*Departments of Perinatology and Gynecology
†Microbiology and Immunology, Medical University of Silesia, Zabrze, Poland
Received 10 July, 2006
Accepted 13 December, 2006
Address correspondence and reprint requests to Anna Oslislo, MD, Department of Perinatology and Gynecology, Plac Traugutta 6, 41-810 Zabrze, Poland (e-mail: firstname.lastname@example.org).
Background and Aim: The aim of the study was to compare epidermal growth factor (EGF) concentration in 81 colostrum samples collected from mothers of newborns in the following growth categories: preterm appropriate for gestational age (AGA), preterm small for gestational age (SGA), and full term (FT).
Results: Significantly higher concentrations of EGF were found in the colostrum of mothers who delivered premature AGA infants at less than 32 weeks of gestation compared with mothers who delivered premature SGA babies at the same gestational age.
Conclusions: We concluded that the maternal compensatory mechanism accelerating the development of immature breast-fed infants may be disturbed when gestation is complicated by intrauterine growth retardation.
Epidermal growth factor (EGF), a small polypeptide mitogen, is present in many human body fluids including plasma, saliva, urine, amniotic fluid, and milk. EGF is responsible for proliferation and differentiation of many tissues and stimulation of DNA synthesis in the gastrointestinal tract (1). The assumption is that amniotic fluid swallowed by a fetus during intrauterine life and the colostrum, which the newborn obtains postpartum, secures physiological continuity of organism growth and development (2). The concentration of EGF in human milk from mothers of premature infants is higher than that from mothers of term infants (3). There are no data available referring to the quantity of this factor in the milk of mothers who delivered preterm neonates who were small-for-gestational-age (SGA) after pregnancy complicated by intrauterine growth retardation (IUGR). The aim of the study was to compare mean EGF concentrations in colostrum collected from mothers of preterm infants in AGA and SGA categories and full-term (FT) infants.
PATIENTS AND METHODS
Eighty-one mothers who delivered newborns in the Department of Perinatology and Gynecology in our institution were included in the study. The inclusion criterion was lactation, which ensures the infant's needs and ability to express excess milk for examination. The exclusion criteria were lack or withdrawal of approval of examinations, medical contraindications to breast-feeding on the mother's and infant's parts, smoking during pregnancy or lactation, the mother's having diabetes, and congenital malformation of the infant. The protocol was approved by the Bioethical Committee of the Medical University of Silesia. IUGR was diagnosed on the grounds of obstetric and ultrasonographic examination (Voluson 730 3D/4D; GE Healthcare, Milwaukee, WI) of the fetus, with biometric evaluation done at least twice. Prenatal diagnosis of IUGR was verified after parturition using the classic definition of IUGR with verification of maturity according to the short Ballard scale (4,5). We used percentile tables for the Polish population designed by Osuch-Jaczewska (6). Mothers of infants with birth weight below the 10th percentile for weeks of gestational age were included in the SGA group. Mothers of infants with birth weight between the 10th and 90th percentiles were included in the AGA group.
Sample Collection and Preparation
Milk samples were expressed manually or by breast pump during the midmorning. Colostrum samples were collected between 1 and 5 days' postpartum from each mother of infants born in 1 of the 5 groups: preterm AGA (22–31 weeks), n = 12; preterm SGA (22–31 weeks), n = 8; preterm AGA (32–36 weeks), n = 13; preterm SGA (32–36 weeks), n = 15; and FT, n = 33. Milk samples were centrifuged soon after collection (5000g, 10 minutes) to remove lipids and cells. Milk whey was frozen (−20° C). Centrifuging was repeated after defrosting before an examination (10,000g, 5 minutes, 4°C).
Concentrations of EGF in milk were measured by enzyme-linked immunosorbent assay (ELISA) using the Quantikine human EGF system (R&D Systems, Minneapolis, MN). Because milk samples generated values higher than the highest standard, we diluted them with the appropriate calibrator diluent (colostrum was diluted 600 times, transitional milk 400 times). Samples were put into microplates with monoclonal antibody (anti-EGF) 200 μL of assay diluent and then 200 μL of standard or sample was added. After being covered with adhesive strip, incubation was performed for 2 hours at room temperature. We aspirated each well and washed it, repeating the process twice for a total of 3 washes. After the last wash, any remaining wash buffer was removed. Next, 200 μL of EGF conjugate was added to each well and the microplate was covered with a new adhesive strip and incubated for 1 hour at room temperature and then washed 3 times. Finally, 200 μL of substrate solution was added to each well and, after 20 minutes, reaction was stopped by 50 μL of stop solution. We determined the optical density of each well within 30 minutes using a microplate reader set to 450 nm (Elx-800; Bio-Tek Instruments, Winooski, VT) with KCjr software (Bio-Tek Instruments).
Statistical analysis was performed by analysis of variance followed by Fisher protected least-significant-difference test. The statistical program Statistica PL (version 6.0; Statsoft Poland, Krakow, Poland) was used. All of the data are expressed as mean ± SD. P < 0.05 was considered significant at the 95% confidence level.
Comparison of mean EGF concentration in colostrum from the 3 groups of mothers (premature AGA [22–31 weeks], premature SGA [22–31 weeks], and FT) shows a statistically significant difference (P = 0.0003; Fig. 1). Concentrations of EGF in colostrum from the preterm AGA group (22–31 weeks; 159.24 ± 51.05 ng/mL) were significantly higher compared with the values from the preterm SGA group (22–31 weeks; 80.69 ± 33.52 ng/mL) and FT group (95.75 ± 48.58 ng/mL; P = 0.0007 and P = 0.0002, respectively). The colostrum EGF levels in the preterm SGA group were similar to those in the FT group. There were no significant differences between EGF concentrations in milk from the preterm AGA group (32–36 weeks; 102.03 ± 55.43 ng/mL), preterm SGA group (128.57 ± 49.29 ng/mL), and FT group. The presence of IUGR in pregnancy and gestational age (< 32 weeks vs ≥32 weeks) interact significantly in their effect on the EGF concentration in colostrum (Fig. 2).
In the group of mothers of AGA babies, mean concentration of EGF in colostrum is higher when delivery occurred at <32 weeks' gestational age compared with the cases of delivery after 32 weeks' gestational age. EGF concentration, which is high in colostrum from mothers of AGA infants, decreases significantly (P = 0.0059) in milk from mothers of AGA infants. There is an opposite observation in the group of mothers of SGA babies when pregnancy was complicated by IUGR. Mean concentration of EGF in colostrum is low when parturition occurred at <32 weeks' gestational age, but in cases of delivery after 32 weeks' gestational age, mean EGF concentration is high. EGF concentration, which is low in colostrum from mothers in the preterm SGA group, increases significantly (P = 0.0321) in the preterm SGA group. The difference between the values of mean colostrum EGF in AGA and SGA groups of infants delivered at <32 weeks' gestational age is highly significant (P = 0.0011), but there is no difference between AGA and SGA groups of infants delivered at 32 weeks' gestational age (P = 0.1633).
In the present study, EGF was estimated by ELISA (7). In most works concerning EGF concentration in bovine and human milk, radioimmunoassays or radioreceptor assays were used (3,8,9). Similar to quantitative radioimmunoassay techniques, ELISA does not show cross-reactivity or interference toward members of the EGF-like peptide family such as transforming growth factor-α1 and heparin-binding EGF (10). An evaluation of milk protein concentration was not done.
Dvorak et al (3) evaluated milk protein concentrations to verify that any changes in peptide growth factor concentration were not a result of differences in total protein content. They stated that there were no statistically significant differences in milk protein concentrations in the experimental group on day 7. On day 14 the group of mothers of extremely preterm infants (<27 weeks) exhibited significantly higher milk protein concentrations compared with mothers of preterm (32–36 weeks) and term-delivered infants.
Our study describes EGF concentrations in colostrum from mothers who delivered infants prematurely after pregnancy complicated by IUGR and compares them with EGF concentrations in colostrum from mothers without this pathology who delivered infants prematurely and full term. The IUGR and gestational age were identified as 2 important factors that interacted and influenced EGF concentration in colostrum. The level of EGF is significantly lower in milk from mothers who delivered premature SGA infants at <32 weeks' gestational age compared with mothers who delivered AGA infants at the same gestational age.
Results from our study indicate that IUGR may be an important factor that influences concentrations of EGF in milk in the first days postpartum. The mechanism of this phenomenon is difficult to explain. The identifiable stages of human milk are colostrum, transitional milk, and mature milk (11). Their relative contents are significant for newborn physiological adaptation to extrauterine life (2,12). In the first postpartum days the residual prepartum milk that presents in the mammary gland and ducts at delivery is progressively mixed with newly secreted milk, forming colostrum. Theoretically it can be assumed that factors that disturbed the course of pregnancy may change the composition of human colostrum as well. The growth, proliferation, and milk secretion in the mammary gland are regulated by hormones secreted by the mother and by the placenta and also by autocrine mechanisms. EGF is known to be synthesized by the mammary gland, like several other hormones (11). The question is which factors that caused IUGR can influence the synthesis of milk-borne EGF.
EGF is responsible for 70% of mitogenic activity of human milk and plays an important role in the development and protection of the gastrointestinal tract (1,13). Salivary and serum EGF levels in neonates with necrotizing enterocolitis are lower compared with healthy infants (14). The incidence of this disease is higher in formula-fed babies than in breast-fed babies (15). EGF has been shown to enhance the growth of the stomach and small intestine and induce maturation of intestinal brush-border disaccharidase activities (9). Premature infants in situations in which enteral feeding is possible profit from a high concentration of this hormone in human milk (11,12). Dvorak and coworkers (3) maintain that EGF concentration is higher in the milk of mothers who delivered extremely preterm newborns (<28 weeks' gestational age) than in the milk of mothers who delivered more mature babies. Our results are compatible with this observation, but only with reference to the mothers who delivered premature AGA infants whose intrauterine development was not restricted.
Researchers who have investigated peptide growth factors in mothers' milk after premature deliveries have not noticed a problem with the coexistence of prematurity and IUGR. Xiao and coworkers (16) showed a negative correlation between EGF concentration in milk and the birth weight of the newborn, but mean gestational age in the preterm group was 32.6 ± 2.7 weeks and there was no information about pregnancy complications. Low levels of EGF in the colostrum of mothers after premature delivery (<32 weeks' gestation) after a pregnancy complicated by IUGR may indicate that the protective effect of EGF can be disturbed. There is a lack of EGF in preterm formulas (16). Supplementation of human milk and formulas using recombinant EGF is considered for treatment of gastrointestinal disorders (17,18). Further study is necessary to determine whether the milk of mothers who had pregnancies complicated by IUGR also needs such supplementation.
We have found high concentrations of EGF in the colostrum of mothers who have delivered premature AGA infants and low concentrations of EGF in the colostrum of mothers who have delivered premature SGA infants (<32 weeks' gestation). We conclude that maternal compensatory mechanism accelerating the development of the digestive tract in immature breast-fed preterm infants may be disturbed when gestation is complicated by IUGR, but this is a preliminary finding and further study is needed. A longitudinal evaluation of the phenomenon would be useful to understand whether this is an isolated feature or is constant.
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