Decreased, ovarian reserve leads to disrupted, ovarian cycle

All primordial follicles are formed during early development (fetal period in humans, perinatally in rodents) and can stay dormant for long periods of time (years in humans, months in rodents). The stock of primordial dormant follicles constitutes the ‘ovarian reserve’. In humans, millions of follicles are formed by mid-gestation (Wallace & Kelsey, 2010). Upon puberty, the hypothalamus-pituitary-ovary (HPO) axis matures enabling the primordial follicles to grow into maturity in a process called folliculogenesis, which serves two functions: i) secretion of steroid hormones that enable pregnancy, and ii) production of mature oocytes that are ovulated for possible fertilization.

Cohorts of primordial follicles continuously enter the growing pool. After puberty, growing antral follicles are recruited for final maturation during each menstrual cycle (estrus cycle in rodents) by gonadotropins secreted from the pituitary gland but only a limited number will reach maturity and ovulate oocytes (typically one in humans and 10-20 in rodents). The majority of follicles never reach maturity and instead die in a process called atresia. Therefore, the ovarian reserve is irreversibly depleted with age. When the reserve is depleted to a level that cannot faithfully maintain steroid production, fertility ceases. In humans, fertility ends in sterility at menopause when less than 1,000 follicles remain (Wallace & Kelsey, 2010).

Regular cycles are considered as an indicator of reproductive health and often used in animal studies as the earliest biomarker to reflect disruption of fertility and ovotoxicity (Hooser et al, 1994). OECD test guidelines 407, 416 and 443 include rodent cyclicity as an endpoint to assess reproductive toxicity (OECD, 2001; OECD, 2008; OECD, 2018). In humans, normal menstrual cycle lasts 28-35 days, and in rodents 4-6 days. When the ovarian reserve is depleted to a critically low level, like naturally during the perimenopausal period in humans, the variability of the cycle length increases with many of the cycles being anovulatory. The lower the ovarian reserve is, the lower the probability of growing follicles to exist at any given time point. Since the growing follicles produce steroid hormones that are essential for cyclicity, the lack of growing follicles leads to disturbances of the HPO axis. Therefore, the lower the ovarian reserve is, the less probable are regular cycles; reflected by regular menstruation in humans (O'Connor et al, 1998; O'Connor et al, 2001).

Supporting evidence exists. Anti-Müllerian hormone (AMH) is a growth factor secreted by growing follicles. Low levels of AMH correlate with longer cycle length (Harris et al, 2021). Other studies have connected AMH or antral follicle count (AFC), another ovarian reserve marker, to shorter cycles (Younis et al, 2020). A systematic review and meta-analysis have revealed in regularly cycling women, a shorter cycle is associated with lower ovarian reserve based on AMH or AFC (Younis et al, 2020). Young women diagnosed with premature ovarian failure have also reported shorter cycles (Guzel et al, 2017). In addition, it is well established in humans that diminishing ovarian reserve leads to perimenopause (a period or irregular cycles) and eventually menopause (complete cessation of cycles).

Stressors that are known to deplete the ovarian reserve include cancer treatments, which kill primordial follicles and disrupt folliculogenesis. Alkylating chemotherapy agents like cyclophosphamide and cisplatin are highly ovotoxic, as well as radiation therapy towards ovaries (Pampanini et al, 2020). Therapies based on high dose alkylators and radiation towards ovaries lead with high likelihood to amenorrhea, infertility and premature ovarian insufficiency in humans due to depletion of ovarian reserve and therefore constitute an indication for clinical fertility preservation (ESHRE_Guideline_Group_on_Female_Fertility_Preservation et al, 2020; Pampanini et al, 2020).

In mice, it has been shown that cisplatin induces primordial follicle loss in a dose-depended manner (Meirow et al, 1999). Cyclophosphamide can accelerate primordial follicle loss in mice and in human ovarian tissue (Kalich-Philosoph et al., 2013; Lande et al., 2017). In human xenografts in mice, the same compound decreased the primordial follicle density(Oktem & Oktay, 2007). These key studies indicate the effect of chemotherapy compounds on the size of the ovarian reserve (Meirow et al, 2010). In treated patients, although there is often no direct information on the size of the ovarian reserve, a rapid decrease of AMH levels has been observed following high risk chemotherapy. Patients receiving chemotherapy have also been shown to have an increased risk of premature ovarian failure. In addition, some studies have shown that the number of healthy follicles is significantly decreased and that of atretic follicles increased in human ovarian tissue following alkylating chemotherapy (Pampanini et al, 2019). These data establish a clear indication of diminished ovarian reserve following chemotherapy. Importantly, chemotherapy compounds also affect the menstrual cycle, with patients experiencing amenorrhea and irregular cycles (Jacobson et al., 2016; Meirow et al., 2010).

Another stressor known to affect the ovarian reserve is smoking. Cigarette smoke contains thousands of chemicals, several of which have been shown to be ovotoxic (Budani & Tiboni, 2017). Mice exposed in vivo to cigarette smoke have significantly fewer primordial follicles compared to the control group (Tuttle et al., 2009). Smoking women display reduced ovarian reserve markers and experience irregular cycles compared to non-smokers of the same age group (El-Nemr et al, 1998; Sharara et al, 1994).

Additional chemical insults affecting the ovarian reserve and causing menstrual cycle irregularities are presented in Table 1. These studies in animal models demonstrate how these chemicals directly target primordial follicles and cause menstrual cycle irregularities. Vinylcyclohexene diepoxide (VCD), metabolite of 4-vinylcyclohexene (VCH), is the most commonly used chemical in these studies and is often used to induce reproductive senescence in model organisms.

Table 1: In vivo studies demonstrating that effects on the KE upstream affect the KE downstream. VCD: vinylcyclohexene diepoxide, VCH: 4-vinylcyclohexene, BPA: bisphenol A, DEHP: bis(2-ethylhexyl) phthalate, B[a]P: Benzo[a]pyrene

As mentioned, several chemotherapy agents damage ovarian reserve and disrupt folliculogenesis. However, it has been shown that regular menses can resume upon treatment cessation (Jacobson et al, 2016). Therefore, in this case reduced ovarian reserve did not lead to permanent irregularities of ovarian cycle. In a systematic review and meta-analysis investigating the connection between the ovarian reserve and the length of the menstrual cycle, studies are mentioned where reduced ovarian reserve markers did not associate with irregular menstrual cycles (Younis et al, 2020). Several factors affect the impact of chemotherapy on ovarian health in humans, including the age at the treatment, size of ovarian reserve at treatment, and treatment regimen. However, late side effects of chemotherapy often include amenorrhea, premature ovarian insufficiency, and infertility.

Menstrual irregularities can be caused by factors other than reduced ovarian reserve. The most common factor affecting cyclicity is HPO axis dysregulation causing hypothalamic amenorrhea (Berga & Naftolin, 2012). Another example is the contraceptive pill that decreases gonadotropin secretion by the pituitary gland, leading to inhibition of folliculogenesis and amenorrhea. Changes in hormone levels produced by the pituitary gland have also been connected to shorter and anovulatory cycles (Mumford et al., 2012). Another factor affecting cyclicity is the thyroid gland function. Thyroid function disturbances, like hypo and hyperthyroidism have been connected to menstrual disturbances (Koutras, 1997).

The timescale at which disruption in cyclicity occurs depends on the type of follicles that are affected, size of the reserve at the time of insult, and the extent of the damage. When a stressor targets selectively the ovarian reserve, it might take months (or years in humans) for the disruptions in cyclicity to be observed (Hoyer & Sipes, 1996). This delay was evident in some of the animal studies mentioned in Table 1 (Hooser et al., 1994; Lohff et al., 2006; Mayer et al., 2002).  

HPO axis regulates estrus/menstrual cycle, and is based on positive and negative feedback loops by ovarian steroids and peptide hormones, and hormones released by the hypothalamus and pituitary gland.