1 Department of Obstetrics and Gynecology, Rabin Medical Center, Petah Tigva, Israel, 2 Department of Obstetrics and Gynecology, Hadassah Medical Center, Jerusalem, Israel, 3 Centre for Reproduction, Growth and Development, University of Leeds, UK and 4 Women's Pavilion, Royal Victoria Hospital, Montreal, Canada
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Abstract |
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Key words: cyclophosphamide/malformation/oocyte/pregnancy/resorption
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Introduction |
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Many studies have demonstrated that cyclophosphamide and many other chemotherapeutic agents cause gene mutations, chromosomal breaks and rearrangements, and aneuploidy in somatic cells, as well as an increased frequency of secondary, treatment-related tumours in human cancer survivors (Sandoval et al., 1993; Povirk and Shuker, 1994
; Ben-Yehuda et al., 1996
). In the gonads cyclophosphamide treatment has been shown to cause primordial follicle destruction, and may, in high doses, result in adverse reproductive consequences, including premature menopause and sterility (Himelstein-Braw et al., 1978
; Apperley and Reddy, 1995
; Meirow et al., 1997
; Chiarelli et al., 1999
). Moreover, animal studies have shown clear evidence that cyclophosphamide causes injury to germ cells as well as induction of transmissible genetic damage (Generoso et al., 1971
; Becker and Schoneich, 1982
; Pdydn and Ataya, 1991
), and this has raised serious concerns regarding the risk of abortions, birth defects, genetic or neoplastic disease in the offspring of cancer survivors who retain fertility after treatment. However, studies on pregnancy outcome in human survivors of cancer treatment have suggested that such concerns are unfounded. Despite the observed increase in germ cell mutations, offspring of women exposed to cancer treatments do not have a greater than normal risk of chromosomal or congenital abnormalities (Hawkins, 1994
; Sanders et al., 1996
). However, these data were collected from women who became pregnant a considerable time (often years) after cessation of therapy. Thus, the lapse of a significant period of time between exposure to the mutagenic drugs and conception may enable the oocytes within the primordial follicles to instigate DNA repair mechanisms to correct any genomic damage (Ashwood-Smith and Edwards, 1996
), or to be eliminated.
With the advent of new reproductive technologies, centres now offer patients the option of oocyte retrieval and embryo cryopreservation before commencement of chemo/radiotherapy in order to preserve fertility. Some centres also offer oocyte retrieval to patients who enter remission following initial chemotherapy treatment cycles, prior to their exposure to more intensive sterilizing treatment. Oocytes retrieved at this point have suffered very recent exposure to chemotherapy and may have been at non-dormant growth stages rather than the primordial stage. It therefore becomes essential to determine if greater risks are associated with oocytes exposed to cytotoxic treatment during or immediately preceding growth stages. The aim of this study was to investigate whether the stage of oocyte development at the time of exposure to cyclophosphamide alters the risks in resultant conceptions. Thus, pregnancy outcome was assessed in terms of fetal viability and teratogenicity in mature female mice exposed to non-sterilizing doses of cyclophosphamide at different intervals prior to conception corresponding to different stages of oocyte maturation.
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Materials and methods |
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The females were examined daily for evidence of vaginal copulatory plugs, and then removed to a separate cage where they were housed for 12 days and then killed by cervical dislocation. It was decided to terminate the pregnancies after 12 days because at this stage it is still possible to count the corpora lutea, allowing for assessment of the number of ovulated oocytes, resorption sites from non-viable fetuses are visible, and the viable fetuses are developed enough to examine for malformations. The uterus and both ovaries were removed and the following measurements taken: the number of corpora lutea were counted under the light binocular microscope, the numbers of viable and non-viable fetuses were noted, each conceptus was weighed (including the placenta, membranes and amniotic fluid). Fetuses were dissected and inspected for gross anomalies under the light microscope (magnification x10x30). Those animals which did not show vaginal plugs were separated from males after 5 days, and killed 9 days later (estimated to be approximately day 12 of pregnancy if conception occurred midway through the 5 day mating period that is the average length of an oestrous cycle). Where non-plugged mice did become pregnant, all of the above parameters were examined except for the fetal weights, since the exact gestational age was unknown.
The data were grouped into three categories, based on the following criteria (Table I): (i) pregnant group (had pregnancy sacs): (a) vaginal plug found (timed mating): (b) no vaginal plug (non-timed mating). (ii) non-pregnant group (no pregnancy sacs): (a) vaginal plugs and corpora lutea; (b) vaginal plugs but no corpora lutea; (c) neither vaginal plug nor corpora lutea.
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Ethical approval for animal experimentation was received from the Ethical Committee of Hadassah Medical CenterHebrew University.
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Results |
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Discussion |
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The higher rate of malformations observed across the groups treated (19 weeks), at least 10 times greater than that seen in the control group, indicates that oocytes exposed to cyclophosphamide during growing stages suffer from increased sublethal damage as a result of the teratogenic effects of cyclophosphamide. The increase in malformation rate was not the same in all the groups. The results showed that the rate of malformations peaked in the group treated 3 weeks prior to mating (oocytes exposed as follicles just beginning the maturation process). Thereafter malformation rate dropped significantly in the group of mice treated 4, 6, and 9 weeks before mating and approached near normal levels at 12 weeks (Figure 3).
This would indicate that oocytes, which began the maturation process during chemotherapy treatment, were most susceptible to non-lethal damage. The subsequent drop in malformation rate following a critical period may be due to the ability of oocytes to repair induced DNA damage (Ashwood-Smith and Edwards, 1996). Another possible option for the reduction in malformation rate is that damaged oocytes were gradually lost during the following weeks. Increased teratogenicity cannot be due to direct effects of cyclophosphamide on the fetus since the drug and its metabolites are cleared from the body within hours (Genka et al., 1990
). This rise in fetal malformations peaking in week 3 and then decreasing again has also been observed in another animal study which looked at the malformation rate following treatment with radiation therapy (Kirk and Lyon, 1982
). In that study the incidence of abnormalities and loss increased with the time interval between exposure and mating, peaking in animals treated 23 weeks before mating, followed by a significant decrease when the interval was between 34 weeks. Other studies (Brewen et al., 1976
; Caine and Lyon, 1977
; Russell, 1977
) also bring evidence that conceptions occurring approximately 3 weeks after exposure are most vulnerable to the mutagenic effects of radiation.
The ability of the oocyte to recover over time may explain the apparent contradiction between the increase in malformations shown in animal studies following recent exposure to chemotherapy, and clinical studies showing that offspring of female chemotherapy survivors have no greater incidence of abnormalities or malformations. Until now, children of cancer therapy survivors were by necessity conceived a considerable length of time after cessation of treatment. Those conceptions resulted from oocytes which had been exposed to therapy in a dormant stage often years earlier (Hawkins, 1994).
Significant numbers of cancer patients suffer infertility post-chemotherapy treatment. In order to preserve fertility, one of the options available is to freeze-bank embryos, which involves follicle aspiration and IVF prior to chemotherapy administration (Apperley and Reddy, 1995). Unfortunately, there are a number of associated difficulties, which prevent widespread use of this procedure for cancer patients. Time is one of the major obstacles, since several weeks of monitoring and ovarian stimulation are often required, and in most cancer patients chemotherapy cannot be delayed. For this reason, some centres tend to offer IVF and embryo cryopreservation during a suitable break in treatment (Brown et al., 1996
) or during first remission prior to implementation of sterilizing protocols (Lipton et al., 1997
).
The safety of using IVF and embryo cryopreservation in cancer patients who have recently undergone chemotherapy treatments is questionable. These agents may cause mutations, DNA adducts and structural breaks as well as oxidative damage. Therefore, concerns have been raised as to the quality of embryos fertilized from oocytes harvested immediately following treatment. The full span of follicle growth from the primordial to Graafian stage in humans is in the order of 612 months (Wasserman, 1996; Gougeon, 1996
) and thus oocytes collected from patients within 612 months of cancer treatment could be compromised. This corresponds to approximately 3 weeks in mice.
This animal model addresses the differences in toxicity response to anti-cancer treatments at different stages of follicular maturation. Results indicate that exposure of mouse oocytes to chemotherapy during different growth stages induces a decrease in implantations and viable pregnancies, and an increase in fetal malformations, which concurs with results from other animal studies (Brewen et al., 1976; Caine and Lyon, 1977
; Russell, 1977
; Kirk and Lyon, 1982
). Although there is not yet any evidence to suggest equivalent treatment-induced genetic abnormalities in human oocytes, it would be unwise to ignore potential risks to the offspring of women exposed to cancer treatments. These results suggest that we cannot necessarily apply the available clinical data concerning pregnancy outcome years after exposure to chemotherapy to pregnancies which result from oocyte collection, IVF and embryo cryopreservation immediately after chemotherapy treatments. Further research is needed to clarify the effects of exposure of human oocytes to cytotoxic treatment during growth stages, in particular whether they are at an increased genetic risk. If so, it will become vital to define a `safety period' between cessation of treatment and oocyte retrieval for IVF. Until definitive data are achieved, it would seem advisable to monitor the pregnancy outcome of all cancer patients who undergo oocyte retrieval and IVF, and possibly to screen fetuses and babies for chromosomal aberrations and birth defects.
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Acknowledgements |
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Notes |
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References |
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Submitted on September 14, 2000; accepted on January 19, 2001.