Decreased, size of the ovarian reserve

Formation of the follicle pool (follicle assembly)

During fetal life, primordial germ cells migrate to the genital ridges where they arrange into germ cell nests and proceed through to meiosis prophase I (Pepling & Spradling, 2001). Assembly into individual follicles occurs via mechanisms that are not well known, but involves germ cell nest break down and a reduction in oocyte numbers via programmed cell death. Somatic pre-granulosa cells infiltrate between the oocytes, arrange around them in a single layer, and establish what is called the primordial follicles (Escobar et al, 2008; Gawriluk et al, 2011; Pepling & Spradling, 2001). The primordial follicles constitute the follicle pool - a limited stock of oocytes that are available for maturation and potential fertilization determining the length of a female’s reproductive life span (Grive & Freiman, 2015).

The timing of follicle assembly differs between mammalian species, but the processes involved seem to be relatively well conserved (Grive & Freiman, 2015). In humans, follicle assembly occurs during mid-gestation whereas in mice and rats it is initiated around the time of birth and continues until approximately six days post partum.

In mammals, the process of forming primordial germ cells within the epiblast, begins in the human ovary at approximately 2 weeks post-conception (wpc), corresponding to 4 weeks of gestation. During this stage, these germ cells migrate to the hindgut and later settle in the gonadal ridges before 7 wpc. Once in the gonadal ridges, the germ cells proliferate to form ovarian cysts or nests as oogonia. These cysts also contain primordial granulosa cells, which derive from the gonadal ridges and contribute to the formation of future ovarian follicle(Hummitzsch K. et al.2013) The meiotic prophase begins around 8.5 wpc, at which point the oogonia can stop mitosis, grow, and mature into primary oocytes. Primordial follicles with a diameter of 30 mm are produced when the cysts break apart, which occurs within 13 and 30 weeks. According to current theories, the newly produced primordial follicles serve as the female's limited supply of resting oocytes for the duration of her reproductive life (Monniaux D et al. 2019,  Hirshfield AN 1991). The establishment and gradual depletion of the ovarian reserve is a fundamental and conserved biological process across mammals. In species such as humans, rodents, sheep, and cattle, primordial follicle formation occurs through similar mechanisms, including oocyte nest breakdown, somatic cell interaction, and the initiation of meiosis. Although the timing of reserve establishment is species-specific—the structural and functional processes are conserved. Cross-species relevance is further supported by the fact that exposure to reproductive toxic environmental chemicals (RTECs) has been demonstrated to impair the development and maintenance of ovarian reserves in both human and animal models. Accordingly, the key event is plausibility extends across the mammalian taxa with ovaries (Monniaux, D. 2018, Ge, W., et al. 2019).

Reduced follicle pool as Key Event

An intact follicle pool is critical for female fertility. Any disruption to the formation of the final pool can have adverse consequences for reproductive capacity, leading to sub- or infertility. Loss of oocytes/follicles can occur during any of the abovementioned stages during the process of follicle assembly – oocyte nest breakdown, programmed cell death or somatic pre-granulosa cell intrusion. Follicle assembly and establishment of the functional follicle pool is also dependent on the stages occurring before this process, e.g. migration of primordial germ cells to the genital ridges, sex determination and meiosis.

In animal studies, counting of follicles of different sizes is included in OECD guidelines: TG 416 (Two-Generation Reproductive Toxicity Study) and TG 443 (Extended One-Generation Reproductive Toxicity Study). It is a time-consuming and labor-intensive method and it is not recommended to compare values between studies (Tilly, 2003).

In humans, there is no direct way to count the follicle pool in vivo. Instead, surrogate markers are used. The most established biomarker for estimation of the follicle pool is anti-Müllerian hormone (AMH). It is readily measured in a blood sample and the levels are rather stable throughout the menstrual cycle (Broer et al, 2014).

The size of the pool can also be measured indirectly by mRNA and protein expression of meiotic markers, or by assessing overall ovary histology by histological assessments (Zhang et al, 2012).

Follicle assembly occur in females during fetal life (humans) or around and after birth (rodents). Many of the mechanisms involved are preserved between mice, rats and humans.

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