Quantitative ELISAs for serum soluble LHCGR and hCG-LHCGR complex: potential diagnostics in first trimester pregnancy screening for stillbirth, Down’s syndrome, preterm delivery and preeclampsia

Abstract

BackgroundSoluble LH/hCG receptor (sLHCGR) released from placental explants and transfected cells can be detected in sera from pregnant women. To determine whether sLHCGR has diagnostic potential, quantitative ELISAs were developed and tested to examine the correlation between pregnancy outcome and levels of serum sLHCGR and hCG-sLHCGR complex.MethodsAnti-LHCGR poly- and monoclonal antibodies recognizing defined LHCGR epitopes, commerical anti-hCGbeta antibody, together with recombinant LHCGR and yoked hCGbeta-LHCGR standard calibrators were used to develop two ELISAs. These assays were employed to quantify serum sLHCGR and hCG-sLHCGR at first trimester human pregnancy.ResultsTwo ELISAs were developed and validated. Unlike any known biomarker, sLHCGR and hCG-sLHCGR are unique because Down’s syndrome (DS), preeclampsia and preterm delivery are linked to both low (less than or equal to 5 pmol/mL), and high (equal to or greater than 170 pmol/mL) concentrations. At these cut-off values, serum hCG-sLHCGR together with PAPP-A detected additional DS pregnancies (21%) which were negative by free hCGbeta plus PAPP-A screening procedure. Therefore, sLHCGR/hCG-sLHCGR has an additive effect on the current primary biochemical screening of aneuploid pregnancies. More than 88% of pregnancies destined to end in fetal demise (stillbirth) exhibited very low serum hCG-sLHCGR(less than or equal to 5 pmol/mL) compared to controls (median 16.15 pmol/mL, n = 390). The frequency of high hCG-sLHCGR concentrations (equal to or greater than 170 pmol/mL) in pathological pregnancies was at least 3-6-fold higher than that of the control, suggesting possible modulation of the thyrotropic effect of hCG by sLHCGR.ConclusionsSerum sLHCGR/hCG-sLHCGR together with PAPP-A, have significant potential as first trimester screening markers for predicting pathological outcomes in pregnancy.

Forms of Circulating Luteinizing Hormone Human Chorionic Gonadotropin Receptor for the Prediction of Early and Late Preeclampsia in the First Trimester of Pregnancy

“…Unlike hCG, very little is known about how LHCGR modulates its ligand activities in human pregnancy. The presence of hCG-LHCGR and sLHCGR in the blood serum of a pregnant woman has recently been investigated [14,15]. Two ELISA assay systems that specifically measure these two hCG receptor forms (sLHCGR and hCG-LHCGR) in human serum have been applied in prenatal screening [14,15].…”

“…Two ELISA assay systems that specifically measure these two hCG receptor forms (sLHCGR and hCG-LHCGR) in human serum have been applied in prenatal screening [14,15]. Chambers et al [14] reported that circulating hCG-LHCGR appeared to be a unique marker due to two cutoff concentrations: pregnancies with a poor outcome had either very low (≤5 pmol/ml) or significantly high (≥170 pmol/ml) concentrations, in combination with low PAPP-A (<0.8 MoM).…”

“…Following 6 washes, the plates were further incubated with 100 µl of TMB for 15-30 min. The intra- and interassay variability of these 2 LHCGR forms is reported elsewhere [14]. …”

“…The hCG-LHCGR and sLHCGR ELISA assays were carried out as reported elsewhere [14], except for the following modifications: 55 µl of 5- to 10-fold diluted plasma was incubated in antibody-coated plates for 15 min prior to adding 100 µl of HRP-labelled detection antibody for another 90 min. Following 6 washes, the plates were further incubated with 100 µl of TMB for 15-30 min.…”

“…The functional state of hCG could also be regulated by the circulating soluble LHCGR (sLHCGR). ELISA methods for the quantitative analysis of circulating hCG-LHCGR and sLHCGR complexes have been developed, and preliminary results suggest that the LHCGR ratio (LHCGR-R), calculated by dividing the values of LHCGR by that of hCG-LHCGR, together with pregnancy-associated plasma protein A (PAPP-A), have a potential role in the first trimester as screening markers for the prediction of PE [14] and Down's syndrome [15]. …”