The Tyrosine Kinase Inhibitor Sunitinib Affects Ovulation but Not Ovarian Reserve in Mouse: A Preclinical Study

Abstract

The aim of the study was to evaluate ovarian toxicity of tyrosine kinase inhibitor (TKI) sunitinib, since only scarce data are available on gonadal function after this treatment. Six-week-old female mice received orally, once daily, vehicle or sunitinib (50 mg/kg/d) during 5 weeks. Fertility parameters were analyzed from ovulation to litter assessment. Sunitinib exposure significantly reduced (i) corpora lutea number per ovary (1.1 ± 0.38 in sunitinib group versus 4 ± 0.79 in control group, p<0.01) and (ii) serum Anti Müllerian hormone (AMH) levels in sunitinib treated mice (12.01 ± 1.16) compared to control mice (14.33 ± 0.87 ng/ml, p< 0.05). However, primordial and growing follicles numbers per ovary were not different in both groups. After treatment withdrawal, female mice in both groups were able to obtain litters. These data could be helpful to counsel clinicians and patients, when fertility preservation methods are discussed, before TKI treatment in girls and young women.

Fig 1. Study design.

Six-week-old female mice received [per os, once daily (5d/wk)] either sunitinib (n = 19) or vehicle (n = 12) during 5 weeks, and were sacrificed at day 35. Another group of 5-weeks-sunitinib (n = 9) or vehicle-treated (n = 5) mice were mated immediately after treatment withdrawal at Day 35 in order to study their fertility. A third group of 3-week-old female mice received sunitinib (n = 8) or vehicle (n = 6) during three weeks, and then were superovulated by intraperitoneal injection of 10 IU pregnant mare’s serum gonadotropin (PMSG) at 6pm, followed by intraperitoneal injection of 5 IU human Chorionic Gonadotropin (hCG) 48 h later. Animals were killed 17 h after the hCG injection.

Fig 2. Mouse cyclicity and histological analysis of ovaries.

A. Representative mean number of estrus along 35 days in the control group (n = 12) and the sunitinib-treated group (n = 15). B. Representative percentage of days in each cycle stage over a 35 days period in both groups. Each bar represents mean with SEM. C. Representative ovarian histological sections from control mice and sunitinib treated mice. Asterisks indicate corpora lutea (CL), reflecting ovulation rate. Bar scale = 500μm. Note the absence of CL in the ovary of a sunitinib-treated mouse. D. Mean number of corpora lutea per ovary after 35 days of vehicle or sunitinib administration. Quantitative analysis revealed a marked decrease in corpora lutea number in the sunitinib group (n = 15) versus the control group (n = 12). **p < 0.01.

Fig 3. Superovulation test.

Number of oocytes obtained after superovulation test in sunitinib-treated (n = 8) and control mice (n = 6).

Fig 4. Ovarian reserve evaluation after sunitinib or vehicle treatment.

A. Expression of ovarian Amh transcripts normalized to 18S RNA in mice treated by sunitinib (n = 15) compared with control mice (n = 12) **p<0.01. B. Serum AMH levels in sunitinib-treated mice (n = 15) compared with control mice (n = 12). *p<0.05. C. Growing follicle counting in sunitinib-treated mice compared with control mice. D. Primordial follicle counting in sunitinib-treated mice compared with control mice. E. Mean delay to obtain first litter in sunitinib (n = 9) and control (n = 6) mice. F. Mean number of pups per litter obtained during 3 months of observation of sunitinib (n = 9) and control (n = 6) mice.

Fig 5. Sunitinib displays an antiproliferative activity.

Sunitinib inhibits p42/44 MAPK activation in response to PDGF on human (A) and mouse (B) granulosa cells in vitro. Cells were treated by sunitinib or vehicle for 4 hours and subsequently stimulated by PDGF for 5, 10 or 30 minutes (min). Lysates were immunoblotted with anti p42/44 and anti-phospho-p42/44 (P-p42/44) indicated by arrows, as described in the Materials and Methods section. Tubulin was used as a loading control. After PDGF treatment, KGN and GRAL cells demonstrated rapid and transient phosphorylation of p42/44 MAPK reaching maximal level by 10 min and declining thereafter to background levels within 30 minutes. This effect was totally abrogated in presence of sunitinib on both cell lines.