Ovarian sections were sampled and follicles were counted according to published methods. Briefly, a stratified sample consisting of every 10th section was used to estimate the total numbers of antral follicles per ovary. The selected sections from one ovary were randomized and the number of antral follicles was counted in the entire section. Only follicles with a visible nucleolus were counted to avoid double counting. Sections were counted without knowledge of treatment.
Ovarian follicles were categorized as described by Flaws et al.. Follicles were classified as antral if they contained five or more layers of granulosa cells and a clearly defined antral space. In some cases, antral follicles showed no antral space in cross section, but were scored as antral if they contained five granulosa cell layers. Antral follicles were classified atretic using standard morphological criteria. Briefly, follicles were considered atretic if 10% of the granulosa cells were apoptotic (defined by the appearance of pyknotic bodies in the granulosa cell layer), the granulosa cell layer was disorganized, the oocyte was degenerating or its nucleus was fragmented.