The Role of Insulin-Like Growth Factor Axis Components in Small for Gestational Age Birth

Abstract

Gestation is a time gap between conception and birth of neonates. The age of the fetus or the number of weeks the pregnancy lasts is called as gestational age. Based on the number of gestational weeks and birth weight, the neonates can be classified as Small for Gestational Age (SGA), Appropriate for Gestational Age (AGA), and Large for Gestational Age (LGA). SGA neonates have a birth weight of less than the 10th percentile, and it is a common problem in developing countries such as India, Pakistan, Nigeria, and Bangladesh. The prevalence of SGA varies from 5.3-41.5% (East-South Asia). The incidence of SGA is 30% in developing countries and 5-7% in developed countries. SGA is one of the leading causes of perinatal morbidity and mortality in neonates. About 85% of the neonates will catch-up with growth at the age of 6 months 2 years. If the SGA neonates do not attain catch-up growth, they remain short stature and have a risk of insulin resistance, high blood pressure, type 2 diabetes, cardiovascular diseases, and mortality in early life. The incidence of SGA births depends on factors such as Intrauterine Growth Restriction (IUGR), preterm birth, environment, nutrition, growth factors, lifestyle, genetic composition, and pregnancy complications such as maternal age, low level of oxygen, low blood sugar, genetic makeup, etc. The Growth Hormone (GH)/Insulin-like Growth Factor (IGF)-axis plays a significant role in the growth and development of the fetus and neonates. Recent studies from the western world highlight that the growth of the fetus depends on the levels of insulin and IGF-axis components such as IGF1, IGF2, IGF binding proteins (IGFBP), IGF receptors (IGFR), gene expression, and epigenetic modification of IGF-axis genes. The altered levels of IGF-axis components may lead to abnormal fetus growth. The studies also highlighted the importance of the IGF-axis in iv GH therapy/monitoring the growth of SGA neonates. The literature on SGA and GH/IGF-axis components revealed contrasting results (higher, lower, or no change), that might depend on the individuals' nutrition, hormone levels, environment, and genetic makeup. To the best of my knowledge, the role of IGF-axis components and the likely factors influencing it was not understood fully in the Indian population. Hence, the thesis aimed to investigate the role of IGF-axis components in SGA neonates born to south Indian women. A total of 98 pregnant women undergoing labor in the department of Obstetrics and Gynecology, RL Jalapa Hospital, Kolar, Karnataka, were recruited in the present study (Ref No: SDUMC/KLR/IEC/32/2019-20). The pregnant women were grouped as AGA (n=49) and SGA (n=49) based on the gestational weeks and birth weight. After delivery, umbilical cord blood samples (5 ml) were collected and used to measure the levels of IGF1, IGF2, and IGFBP3 proteins using the ELISA method and cultured in vitro to calculate the mitotic index. In addition to cord blood, approximately 150-200 mg of placental tissue samples were collected using a sterile surgical blade. The placental tissues were used to measure the IGFR1 and IGFR2 gene expression using qPCR and methylation status of IGF2, H19, IGF1, and IGFR1 genes using methylation specific PCR. The mean±SD (range) of IGF1, IGF2, and IGFBP3 proteins in AGA neonates is 118±33 (54-210), 124±10 (103-146), and 1606±277 (1032-2006), and in SGA neonates, is 118±47 (4-294), 123±12 (92-154), and 1432±387 (315-2137) (ng/ml) respectively. The levels of IGF1 (p=1) and IGF2 (p=0.69) protein did not differ between AGA and SGA neonates, whereas the IGFBP3 protein is significantly (p=0.023) less in SGA compared to AGA neonates. The plasma levels of IGF1, IGF2, and IGFBP3 proteins v did not correlate (r=0.01) with gestational weeks and birthweight of neonates. Further, the study was extended to find the expression of IGFR1 and IGFR2 genes in the placenta of AGA and SGA neonates. The mean±SD (range) of ∆ cycle threshold (Ct) of the IGFR1 gene obtained from AGA neonates is 5.06±4.28 (-4.82-10.63), and SGA neonates are 7.03±3.82 (1.44-13.20). The mean±SD (range) of ∆Ct of the IGFR2 gene obtained from AGA neonates is 2.45±3.56 (-7.67-6.04), and SGA neonates are 3.94±1.59 (1.66-7.96). The fold change expression of IGFR1 and IGFR2 genes in SGA neonates was calculated (2-∆∆Ct method) by subtracting the ∆Ct of AGA from SGA neonates. The IGFR1 gene is down-regulated 3.9-folds, whereas the IGFR2 gene is down-regulated 2.8-folds in SGA compared to AGA neonates. Further, there is no correlation between IGFR1 and IGFR2 gene expression with gestational weeks and the birth weight of neonates. The results indicate that the levels of IGFBP3 protein were significantly lower, and the expression of IGFR1 and IGFR2 genes were downregulated in SGA compared to AGA neonates. Further, the study was extended to find the methylation status of the IGF-axis of imprinted (IGF2 and H19) and non-imprinted (IGF1 and IGFR1) gene promoters in the placenta of AGA and SGA neonates. The methylation of imprinted and non-imprinted genes is 2.5, 1.5, 5, and 7.5% lower in SGA compared to AGA neonates. The co-methylation of IGF-axis gene promoters [imprinted: 7.5% lower, non imprinted: 15% lower, and both imprinted and non-imprinted: 20% lower] were lower in SGA compared to AGA neonates. The methylation of imprinted (IGF2 and H19) and non-imprinted (IGF1 and IGFR1) genes significantly reduced (p<0.05) the levels of IGFBP3 protein, IGFR1 and IGFR2 gene expression, gestational weeks, and birth weight of SGA compared to AGA neonates. Further, the study was extended to find the difference in the intrinsic division capacity of the cord blood lymphocytes of AGA and vi SGA neonates. The range (mean±SD) of the mitotic index obtained from AGA neonates is 12-22% (15.71±1.81), and SGA neonates are 10-16% (12.60±1.16). The mitotic index of SGA neonates is significantly (p=0.0001) less compared to AGA neonates. The mitotic index is significantly (p<0.05) less in methylated positive SGA compared to AGA neonates. The levels of IGF-axis proteins and gene expression, gestational weeks, and birth weight did not correlate with the mitotic index of cord blood lymphocytes. Overall, the thesis generated evidence on the role of IGF-axis components (IGF1, IGF2, IGFBP3 proteins, IGFR1 and IGFR2 gene expression, methylation of IGF2, H19, IGF1, and IGFR1 gene promoter, intrinsic division capacity of cord blood lymphocytes) in the SGA neonates born to Indian women. The information might be helpful for further studies to explore the potential markers to screen/monitor/diagnose/management of SGA neonates/babies born to Indian women.