Subject recruitment and follow-up
Singleton pregnant women who attended regular antenatal examinations at one obstetric-pediatric clinic in Tainan City, Taiwan, between January 2014 and January 2015 were identified and recruited into this study. Eligibility was then assessed between 28 and 32 weeks of gestation. Individuals with underlying medical conditions such as chronic hypertension, pre-gestational diabetes mellitus, chronic lung disease, renal disease, major cardiac disease, autoimmune conditions, thyroid disease, malignancy, and uterine malformations were excluded. Individuals that had a history of illicit drug abuse and those whose fetuses had chromosomal abnormalities, congenital malformations or evident congenital infections (TORCH) were also excluded. Follow-up assessments were carried out at the time of delivery, and at 1, 2, and 3 years after delivery.
This study was approved by the Ethics Committee (B-BR102-001) of National Cheng Kung University Hospital, Tainan, Taiwan, and written informed consent was obtained from each participant and her spouse. The demographic characteristics, anthropometric data, and common risk factors of SGA were collected and assessed, including maternal age, body height, body weight before pregnancy, body mass index (BMI) before pregnancy, smoking tobacco, alcohol use, maternal educational attainment, annual household income, and pregnancy complications such as antepartum bleeding, anemia, pregnancy-induced hypertension (PIH), and PE. Anemia was defined as a hemoglobin concentration of < 11 g/dL. PIH was defined as any new onset of hypertension (systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg) after 20 weeks of gestation. PE was defined as a combination of PIH and proteinuria or signs of end-organ dysfunction.
The corresponding neonates enrolled in the follow-up were full-term (gestational age 37–40 weeks) and healthy. Parameters recorded at birth included gestational age, body weight and length, head circumference, and Apgar score at 1 and 5 minutes post-delivery. Neonates that required post-delivery intensive care were excluded from the follow-up study.
Maternal serum collection and testing for anti-H. pylori IgG
The status of H. pylori infection was assessed by measuring serum IgG against H. pylori using a commercial H. pylori IgG ELISA kit (IBL, Hamburg, Germany) at 28–32 weeks of gestation, the period when a routine screening test for hepatitis B surface antigen is commonly conducted in Taiwan. Anti-H. pylori IgG titers > 12 U/ml were considered to be positive, while titers < 8 U/ml were considered to be negative. Titer values between 8 and 12 U/ml were considered to be equivocal. The mothers were subsequently categorized as being either H. pylori-seropositive or H. pylori-seronegative according to the ELISA results.
Cord blood levels of IGF-1, IGFBP-3, insulin, and ghrelin
Cord venous blood samples were collected at delivery and centrifuged at 3500 x g for 30 minutes at 4°C to separate the serum. The serum samples were stored at -80°C. IGF-I (R&D Systems, Inc. Minneapolis, MN, USA), IGFBP-3 (R&D Systems, Inc. Minneapolis, MN, USA), insulin ((R&D Systems, Inc. Minneapolis, MN, USA) and ghrelin (EMD Millipore Corporation, St. Charles, MO, USA) levels were measured using ELISA following the manufacturers’ instructions.
Assessment of anthropometric parameters and cognitive development of newborns
The enrolled newborns were studied longitudinally for up to 3 years. The weight and length of each child were measured at birth and then annually. According to the gestational age of infants born in Taiwan, SGA was defined by a birth weight below the 10th percentile .
Cognitive development was assessed using the comprehensive developmental inventory for infants and toddlers (CDIIT) test at 3 years of age. The CDIIT is a reliable pediatric norm-referenced assessment tool that is widely used for the clinical diagnosis of developmental delays in five major developmental areas, including cognition, language, motor, social and self-care skills [25, 26]. The CDIIT test consists of a diagnostic test (CDIIT-DT) and a screening test (CDIIT-ST). In this study, we applied the cognition subtest of the CDIIT-DT and assessed five aspects of a child’s mental capacity, including attention, perception, memory, reasoning and concepts of color, shape, size, and number. The evaluations were conducted by a trained administrator.
H. pylori stool antigen test (HPSA) and definition of H. pylori infection in children
Stool samples were collected from the enrolled infants at 1, 2 and 3 years after birth to detect new H. pylori infections using the HPSA test. The HPSA test (Meridian Diagnostic Inc., Cincinnati, Ohio, USA) uses a plurality of monoclonal anti-H. pylori antibodies adsorbed to microwells. The results were interpreted spectrophotometrically, and the cutoff optical density at 450 nm for a positive outcome was set at 0.14. Children with a positive HPSA test in any one of the three samples were considered to be infected with H. pylori, while those who had a negative HPSA test result following a previous positive result were defined as having spontaneous elimination of H. pylori infection. A minimum of two consecutive positive HPSA tests during the follow-up period was considered to indicate persistent H. pylori infection. Children with negative HPSA tests throughout the follow-up period were considered to be non-infected.
Demographic data and measurable parameters were presented as frequencies and means ± standard deviations (SDs). Significance of association was determined using the Pearson chi-square (χ2) test for categorical variables and the independent sample t-test for continuous variables. As ELISA tests tend to produce high SD values which may give rise to type II statistical errors, receiver operating characteristic (ROC) curve analysis in conjunction with Youden's index was used to determine the best cutoff values of cord blood IGF-1, IGFBP-3, insulin and ghrelin levels to differentiate H. pylori-seropositive and H. pylori-seronegative mothers. A p value of less than 0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS Statistics V.17.0.