P–591 Mass spectrometry-based glycomic profiling of the follicular fluid total immunoglobulin G and proteome N-glycomes reveals deregulated inflammatory processes associated with specific controlled ovarian stimulation protocols
Authors: Devi. Pavlić S , Klobučar M , Smilja. Severinski N , Radojči. Badovinac A 
Published: August 2021

Abstract

Study question
Is there a significant inflammatory-related difference between analyzed follicular fluid (FF) glycome profiles regarding the ovarian stimulation protocol used in patients?

Summary answer
Observed differences between analyzed glycome profiles from patients that underwent different controlled ovarian stimulation (COS) protocols point to deregulated inflammatory processes associated with specific COS.

What is known already
Successful physiological folliculogenesis and ovulation require an adequate inflammatory response. On the other hand, COS application in ART relies on induced hormonal activation of systemic inflammatory processes. Several studies have confirmed a rise in inflammatory cytokines, CRP, and other markers of inflammation in patients subjected to different COS protocols, pointing to an enhanced inflammatory response during ovulation stimulation. Glycoproteins and glycans have an indisputable role in immune response modulation: proper glycosylation of glycoproteins plays a pivotal role in the regulation of normal physiological processes, and aberrant glycosylation of glycoproteins has been associated with various pathological states, including inflammation.

Study design, size, duration
Study design: Cross sectional – FFs from patients that underwent ART in modified natural cycle (MNC group) versus FFs from patients that underwent ART under GnRH antagonist COS (COS group). Size: 20 FFs from 20 patients undergoing ART. Duration: One year. Sampling procedure: Each FF was aspirated from the dominant follicle. In the COS group, only the fluid from the first aspirated follicle of each patient was collected.

Participants/materials, setting, methods
Study included 20 FF samples from 20 patients divided into two groups according to the applied ovarian stimulation protocol: MNC group (n = 10) and COS group (n = 10). The immunoglobulin G (IgG) was isolated from FF samples by immunoaffinity chromatography. The N-linked glycans derived from IgG molecule and the remaining FF total proteomes were enzymatically cleaved and subjected to derivatization procedure. N-glycomes of FF-isolated IgG and total proteomes were analyzed separately by MALDI-TOF-MS.

Main results and the role of chance
FF IgG N-glycome profiling The MALDI-TOF-MS based comparative analysis of the individual glycan relative abundances, revealed several significantly deregulated glycoforms between analyzed groups whose levels were significantly elevated (p˂0.05) in the COS vs. MNC group. Furthermore, additional low abundant N-glycan species were also found to be deregulated between the analyzed groups: two monogalactolysed and monosialylated N-glycan compositions were only identified in the COS group. The comparative analysis of FF IgG N-glycome features revealed statistically relevant differences in the levels of two derived traits: galactosylation and bigalactosylation levels of the FF IgG N-glycome, both significantly downregulated (p˂0.05) in the MNC vs. COS profile. Comparative analysis of FF total proteome N-glycome The majority of identified glycan compositions were complex type N-glycans representing more than 98% of the total N-glycome profiles in both analyzed groups. The comparative analysis of individual glycan relative abundances revealed relevant differences in regulation of ten N-glycan species between the two analyzed profiles. In the MNC group, six N-glycan species showed significantly increased abundances (p˂0.05) compared with the COS group. Moreover, two compositions were exclusively identified in the MNC group, while two compositions were identified only in the COS group.

Limitations, reasons for caution
Since this preliminary study was conducted on relatively small sample size, all results should be verified on a larger sample set. Moreover, focused glycosylation analysis of a panel of individual FF acute phase blood serum derived proteins and immunoglobulins, might additionally clarify the inflammatory mechanisms underlying different ART stimulation protocols.
Wider implications of the findings: While glycome profiling of human FFs was conducted for the first time, previous evidence supports the shown association of aberrant inflammation in diverse ART stimulation protocols and in development of various pathological states (i.e. OHSS). Obtained results are in line with previous similar studies performed in the human plasma.

Trial registration number
uniri-biomed–18–161 1310