# Modulation of test anxiety-induced salivary protein secretion by ovarian steroid hormones: a preliminary study

## TL;DR

This study suggests that ovarian hormones may influence how women's saliva proteins respond to test anxiety, with different patterns in hormone levels affecting specific proteins.

## Contribution

The study provides preliminary evidence that ovarian steroid hormones modulate salivary protein secretion during test anxiety.

## Key findings

- Test anxiety increased alpha-amylase, carbonic anhydrase, and cystatin S, while decreasing immunoglobulin light/J chains in both groups.
- Cystatins and 14-3-3 protein changes occurred only in the Pre-Ov group, while other proteins changed only in the Post-Ov group.
- Ovarian steroid hormones may drive differences in physiological responses to test anxiety.

## Abstract

In women the menstrual cycle influences mood and anxiety. Aim of this study was to preliminarily investigate whether different ovarian steroid hormone levels may modulate the psychophysiological responses elicited by test anxiety. Specifically, we compared the secretion of anxiety-induced salivary proteins of healthy women in the early follicular (Pre-Ov group) (low ovarian steroid hormones levels) and mid-luteal (Post-Ov group) (medium/high ovarian steroid hormones levels) phase of the menstrual cycle, during the simulation of an oral examination. Saliva samples were collected before and after a relaxation period and at two post-simulation times and analyzed by two-dimensional electrophoresis and western blot. Proteins corresponding to spots differentially expressed in the two groups across the session were identified through mass spectrometry and most of them corresponded to acute stress and/or oral mucosa immunity biomarkers. The task induced an increase in alpha-amylase, carbonic anhydrase and cystatin S, and a decrease in immunoglobulin light/J chains in both groups. Analogous changes in these proteins have previously been linked to psychological or physical stress. However, specific spots corresponding, for example, to cystatins and 14-3-3 protein, changed exclusively in the Pre-Ov group, while prolactin-inducible protein, polymeric immunoglobulin receptor, fragments of alpha-amylase and immunoglobulins only in the Post-Ov group, indicating a potential modulation of their secretion by ovarian steroid hormones. Overall, the results provide preliminary evidence that ovarian steroid hormones may be a driving factor for differences in physiological responses induced by test anxiety. The results are promising, but further validation in a larger sample is needed.

The online version contains supplementary material available at 10.1007/s13105-025-01067-w.

● Text anxiety prompts changes in the secretion of various salivary proteins.

● We studied how these protein changes relate to ovarian steroid hormone levels.

● Many anxiety-modulated proteins were exclusive to women with high hormone levels.

● Ovarian steroid hormones modulate the physiological biomarkers of test anxiety.

The online version contains supplementary material available at 10.1007/s13105-025-01067-w.