Introduction: Abnormal fetal growth remains a major problem in pregnancies complicated by diabetes and is associated with increased maternal and offspring mortality and morbidity. Insulin-like growth factors (IGFs) stimulate fetal growth while their effects are inhibited by binding proteins (IGFBPs). IGFBP-1 is a significant IGFBP in maternal and fetal circulation and the only binding protein acutely affected by glucoregulatory hormones; as such, IGFBP-1 is particularly important in pregnancy with diabetes. In plasma of healthy human adults, the fully phosphorylated (pIGFBP-1) isoform is predominant. During pregnancy the phosphorylation status of IGFBP-1 changes; in addition to pIGFBP-1, non-phosphorylated (np) IGFBP-1 and 3 lesser phosphorylated (lp) IGFBP-1 variants with lower affinity for IGF-I are detected in the maternal circulation. The change in the phosphorylation status of IGFBP-1 in pregnancy may provide a physiological mechanism for the increased IGF-I bioavailability at the maternal/fetal interface required for placental and fetal growth.Hypothesis: IGFBP-1 de-phosphorylation occurs at the maternal/fetal interface and this process is catalyzed by placental alkaline phosphatase (PLAP). Fetal overgrowth (macrosomia) in pregnancy with diabetes may be a consequence of elevated IGF-I action at the placenta secondary to increased PLAP activity.Methods and Results: In vitro: Explants of human term placentas from normal pregnancies (n=5), or their conditioned media (CM), were incubated with pIGFBP-1 in the presence or absence of an anti-PLAP function blocking antibody. Addition of pIGFBP-1 to explants resulted in its binding to the tissue and de-phosphorylation, with npIGFBP-1 isoforms appearing in the medium. pIGFBP-1 was not de-phosphorylated when cultures were carried out in the presence of anti-PLAP antibody. In solution phase assays, PLAP failed to de-phosphorylate pIGFBP-1. Thus, placenta de-phosphorylates IGFBP-1 as a result of PLAP activity, and this requires its binding to the tissue.To investigate factors which may affect the activity of PLAP, placental explants (n=3 for each series of experiments) were incubated with pIGFBP-1 in the presence of insulin, IGF-I/-II or under hyperglycemic or hypoxic conditions. Following incubation, the phosphorylation status of IGFBP-1 present in placental-CM was assessed by native electrophoresis and western blot. PLAP-mediated IGFBP-1 de-phosphorylation was not affected in vitro by hyperglycemia, hypoxia, insulin or IGF-I/-II. In vivo: 30 patients with any type of diabetes in pregnancy and 20 controls were recruited. Maternal/cord blood was collected at term and analysed for IGF-I/-II, total IGFBP-1 and the phosphorylation status of IGFBP-1. Placentas were analysed for PLAP expression and activity. The maternal blood levels of both IGFs and total IGFBP-1 were similar in the diabetes and control groups, while cord IGF-II was elevated in diabetes. Unexpectedly, the p/npIGFBP-1 ratio in maternal serum was elevated in patients with diabetes, which may be a result of decreased IGFBP-1 de-phosphorylation. In controls, maternal p/npIGFBP-1 ratio correlated with infant weight, whilst this correlation was not demonstrated in women with diabetes. Placental PLAP expression/ex-vivo activity and total IGFBP-1 levels in maternal serum were unaltered in diabetes and did not relate to fetal growth in either diabetes or control groups. Conclusions: The hypothesis that activated PLAP and therefore enhanced IGFBP-1 de-phosphorylation may increase the effect of IGF-I on placental cell turnover and accelerate fetal growth in diabetes was not supported by the results of this study. Further work is required to reveal mechanisms by which the maternal/placental/fetal IGF-IGFBP-PLAP pathways modulate fetal growth in normal and compromised pregnancy.