Folliculogenesis Is Not Fully Inhibited during GnRH Analogues Treatment in Mice Challenging Their Efficiency to Preserve the Ovarian Reserve during Chemotherapy in This Model

Abstract

As many chemotherapy regimens induce follicular depletion, fertility preservation became a major concern in young cancer patients. By maintaining follicles at the resting stage, gonadotropin-releasing hormone analogues (GnRHa) were proposed as an ovarian-protective option during chemotherapy. However, their efficacy and mechanisms of action remain to be elucidated. Mice were dosed with cyclophosphamide (Cy, 100-500 mg/kg i.p) to quantify follicular depletion and evaluate apoptosis at different times. We observed a dose-dependent depletion of the follicular reserve within 24 hours after Cy injection with a mean follicular loss of 45% at the dose of 200mg/kg. Apoptosis occurs in the granulosa cells of growing follicles within 12 hours after Cy treatment, while no apoptosis was detected in resting follicles suggesting that chemotherapy acutely affects both resting and growing follicles through different mechanisms. We further tested the ability of both GnRH agonist and antagonist to inhibit oestrus cycles, follicular growth and FSH secretion in mice and to protect ovarian reserve against chemotherapy. Although GnRHa were efficient to disrupt oestrus cycles, they failed to inhibit follicular development, irrespective of the doses and injection sites (sc or im). Around 20% of healthy growing follicles were still observed during GnRHa treatment and serum FSH levels were not reduced either by antagonist or agonist. GnRHa had no effect on Cy-induced follicular damages. Thus, we showed that GnRHa were not as efficient at inhibiting the pituitary-gonadal axis in mice as in human. Furthermore, the acute depletion of primordial follicles observed after chemotherapy does not support the hypothesis that the ovary may be protected by gonadotropin suppression.

Fig 1. Dose- and time-related follicular depletion induced by chemotherapy.

The follicular population was evaluated by counting all the follicles in every fifth section of each whole serial sectioned ovary from mice treated with (a) 100, 200, or 500 mg/kg of Cy and sacrificed after 7 days or (b) 200 mg/kg of Cy and sacrificed after 1 or 7 days. Results are expressed as mean ± SD; *p < 0.05 compared with controls. (c) Representative immunohistological sections showing apoptotic follicles (stained by TUNEL and CASPASE-3) in 200mg/kg Cy-treated mice sacrificed at different time points. TUNEL staining peaked at 12–18 hours, whereas CASPASE-3 peaked at 8–12 hours. Scale bar = 100 μm.

Fig 2. Fertility impact of chemotherapy.

Mice were treated with Cy (200 or 500mg/kg) or saline and mated for 36 weeks. The number of litter per month, of pups per litter and the number of days between two consecutive litters (DBL) were calculated. Results are expressed as mean ± SD; N = 2 mice per condition.

Fig 3. Inhibitory effect of GnRHa on follicular development.

(a) The oestrus stages were determined by evaluating the vaginal cytology (pro-oestrus (P), oestrus (E), metoestrus (M) and dioestrus (D). The oestrus cycle is partially blocked according to the type of GnRHa, irrespective to the dose. Here oestrus cycle during treatment with antagonist 20μg/kg, agonist 2μg/kg and agonist im 4mg/kg are represented (b) The growing follicles ratio was evaluated by counting follicles in every fifth section of each whole serial sectioned ovary from different experimental conditions (2, 20, 200 or 500 μg/kg/day or im agonist injection of 4mg/kg). Results are expressed as mean ± SD. (c) Representative immunohistological sections showing growing follicles (stained with KI-67) without apoptosis staining in granulosa cells (TUNEL) after 15 days of GnRHa treatment. Scale bar = 100 μm. (d) Serum FSH levels (ng/ml) according to the doses and injection site of GnRHa. Each symbol represents one animal.

Fig 4. Evaluation of the protective effect of GnRHa on Cy-induced follicular depletion.

The follicular population was evaluated by counting all follicles in every fifth section of whole serial sectioned ovaries of mice injected daily with 500μg/kg of GnRH agonist or antagonist subcutaneously for 21 days and treated with 200mg/kg of Cy on day 15. The different conditions are control, Cy alone (Cy), GnRH agonist alone (Ago), or with Cy (Ago + Cy) and GnRH antagonist alone, (Ant) or with Cy (Ant + Cy). Results are expressed as mean ± SD, *p < 0.05 compared with controls. Statistical difference was observed between Cy-treated groups and control but not with GnRHa treatments.