Ovarian Follicle Quantification and Stromal Analysis

Fixed ovarian fragments were dehydrated in increasing concentrations of ethanol (70–100%), embedded in paraffin and serially sectioned at 5 μm thickness. Every fifth section was stained with haematoxylin and eosin, and both follicle counts and stromal cell density were assessed. Follicles were classified according to their developmental stage as primordial follicles (oocyte surrounded by a few flattened granulosa cells), transitory follicles (oocyte surrounded by flattened and at least one cuboidal granulosa cell), or growing follicles (oocyte surrounded by one or more complete layer(s) of cuboidal granulosa cells). Only follicles that contained an oocyte nucleus were counted to prevent double counting. Follicle spatial distribution within each sub-region of the ovarian cortex, namely the outer cortex, the mid-cortex, and the cortex–medulla junction, was assessed by dividing the cortex into three equal layers of 300 µm each from the epithelium surface to the medulla side. The outer cortex and cortex–medulla junction regions were defined in the sections based on the identification of the surface epithelium and tunica albuginea layers on one side, and small cortical arterioles on the opposite side, respectively.
For PicroSirius Red (PSR) staining, sections were deparaffinized and rehydrated in a series of ethanol baths of decreasing concentrations. The slides were immersed in a PSR staining solution (ab246832, Abcam, UK) for 1 h at room temperature, then washed twice with 0.5% glacial acetic acid and three times with 100% ethanol. The slides were cleared in xylene and mounted with DPX. All slides from the same patient were processed at the same time to minimize staining variation.