Department of Obstetrics and Gynaecology, Hadassah University Hospital, PO Box 12000, Jerusalem, Israel, Institute for reproduction and development, Monash Medical Centre, Melbourne, Australia and Division of Obstetrics and Gynaecology, University of Leeds, West Yorkshire, UK
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Abstract |
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Key words: chemotherapy/mouse/ovarian failure/primordial follicles/reproduction
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Introduction |
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Histological studies of human ovarian tissue, which examined the effects of chemotherapy on human ovaries following treatment, have shown that the end result of therapy is ovarian atrophy with a marked loss of PMF (Warne et al., 1973; Himelstein-Braw et al., 1978
; Marcello et al., 1990
; Familiari et al., 1993
).
Animal studies have demonstrated that irradiation at higher intensities caused depletion of PMF reserve in a dose-related manner (Gosden et al., 1997).
The aims of this study were to analyse the effects of cyclophosphamide administration at different doses on the ovarian PMF population in a uniform highly inbred cohort of young mice, in order to isolate the doseeffect relationship from other confounding factors such as animal age. In addition, the correlation between significant reduction in PMF number post-chemotherapy and reproductive performance was investigated following maturation of the exposed follicle population, as expressed by ovulation, mating and pregnancy rates. Data of this nature reflect directly the relationship between ovarian injury as expressed by clinical observations, and the loss of PMF which was counted directly. This experiment may indicate whether regular ovulation post-treatment attests to the fact that the ovaries remained uninjured despite exposure to chemotherapy.
For this study, the alkylating agent cyclophosphamide was used. Cyclophosphamide is commonly employed in the treatment of many malignancies including lymphomasHodgkin's and non-Hodgkin's, leukaemias and many solid tumours, and results in a high rate of ovarian failure. Alkylating agents act by transferring alkyl groups to biologically important cellular constituents, in particular alkylation of guanine compound of the DNA which results in miscoding, crosslinking and DNA breakage. The doses used in this study were within the regular experimental range used for single dose intraperitoneal injection in animal studies.
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Materials and methods |
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Results |
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Discussion |
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Clinical studies on the effects of chemotherapy on female fertility which have defined the outcome in dichotomous terms, whether ovarian function was regained or not, have highlighted age as a significant factor in determining the effects of the chemotherapy on subsequent ovarian function. Older women had a much higher incidence of complete ovarian failure and permanent infertility than did younger women (Kumar et al., 1972; Uldall et al., 1972
; Koyama et al., 1977
; Shalet, 1980
; Fisher and Cheung 1984
; Sanders et al., 1996
).
The young age of the mice used in this study, and the results that indicate follicular destruction at all levels of exposure, suggest that ovarian damage occurs in all age groups and is not restricted to older females. The clinical observation that older females appear to be more affected by exposure to chemotherapy can be explained by the fact that older females naturally have a smaller ovarian reserve of follicles. At birth in the human female, there are ~2x106 follicles in the ovary; however, due to a continual process of atresia this reserve is progressively eroded over time (Gosden and Faddy, 1994). At the age of 4550 years, the PMF population falls below a key threshold number required for ovarian function, at which point the menstrual cycle ceases and natural menopause occurs. The chemotherapeutic destruction of an already low follicular reserve reduces the number of follicles in the ovary below a certain `threshold' number required to sustain ovarian function, thus resulting in ovarian failure, that is menopause. Thus the depletion of the PMF store, as presented in this study, may explain the higher incidence of ovarian failure in older women treated with chemotherapy.
Several studies have observed that a significant proportion of younger patients who did regain ovarian function after chemotherapy were at risk of undergoing premature menopause a number of years after treatment (Rose and Davis, 1977; Chapman et al., 1979
; Byrne et al., 1992
; Wallace et al., 1993
), indicating that their ovaries sustained damage which became evident years later. It is this particular observation which indicates that the effects of chemotherapy on the ovary can be concealed and require more research beyond the limitation of clinical studies. In young females, with a larger reserve of follicles, the chemotherapy-induced loss of PMF may not be proportionally enough to cause immediate ovarian failure. However, the reduction in reserve in addition to natural atretic follicular loss, is the explanation for the increased risk of premature menopause in these patients.
As the results of this study clearly demonstrate, exposure to doses of chemotherapy strong enough to destroy half of the ovarian PMF reserve, does not affect reproductive performance following short term maturation of the exposed primordial follicles (week 3 and 4). There was no influence of treatment on ovulation, mating and pregnancy rates. These results strongly indicate that clinical information such as regular menses and normal reproductive outcome after chemotherapy are not reassuring parameters from which it is possible to conclude that the ovaries survived treatment unaffected (Figure 4).
The results of this study support the recommendation that patients who regain ovarian function post-radiotherapy or chemotherapy treatments should not delay child bearing, given the very real risk of premature ovarian failure. An alternative is to try and preserve the future fertility of young cancer patients through cryopreservation and storage of ovarian tissue, containing many PMF, or embryos prior to radiochemotherapy treatments and consequent PMF destruction.
The significant reduction in the number of pregnancy sacs and the reduced number of corpora lutea that were observed in females mated 1 week following the chemotherapy injection, might suggest that mature follicles are more sensitive to chemotherapy-induced damage. Alternatively, this short interval of time between exposure to cyclophosphamide and conception may in itself inhibit conception due to general effects of the toxin on the mother, in particular on uterine receptivity. These results require further investigation, and are in accordance with observations made by other studies (Kuhajda et al., 1982; Familiari, 1993
; Gosden et al., 1997
).
The direct mechanisms of PMF destruction by chemotherapy are unclear. A possible explanation is that the drug induces apoptosis in the supporting granulosa cells of the follicle, without which it cannot survive. In-vitro studies on human ovarian tissue show that granulosa cells undergo apoptosis as a result of exposure to chemotherapy (Meirow et al., 1997b). Future research is needed to confirm the exact mechanisms of PMF destruction and to determine the apoptotic pathways induced by chemotherapy.
New treatment regimens have and will continue to emerge, and will continue to increase survival rates among young cancer patients. The described animal model may serve as a more accurate tool to study the effects of existing and newly developed chemotherapeutic agents, as well as different protocols, on the gonads. Currently, the standard method for assessing the toxic effects of a new agent on fertility is to check the ability of treated females to conceive and carry pregnancies post-treatment. As this study has shown, conception and pregnancy are not accurate parameters by which to assess the damage caused by a drug to the ovary. Histological counting of primordial follicle number, as described in this study, reflects directly the effects of chemotherapy on the ovaries. This animal assay is a sensitive and inexpensive method of gauging and comparing the damage to fertility caused by new agents or combination of agents, and determining the least toxic agent or protocol before it is used in a clinical setting. This method can also be used to evaluate the practical effectiveness of proposed protectants, such as gonadotrophin-releasing hormone antagonists (Gosden et al., 1997) or antioxidants in circumventing chemotherapy induced gonadal failure.
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Acknowledgments |
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Notes |
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References |
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Submitted on December 21, 1998; accepted on January 1, 1999.