Influence of female sex hormones on salivary protein secretion in test anxiety

Social anxiety is a psychological disorder derived from the fear of being judged by others that leads to a strong state of stress. The physiological response to a stressful condition occurs mainly through the involvement of the endocrine, nervous and immune systems. In particular, the hypothalamic-pituitary-gonadal and hypothalamus-pituitary- adrenal axis are involved. This study aims to investigate the modulation of salivary protein secretion by female sex hormones, during an acute psychological stress. Twenty healthy females recruited either in the pre-ovulatory (Pre-Ov) or post-ovulatory (Post-Ov) phase of the menstrual cycle, characterized, respectively, by low and medium-high levels of estradiol and progesterone, participated to a test anxiety task. The task simulated an oral exam and consisted of 3 phases: relaxation, study of a written text, and oral exposition in front of a “professor” of the studied text. Four saliva samples were collected: before starting the test (T1), after the relaxation phase (T2), after the exam simulation (T3), and 20 minutes after the exam end (T4). Anxiety perception during the various phases was evaluated by means of specific questionnaires. Progesterone and Estradiol concentrations were analyzed through kit ELISA. Comparative proteomic analysis of saliva samples was performed by 2-DE/MS. Significant differences of protein expression were observed at different times both in Pre-Ov and Post-Ov groups suggesting a different response to acute stress linked to sex hormone levels. Furthermore, for all the observed spots, correlations were carried out between the ratios T3/T2 and T4/T2 of the normalized optical densities and the levels of estradiol and progesterone. The comparisons between Pre-Ov and Post-Ov highlighted 29 spots in common between groups, 17 spots exclusively dysregulated in Pre-Ov (i.e., YWHAE, YWHAZ, SFN, CST2, and CST4) and 53 spots exclusively dysregulated in Post-Ov (i.e., PIGR, IGJ, IGLC/IGKC, PIP, and PFN1). The correlation analysis pointed out a link between progesterone, estradiol and protein levels of 8 (i.e., AMY1, and CST1) and 11 spots (i.e., CA), respectively. In conclusion, this study highlighted that female sex hormone levels modulate the secretion of salivary proteins during an acute psychological stress. This research was funded by PRA 2020–2021, grant numb