An accurate, simple method for unbiased determination of primordial follicle number in the primate ovary

Previous investigations of primordial follicle (PF) number in primate ovaries have used biased, model-based techniques that require correction factors based on assumptions regarding cell size, orientation, and shape. We sought to apply several techniques from the "new stereology" to obtain unbiased number estimates. This method involves a hierarchy of systematic random sampling combined with the physical disector and fractionator techniques. The method readily allows the estimation of the coefficient of error (CE) of each sampling level as it contributes to the observed variance of the overall number estimate. We examined one ovary from each of five pigtailed monkeys (Macaca nemestrina). The mean number of PF was 15,735 +/- 6214 (mean +/- SD). The mean CE for the individual number estimates was 0.085, contributing minimally to the inter-individual coefficient of variation (CV) of the primordial follicle numbers (CV = 0.395). The correlation between age and PF number was not significant (r = -0.74, p > 0.1). The total time taken to count the 100-200 PF necessary per ovary was 4-5 h. We conclude that this method produces reliable, unbiased estimates with measurable and acceptable accuracy in a robust, efficient manner.