Ovarian Follicle Histomorphometry and Classification

Histological assessment and classification of follicle structure were conducted as described previously with slight modification [18 (link),21 (link)]. Briefly, at least three pieces of ovarian tissue in each group were fixed with Bouin’s solution (FUJIFILM Wako Pure Chemical Corporation), kept at 4 °C overnight, dehydrated in a series of ethanol solutions (70–100%), and embedded in paraffin. Serial sections (5 μm thick) of ovarian tissues were prepared and stained with haematoxylin and eosin (both Muto Pure Chemicals CO. Tokyo, Japan). To avoid double counting, three sections, each at least 20 µm apart, were assessed by light microscopy (Nikon ECLIPE E600, Nikon).
Follicles within the ovarian tissues were classified as primordial (<80 um), primary (0.08–1 mm), or pre-hierarchical (>1 mm) [8 (link)]. All the follicles were further characterized as follows: ‘normal’, when the layer of granulosa cells is attached to the spherical oocyte surrounding it and the homogenous ooplasm contains a tiny granulated nucleus, or ‘abnormal’, wherein aggregation and shrinkage of nuclear chromatin and wrinkling of the nuclear membrane were regarded as signs of atresia [6 (link)]. The proportion of morphologically normal follicles per section was calculated by dividing the number of normal follicles by the total number of assessed follicles.