Analysis and characterisation of human chorionic gonadotropin glycoforms in pregnancy and trophoblastic disorders

Human chorionic gonadotropin (hCG) is a heterogeneous glycoprotein hormone with a varying degree of carbohydrate moieties. Its glycosylation arrangements are increasingly recognized as a common and important element of disease pathophysiology and are associated with many disorders including gestational trophoblastic diseases (GTDs). This study aimed to optimise methodologies to permit the characterisation of hCG N-linked glycans from urine samples collected throughout normal pregnancy and GTD using matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS). hCG isolated from pooled pregnancy urine was used in this study. All the stages in pregnancy urine preparation were optimised; including conditions for hCG immunopurification, deglycosylation, solid-phase extraction of resulting glycan:protein mixture and application of N-glycans for MALDI-TOF MS analysis. GlycoQuest software was used to characterise specific N-glycans configurations from the resulting MALDI-TOF MS spectra. This methodology was then applied to urine samples collected throughout normal singleton pregnancy and urine collected from patients with choriocarcinoma. The relative abundance of glycans of different molecular weights and specific types (i.e. fucosylated, sialylated, bisected and sulphated) at each stage of normal pregnancy and in GTD were compared. Each stage of optimisation increased the number of N-glycans detected such that we were ultimately able to detect 50 different glycans in normal pregnancy urine. In these samples, advancing gestation was associated with an increase in the proportion of branched N-glycans and multi-fucosylated N-glycans. Also, a significant increase in the proportion of high molecular weight glycans (>2100 Da) between choriocarcinoma and first-trimester normal pregnancy was observed. Further striking differences in the repertoire of glycan expression were also seen in choriocarcinoma urine compared with first-trimester pregnancy urine. The proportion of multi-fucosylated and tri-and tetra antennal, glycans was increased 3 and 2 fold respectively. In addition, 14 unique N-linked glycan structures were identified in choriocarcinoma samples. These included hyperfucosylated (7 fucose groups) and hypersialylated (4 sialic acid groups) glycans. A feature of this unique set of glycans was that they contained a combination of multiple branching, fucosylation, sialylation, sulphation and glycans with Lewis X terminal epitopes. In summary, we have successfully developed a methodology for the detection of a diverse range of N-linked glycans from hCG. These results suggest that this approach can be successfully used for the detection of novel glyco-biomarkers for the early detection of choriocarcinoma and may be applied to other GTDs associated with a dysregulation of hCG expression.