Abstract
Objective The follicular fluid (FF) plays an essential role in the physiology of the follicle and the oocyte. Gonadotropin stimulation affects the FF steroid hormone and anti-Mullerian hormone (AMH) concentrations, which has been suggested to be the reason for lower oocyte competence in conventional gonadotropin stimulated in vitro fertilisation (cIVF) compared to natural cycle IVF (NC-IVF). To analyse the effect of gonadotropin stimulation on a broad spectrum of signalling proteins, we ran proteomic antibody arrays on FF of women undergoing both treatments NC-IVF and cIVF. Method Twenty women underwent one NC-IVF and one cIVF treatment cycle. Follicular fluids of the first aspirated follicle were compared between the two groups using a protein microarray which included antibodies against 224 proteins related to cell signalling and reference proteins. Each of the 40 albumin-stripped, matched-pair samples was labelled in the reverse-dye (Cy3/Cy5) procedure before undergoing array hybridisation. Signal analysis was performed using normalisation algorithms in dedicated software. Five proteins yielding a value of P < 0.05 in the array experiment (Cystatin A, Caspase-3, GAD65/67, ERK-1, and ERK-2) were then submitted to quantitative determination by ELISA in the same follicular fluids. Results Array analysis yielded only a small number of differentially expressed signalling markers by unadjusted P values. Adjustment as a consequence of multiple determinations resulted in the absence of any significant differential marker expression on the array. Five unadjusted differentially expressed proteins were quantified immunometrically with antibodies from different sources. Follicular fluid concentrations of Cystatin A and MAP kinase ERK-1 concentrations were significantly higher in the cIVF than in the NC-IVF follicles, while GAD-2 (GAD65/67) did not differ. The assays for Caspase-3 and MAP kinase ERK-2 did not have the required sensitivities. Conclusion In contrast to FF steroid hormones and AMH, FF concentrations of signalling proteins are not or only marginally altered by gonadotropin stimulation.
Highlights
Follicular fluid (FF) is composed of different elements such as hormones, enzymes, anticoagulants, electrolytes, reactive oxygen species, and antioxidants [1]
Several studies revealed that gonadotropin stimulation alters the concentration of several FF protein components such as cytokines including leukaemia inhibitory factor (LIF) [3,4,5], steroid hormones [3, 6], and antiMullerian hormone (AMH) [6, 7]
The analysis of the follicular fluid samples, performed over 15 matched NC-IVF/conventional in vitro fertilisation (cIVF) pairs after extensive internal normalisation using a large number of spotted reference, mostly cytoskeleton proteins, yielded a small number of differentially expressed markers amongst the remaining 179 “unknown” proteins in the array: applying a nonadjusted P value of 0.05, seven showed a higher and eight a lower expression level in the NC-IVF when compared to cIVF treatment cycle in one or both label combination replicates
Summary
Follicular fluid (FF) is composed of different elements such as hormones, enzymes, anticoagulants, electrolytes, reactive oxygen species, and antioxidants [1]. It provides the oocyte with nutrients and mediates its communication with the follicle and thereby with the female endocrine system. The gonadotropin stimulation has been suggested to be the reason for the lower oocyte competence in cIVF [2]. Several studies revealed that gonadotropin stimulation alters the concentration of several FF protein components such as cytokines including leukaemia inhibitory factor (LIF) [3,4,5], steroid hormones [3, 6], and antiMullerian hormone (AMH) [6, 7].
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