Insulin-like growth factor axis in pregnancies affected by fetal growth disorders

Abstract

Background: Insulin-like growth factors 1 and 2 (IGF1 and IGF2) and their binding proteins (IGFBPs) are expressed in the placenta and known to regulate fetal growth. DNA methylation is an epigenetic mechanism which involves addition of methyl group to a cytosine base in the DNA forming a methylated cytosine-phosphate-guanine (CpG) dinucleotide which is known to silence gene expression. This silences gene expression, potentially altering the expression of IGFs and their binding proteins. This study investigates the relationship between DNA methylation of components of the IGF axis in the placenta and disorders in fetal growth. Placental samples were obtained from cord insertions immediately after delivery from appropriate, small (defined as birthweight <10th percentile for the gestation [SGA]) and macrosomic (defined as birthweight > the 90th percentile for the gestation [LGA]) neonates. Placental DNA methylation, mRNA expression and protein levels of components of the IGF axis were determined by pyrosequencing, rtPCR and Western blotting.

Results: In the placenta from small for gestational age (SGA) neonates (n = 16), mRNA and protein levels of IGF1 were lower and of IGFBPs (1, 2, 3, 4 and 7) were higher (p < 0.05) compared to appropriately grown neonates (n = 37). In contrast, in the placenta from large for gestational age (LGA) neonates (n = 20), mRNA and protein levels of IGF1 was not different and those of IGFBPs (1, 2, 3 and 4) were lower (p < 0.05) compared to appropriately grown neonates. Compared to appropriately grown neonates, CpG methylation of the promoter regions of IGF1 was higher in SGA neonates. The CpG methylation of the promoter regions of IGFBP1, IGFBP2, IGFBP3, IGFBP4 and IGFBP7 was lower in the placenta from SGA neonates as compared to appropriately grown neonates, but was unchanged in the placenta from LGA neonates.

Conclusions: Our results suggest that changes in CpG methylation contribute to the changes in gene expression of components of the IGF axis in fetal growth disorders. Differential methylation of the IGF1 gene and its binding proteins is likely to play a role in the pathogenesis of SGA neonates.

Keywords: DNA methylation; Insulin growth factor; Large fetus; Placental expression; Small fetus.

Fig. 1

Placental mRNA expression of the IGF axis in appropriately grown, SGA and LGA neonates. a IGF1 expression, b IGF2 expression, c IGFBP1 expression, d IGFBP2 expression, e IGFBP3 expression, f IGFBP4 expression, g IGFBP7 expression. *p < 0.05, **p < 0.005, ***p < 0.0005 when compared to appropriately grown neonates; SGA small for gestational age, LGA large for gestational age

Fig. 2

Spearman’s correlation between placental mRNA expression and increasing order of birthweight centiles. a IGF1 expression vs. birthweight centiles, b IGFBP1 expression vs. birthweight centiles, c IGFBP2 expression vs. birthweight centiles, d IGFBP3 expression vs. birthweight centiles, e IGFBP4 expression vs. birthweight centiles, f IGFBP7 expression vs. birthweight centiles. A significant positive correlation between IGF1 mRNA expression and a significant negative correlation between the binding protein expression with increasing order of birthweight centiles suggest the crucial role played by their inverse relationship in controlling fetal growth

Fig. 3

Placental protein expression of the IGF axis in appropriately grown, SGA and LGA neonates. a IGF1 expression, b IGFBP1 expression, c IGFBP2 expression, d IGFBP3 expression, e IGFBP4 expression, f IGFBP7 expression, g IGFBP1 expression, h IGFBP2 expression, i IGFBP3 expression, j IGFBP4 expression. *p < 0.05, **p < 0.005, ***p < 0.0005, when compared to appropriately grown neonates. SGA small for gestational age, LGA large for gestational age. Increased protein expression of IGFBP1, IGFBP2, IGFBP3, IGFBP4 and IGFBP7 in SGAs and decreased expression of IGFBP1, IGFBP2, IGFBP3 and IGFBP4 in LGA neonates suggest inverse relationship of the binding protein expression with fetal growth

Fig. 4

Placental promoter methylation in appropriately grown, SGA and LGA neonates. a IGFF1 promoter methylation—CpG sites 1, 2 and 3; b IGFBP1 promoter methylation—CpG sites 11, 12 and 13, c IGFBP2 promoter methylation—CpG sites 9, 10, 11; d IGFBP4 promoter methylation—CpG sites 23, 24, 25, 26, 27, 28 and 29; e IGFBP3 promoter methylation—CpG sites 46, 47, 48, 49 and 50; f IGFBP7 promoter methylation—CpG sites 8, 9, 10, 11 and 12. *p < 0.05 when compared to appropriately grown, SGA small for gestational age, LGA large for gestational age

Fig. 5

Spearman’s correlation between placental promoter CpG methylation and increasing order of birthweight centiles. a IGF1 methylation vs. birthweight centiles, b IGFBP1 methylation vs. birthweight centiles, c IGFBP2 methylation vs. birthweight centiles, d IGFBP3 methylation vs. birthweight centiles, e IGFBP4 methylation vs. birthweight centiles, f IGFBP7 methylation vs. birthweight centiles. A significant negative correlation between IGF1 mRNA expression and a significant positive correlation between the binding protein expression with increasing order of birthweight centiles could reflect the indirect role played by DNA methylation relationship in controlling fetal growth by controlling gene expression of the IGF axis