Pro-EPIL forms are present in amniotic fluid and maternal serum during normal pregnancy

Recent observations based on immunohistochemistry and RT-PCR demonstrate that early placenta insulin-like peptide (EPIL), encoded by the INSL4 gene, is present in the placenta during gestation. In the present study, we report on the development of a specific immunoassay, entirely based on two monoclonal anti-peptide antibodies (mAbs), and its use for the detection of pro-EPIL forms in biological fluids during pregnancy. One mAb directed against the C-connecting peptide was used to capture pro-EPIL forms and their binding was revealed by a radiolabeled anti-A chain mAb as the indicator. A composite synthetic peptide, encompassing the C- and A-domains, was utilized as the standard. Under these experimental conditions, the assay displays a sensitivity limit of 2 ng/mL. Pro-EPIL molecular forms were detected in both amniotic fluid and maternal serum of pregnant women. At 12 to 16 weeks of pregnancy, the pro-EPIL level was higher in amniotic fluid (246 +/- 50.8 ng/mL) than in maternal serum (5 +/- 2.0 ng/mL). As gestation advanced, so the concentration of pro-EPIL forms decreased in amniotic fluid while its level increased in maternal serum. Interestingly, in amniotic fluid, the pattern of pro-EPIL concentration during pregnancy is very similar to that observed for human chorionic gonadotropin (hCG) and its free subunits. The pattern of serum pro-EPIL concentration is similar to that of the free alpha-subunit. Together with our previous immunohistochemical observations, these results indicate that pro-EPIL is preferentially secreted by cytotrophoblasts in amniotic fluid and that the biosynthesis of hCG subunits and EPIL may be regulated by common pathways. Overall, our observations strongly suggest that EPIL may play a critical physiological role during embryonic and foetal development.