Melchiorre, Chiara (2021) Development of a new method for identification of FSH glycoforms involved in human fertility. [Tesi di dottorato]

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Follicle Stimulating Hormone (FSH) is one of the most important glycohormones involved in human fertility and it is widely used to assess the ovarian reserve in women for in vitro fertilization. FSH is a heterodimeric glycohormone composed by two polypeptide subunits, the alpha-subunit (common to all gonadotropins) and a FSH specific beta-subunit. The molecule has 4 glycosylation site, Asn52-Asn78 of alpha-subunit and Asn7-Asn24 of beta-subunit. Each glycosite hosts a wide microheterogeneity of glycans, thus generating a heterogeneous population of different glycoforms. The biological activity of human hFSH is highly regulated by its glycosylation, but the role of the different glycoforms in FSH mechanism of action is still poorly understood. The development of new methodologies aimed at characterizing and identifying the site-specific glycosidic profile of hFSH could contribute to understanding how the glycosylation pattern modulates hFSH function. Glycomic studies on serum hFSH are very challenging because of the huge heterogeneity of glycoforms and their very low concentration in serum. The current reference method for serum hFSH dosage is ELISA, but it fails in distinguishing proteoforms because of the lack of specific antibodies. A combination of mass spectrometry approaches was used here to characterise recombinant r-FSH and ultimately quantify endogenous hFSH proteoforms in serum. Site-specific glycosylation profiles of different commercialised r-FSH molecules (GONAL-F ®, BEMFOLA, OVALEAP, FOSTIMON) were determined by untargeted proteomics and glycan analysis. The MS analysis led to the identification of at least 5 known glycoforms for each of 4 glycosylation sites of the molecule. In order to find higher efficiency r-FSH isoforms of the molecule, artificial glycovariant were produced by stress conditions (temperature, pH, etc.) and analysed in the same way. Glycosylation of the molecule subject to pH stress condition shows a slight decrease in the sialic acid content as well as in hyper O-Acetylated species. AEX-pH acidic fraction was characterised by highly sialylated end-capping species, while the basic fraction showed the presence of neutral galactosylated species and a lower content of sialylated moieties. Totally de-sialylated and partially de-galactosylated FSH glycovariants were obtained by enzymatic treatments. In vivo and in vitro experiments will be conducted by Merck (www.merckgroup.com) to determine the efficiency of these glycovariants in fertility therapy. On the basis of mass spectral r-FSH characterisation, two innovative targeted MS methods were developed to identify and quantify the global amount of hFSH as well as different glycoforms in woman serum. A Multiple Reaction Monitoring (MRM) MS-based method for the absolute quantification of circulating hFSH molecule was optimised, and it was successfully applied to the analysis of 10 women sera samples of different ages showing a great variability of the amount of the hormone. Results show comparable sensitivity (>1 ng/ml) and higher selectivity and specificity than conventional ELISA assays, and therefore this MRM-MS method could be proposed as an alternative for the quantification of hFSH. As for glycoforms detection and dosage, a method based on high-resolution mass spectrometry in Parallel Reaction Monitoring (PRM-MS) mode was developed to monitor the glycosylation profile of hFSH in pooled sera sample. The sensitivity was boosted by an optimised sample preparation involving immunoaffinity purification of hFSH from serum. The 9 most abundant hFSH site-specific glycoforms were identified in a very complex matrix such as serum. Moreover, a relative quantification of the identified glycoforms was obtained by post acquisition data processing using Skyline software. In both recombinant and serum hFSH the same most abundant glycoforms were found, the biantennary fully sialylated (A2G2S2) on alpha-Asn52, the mono-sialylated (A2G2S1) on alpha-Asn78 and fucosylated biantennary (FA2G2S2 and FA2G2S1) structures on beta-Asn 24. Glycopeptide spectra containing beta-Asn 7 glycosite didn't pass our quality criteria for quantitative analysis. Although the importance of glycosylation in the biological activity of FSH is well documented, this is the first study reporting site-specific characterization of circulating h-FSH glycoforms by MS. The proposed strategy, if applied to a large cohort of women, can be useful to investigate the existing relations between the hFSH glycan moieties and the hormone function. Such knowledge will bring new insights on the complex mechanisms in which hFSH glycoforms are involved and, ultimately, will improve its usage in infertility treatments. Furthermore, these targeted MS methods can be easily implemented for a simultaneous analysis of several serum proteins, making the PRM/MRM-MS methodology advantageous in terms of time and cost.

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