1 Department of Reproductive and Developmental Sciences, University of Edinburgh and 2 MRC Human Reproductive Sciences Unit, University of Edinburgh Chancellors Building, Edinburgh, UK
3 To whom correspondence should be addressed at: Department of Reproductive and Developmental Sciences, University of Edinburgh, Edinburgh, UK. e-mail: Louise.Bath{at}luht.scot.nhs.uk
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Abstract |
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Key words: anti-Müllerian hormone/antral follicle count/chemotherapy/inhibin B/ovarian reserve
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Introduction |
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The ability to predict a womans reproductive lifespan would be of considerable value to those long-term survivors of childhood cancer who may be counselled not to delay childbearing. Current fertility prediction is limited. FSH is widely used in clinical practice, but shows considerable intercycle variability (Sherman et al., 1976; Ahmed Eddiary et al., 1994
; Wallach, 1995
) and reflects the sum of both central hypothalamic drive and ovarian feedback. Direct products of the ovary, including inhibin B and anti-Müllerian hormone (AMH), have been investigated as markers of ovarian reserve (Seifer et al., 1997
; Tinkanen et al., 1999
; Creus et al., 2000
; Dumesic et al., 2001
; de Vet et al., 2002
), the latter showing particular promise as a marker of the earliest growing follicles (Fanchin et al., 2003b
). Biophysical measures including ovarian volume and antral follicle count (AFC) have also been shown to correlate with reproductive potential (Tomas et al., 1997
; Scheffer et al., 1999
; 2003; Syrop et al., 1999
; Bancsi et al., 2002
; Yong et al., 2003
). AFC has recently been shown to be reduced in women who have survived childhood leukaemia (Larsen et al., 2003
), but no hormonal effects were detected in that group.
The assessment of reproductive potential is complicated in those women taking the combined oral contraceptive pill (COCP). To evaluate ovarian function it has been necessary to stop the COCP, which is both inconvenient and impractical in young women relying on this method of contraception. While it is likely that assessment of ovarian function during COCP administration will remain less accurate than in women cycling spontaneously, it would be of value to assess markers of ovarian function under these conditions.
The objective of the present study was to investigate basal and stimulated hormone production by the ovary in women who have survived cancer treatment as children to detect and assess the degree of loss of ovarian reserve. The relative value of endocrine markers and ultrasound investigation was compared, both in women with regular menstrual cycles and in those taking the COCP.
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Materials and methods |
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Study design
Women with spontaneous menstrual cycles attended during the early follicular phase (days 35), in addition to those taking the COCP during the third week of their pill cycle (i.e. between days 14 and 20). Venesection was performed and the serum was separated and stored at 20°C for subsequent assay. Transvaginal ultrasonography was used to determine ovarian volume and the number of small antral follicles. All women were then administered an injection of 225 IU recombinant human FSH (rhFSH, Gonal F; Serono, Welwyn Garden City, UK) subcutaneously, and returned 24 h later for a further blood sample to be taken. Patients taking the COCP provided a further blood sample on day 7 of a subsequent pill-free week for measurement of FSH to exclude occult ovarian failure: this was in the normal range (<10 IU/l) in all women.
Outcome measures
Immunoassays for FSH (determined as a single value), estradiol, inhibin A, inhibin B and pro-C inhibin forms and ultrasound examinations were carried out as described previously (Yong et al., 2003
). All examinations were performed by the same investigator, and using the same equipment (7 MHz probe; Toshiba Eccocee, Stirling, UK). AMH was measured in a single assay (AMH ELISA; Beckman Coulter, High Wycombe, UK); the intra-assay coefficient of variation was 7%. Ovarian volume was calculated from three orthogonal diameters using the formula for a prolate ellipsoid (
/6xd1xd2xd3), and the mean volume of the two ovaries was calculated. AFC was determined as the mean of the number of follicles 210 mm diameter in the two ovaries.
Statistical analysis
The day of onset of menses was defined as day 1 of the cycle. Hormonal data were presented as mean ± SEM, and compared using Students t-test after log transformation to correct for heterogeneity of variance. Ovarian volume and AFC were compared using the MannWhitney U-test.
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Results |
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Discussion |
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Two hormonal differences were detected between cancer survivors and controls. First, early follicular FSH was significantly elevated, but was high in only one patient (18.5 IU/l, others all <10.2 IU/l). However, there was a striking fall in serum AMH concentrations, while the other direct products of the ovaryestradiol and the inhibins A, B and pro-Cwere unchanged. AMH is produced by the granulosa cells of small growing follicles (Baarends et al., 1995
), is involved in the regulation of primordial follicle recruitment (Durlinger et al., 1999
), and has recently been suggested as a marker of ovarian ageing (de Vet et al., 2002
). The circulating concentration shows little fluctuation over the menstrual cycle (Cook et al., 2000
), but is a relatively good predictor of the number of small FSH-sensitive follicles and thus of the number of oocytes recovered following controlled ovarian stimulation (Seifer et al., 2002
; Van Rooij et al., 2002
; Fanchin et al., 2003a
). Consistent with previous reports, serum AMH was unchanged by administration of a single dose of FSH. The present data are thus consistent with a partial depletion of the follicular reserve in these patients and provide the first demonstration of a fall in an ovarian hormone in such cancer survivors with regular menstrual cycles.
Ovarian volume, but not AFC, was also reduced in the cancer survivors compared with controls. Both ovarian volume and AFC are indirect markers of ovarian reserve (Tomas et al., 1997; Scheffer et al., 1999
; 2003; Syrop et al., 1999
; Bancsi et al., 2002
; Yong et al., 2003
), and have been reported to be reduced in female survivors of childhood cancer (Larsen et al., 2003
). Inhibin B, a product of these follicles, was also normal. It has recently been shown that the value of inhibin B as a measure of the ovarian reserve is greatly increased following administration of a single dose of FSH to stimulate granulosa cell function in small healthy follicles (Yong et al., 2003
). While these results show the expected increase in inhibin B following FSH administration, there remained no difference between patients and controls. Taken together with the AFC results, these data are consistent with these cancer survivors having a near-normal compliment of small antral follicles. The discrepancy between these normal results and the clearly reduced AMH data may illustrate the problems inherent in trying to assess the number of primordial follicles present using only indirect means. As AMH is the product of the smallest growing follicles, it may more accurately reflect the ovarian reserve than AFC and basal and stimulated inhibin B. The latter two markers reflect the number of FSH-sensitive small antral follicles and therefore show relationships to the number of oocytes recovered following controlled ovarian stimulation, but it appears they are unable to reflect the reduced ovarian reserve exhibited by the cancer survivors investigated herein.
Groups of cancer survivors and controls taking the COCP were also investigated in the present study. The ability to carry out accurate assessment of the ovarian reserve under such conditions would be of great practical value in light of the high prevalence of oral contraceptive use among young women. Assessment under hypogonadotrophic conditions might also be advantageous: it has been demonstrated that (GnRH analogue-induced) hypogonadotrophism with subsequent FSH stimulation greatly increased the correlation between inhibin B and oocyte recovery following controlled ovarian stimulation (Yong et al., 2003). As with the spontaneously cycling groups, cancer survivors taking the COCP showed both hormonal and biophysical differences from the control group, although the differences were in distinct measures of ovarian function. During COCP administration, inhibin B was suppressed to undetectable concentrations in both cancer survivors and controls. In response to FSH administration, the two groups showed similar responses overall, but analysis of individual responses showed that whereas all controls showed a response to FSH, only six of 10 cancer survivors showed a response, with inhibin B remaining undetectable in the others. AFC was lower in women taking the COCP (significantly so for the cancer-survivor groups), but there was also a significant difference in AFC between COCP-taking cancer survivors and COCP-taking controls. Thus, in contrast to the spontaneously cycling groups, during COCP administration there were differences between cancer survivors and controls in AFC and inhibin B. These results again suggest that cancer survivors show a degree of depletion of ovarian reserve and, under conditions of hypogonadotrophism, this is reflected in the number of FSH-sensitive antral follicles. As the degree of loss of ovarian reserve is modest, it may be that under normal conditions the larger number of growing follicles has obscured an ability to detect differences between the groups.
Ovarian volume and serum AMH were however not significantly different between COCP-taking cancer survivors and controls. Both these markers were significantly reduced in the COCP controls compared with the spontaneously cycling controls, indeed to values similar to those found in the spontaneously cycling cancer survivors. It appears likely that the effect of the COCP has obscured the value of these markers of the ovarian reserve. Ovarian volume, AFC, inhibin B and AMH are all to greater or lesser extents markers of ovarian reserve. They are however both indirect and, while inter-related, reflect slightly different aspects of ovarian function. The present results further illustrate the relationships between these markers under different conditions, and confirm the need for a range of markers for a full assessment of the ovarian reserve.
Some chemotherapy agents are believed to be more gonadotoxic than others (Whitehead et al., 1983; Mackie et al, 1996
). In particular, alkylating agents are recognized to carry a significant risk of premature ovarian failure. Greater than half the group of cancer survivors [acute lymphoblastic leukaemia (ALL) and Wilms tumour survivors] have received treatment believed to have little or no ovarian toxicity (Wallace et al., 1993
), although craniospinal irradiation may have indirect adverse effects on ovarian function (Bath et al., 2001
). However, of the six patients taking the COCP who showed no inhibin B response to FSH, three had been treated for ALL. Analysis is limited by the small number of patients treated for a variety of primary diagnoses with different chemotherapy regimens, but these data show that even these therapies are associated with a detectable loss of ovarian reserve.
In conclusion, survivors of childhood cancer were demonstrated to have suffered a depletion of the ovarian reserve despite maintaining regular menstrual cycles. Serum AMH concentrations and measurement of ovarian volume by transvaginal ultrasound scan were found to be the clearest indicators of this. Evidence of a similar effect was also detected despite COCP administration using different markers. Regular menstrual cycles and normal early follicular phase FSH do not therefore confirm the absence of damage to the ovary, and such patients should be advised accordingly. Long-term follow-up of these patients is necessary to evaluate further ovarian function and definitively correlate these indirect markers of ovarian reserve investigated here with a true reproductive lifespan. The prospective, systematic collection of data will also, in the future, allow a more accurate prediction of reproductive potential.
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Acknowledgements |
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References |
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Submitted on June 23, 2003; accepted on August 6, 2003.