The effect of cyclophosphamide on ovarian reserve
As one of the most commonly used antineoplastic drugs with a potential risk to the gonad, cyclophosphamide imposes a negative effect on gonad in a dose- and age-related manner as demonstrated by the observed higher risk of amenorrhea in aged patients or those on a larger dose of chemotherapeutic regimen. Study of the pharmacological mechanism of cyclophosphamide has revealed that it primarily targets the cell in proliferating phase by intracellular DNA crosslinking, which results in an effect of inhibiting cell growth and promoting apoptosis. Therefore, the major consequence of the impairment to ovaries is considered to be the atresia of follicles in proliferation, whereas the toxicity of cyclophosphamide to quiescent primordial follicles is still unclear. The preliminary results of our study have shown that CY-induced POF do lead to the atresia of the follicles in proliferation phase as well as ovarian interstitial fibrosis. We also found that despite of the increased ratio of early follicles to primordial follicles, apoptosis of the primordial follicles in ovarian cortex was not observed by TUNEL test, which suggested a relative increase in the number of the early follicles since they can only be formed via the recruitment of primordial follicles. These findings have provided indirect evidence supporting that chemotherapy drugs may prematurely induce the primordial follicles into the recruitment stage. The premature recruitment of primordial follicles into the growing follicular pool may be caused by a direct CY-induced apoptosis of granulosa cells of mature antral follicles and a subsequent decrease in the negative regulatory factors for primordial follicle recruitment [
16]. Since the growing follicles are more sensitive to chemotherapy agents, the apoptosis process is accelerated by cyclophosphamide, eventually precipitating a rapid decline in ovarian reserve and, as a result, POF. The other possibility is that the direct chemotherapeutics-induced injuries to the DNA of oocytes in primordial follicles may accelerate the repair of defective DNA, and then trigger the premature recruitment of primordial follicles [
25].
Oocytes and granulosa cells are different in terms of their susceptibility to chemotherapy drugs, as proliferation and differentiation would not occur in the oocytes even in the follicles at a rapid growth stage, which is contrasted with the continuous proliferation of granulosa cells. In vitro studies have shown that the target of cyclophosphamide leading to ovarian impairment is the granulosa cell whose apoptosis induced by activation of some certain signals in follicles will precipitate POF characterized by the loss of reproductive and endocrine functions of the ovary [
26,
27]. So, the first explanation as described above appears to be a more plausible mechanism that the direct CY-induced mature antral follicle apoptosis contributes to the attenuation of the negative regulation factors for primordial follicle recruitment (such as AMH), which, in turn, can cause the premature recruitment of primordial follicles.
8-week old adult BALB/c mice were used in the present study, as the ovarian morphology and follicular development in mouse share a high similarity with those of human. Both of single administration and consecutive 4-week multiple dosing were employed for POF model group to observe the acute effects of cyclophosphamide on ovary as well as to mimic the clinical ovarian changes of patients after repeated dosing of chemotherapeutics. The process of recruitment of primordial follicles to ovulation takes about 9 months, during which several courses of chemotherapy may have been conducted for a patient while it takes only 3 weeks for the mice to go through the same process. Therefore, the models established by consecutive 4-week chemotherapy could be used to observe the effect on the growth and apoptosis of the follicles.
HE staining follicle counts showed that with the increased dose of cyclophosphamide, a constant shrink in the number of antral follicles was observed in contrast to an elevating tendency in the ratio of early ovarian follicles to primordial follicles, which was the most significant in the 75 mg/kg × 4 weeks group. This finding suggests that the damage to ovarian tissues and the decline in the number of follicles, particularly the mature antral follicles, are correlated with the dose of cyclophosphamide. Different degree of fibrosis was also observed in ovarian cortex, which is consistent with the ovarian toxicity from previous reports [
2]. Ovarian morphological study for different dose groups revealed that the mice in 150 mg/kg group had the most significant decrease in the number of mature antral follicles and ovarian fibrosis changes as pathologically defined by POF, so 150 mg/kg was selected as the drug concentration for the following mechanism study. It is believed that the models were well-established due to the short modeling time and the ovarian histological changes similar to human POF, and could also be employed in the following studies.
A number of studies have shown that [
28,
29] apoptosis of growing cells plays a key role in chemotherapeutics-induced impairment on ovarian structure and function. The TUNEL staining in this experiment also showed that apoptosis of the granulosa cells in antral follicles initiated in CY group within 12 h after drug administration, and significantly larger number of apoptotic follicular granulosa cells were observed compared with the PBS control group after repeated drug administration. However, apoptosis of primordial follicles was not found in any of the groups with different dosing duration or at different doses. In Ki67 immunohistochemical test, high magnification microscopy for the parafin sections showed the most evident DAB staining of Ki67 in granumosa cells of early follicles in CY group, but no DAB stains in the granumosa cells of the follicles at the same stage in PBS group. These findings suggest that cyclophosphamide may directly trigger mature follicle apoptosis, accompanied by an increase of the ratio of early follicles to primordial follicles. The absence of primordial follicle apoptosis and the significant rise in the number of early follicles also indicate that cyclophosphamide may prematurely induce primordial follicles into the growing follicle pool and eventually result in the shrink of ovarian reserve.
Ovarian tissue proteins of the mice in 150 mg/kg CY group were extracted for quantitative determination to explore the correlation between CY-induced effect on ovarian reserve and the signaling pathway closely related to follicular growth. The results demonstrated an increase in phosphorylation of signaling pathway proteins AKT and mTOR but with the absence of statistical difference, as well as a statistically significant elevation in phosphorylation of rpS6, the downstream protein of mTOR (P = 0.025), which indicates that cyclophosphamide may cause over-activation of primordial follicles by activating mTOR signaling pathway for follicle growth regulation. However, this conclusion is subject to a certain level of limitations, as the experiment techniques are insufficient to separate the primordial follicles and the early growing follicles in ovarian cortex for protein quantitative determination. Immunohistochemistry and immunofluorescence localization tests were also performed to screen the confounding results caused by mature follicles. Evident staining was observed in the proteins related to this signal pathway in ovarian granulosa cells but not in ovarian interstitial cells, indicating that the follicular granulosa cells may contribute to the enhanced protein phosphorylation.
The inhibitory effect of rapamycin on chemotherapy-induced ovarian reserve decline
Rapamycin (Rapa), a specific inhibitor of mammalian target protein (mTOR), controls cell proliferation and apoptosis by regulating a variety of biosignals such as saccharides, amino acids and insulin [
30]. There are two isomers of mTOR playing different roles in the cells, with mTOR1 mainly responsible for the control of cell growth and mTOR2 for the regulation of cell survival. It has been proved that only mTOR1 can inhibit the activation of downstream signaling molecules and the subsequent cell proliferation by specific binding with rapamycin. Adhikari et al. found that knockout of tuberous sclerosis complex (Tsc) 1 and 2 in oocytes of mice was associated with the elevation in mTOR1 activity and a rapid transform of primordial follicles into growing follicles ultimately leading to premature ovarian follicle depletion and POF [
31]. This study suggests that rapamycin may participate in the regulation on the recruitment, proliferation and differentiation of primordial follicles. In 2009, Randy et al. reported that rapamycin may have an anti-aging effect as demonstrated by 9% lifetime extension observed in male mice and 14% in female mice, which was considered as the evidence to support that mTOR signaling pathway may be involved in puberty initiation and lifetime extension [
32,
33]. All these findings suggest that rapamycin is closely associated with the recruitment of primordial ovarian follicles, proliferation and apoptosis of follicles, as well as the anti-aging mechanisms.
In vivo study using gene knockout mice has shown that rapamycin as the specific blocker of mTORC1 can inhibit the recruitment of primordial follicles in the ovaries of mice [
21]. In the present study, comparison between concomitant rapamycin administration 1 week before and after chemotherapy and cyclophosphamide alone regimen revealed a statistically significant decline in the ratio of early growth follicles to primordial follicles in mouse ovaries, suggesting that rapamycin may inhibit the recruitment of primordial follicles in chemotherapeutics-treated mouse models. According to the references, the anti-apoptosis effect of rapamycin is considered to be achieved by involving the cell division cycle from G1 phase to S phase [
34]. The binding of rapamycin with FKBP (FK506 binding protein) will inhibit the activity of mTOR and block PI3K/AKT/mTOR signaling pathway, thereby preventing phosphorylation of S6 K1 and 4E–BP1. As a result, translation of a portion of intracellular proteins is depressed, accompanied by a disturbed cycline protein expression as well as the inhibition on the activity of cyclin-dependent kinase (CDK), which will eventually lead to the block of late G1 phase-S phase transition. In this study, TUNEL staining showed an evidently suppressed apoptosis of granulosa cells in mature antral follicles in CY + Rapa group as compared to CY alone group, along with the absence of apoptotic death of primordial follicles, indicating that rapamycin can inhibit CY-induced apoptosis of follicular granulosa cells in ovaries of mice.
AMH, a glycoprotein secreted by granulosa cells of preantral follicles and small antral follicles, can prevent premature follicle depletion and protect ovarian reserve by regulating oocyte maturation and inhibiting follicular development. The level of serum AMH can be used to evaluate the number of follicles in growing stage and, as an indirect parameter, to estimate the number of primordial follicles. AMH is expected to be employed as a new index for assessment of ovarian toxicity of chemotherapeutics, as a sharp decline even larger than that of estradiol and inhibin B which are usually observed during chemotherapy [
35]. Besides, the high sensitivity and specificity as well as the satisfying intraperiodic and interperiodic stability make AMH the only ovarian reserve marker that can be determined in both of follicular phase and luteal phase. Serum AMH determination using ELISA in the present study revealed insignificant inter-group AMH changes among different CY dose groups, in contrast to a dramatic drop in serum AMH level after 4-week CY treatment (
P < 0.05), suggesting that chemotherapeutics generally show an impact on ovarian reserve only at a certain level of accumulated dose. An insignificant decrease without statistical significance (
P > 0.05) in serum AMH was observed after concomitant use of cyclophosphamide and rapamycin, which may be associated with the sensitivity and specificity of ELISA as well as some other factors. Therefore, more parameters should be included and verified to evaluate the post-chemotherapeutic protective effect of rapamycin on ovarian reserve.
In this study, determination of the proteins involved in signaling pathways was also performed in an attempt to further explore the potential mechanism of rapamycin on ovarian reserve protection, with the results showing no significant inter-group difference in p-Akt/Akt ratio and p-mTOR/mTOR ratio but an evidently decreased p-rpS6 phosphorylation in CY + Rapa groups as compared to CY groups (P = 0.001). These results imply mTOR as the action site through which rapamycin can inhibit its activity for phosphorylation, block the mTOR signaling pathway, prevent the phosphorylation of S6 K and other relevant downstream proteins, and restrain the excessive cell growth and proliferation. Also, these findings are considered as an indirect prove for the hypothesis that cyclophosphamide may lead to excessive activation of primordial follicles through over-activating this signaling pathway.
In addition, we conducted a control experiment using in-vitro cultured ovarian tissues from neonatal mice to investigate the direct impact of rapamycin on the recruitment of primordial follicles. After 1 week of in vitro culture in our preliminary test using 3-day old mice, recruitment of a small number of primordial follicles into primary follicular phase was observed in the ovarian tissues cultured under a natural in vitro condition, which was a striking contrast to the resting state observed in all primordial follicles cultured in the media with rapamycin added. These findings provide a further support for the hypothesis that rapamycin can protect ovarian reserve by inhibiting the activation of primordial follicles.
Studies have reported that single use of rapamycin can impose an anti-tumor effect, whereas combined chemotherapy can further increase the sensitivity of tumor cells to chemotherapy [
36], and thus always demonstrates a better antitumor and chemosensitization effect. However, considering the complexity and variability of intracellular signaling pathways, the interaction between rapamycin and antineoplastic agents, as well as the side effects of rapamycin, more fundamental and clinical research should be carried out to verify the in vivo effect of rapamycin.
The unremitting exploration on rapamycin used for tumor treatment and follicle growth regulation is likely to unfold a new path for antitumor treatment and control of chemotherapy-induced POF. Indepth and comprehensive investigations in this field may create a promising future for females undergoing chemotherapy due to the possibility that the fertility of the patients may be preserved without compromising the treatment effect.