5.2.1

Chemotherapy-Induced Follicular Depletion

Foundational chemotherapy studies measured ovotoxicity as follicle loss, leading to three models describing follicular depletion. In the first model, chemotherapy directly destroys primordial follicles, depleting the ovarian reserve. For example, cisplatin depletes primordial and primary follicles in mice treated at postnatal day (PND)5 (i.e., paediatric mice). Secondary follicle counts are unchanged at PND9, however, demonstrating relative resistance.

In the second model, chemotherapy induces apoptosis only in follicles containing actively dividing granulosa cells. Doxorubicin (DXR) induces widespread apoptosis in granulosa cells of growing follicles within 12 hours, but primordial follicles do not exhibit apoptosis until 48 hours post-treatment, and then only in small numbers consistent with the primordial follicle recruitment rate in mice.

The third, not mutually exclusive, “burnout” model postulates that cyclophosphamide first depletes growing follicles, which promotes primordial follicle recruitment, explaining the observed time-dependent follicular depletion followed by restoration of growing follicle numbers. This model predicts that consecutive chemotherapy treatments compound depletion as primordial follicles prematurely activated by the first round of chemotherapy become the growing follicle pool destroyed by the following chemotherapy dose. Future studies tagging chemotherapy-injured follicles to quantify growth and atresia in vivo may allow clear delineation of ovarian remodelling.

5.2.2

Cell-Type Specificity of Chemotherapy Toxicity

Early studies of chemotherapy toxicity placed significant focus on oocytes, including cisplatin destruction of primordial follicle oocytes. Distinct chemotherapy agents exhibit differential toxicity profiles, however, with respect to which cell and follicle types are the primary targets for insult and demise. Stromal and thecal cells in direct contact with the blood supply are the first cells to exhibit DXR-induced DNA damage, followed by subsequent damage to follicular cells ( Figure 5.1 ). Identifying the cells that receive the first insult for each chemotherapy agent is an important step toward designing protective approaches that prevent ovotoxicity.

Temporal accumulation of doxorubicin (DXR)-induced double-strand DNA breaks is cell-type specific.

Summary data quantify double-stranded DNA damage as the comet moment utilizing the comet assay. Panels summarize DNA damage in stromal/thecal cells (A), granulosa cells (B), and oocytes (C) as hours post-DXR injection plotted against comet moment. n=3 animals/group/time point/replicate, >100 cells/point, 4 replicates total. *p < 0.05, one-way analysis of variance.

Adapted from .

5.2.3

Ovarian Toxicity Lessons from Ovoprotection Studies

Mechanistic details of how chemotherapy causes ovarian damage continue to expand through studies testing ovoprotective agents. Here we highlight key considerations for rodent models, including animal age at treatment, drug preparation methods and drug dose, alongside the need to develop robust chemoprotective agents that provide protection across a range of chemotherapy doses.

The importance of drug preparation was revealed by conflicting results in cisplatin-induced ovarian damage in studies by Gonfloni and Kerr. A thorough and mechanistic study by Gonfloni et al. demonstrating that imantinib shields the ovary from cisplatin damage showed an initial 40–50% loss of fertility and pups per litter following cisplatin. In replicating that study, however, Kerr et al. observed almost complete loss of both. Given identical calculated drug doses, mouse treatment and mating age, and that the Gonfloni group replicated their own results, differences in cisplatin formulations may account for higher level of toxicity and the resultant lack of imatinib protection observed by Kerr et al.

Comparing ovotoxicity across studies can be challenging when different chemotherapy doses are used. Cyclophosphamide at 50 mg/kg causes 60% primordial follicle loss in mice treated at 5–6 weeks of age. Higher cyclophosphamide doses (120 mg/kg) combined with busulfan (12 mg/kg) cause a striking >95% reduction in primordial follicle counts. While greater toxicity with higher dose is not surprising, these studies demonstrate challenges in cross-study comparisons and effective ovoprotective agents will need to shield across a range of chemotherapy doses.

Similar to cancer patients, age of mice at time of treatment alters the characteristics of chemotherapy ovotoxicity. The burnout model explains dynamics of initial secondary follicle depletion and restoration in PND5 mice treated with 150 mg/kg cyclophosphamide. In contrast, primordial follicles from mice treated at 4.5 weeks of age with 50 mg/kg cyclophosphamide were depleted prior to loss of growing follicles. While the chemotherapy dose was different, altered follicular dynamics are likely age-dependent. Similarly, 4-week-old (adolescent) mice pretreated with granulocyte colony stimulating factor as an ovoprotective agent exhibited >80% follicle loss following cyclophosphamide/busulfan, but breeding mice only showed a 30% reduction in litter size. These discrepancies may be due to the treatment of 8-week-old (reproductively mature) mice for fertility assessment. As the field moves forward, systematic assessment of age-related chemotoxicity and efficacy of ovoprotective agents will aid translation of fertility interventions to cancer patients.

5.2.4

Rodent Studies of Ovarian Chemotherapy Toxicity Compared to Data from Clinical Studies

Chemotherapy agents have been classified as differentially ovotoxic based on retrospective analysis of female cancer survivors who received multi-drug treatments. Alkylating agents like cyclophosphamide are considered the most detrimentally ovotoxic. Platinum drugs, including cisplatin, are classified as moderately ovotoxic, and anthracyclines like DXR, mildly ovotoxic. Rodent studies of cisplatin and cyclophosphamide confirm the human ovotoxicity, but mice receiving a single human equivalent dose of DXR exhibit a 50% decrease in ovarian mass and decline in fertility rates much greater than “mildly ovotoxic” would predict. It is unclear whether the disparities are due to species differences or the fact that infertility post-chemotherapy is underestimated, given that it requires self-reporting from patients, and that follow-up is usually for a relatively short period of time. In addition, clinical ovotoxicity has thus far only been scored according to infertility, not accounting for additional negative ramifications in females who do achieve pregnancy, including delivery complications and low birth weights. Sibling studies conclude that while alkylating agents (considered highly ovotoxic) do not affect birth weight, non-alkylating agents, particularly DXR, carry high risk for low birth-weight babies in subsequent pregnancies. These data highlight the need for a comprehensive classification of chemotherapy ovotoxicity.

Cancer patients receive chemotherapy cocktails, but most animal models have assessed ovotoxicity of each chemotherapy agent in isolation. Advancing toward the goal of cross-chemotherapy ovoprotection, tamoxifen shields the ovary from both cyclophosphamide ( in vivo ) and doxorubicin ( in vitro ). This study provides an important step toward animal models of combined chemotherapy regimens.

Another critical question in predicting POI following chemotherapy is determining how follicle counts predict POI and developing in vivo follicle assessment tools. Mice treated with cyclophosphamide exhibit a linear relationship between dose and primordial follicle depletion but, surprisingly, depleting 60% of primordial follicles does not change ovulation, fertilization, or pregnancy rates. Certainly follicle depletion reduces the fertile window, but in women, removing one ovary does not dramatically change the age of menopause. The processes allowing one ovary to compensate for the loss of the contralateral ovary are unknown, and may reveal novel ways to prolong fertility despite follicular depletion. Magnetic resonance imaging (MRI) has revealed DXR-induced ovarian remodelling in mice as a decrease in ovarian dimensions through 1-month post-DXR treatment. Future studies correlating follicle counts with ovarian changes measured by MRI may provide a non-invasive measure of ovarian remodelling and POI assessment post-chemotherapy in patients.

5.2.5

Transgenerational Toxicity of Chemotherapy

Current dogma suggests that women who become pregnant post-cancer are at no increased risk for offspring with congenital defects. Data from mice, however, demonstrate that while the first generation post-chemotherapy manifests no gross defects, chromosomal abnormalities appear in later generations. In a transplant model that distinguishes between effects on the ovary itself and general physiological decline of the dam, ovaries from DXR-treated mice were transplanted into naïve mice, which were subsequently mated. Starting at generation (G)4, DXR offspring exhibited increased neonatal death and physical abnormalities due to deletions on chromosome 10. Dams receiving DXR ovaries suffered frequent dystocia not observed in mice receiving control ovaries, and died due to delivery complications in G4–G6. Similarly, oocytes retrieved from cyclophosphamide-treated mice exhibited abnormalities that led to reduced fertilization and embryonic development, and increased aneuploidy. These data suggest transgenerational effects of chemotherapy may be underestimated in human cancer survivors, as society has not reached five generations post-chemotherapy (as these agents were first developed in the 1960s) and demonstrate that successful ovoprotective agents will need to prevent chemotherapy-induced genotoxicity, diminishing risk for propagating chemotherapy-induced genetic abnormalities.

Mice provide the ideal model for initiating studies to determine whether mechanistic ovoprotective agents like dexrazoxane and bortezomib can prevent chemotherapy-induced genotoxicity and limit risk for future generations. Demonstrating the strengths of an in vivo model, including thorough analysis of acute insult to all ovarian cell types combined with long-term fertility assessment, a recent study by the authors showed that bortezomib shields granulosa, stromal/thecal cells and oocytes of adolescent mice (4 weeks of age) from DXR-induced DNA damage in a temporal fashion, preserving the growing follicle pool. While bortezomib did not alter initial DXR-induced loss, litter size recovered over time compared to DXR alone ( Figure 5.2 ). The temporal data provided a thorough assessment of fertility across the reproductive lifespan of the mice and revealed that initial fertility loss can be recovered. Future studies will determine whether mechanistic ovoprotective agents fulfil the potential of diminishing the transgenerational consequences of chemotherapy.

Bort pretreatment improves the infertility index following doxorubicin (DXR) treatment across six rounds of continuous breeding.

Plot represents the “infertility index” plotted as the percentage of surviving animals from each treatment group that fail to achieve the next birth as a function of birth (litter) number. Symbols correspond to control, DXR, Bort-DXR and Bort treatment groups as indicated. DXR points in the linear range were statistically different from Bort-DXR, Bort, and control (p < 0.05, one-way analysis of variance).

Adapted from .