Several different factors have been shown to prematurely deplete primordial follicle numbers, including cancer treatments, environmental toxicants, infection and inflammation, with implications for fertility, depending on the extent of primordial follicle loss. Therefore, the length of the female fertile lifespan, from puberty to menopause, is determined by 1) the maximal number of primordial follicles initially formed in the ovary, 2) the rate of primordial follicle activation and 3) the rate of primordial follicle death. Once exhausted, there is no replenishment of the ovarian reserve.
This process causes a slow decline in the supply of primordial follicles until exhaustion of the reserve, leading to infertility followed by menopause. These remain in a quiescent state until recruited to undergo a folliculogenesis, ultimately culminating in follicular atresia or ovulation of a mature oocyte. Primordial follicles consist of a singular oocyte arrested at diplotene of meiotic prophase 1, surrounded by one layer of squamous granulosa cells.
The ovarian reserve of primordial follicles represents the entire stockpile of gametes available to females. The findings presented here will assist others to select the most appropriate experimental approach for accurate follicle enumeration, depending on whether the primary objective of the study is to determine absolute primordial follicle numbers or relative differences between groups. This work indicates that the direct count method can produce similar results to stereology when Bouin’s fixative is used instead of formalin. However, when Bouin’s fixative was used, direct counts and stereology were comparable in their ability to detect follicle depletion caused by cyclophosphamide. When applied to formalin fixed tissue, the direct count method did not detect differences in follicle numbers between saline and cyclophosphamide treated groups to the same degree of sensitivity as the gold standard stereology method (referred to as the Reference standard). Resultsĭirect counts resulted in primordial follicle numbers that were significantly lower than those obtained by stereology, irrespective of fixation and embedding material. We also compared the impacts of different fixatives, embedding material and section thickness on the overall results for each method.
The second method was the more widely used serial section and direct count approach, which is relatively quick and easy.
While accurate, highly reproducible and sensitive, this method relies on specialist microscopy equipment and software, requires specific fixation, embedding and sectioning parameters to be followed, and is largely a manual process that is tedious and time-consuming. The first was the fractionator/optical dissector technique, an unbiased, assumption-free stereological approach for quantification of primordial follicle numbers. In this study, we compared and contrasted two methods for counting healthy primordial follicles following exposure to cyclophosphamide (75 mg/kg), a well-established model of follicle depletion.
Accurate evaluation of primordial follicle numbers in mouse ovaries is an essential endpoint for studies investigating how endogenous and exogenous insults, such as maternal aging and chemotherapy, impact the ovarian reserve.