Center for Fertility and Reproductive Endocrinology, Virginia Mason Medical Center, Seattle, Washington, USACorresponding address: Virginia Mason Medical Center, 1100 9th Avenue (X11-FC), Seattle, WA 98110, USA. e-mail: obsgsl{at}vmmc.org
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Abstract |
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Key words: chemotherapy/cyclophosphamide/cytotoxic therapy/oocyte apoptosis/ovarian failure
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Introduction |
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The purposes of the present investigation using the rat model are 3-fold: (i) to determine the maximum tolerated dose(s) and cytotoxic effects of cyclophosphamide on ovarian follicles in a doseresponse group; (ii) to compare the effectiveness of two dosages of two hormonal regimens in achieving anovulation; and (iii) to determine if the induction of an anovulatory state by two different hormonal methods may spare the ovary the cytotoxic effects of cyclophosphamide. The investigation was performed in two parts as a doseresponse study and as an experimental study.
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Materials and methods |
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Drug treatment
Cyclophosphamide was administered by daily i.p. injection. The combination of ethinyl estradiol and norgestrel was delivered using time-released, subdermally placed capsules. The capsules consisted of the active hormones embedded in a methyl cellulose matrix and were designed to deliver constant, fixed daily doses for up to 60 days per capsule. All capsules were placed s.c. through a 1 cm incision in the right paravertebral area under light general anaesthesia achieved with metofane. Leuprolide acetate was injected s.c. Control animals were injected i.p with sterile saline solution.
Treatment protocols
The investigation was performed as a doseresponse study and experimental study. In the doseresponse study, the maximum tolerated dose of cyclophosphamide and the anovulatory doses for the combination of ethinyl estradiol/norgestrel and the GnRH agonist, leuprolide acetate, were studied. Cyclophosphamide was chosen as the alkylating agent because of its known impact on gonadal function and its prevalence in clinical practice. The combination of ethinyl estradiol/norgestrel and leuprolide acetate were selected as anovulatory agents to study the impact of two different hormonal environments on cyclophosphamide toxicity, namely an estrogen/progestin-rich environment and a hypoestrogenic environment secondary to the ethinyl estradiol/norgestrel combination and leuprolide acetate, respectively. Toxicity was assessed by monitoring weight weekly and physical well-being daily and by measuring complete blood counts after sacrifice. Haematological function was assessed to determine whether the varying hormonal milieu induced by the two anovulatory protocols altered cyclophosphamide toxicity. The least toxic, effective dose of cyclophosphamide and lowest effective dose of the two anovulatory regimens were used in the experimental study. The experimental study sought to investigate if there was any protective effect of these two anovulatory regimens on follicular number after treatment with cyclophosphamide.
Doseresponse study
To determine the maximum tolerated dose of cyclophosphamide needed to induce follicular attrition, the number of follicles as determined by ovarian histology was studied after treatment. Sixty rats were divided into three groups. All rats were given a 50 mg/kg i.p. loading dose of cyclophosphamide and maintenance doses of cyclophosphamide of either 5, 10 or 15 mg/kg/day i.p. After 6 weeks respectively of treatment, animals were sacrificed.
To compare the anovulatory doses of the combination of ethinyl estradiol/norgestrel and the GnRH agonist, leuprolide acetate, 40 rats were divided into two groups. Combinations of 100 µg of ethinyl estradiol/4 mg of norgestrel, and 50 µg of ethinyl estradiol/2 mg of norgestrel were administered s.c using time-release capsules to 20 rats in each group. In the second group, 2.5 µg of leuprolide acetate s.c. twice daily was compared with 2.5 µg s.c. daily in 20 rats in each group. Ovulatory status was assessed by examination of vaginal cytology. For analysis of estrous cycles, vaginal smears were obtained on a daily basis, stained with haematoxylin and eosin and examined for cellular content. Vaginal smears were indexed according to cellularity to determine ovulatory status. Rats were assessed as being in one of four stages of the cycle: proestrus, estrus, metestrus or diestrus. All animals were cycling for a 2 week observation period prior to capsule placement or s.c. injection. Anovulatory smears were reliably induced within 6 days in all animals after capsule placement and 3 weeks after the first injection of leuprolide acetate, and maintained for the duration of observation.
Experimental study
In the second part of the investigation, the impact of cyclophosphamide on follicular number without treatment with anovulatory agents and after induction of anovulation was studied and compared with saline-treated controls. All experimental animals with the exception of the control group were treated with i.p. cyclophosphamide alone or in combination with one of the two anovulatory agents. Four groups of 20 mature SpragueDawley female rats in each group were studied as follows: saline-treated control (group I), cyclophosphamide only (group II), cyclophosphamide and ethinyl estradiol/norgestrel (group III) and cyclophosphamide and GnRH analogue (group IV). Treatment with cyclophosphamide was started after an observation period in groups I and II and a treatment in groups III and IV with ethinyl estradiol/norgestrel and leuprolide of 3 weeks. Anovulation was documented using vaginal cytology prior to starting treatment and assessed weekly during treatment. Animals were sacrificed after 4 weeks of treatment and the ovaries fixed. Capsules for hormone delivery were identified in all animals in group III at time of death. If no capsule could be found, the animal was excluded from the study. All animals were sacrificed between 08.00 and 09.00 h.
Haematological studies
All animals were sacrificed by cervical dislocation. Blood was drawn by cardiac puncture for complete blood counts and analysed for total leukocytes, differential count and haematocrit. Haematological studies were performed as a secondary measure of the impact of cyclophosphamide on metabolic function, well-being and toxicity, and to assess any differential effect that the anovulatory agents may have had on cyclophosphamide toxicity.
Histological analysis of ovarian tissue
Both ovaries were removed in their entirety for processing. After removal, the ovaries were placed in formalin. The tissue was embedded in paraffin, step-sectioned and stained with haematoxylin and eosin. Among the various parameters, follicular diameter was chosen as the parameter to classify follicles and assess the impact of treatments (Pedersen and Peters, 1968; Hirshfield and Midgley, 1978
). All follicles were measured in two dimensions. The maximum follicular diameter and a diameter at right angles to it were used to calculate a mean diameter for each follicle. Only those follicles that contained the nucleolus of the oocyte were included in the final count as a safeguard. The ovaries were examined at 5 µm sections and studied for the number of medium (300450 µm) and large (>450 µm) follicles per section of ovary. An average of 50 sections per ovary were studied (range 3763). The degree of follicular atresia and primordial follicle count were not assessed.
Statistical analysis
Statistical analysis was performed using a two-way analysis of variance and Bonferroni correction. Level of significance was considered to be P 0.05 (InStat Instant Biostatistics, Graph Pad Software, San Diego, CA).
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Results |
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Discussion |
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Cyclophosphamide appears to influence the number of growing follicles. The drug may induce a discontinuous movement of follicles into the next class size (from small <200 to medium 300450 µm). These unique observations suggest that the alkylating agent, cyclophosphamide, and/or its metabolites, in addition to gonadotropins and anti-estrogens, are unique among drugs in their ability to increase the maturation of follicles and movement toward ovulation. Cyclophosphamide may force smaller follicles beyond a critical stage into the next pool of growing and developing follicles. The observations of ovarian stimulation noted in the present study are restricted to medium and large follicles. It is unclear whether primordial follicles were affected. By study design, any possible impact on primordial follicles by and/or differential sensitivity to cyclophosphamide were not evaluated. Given the fixed, limited store of follicles in an ovary, it is tempting to infer, oversimplistically, that movement of follicles into a larger size category occurs at the expense of the pool of primordial follicles.
Prior studies together with the data of the present study suggest that follicle attrition and gonadal failure secondary to cyclophosphamide may be due to a marked acceleration in follicular maturation, depletion and eventual exhaustion. The observation interval in the present study was long enough to induce movement of follicles to larger size classes. However, it may have been too brief to observe follicular exhaustion noted in prior studies (Meirow et al., 1999). Morbidity and mortality observed during the doseresponse study precluded a longer duration of treatment. This impact on follicular activity may be due to an effect of cyclophosphamide or, after hepatic oxidation, its aldehyde derivatives on the follicle itself, the granulosa cell or the basement membrane. Two laboratory models support this concept. Cumulus-enclosed germinal vesicle stage porcine oocytes demonstrate a doseresponse inhibition of nuclear maturation when cultured with cyclophosphamide (Chen et al., 1998
). Data also suggest that the 4-hydroperoxycyclophosphamide-activated metabolite of cyclophosphamide directly decreases granulosa cell survival and function (Ramahi-Ataya et al., 1988
). Electron microscopy of human ovarian cortical tissue cultured with cyclophosphamide suggests toxicity to follicles possibly mediated through damage to both granulosa cells and basement membrane (Raz et al., 2002
).
Age and hormonal status appear to correlate with the effect of cyclophosphamide on follicles (Marcello et al., 1990; Familiari et al., 1993
). Observational studies suggest that prepubertal patients administered alkylating agents may be spared gonadal damage. In this age group, protection may be afforded secondary to a larger reservoir of primordial oocytes (Faddy et al., 1992
). These observations suggest a possible role for hormonal suppression of gonadal function to minimize the impact alkylating agents may have on gonadal function (Chapman and Sutcliffe, 1981
; Horning et al., 1981
). In the present study, cyclophosphamide induced ovarian damage regardless of ovulatory or hormonal status. The impact of cyclophosphamide was observed in spite of varying the hormonal milieu by inducing an estrogen/progestin steady state with the combination ethinyl estradiol/norgestrel or a hypoestrogenic environment using the GnRH analogue leuprolide acetate.
Previous studies of the impact of hormonal suppressive therapy in males and females of different species on gonadal protection during treatment with cytotoxic agents have been conflicting (Fossa et al., 1988; Blumenfeld et al., 1996, 2000). Testicular suppression with the GnRH analogue triptorelin was protective in rats (Karashima et al., 1988
). In contrast, nafarelin potentiated testicular damage in dogs (Goodpasture et al., 1988
). In humans, results after treatment with both oral contraceptives and a variety of GnRH analogues have been inconclusive, in part due to study design and low numbers of patients studied (Johnson et al., 1985
; Thibaud et al., 1998
). In the present study, no protective effect of anovulation against follicular attrition using two protocols was observed in the rat model. Pathways of oocyte loss are complex and may involve factors other than the degree of follicular stimulation and the hormonal environment of the follicle (Gougeon, 1996
). In a limited comparative study in humans, ovarian suppression with GnRH analogues was not an effective method to prevent ovarian failure (Waxman et al., 1987
). The hypothesis that any reduction in endogenous follicular stimulation may spare the ovary the impact of cyclophosphamide may be an oversimplification.
Results of the present study suggest that induction of anovulation in the rat model, whether by combination ethinyl estradiol/norgestrel or a GnRH analogue, may not provide a reliable method of maintaining ovarian function and reproductive potential for patients using cyclophosphamide regardless of indication. Assisted reproductive technologies such as IVF, reimplantation of cryopreserved ovarian tissue, or cryopreservation of oocytes may be more effective for this purpose. Several reports describe favourable outcomes with the second of these techniques, first harvesting, cryopreserving and then reimplanting ovarian cortical strips (Gosden, 1990; Oktay and Karlikaya, 2000
; Radford et al., 2001
). In these cases, estradiol secretion and folliculogenesis were resumed after transplantation. Although cryopreservation may induce damage on chromosomes X, 16 and 18, functional ovarian tissue and a cohort of chromosomally competent oocytes remain within the ovarian substance, offering the possibility of continued secretion of estradiol and progesterone and reproduction (Poirot et al., 2002
). A second option for patients confronting cytotoxic chemotherapy is cryopreservation of oocytes. In this circumstance, immature oocytes may be retrieved, cryopreserved and stored until needed (Yoon et al., 2000
). Early studies with vitrified oocytes have resulted in live births (Hong et al., 1999
). Both techniques offer viable options but require further study before considered acceptable clinically.
Recent data suggest that interference with the steps of apoptosis may reduce follicular attrition induced by anticancer therapy. This approach may be more effective than ovulation prevention in reducing follicular loss in this setting. Apoptosis has been identified recently as a possible mechanism for oocyte depletion associated with both ageing and cytotoxic therapies (Morita and Tilly, 1999). Sphingomyelin-derived second messengers may be key in the programmed loss of oocytes (Kolesnick and Kronke, 1998
). Inhibition of the enzyme sphingomyelin phosphodiesterase results in disruption of ceramide, a sphingolipid-based second messenger. Manipulation of these intracellular events may offer an opportunity to alter the rate of oocyte attrition. Data from animal studies suggest that normal apoptotic oocyte loss may be suppressed by disruption of the gene for acid sphingomyelinase (Morita et al., 2000
). Furthermore, radiation-induced oocyte loss may be prevented by treatment with the small lipid molecule sphingosine-1-phosphate. These studies suggest that manipulation of second messengers and treatment with small lipid molecules may be an effective and efficient treatment to prevent oocyte loss associated with cytotoxic therapy.
In conclusion, cyclophosphamide may result in ovarian failure by rapid development and attrition of medium follicles in the rat model. These data support previous studies suggesting that cyclophosphamide may have a unique effect of superovulation in this species. No clearly protective effect is noted on either medium or large follicles in this study in the rat model by an induction of anovulation using either the combination of ethinyl estradiol and norgestrel or the GnRH analogue leuprolide acetate. These results further suggest that this impact may occur whether the hormonal milieu is estrogen/progestin dominant or hypoestrogenic. Cryopreservation of either ovarian tissue or immature oocytes and, in the future, manipulation of molecular events critical to oocyte depletion may be more effective and reliable techniques for preservation of gonadal function during cytotoxic therapy.
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Acknowledgements |
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References |
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Submitted on January 27, 2003; resubmitted on October 29, 2003; accepted on November 10, 2003.
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