGF is abundant in many fluids bathing the fetal and neonatal gastrointestinal tract, including amniotic fluid, saliva, and breast milk. It is important for normal intestinal development as well as repair following injury to the gastrointestinal mucosa [13]. The significant effects of EGF on the healing of damaged gastrointestinal mucosa or on intestinal adaptation after injury have been reported in a number of studies [14]. The biological actions of EGF are mediated through binding to its specific receptor, EGF-R, which is distributed throughout the fetal and neonatal gastrointestinal tract [15, 16]. During the past three decades, several animal models have been developed to study the etiology of NEC, with the neonatal rat model considered one of the best means to study this disease. In this model, EGF reduces the development of NEC [14]. The molecular mechanism underlying EGF’s protective effects against NEC is complex, including the reduction of inflammation in the ileum [16], maintenance of bile acid transport [17], protection of integrity of intestinal tight junctions [18], and regulation of epithelial apoptosis [19]. Maynard et al. demonstrated a new role of EGF in the prevention of NEC. They speculated that EGF treatment of experimental NEC downregulates the expression of autophagy proteins and blocks autophagy in the site of injury. This might be an additional molecular mechanism by which EGF downregulates epithelial cell death and thus protects the intestinal epithelium against mucosal injury [20]. Diminished levels of salivary EGF in human babies with NEC [21] and the development of hemorrhagic enteritis pathologically similar to NEC in EGF-R knockout mice [22] suggest an important role of EGF insufficiency in the pathogenesis of NEC. The aim of the current study was to evaluate EGF concentration
in cord blood of VLBW infants and examine its utility in the prediction of subsequent development of NEC in VLBW infants.
2. Patients and methods 2.1. Study design A prospective study was conducted at the NICU of Khamis Civil Hospital, Saudi Arabia between September 2010 and August 2012. The study was approved by the ethics committee of the Hospital. Written informed consent was obtained for each patient before enrollment. The study included all VLBW infants ( 0.05). However, the time to reach full enteral feeding was significantly shorter in infants who developed NEC than those who did not (15.1 ± 3.2, 16.7 ± 4.7 days, respectively, p = 0.04). The mean age at onset of NEC was 17.3 ± 2.2 days. The baseline and clinical characteristics of study participants are summarized in Table 1. Clinical manifestations in the NEC group included: gross blood in the stool 31 (81.2%) and abdominal distension 28 (73.7%). Thirteen (34.2%) infants with NEC required increased respiratory support, 11 (28.9%) developed hypotension treated with fluid boluses and vasopressors, and 6 (15.8%) developed disseminated intravascular coagulation, treated with
260
W.A. Wahab Mohamed and A.M. Aseeri / Epidermal growth factor and NEC Table 1 Baseline and clinical characteristics of preterm infants with or without necrotizing enterocolitis Gestational age (wk)a Birth weight (g)a Gender (Male)b Caesarean deliveryb Prenatal steroidsb Apgar score at 1 mina Apgar score at 5 mina Respiratory distress syndromeb UACb UVCb Patent ductus arteriosusb Indomethacinb Packed red blood cell or plasma product transfusionb Time of first feed (d)a Time to reach full enteral feeding (d)a Type of feeding Breast milkb Formulab Combined Deathb
NEC (n = 38)
No NEC (n = 203)
p value
28.1 ± 1.2 1083.1 ± 184.2 22 (57.9) 5 (13.1) 29 (76.3) 5.1 ± 0.1 7.1 ± 1.0 29 (76.3) 4 (10.5) 11 (28.9) 6 (15.8) 4 (10.5) 33 (86.8) 6.5 ± 2.4 15.1 ± 3.2
28.6 ± 1.7 1121.2 ± 224.6 98 (48.3) 24 (11.8) 139 (68.5) 5.3 ± 0.9 6.9 ± 0.9 148 (72.9) 18 (8.9) 41 (20.2) 24 (11.8) 20 (9.8) 168 (82.7) 5.9 ± 2.1 16.7 ± 4.7
0.08 0.32 0.76 0.47 0.69 0.17 0.21 0.58 0.45 0.78 0.60 0.41 0.54 0.11 0.04
4 (10.5) 13 (34.2) 21 (55.3) 5 (13.1)
25 (12.3) 86 (42.4) 92 (45.3) 8 (3.9)
0.32 0.40 0.77 0.03
are means ± SD. b Number (percent). UAC: Umbilical arterial catheterization. UVA: Umbilical venous catheterization.
a Values
Fig. 1. Cord blood levels of epidermal growth factor in neonates who developed NEC and those who did not.
Fig. 2. Cord blood levels of epidermal growth factor among infants with NEC.
transfusion of blood products. Culture-proven sepsis was detected in 7 (18.4%) infants with NEC, (Klebsiela pneumoniae [n = 4], Enterobacter sp [n = 3], and Escherichia coli [n = 1]). Of the 38 infants with NEC, 23 (60.5%) had suspected NEC (stage I) and 15 (39.5%) had confirmed NEC (Bell’s stage ≥2), of whom 9 (23.7 %) had stage II, and 6 (15.8 %) had stage III. Radiographic findings included pneumatosis intestinalis (n = 9), and intestinal perforation and/or ascites (n = 6). Six (15.8%) had placement of surgical drain and/or exploratory laparotomy of whom, 4 had
positive peritoneal fluid cultures with Klebsiela pneumoniae (n = 4). Five (13.1%) infants died (3 had stage III-NEC, and 2 had stage II-NEC). VLBW infants who subsequently developed NEC had significantly lower cord serum levels of EGF than those who did not (13.7 ± 2.0 vs. 39.2 ± 1.5 pg/mL, p < 0.0001; Fig. 1). Moreover, cord serum levels of EGF were significantly lower in infants with stage III than those with stage II or I NEC (10.2 ± 0.4, 12.2 ± 0.9, 14.7 ± 1.5 pg/mL respectively, p < 0.001; Fig. 2). In NEC group, cord blood levels of EGF
W.A. Wahab Mohamed and A.M. Aseeri / Epidermal growth factor and NEC
were significantly lower in non-survivors than in survivors (10.3 ± 0.4 vs. 13.8 ± 1.9 pg/mL, p < 0.001). Our results showed a significant negative correlation between cord blood concentrations of EGF and the severity of NEC (r = −0.57, p = 0.01). Cord blood concentrations of EGF were significantly lower in preterm infants with gestational age ≤28 weeks (n = 63) than in those with older gestational age (n = 178) [33.09 ± 1.4 vs. 36.07 ± 1.7 pg/mL, p < 0.001]. We detected a significant positive correlation between cord blood concentrations of EGF and the gestational age (r = 0.65, p < 0.01). Multivariable logistic regression analysis, with NEC as the dependant variable and gestational age, birth weight, prenatal steroids administration, RDS, PDA, indomethacin use, and cow’s milk formula feeding as risk factors demonstrated that only low cord blood EGF concentration was independently associated with the subsequent risk of NEC (OR = 0.978 [95% CI: 0.959–0.997]; p = 0.02).
4. Discussion Necrotizing enterocolitis is the leading cause of death from gastrointestinal disease in preterm infants, and remains a major cause of long-term disability in those who survive the disease. Early warning signs and symptoms of NEC are nonspecific, often inconspicuous and may be difficult to distinguish from other non-specific abdominal disorders such as gastrointestinal dysmotility [10]. Thus, early identification of NEC in VLBW infants remains a major diagnostic challenge. No reliable early biomarkers have demonstrated sufficient positive and negative predictive values to be clinically useful [25]. Only few clinical studies measured the serum concentrations of EGF in preterm infants with NEC. Shin et al., reported diminished concentrations of salivary and serum EGF in preterm infants with NEC [21]. A recent clinical trial with 327 preterm and term neonates evaluated the ontogeny of salivary EGF and its relation to the development of NEC [26]. The concentration of salivary EGF positively correlated with gestation age; infants at earlier gestational ages had significantly lower EGF concentrations in their saliva compared with term neonates. Importantly, lower concentrations of salivary EGF during the first week of life were associated with an increased incidence of NEC [26].
261
To the best of our knowledge, this is the first reported prospective study that measured the cord blood concentrations of EGF in VLBW infants. Our results showed that VLBW infants who subsequently developed NEC had significantly lower cord blood concentrations of EGF than those who did not. These results suggest that VLBW infants who have low concentrations of EGF at birth, are at substantially increased risk of developing NEC. This holds as true even after correcting for the known risk factors for NEC, including low gestational age, sex, RDS, PDA and cow’s milk formula feeding. Moreover, the lowest EGF concentrations were detected in infants who developed severe NEC, particularly those who died. We speculate that EGF not only plays a role in predicting NEC, but also might have a prognostic value in this disease. Further studies with larger sample sizes are needed to examine such hypothesis. Indeed, the protective role of maternal milk in NEC pathogenesis has been reported both in human [9] and animal studies [14, 27]. These findings stimulated the search for various components of milk that might be responsible for protection against NEC. Mammalian milk contains a large number of biologically active substances that directly affect gut maturation and mucosal protection. One of these substances is EGF, which was detected in many body fluids, including colostrum and milk during the early postnatal period. EGF has been found at high concentrations in the maternal colostrum [28]. In our study, only four infants in the NEC group were on breast milk feeding and none of them received colostrum. Interestingly, none of infants with severe (stage III) NEC was on breast milk feeding. Dvorak et al. [14] examined the effect of orally administered EGF on the incidence of NEC in a neonatal rat model. They showed that EGF supplementation of formula reduced the incidence and severity of NEC in rats. These data suggest a new potential therapeutic approach for the prevention and treatment of NEC in neonates. Our study has important limitations that require consideration. First, the total number of patients was small; therefore, further studies with larger sample sizes should be undertaken. Second, single measurement of serum EGF concentrations at birth, so another measurement of EGF when the patient is ill might be more useful. The association between low EGF concentrations and NEC may provide the rational for a controlled trial to evaluate the efficacy of recombinant EGF fortified milk formula in preventing the development of NEC in VLBW neonates.
262
W.A. Wahab Mohamed and A.M. Aseeri / Epidermal growth factor and NEC
5. Conclusion Low cord blood EGF concentrations may predict the subsequent development of NEC in very low birth weight infants. The measurement of cord blood EGF concentration at birth might be a biological marker to permit early intervention and might provide a new therapeutic target to prevent NEC. Acknowledgments
[13]
[14]
[15]
[16]
The authors acknowledge the major contribution of the staff at the NICU, Khamis Civil Hospital. [17]
Financial disclosure statement The authors report no conflicts of interest.
[18]
References [19] [1]
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Kafetzis DA, Skevaki C, Costalos C. Neonatal necrotizing enterocolitis: An overview. Curr Opin Infect Dis 2003;16:34955. Sharma R, Tepas J Jr, Hudak ML, Mollitt DL, Wludyka PS, Tseng RJ, et al. Neonatal gut barrier and multiple organ failure: Role of endotoxin and proinflammatory cytokines in sepsis and necrotizing enterocolitis. J Pediatr Surg 2007;42:454-61. Guthrie SO, Gordon PV, Thomas V, Thorp JA, Peabody J, Clark RH. Necrotizing enterocolitis among neonates in the United States. J Perinatol 2003;23:278-85. Hsueh W, Caplan MS, Qu XW, Tan XD, De Plaen IG, Gonzalez-Crussi F. Neonatal necrotizing enterocolitis: Clinical considerations and pathogenetic concepts. Pediatr Dev Pathol 2003;6:6-23. Lin PW, Stoll BJ. Necrotising enterocolitis. Lancet 2006;368:1271-83. Feng J, El-Assal ON, Besner GE. Heparin-binding EGF-like growth factor (HB-EGF) and necrotizing enterocolitis. Semin Pediatr Surg 2005;14:167-74. Lin HC, Su BH, Chen AC, Lin TW, Tsai CH, Yeh TF, et al. Oral probiotics reduce the incidence and severity of necrotizing enterocolitis in very low birth weight infants. Pediatrics 2005;115:1-4. Bhatia J. Strategies to prevent necrotising enterocolitis. Chin Med J (Engl) 2010;123:2759-65. Lee JH. An update on necrotizing enterocolitis: Pathogenesis and preventive strategies. Korean J Pediatr 2011;54:368-72. Young C, Sharma R, Handfield M, Mai V, Neu J. Biomarkers for infants at risk for necrotizing enterocolitis: Clues to prevention? Pediatr Res 2009;65:91-7. Frost BL, Jilling T, Caplan MS. The importance of proinflammatory signaling in neonatal necrotizing enterocolitis. Semin Perinatol 2008;32:100-6. Herbst RS. Review of epidermal growth factor receptor biology. Int J Radiat Oncol Biol Phys 2004;59:21-6.
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Warner BW, Warner BB. Role of epidermal growth factor in the pathogenesis of neonatal necrotizing enterocolitis. Semin Pediatr Surg 2005;14:175-80. Dvorak B, Halpern MD, Holubec H, Williams CS, McWilliam DL, Dominguez JA, et al. Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model. Am J Physiol Gastrointest Liver Physiol 2002;282:156-64. Fagbemi AO, Wright N, Lakhoo K, Edwards AD. Immunoreactive epidermal growth factor receptors are present in gastrointestinal epithelial cells of preterm infants with necrotising enterocolitis. Early Hum Dev 2001;65:1-9. Halpern MD, Dominguez JA, Dvorakova K, Holubec H, Williams CS, Meza YG, et al. Ileal cytokine dysregulation in experimental necrotizing enterocolitis is reduced by epidermal growth factor. J Pediatr Gastroenterol Nutr 2003;36:126-33. Halpern MD, Holubec H, Saunders TA, Dvorak K, Clark JA, Doelle SM, et al. Bile acids induce ileal damage during experimental necrotizing enterocolitis. Gastroenterol 2006;130:359-72. Clark JA, Doelle SM, Halpern MD, Saunders TA, Holubec H, Dvorak K, et al. Intestinal barrier failure during experimental necrotizing enterocolitis: Protective effect of EGF treatment. Am J Physiol Gastrointest Liver Physiol 2006;291:938-49. Clark JA, Lane RH, Maclennan NK, Holubec H, Dvorakova K, Halpern MD, et al. Epidermal growth factor reduces intestinal apoptosis in an experimental model of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 2005;288:755-62. Maynard AA, Dvorak K, Khailova L, Dobrenen H, Arganbright KM, Halpern MD, et al. Epidermal growth factor reduces autophagy in intestinal epithelium and in the rat model of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 2010;299:614-22. Shin CE, Falcone RA Jr, Stuart L, Erwin CR, Warner BW. Diminished epidermal growth factor levels in infants with necrotizing enterocolitis. J Pediatr Surg 2000;35:173-76. Miettinen PJ, Berger JE, Meneses J, Phung Y, Pedersen RA, Werb Z, et al. Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature 1995;376:337-41. Lyra PP, Diniz EM, Abe-Sandes K, Angelo AL, Machado TM, Cardeal M. Surfactant protein B gene polymorphism in preterm babies with respiratory distress syndrome. Braz J Med Biol Res 2011;44:66-72. Walsh MC, Kliegman RM. Necrotizing enterocolitis: Treatment based on staging criteria. Pediatr Clin North Am 1986;33:179-201. Chaaban H, Shin M, Sirya E, Lim YP, Caplan M, Padbury JF. Inter-alpha inhibitor protein level in neonates predicts necrotizing enterocolitis. J Pediatr 2010;157:757-61. Warner BB, Ryan AL, Seeger K, Leonard AC, Erwin CR, Warner BW. Ontogeny of salivary epidermal growth factor and necrotizing enterocolitis. J Pediatr 2007;150:358-63. Crissinger KD, Burney DL, Velasquez OR, Gonzalez E. An animal model of necrotizing enterocolitis induced by infant formula and ischemia in developing piglets. Gastroenterol 1994;106:1215-22. Xu RJ. Development of the newborn GI tract and its relation to colostrum/milk intake: A review. Reprod Fertil Dev 1996;8:35-48.
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Journal of Neonatal-Perinatal Medicine 6 (2013) 257–262 DOI 10.3233/NPM-1370813 IOS Press
Original Research
Cord blood epidermal growth factor as a possible predictor of necrotizing enterocolitis in very low birth weight infants W.A. Wahab Mohameda,b,∗ and A.M. Aseeric a Department
of Pediatrics, Faculty of Medicine, Minia University, Minia, Egypt of Pediatrics, College of Medicine, King Khalid University, Abha, Saudi Arabia c Department of Biochemistry, Khamis Civil Hospital, Khamis Mushayt, Saudi Arabia b Department
Received 15 February 2013 Revised 06 May 2013 Accepted 13 June 2013
Abstract. OBJECTIVE: To evaluate epidermal growth factor (EGF) levels in cord blood as a possible marker for predicting the subsequent development of necrotizing enterocolitis (NEC) in very low birth weight (VLBW) infants. PATIENTS AND METHODS: A total of 241 VLBW infants (38 infants developed NEC and 203 had no NEC) were enrolled. EGF concentrations were measured by enzyme-linked immunosorbent assay. RESULTS: VLBW infants who subsequently developed NEC had significantly lower cord serum concentrations of EGF than those who did not (p < 0.0001). Moreover, cord blood concentrations of EGF were significantly lower in infants with stage III than those with stage II or I NEC (p < 0.001). Logistic regression analysis demonstrated that low cord blood EGF was independently associated with the subsequent risk of NEC (OR = 0.978 [95% CI: 0.959–0.997]; p = 0.02). CONCLUSION: Low cord blood EGF concentrations may predict the subsequent development of NEC in VLBW infants. Keywords: Epidermal growth factor, necrotizing enterocolitis, very low birth weight infants
1. Introduction Despite modern medical advances, necrotizing enterocolitis (NEC) remains a significant cause of morbidity and mortality in neonatal intensive care units (NICUs). It is more common in very low birth weight (VLBW) infants, with highly variable incidence affecting 2.6% to 28% of these infants [1, 2]. Mortality rate ranges from 20% to 40%, and survivors after either ∗ Corresponding
author: Dr. Walid Abdel Wahab Mohamed, Department of Pediatrics, College of Medicine, King Khalid University, Abha P.O. Box 941, Saudi Arabia. Tel.: +966 502507894; Fax: +966 72418194; E-mail: [email protected].
medical or surgical therapy can present with failure to thrive, feeding abnormalities, diarrhea, bowel obstruction or neurodevelopmental delay [3–5]. In spite of extensive epidemiological, clinical, and basic research, the pathogenesis of NEC remains unknown, and subsequently efforts to prevent the disease remain ineffective. Several potential preventive strategies have aimed at induction of gastrointestinal maturation in premature infants, however, none of them have led to consistently positive results [6–9]. Identification of a predictive marker would provide the opportunity for both early prevention and possibly a treatment specifically directed at one or more
1934-5798/13/$27.50 © 2013 – IOS Press and the authors. All rights reserved
258
W.A. Wahab Mohamed and A.M. Aseeri / Epidermal growth factor and NEC
steps in the pathophysiologic cascade leading to NEC. The search for useful biomarkers based on cytokines or other mediators continues but none have shown sufficient sensitivity and specificity for clinical use [10]. In recent years, studies have focused on the role of the inflammatory cascade and its impact on the disease process, and investigators are evaluating strategies to attenuate inflammatory signaling that might prevent and/or ameliorate neonatal NEC [11]. Epidermal growth factor (EGF), is a potent peptide that stimulates cell growth, proliferation and differentiation. EGF was first purified from the mouse submandibular gland, but since then found in many human tissues and fluids including submandibular and parotid glands, human platelets, macrophages, urine, saliva, and plasma [12]. EGF is abundant in many fluids bathing the fetal and neonatal gastrointestinal tract, including amniotic fluid, saliva, and breast milk. It is important for normal intestinal development as well as repair following injury to the gastrointestinal mucosa [13]. The significant effects of EGF on the healing of damaged gastrointestinal mucosa or on intestinal adaptation after injury have been reported in a number of studies [14]. The biological actions of EGF are mediated through binding to its specific receptor, EGF-R, which is distributed throughout the fetal and neonatal gastrointestinal tract [15, 16]. During the past three decades, several animal models have been developed to study the etiology of NEC, with the neonatal rat model considered one of the best means to study this disease. In this model, EGF reduces the development of NEC [14]. The molecular mechanism underlying EGF’s protective effects against NEC is complex, including the reduction of inflammation in the ileum [16], maintenance of bile acid transport [17], protection of integrity of intestinal tight junctions [18], and regulation of epithelial apoptosis [19]. Maynard et al. demonstrated a new role of EGF in the prevention of NEC. They speculated that EGF treatment of experimental NEC downregulates the expression of autophagy proteins and blocks autophagy in the site of injury. This might be an additional molecular mechanism by which EGF downregulates epithelial cell death and thus protects the intestinal epithelium against mucosal injury [20]. Diminished levels of salivary EGF in human babies with NEC [21] and the development of hemorrhagic enteritis pathologically similar to NEC in EGF-R knockout mice [22] suggest an important role of EGF insufficiency in the pathogenesis of NEC. The aim of the current study was to evaluate EGF concentration
in cord blood of VLBW infants and examine its utility in the prediction of subsequent development of NEC in VLBW infants.
2. Patients and methods 2.1. Study design A prospective study was conducted at the NICU of Khamis Civil Hospital, Saudi Arabia between September 2010 and August 2012. The study was approved by the ethics committee of the Hospital. Written informed consent was obtained for each patient before enrollment. The study included all VLBW infants ( 0.05). However, the time to reach full enteral feeding was significantly shorter in infants who developed NEC than those who did not (15.1 ± 3.2, 16.7 ± 4.7 days, respectively, p = 0.04). The mean age at onset of NEC was 17.3 ± 2.2 days. The baseline and clinical characteristics of study participants are summarized in Table 1. Clinical manifestations in the NEC group included: gross blood in the stool 31 (81.2%) and abdominal distension 28 (73.7%). Thirteen (34.2%) infants with NEC required increased respiratory support, 11 (28.9%) developed hypotension treated with fluid boluses and vasopressors, and 6 (15.8%) developed disseminated intravascular coagulation, treated with
260
W.A. Wahab Mohamed and A.M. Aseeri / Epidermal growth factor and NEC Table 1 Baseline and clinical characteristics of preterm infants with or without necrotizing enterocolitis Gestational age (wk)a Birth weight (g)a Gender (Male)b Caesarean deliveryb Prenatal steroidsb Apgar score at 1 mina Apgar score at 5 mina Respiratory distress syndromeb UACb UVCb Patent ductus arteriosusb Indomethacinb Packed red blood cell or plasma product transfusionb Time of first feed (d)a Time to reach full enteral feeding (d)a Type of feeding Breast milkb Formulab Combined Deathb
NEC (n = 38)
No NEC (n = 203)
p value
28.1 ± 1.2 1083.1 ± 184.2 22 (57.9) 5 (13.1) 29 (76.3) 5.1 ± 0.1 7.1 ± 1.0 29 (76.3) 4 (10.5) 11 (28.9) 6 (15.8) 4 (10.5) 33 (86.8) 6.5 ± 2.4 15.1 ± 3.2
28.6 ± 1.7 1121.2 ± 224.6 98 (48.3) 24 (11.8) 139 (68.5) 5.3 ± 0.9 6.9 ± 0.9 148 (72.9) 18 (8.9) 41 (20.2) 24 (11.8) 20 (9.8) 168 (82.7) 5.9 ± 2.1 16.7 ± 4.7
0.08 0.32 0.76 0.47 0.69 0.17 0.21 0.58 0.45 0.78 0.60 0.41 0.54 0.11 0.04
4 (10.5) 13 (34.2) 21 (55.3) 5 (13.1)
25 (12.3) 86 (42.4) 92 (45.3) 8 (3.9)
0.32 0.40 0.77 0.03
are means ± SD. b Number (percent). UAC: Umbilical arterial catheterization. UVA: Umbilical venous catheterization.
a Values
Fig. 1. Cord blood levels of epidermal growth factor in neonates who developed NEC and those who did not.
Fig. 2. Cord blood levels of epidermal growth factor among infants with NEC.
transfusion of blood products. Culture-proven sepsis was detected in 7 (18.4%) infants with NEC, (Klebsiela pneumoniae [n = 4], Enterobacter sp [n = 3], and Escherichia coli [n = 1]). Of the 38 infants with NEC, 23 (60.5%) had suspected NEC (stage I) and 15 (39.5%) had confirmed NEC (Bell’s stage ≥2), of whom 9 (23.7 %) had stage II, and 6 (15.8 %) had stage III. Radiographic findings included pneumatosis intestinalis (n = 9), and intestinal perforation and/or ascites (n = 6). Six (15.8%) had placement of surgical drain and/or exploratory laparotomy of whom, 4 had
positive peritoneal fluid cultures with Klebsiela pneumoniae (n = 4). Five (13.1%) infants died (3 had stage III-NEC, and 2 had stage II-NEC). VLBW infants who subsequently developed NEC had significantly lower cord serum levels of EGF than those who did not (13.7 ± 2.0 vs. 39.2 ± 1.5 pg/mL, p < 0.0001; Fig. 1). Moreover, cord serum levels of EGF were significantly lower in infants with stage III than those with stage II or I NEC (10.2 ± 0.4, 12.2 ± 0.9, 14.7 ± 1.5 pg/mL respectively, p < 0.001; Fig. 2). In NEC group, cord blood levels of EGF
W.A. Wahab Mohamed and A.M. Aseeri / Epidermal growth factor and NEC
were significantly lower in non-survivors than in survivors (10.3 ± 0.4 vs. 13.8 ± 1.9 pg/mL, p < 0.001). Our results showed a significant negative correlation between cord blood concentrations of EGF and the severity of NEC (r = −0.57, p = 0.01). Cord blood concentrations of EGF were significantly lower in preterm infants with gestational age ≤28 weeks (n = 63) than in those with older gestational age (n = 178) [33.09 ± 1.4 vs. 36.07 ± 1.7 pg/mL, p < 0.001]. We detected a significant positive correlation between cord blood concentrations of EGF and the gestational age (r = 0.65, p < 0.01). Multivariable logistic regression analysis, with NEC as the dependant variable and gestational age, birth weight, prenatal steroids administration, RDS, PDA, indomethacin use, and cow’s milk formula feeding as risk factors demonstrated that only low cord blood EGF concentration was independently associated with the subsequent risk of NEC (OR = 0.978 [95% CI: 0.959–0.997]; p = 0.02).
4. Discussion Necrotizing enterocolitis is the leading cause of death from gastrointestinal disease in preterm infants, and remains a major cause of long-term disability in those who survive the disease. Early warning signs and symptoms of NEC are nonspecific, often inconspicuous and may be difficult to distinguish from other non-specific abdominal disorders such as gastrointestinal dysmotility [10]. Thus, early identification of NEC in VLBW infants remains a major diagnostic challenge. No reliable early biomarkers have demonstrated sufficient positive and negative predictive values to be clinically useful [25]. Only few clinical studies measured the serum concentrations of EGF in preterm infants with NEC. Shin et al., reported diminished concentrations of salivary and serum EGF in preterm infants with NEC [21]. A recent clinical trial with 327 preterm and term neonates evaluated the ontogeny of salivary EGF and its relation to the development of NEC [26]. The concentration of salivary EGF positively correlated with gestation age; infants at earlier gestational ages had significantly lower EGF concentrations in their saliva compared with term neonates. Importantly, lower concentrations of salivary EGF during the first week of life were associated with an increased incidence of NEC [26].
261
To the best of our knowledge, this is the first reported prospective study that measured the cord blood concentrations of EGF in VLBW infants. Our results showed that VLBW infants who subsequently developed NEC had significantly lower cord blood concentrations of EGF than those who did not. These results suggest that VLBW infants who have low concentrations of EGF at birth, are at substantially increased risk of developing NEC. This holds as true even after correcting for the known risk factors for NEC, including low gestational age, sex, RDS, PDA and cow’s milk formula feeding. Moreover, the lowest EGF concentrations were detected in infants who developed severe NEC, particularly those who died. We speculate that EGF not only plays a role in predicting NEC, but also might have a prognostic value in this disease. Further studies with larger sample sizes are needed to examine such hypothesis. Indeed, the protective role of maternal milk in NEC pathogenesis has been reported both in human [9] and animal studies [14, 27]. These findings stimulated the search for various components of milk that might be responsible for protection against NEC. Mammalian milk contains a large number of biologically active substances that directly affect gut maturation and mucosal protection. One of these substances is EGF, which was detected in many body fluids, including colostrum and milk during the early postnatal period. EGF has been found at high concentrations in the maternal colostrum [28]. In our study, only four infants in the NEC group were on breast milk feeding and none of them received colostrum. Interestingly, none of infants with severe (stage III) NEC was on breast milk feeding. Dvorak et al. [14] examined the effect of orally administered EGF on the incidence of NEC in a neonatal rat model. They showed that EGF supplementation of formula reduced the incidence and severity of NEC in rats. These data suggest a new potential therapeutic approach for the prevention and treatment of NEC in neonates. Our study has important limitations that require consideration. First, the total number of patients was small; therefore, further studies with larger sample sizes should be undertaken. Second, single measurement of serum EGF concentrations at birth, so another measurement of EGF when the patient is ill might be more useful. The association between low EGF concentrations and NEC may provide the rational for a controlled trial to evaluate the efficacy of recombinant EGF fortified milk formula in preventing the development of NEC in VLBW neonates.
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W.A. Wahab Mohamed and A.M. Aseeri / Epidermal growth factor and NEC
5. Conclusion Low cord blood EGF concentrations may predict the subsequent development of NEC in very low birth weight infants. The measurement of cord blood EGF concentration at birth might be a biological marker to permit early intervention and might provide a new therapeutic target to prevent NEC. Acknowledgments
[13]
[14]
[15]
[16]
The authors acknowledge the major contribution of the staff at the NICU, Khamis Civil Hospital. [17]
Financial disclosure statement The authors report no conflicts of interest.
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