Mass spectrometric investigation into glycomes implicated in human reproductive biology

Glycosylation is a complex biological process where enzymes attach glycans to proteins, altering their structure and function. In reproduction, glycoproteins support sperm immunoprotection, modulate sperm-oocyte interactions, and influence implantation. Glycan sequences on cell surfaces, such as uterine epithelial cells, facilitate cell-to-cell communication, playing roles in both normal physiology and pathological conditions. Understanding glycan structures and functions enhances our knowledge of glycosylation in reproduction and its potential therapeutic and diagnostic applications. This project examined the differential glycosylation of human chorionic gonadotrophin (hCG) in normal and abnormal pregnancies, as well as across pregnancy stages. Additionally, we analysed glycomic profiles of normal endometrial tissue and tissue affected by endometriosis. Structural characterisation of glycans was performed using Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (MALDI-TOF MS), Tandem Mass Spectrometry (MS/MS), and enzymatic digestion. Enzymatic digestion allowed for the breakdown of glycans into smaller components, facilitating their structural identification. Our findings reveal that βhCG N-glycans exhibit high mannose, bi-, tri-, and tetraantennary structures. The novel identification of bisecting N-glycans in β-hCG, alongside their increased expression in late pregnancy, suggests a role in immune defence, possibly supporting immune tolerance. The data confirm that hCG undergoes differential glycosylation throughout pregnancy and that abnormal glycosylation is linked to pathological pregnancies. Analysis of endometrial tissue N-glycans showed that endometriosis is associated with altered glycan profiles, likely due to hyposialylation. The N-glycan profiles presented here allow for direct comparisons between normal and pathological states. Changes in glycan composition and structure, particularly those with immunomodulatory potential, may serve as targets for nonsurgical endometriosis management through diagnostic and therapeutic strategies.