Guidelines for quantifying ovarian follicles: every follicle counts

Accurate quantification of follicle number is critical for assessing ovarian health and function; however, variability in methodology and subjective interpretation compromise data accuracy and reproducibility. Here, we present standardised guidelines for follicle counting in ovarian histological sections. Abstract Quantifying ovarian follicles is a fundamental tool in reproductive biology, providing the only direct measure of the ovarian reserve. The characterisation of follicle dynamics and depletion across the reproductive lifespan, and in response to the compounding effects of pathological insults, is essential for understanding women's reproductive, endocrine, and systemic health. Manually tallying follicles across serial histological sections is the most widely used method for quantifying ovarian follicles. However, this approach is prone to protocol inconsistencies and interpretation bias. Consequently, the accuracy, reproducibility, and suitability of the data for cross-study comparisons or meta-analyses are often compromised. In this protocol, we outline key considerations for follicle counting and provide standardized approaches using the two principal methods: direct counting with paraffin-embedded tissue and stereological analysis with glycol methyl-acrylate sections. These guidelines are intended to ensure consistent, accurate, and objective follicle quantification, and to support researchers across disciplines in adopting ovarian health and function, measured by follicle number, as a routine outcome in preclinical animal studies.