Doxorubicin-induced ovarian toxicity

Abstract

Background: Young cancer patients may occasionally face infertility and premature gonadal failure. Apart from its direct effect on follicles and oocytes, chemotherapy may induce ovarian toxicity via an impact on the entire ovary. The role of doxorubicin in potential ovarian failure remains obscure. Our intention was to elucidate doxorubicin-related toxicity within ovaries.

Methods: Female mice were injected intraperitoneally with 7.5 or 10 mg/kg doxorubicin and their ovaries were visualized in vivo by high resolution MRI, one day and one month following treatment. Ovaries of other treated mice were excised and weighed at the same post-treatment intervals. Ovarian histological sections were stained for TUNEL or active caspase-3 and follicles were counted and categorized. Ovulation rates were evaluated in superovulated female mice treated with doxorubicin.

Results: A single injection of doxorubicin resulted in a major reduction in both ovarian size and weight that lasted even one month post treatment. A dramatic reduction in ovulation rate was observed one week after treatment, followed by a partial recovery at one month. Histological examination revealed positive staining of TUNEL and active caspase-3. We observed a significant reduction in the population of secondary and primordial follicles one month following treatment.

Conclusions: Our results may imply a mechanism of chemotherapy-induced ovarian toxicity, manifested by reduced ovulation and accompanied by a reduction in ovarian size, caused probably by an acute insult to the ovary.

Figure 1

Changes in ovarian weight induced by doxorubicin. Ovaries of female mice injected with doxorubicin were excised, weighed one week or one month after injection and compared with those of saline-injected control mice. Bars represent weight (mean ± SEM) of ovaries, (*)-significantly different from control value (P < 0.05); N = number of ovaries.

Figure 2

Ovulation rate following doxorubicin treatment. Female mice were injected with doxorubicin or with saline. Ovulation was induced by injecting the mice with 5 IU hCG 48 hours after administration of 10 IU of PMSG. The mice were sacrificed 16-17 hours after hCG administration at a time coinciding with either 48 hours, one week or one month after the doxorubicin or saline injections. The number of ovulated oocytes was recorded. Bars represent percent of ovulated control oocytes (mean ± SEM), (*)-significantly different from control value (P < 0.05); N = number of mice.

Figure 3

The effect of doxorubicin on the size of ovaries. The ovaries of anaesthetized mice injected with doxorubicin or with saline were imaged with a high-resolution coronal T2-weighted MRI 24 hours and one month post injection. (A) A relative decline in ovaries size was observed already 24 hours post treatment and persisted also one month post treatment. Bars represent percent of control (mean ± SEM), (*)-significantly different from control value (P < 0.05); N = number of mice. (B) Representative images of ovaries from saline-injected (a) and doxorubicin-injected (b) mice at one month post treatment.

Figure 4

Doxorubicin induced ovarian apoptosis. Confocal micrographs of ovaries excised from doxorubicin or saline-injected mice, 12 or 24 hours after treatment. Ovaries of saline-treated mice (A, A'A"). Ovaries of doxorubicin-treated mice 12 hours (B, B'B") or 24 hours (C, C'C") after treatment. Magnification: ×10 (A-C), ×20 (A'-C'), ×63 (A"-C"). (A) TUNNEL staining (green), (B) caspase-3 staining (red). Blue staining represents DNA labeling by Heochst 32242.

Figure 5

Depletion of ovarian follicles reservoir by doxorubicin. The number of ovarian follicles (mean ± SEM) at various developmental stages, one month after injection of doxorubicin or saline. Bars represent mean ± SEM, (*)-significantly different from control value (P < 0.05).