Hypothalamic–pituitary–ovarian dysfunction after prepubertal chemotherapy and cranial irradiation for acute leukaemia

Louise E. Bath1, Richard A. Anderson2,4, Hilary O.D. Critchley3, Christopher J.H. Kelnar1 and W.Hamish B. Wallace1

1 Section of Child Life and Health, Department of Reproductive and Developmental Sciences, University of Edinburgh, 2 MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology and 3 Section of Obstetrics and Gynaecology, Department of Reproductive and Developmental Sciences, University of Edinburgh, Edinburgh, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: We assessed adult hypothalamic–pituitary–ovarian function following treatment with chemotherapy and cranial irradiation for childhood acute lymphoblastic leukaemia. METHODS: The patients (n = 12) had median age at diagnosis of 4.7 years, and at assessment of 20.8 years. They collected a daily urine sample over two to five consecutive menstrual cycles (total of 41 cycles) for analysis of LH and steroid excretion. Blood sampling and ovarian ultrasound examination was performed in the early follicular phase. Sixteen healthy women with regular menstrual cycles were recruited as controls. RESULTS: Urinary LH excretion was significantly lower in patients throughout the cycle, particularly during the LH surge (P < 0.0001). The length of the luteal phase was significantly shorter in patients than in normal controls (12.2 ± 0.3 versus 13.6 ± 0.4 days, P = 0.01) with a high prevalence of short (<=11 days) luteal phases (15/39 cycles). Luteal phase pregnanediol excretion was slightly but not significantly lower. Follicular and luteal phase excretion of oestrone was lower in patients than in controls (P = 0.01). Early follicular phase plasma oestradiol was also lower in the patient group (P = 0.032) although LH, FSH, inhibin A and B concentrations were similar. CONCLUSIONS: These data indicate that treatment for childhood leukaemia results in subtle ovulatory disorder in some patients, probably related to cranial irradiation. Follow-up of these women is required to detect any effect on reproductive potential.

Key words: chemotherapy/leukaemia/ovary/pituitary/radiotherapy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The majority of children presenting now with acute lymphoblastic leukaemia (ALL) will be cured (Chessells et al., 1995Go). In the 1960s the cure rate was <5%. Increased survival over the following 20 years was achieved with chemotherapy and radiotherapy and by the 1980s the survival rate had risen to 70%. This allowed a shift in aim over the last 20 years to reducing treatment-related effects whilst further improving the survival rate. At the start of the 21st century, one in 600 young adults is a survivor of childhood cancer and while the challenge of the next decade is to improve further the high survival rates, increasing attention is being focused on minimizing the late effects of treatment. Central to this is the need to identify these late effects, which may only become apparent many years after successful treatment (Hawkins and Stevens, 1996Go). This is essential not only to reduce the risk for future survivors of childhood cancer, but also to address the current needs of the young adult survivors.

The reproductive system is one of the major sites of secondary effects of anti-cancer treatment (Ogilvy-Stuart and Shalet, 1993Go). Potential adverse effects on reproductive function in the female may be mediated through effects at one or more levels of the hypothalamic–pituitary–ovarian axis (Wallace et al., 1989aGo), or at the uterus (Critchley et al., 1992Go; Bath et al., 1999Go). One of the most commonly recognized adverse effects of anti-cancer treatments is on the ovary, by depletion of the stock of primordial follicles and thus hastening or inducing the menopause. This effect is well-documented with certain chemotherapy drugs, in particular the alkylating agents, and with direct or scatter radiation to the ovary (Wallace et al., 1989bGo; Ogilvy-Stuart and Shalet, 1993Go). The effects on the hypothalamus or pituitary are less obvious. While high dose cranial radiation is recognized to have direct damaging effects, the effects of the relatively low dose used for childhood leukaemia are uncertain. Reports of ovarian function after treatment of standard risk childhood leukaemia have been reassuring (Wallace et al., 1993Go); however, the relatively recent advances in treatment, and thus survival, mean that few patients are >30 years of age. We have therefore carried out a detailed investigation of hypothalamic–pituitary–ovarian function in long-term survivors of childhood ALL.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Women were eligible for recruitment to this study if they were in first clinical remission following treatment for ALL (chemotherapy with cranial irradiation) before puberty, >=5 years out from end of treatment and were post-pubertal at the time of recruitment. A total of 30 such women was identified from the long-term follow-up clinic at The Royal Hospital for Sick Children, Edinburgh. Eighteen were excluded from assessment: one woman was pregnant, eight were using hormonal contraception, seven declined to take part and two had ongoing medical problems. Twelve women were therefore recruited for participation in the study. Sixteen healthy women with regular menstrual cycles were recruited as normal controls.

The combination chemotherapy schedules were the Medical Research Council studies UKALL I, II, V, VIII and X. All these protocols included the use of vincristine, 6-mercaptopurine, prednisolone and methotrexate. Other drugs used in some schedules included asparaginase, cytosine arabinoside, doxorubicin and cyclophosphamide. The protocols evolved with increasing intensity of chemotherapy with subsequent protocols. All patients received cranial irradiation in a total dose of 18–24 Gy, depending on the protocol, and two patients received in addition spinal irradiation (dose 10–14 Gy).

Subject details
The 12 women recruited following treatment for ALL had median age at diagnosis of 4.7 years (range 1.9–13.1), and at assessment 20.8 years (range 15.8–32.8). Mean body mass index (BMI) was 25.8 ± 1.2 kg/m2. The controls were aged 17.3–29.0 years (median 24.1), with regular menstrual cycles (25–32 days) and had not used hormonal contraception in the preceding 3 months. Mean BMI was 23.3 ± 1.2 kg/m2. A full menstrual and pregnancy history was taken from subjects and controls, and all were confirmed to be euthyroid and have normal prolactin concentrations.

The local ethics committee gave approval for the study and informed consent was obtained from all women.

Study design
The women collected a daily early morning urine sample from day 1 of the cycle for a minimum of two cycles in ALL patients (total of 41 cycles) and one cycle in controls. Preliminary data suggested that cycle characteristics were more variable in ALL patients than controls, thus repeated cycles were collected and analysed where possible. At least three cycles were collected by nine of the ALL patients. While control women initially collected two consecutive cycles, in view of the consistency of cycle characteristics in these women, only one cycle per woman was included in the detailed analysis to give the normal comparison group.

All women also attended during the early follicular phase (day 3–5) of a cycle for blood sampling and ultrasound scan of the ovaries. Blood samples were analysed for plasma oestradiol, progesterone, LH, FSH, inhibin A and B, insulin-like growth factor 1 (IGF-1), insulin-like binding protein 3 (IGFBP-3) and prolactin. All ultrasound examinations were performed by one of two radiologists trained in gynaecological scanning using a Hitachi EUB 555. Scans were performed transvaginally (6.5 MHz transducer) in sexually active women and transabdominally (3.5 MHz transducer) in those who were not. Ovarian volume was measured in three orthogonal diameters and using the formula for a prolate ellipsoid (d1xd2xd3)x0.523 (Holm et al. 1995Go).

Hormone analyses
Urine samples were analysed for LH, oestrone conjugates and pregnanediol-3-glucuronide (P3G). Urinary oestrone conjugates and P3G were measured using an `in-house' enzyme-linked immunoabsorbent assay (ELISA) using horseradish peroxidase-conjugate as label and solid-phase second antibody separation (for oestrone conjugates, CV <4%; for P3G, CV <13%). Urinary LH was measured by a two-site immunoradiometric assay (Serono; MAIAclone, CV <12%). Urinary hormone concentrations were corrected for creatinine concentration. Serum oestradiol was measured by competitive immunoassay using the Boehringer Mannheim Elecsys (Mannheim, Germany). Progesterone, FSH and LH were measured by microparticle enzyme immunoassay (Abbott Axsym, Chicago, IL, USA). Inhibin A and B were measured by two-site ELISAs as previously reported (Groome et al., 1994Go). For inhibin B, assay sensitivity was 7.8 pg/ml, intra- and inter-plate CVs were 10.6 and 11.4% respectively and for inhibin A, intra- and inter-plate CVs were 5.0 and 12.7% respectively. IGF1 and IGFBP3 were measured as previously described (Blum, 1996Go).

Statistical analysis
The day of onset of menses was defined as cycle day 1, and the last day of the cycle was that preceding the next menstruation, giving cycle length. The last day of the follicular phase was the day of the onset of the LH surge, defined as a minimum of a 2.5-fold rise in LH excretion over the preceding 4-day mean. Follicular and luteal steroid excretion was calculated as the integrated area under the curve using the trapezoid method over the relevant part of the cycle (days 2–5 and days 6–12 to give measures of early and late follicular phases, and days 2–12 for total luteal phase). Peak luteal P3G was calculated as the mean of the 3 consecutive days of maximal excretion, and the LH surge was quantified as the sum of values of the 3 days from the onset of the LH surge. Follicular LH excretion was calculated as the mean of the first 7 days of the cycle.

Hormonal data are presented as mean ± SEM. Data were log transformed to correct non-equality of variance and compared by analysis of variance (ANOVA) and Student's t-test. Initial comparisons investigated differences between controls and all ALL patient cycles. If this indicated a significant difference, data were further analysed by ANOVA after subdivision of ALL patient cycles into those with normal or short luteal phases, cycles with a luteal phase length of <=11 days being defined as short. Data showing significant non-equality of variance (cycle characteristics and ovarian volume) were analysed using the Mann–Whitney test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Cycle characteristics
All 12 women treated for childhood ALL had achieved adult sexual development and the onset of menses. They reported regular menstrual cycles (26–30 days), and this was confirmed during the study when ovulatory cycles were demonstrated in all subjects. None had been pregnant. A total of 41 cycles from ALL patients was analysed. Two cycles from ALL patients were anovulatory (no LH surge and no rise in P3G), both in women who also had normal ovulatory cycles. These two cycles were excluded from further analysis. No anovulatory cycles were seen in the control women.

Overall cycle length was not different in ALL patients compared with controls (28.1 ± 0.5 versus 28.7 ± 0.7 days, Figure 1Go). Length of the follicular phase was also similar (15.9 ± 0.4 versus 15.2 ± 0.6 days) but the length of the luteal phase was significantly shorter in ALL patients (12.2 ± 0.3 versus 13.6 ± 0.4 days, P = 0.01). Closer analysis of luteal phase length indicated a high prevalence of short (<=11 days) luteal phases in ALL patients, whereas only one such cycle was seen in controls. Overall, 15 out of 39 apparently ovulatory cycles in ALL patients showed short luteal phases, in five of the 12 patients. Short luteal phases were not always consistently seen in those ALL patients who demonstrated them: all four cycles from one subject showed short luteal phases, but cycles with normal luteal length were also seen in the other four patients. These cycles are hereafter referred to as `short' and `normal' ALL cycles in distinction to control cycles (i.e. in normal, control women).



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Figure 1. Cycle length in controls (open bars) and ALL subjects (hatched bars). Total cycle length, and duration of the follicular and luteal phases. *P = 0.01. n = 16, control; n = 12, ALL subjects, mean ± SEM.

 
ALL patients with short luteal phases did not differ in age at initial treatment or age at present investigation from those who only showed normal luteal phase length (Figure 2Go). There was, however, a significantly greater interval between initial treatment and time of present investigation in those showing short luteal phases (P = 0.04), consistent with a progressive effect of treatment. BMI did not differ between the two groups (26.1 ± 0.6 kg/m2, normal cycle group; 25.4 ± 1.9 kg/m2, short luteal cycle group).



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Figure 2. Age at initial treatment, present investigation, and interval since treatment in ALL patients who showed cycles with short luteal phases (n = 5, open bars) and those who only showed cycles with normal luteal phases (n = 7, hatched bars). *P = 0.04. Mean ± SEM.

 
Urinary hormone excretion
Detailed analysis of daily urinary hormone excretion revealed several differences between ALL patients and controls. During the follicular phase, LH excretion was lower in ALL patients than controls (P < 0.002, Figure 3aGo), with lowest concentrations in short luteal phase cycles which were significantly different both from controls (P < 0.002) and ALL cycles with normal luteal phase length (P = 0.03). Normal length ALL cycles had lower LH concentrations than control cycles (P < 0.05). A more marked difference in LH excretion was seen during the LH surge (Figure 3bGo). Mean 3-day LH excretion was significantly lower in ALL cycles than controls (P < 0.0001), with both short and normal ALL cycles showing this difference (P = 0.0005 and P = 0.004 respectively versus control). There was no significant difference between normal and short ALL cycles in this respect. LH excretion remained lower in ALL patients than controls throughout the luteal phase (Figure 3bGo).



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Figure 3. Urinary LH excretion in control and ALL subject cycles. (a) Mean day 1–7 LH excretion in controls (open bars, n = 16) and ALL subjects (filled bars, n = 39). The stippled and hatched bars represent LH excretion in the ALL subgroups of cycles with short(n = 15) and normal (n = 24) luteal phase length respectively. *P < 0.05; **P < 0.002 versus control cycles; {dagger}P < 0.05 short versus normal ALL cycles. (b) Daily LH excretion centred on the day of onset of the LH surge (cycle day 0) in control cycles (filled circles,n = 16) and cycles for ALL subjects with normal (open triangles, n = 24) and short (open squares, n = 15) luteal lengths. Mean ± SEM.

 
Follicular phase oestrone conjugates excretion during both early (days 2–5) and late (days 6–12) follicular phase was lower in ALL cycles (P = 0.01 in both cases, Figure 4a and bGo). Reduced excretion was only lower than control in normal ALL cycles (P <= 0.001, early and late follicular phases) whereas there were no differences between control and short ALL cycles. Oestrone conjugates excretion was lower in normal than short ALL cycles at both stages of the follicular stage (P < 0.05 in both cases).



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Figure 4. Urinary steroid excretion in control and ALL subject cycles. (a) Early (days 2–5) and (b) late (days 6–12) follicular phase E1C excretion; (c) luteal phase E1C excretion; (d) luteal phase P3G excretion. Controls (open bars, n = 16), ALL subjects (filled bars, n = 39). The stippled and hatched bars represent LH excretion in the subgroups of ALL patient cycles with short(n = 15) and normal (n = 24) luteal phase length respectively.**P < 0.001 versus control; {dagger}P < 0.05 versus short luteal length ALL cycles.

 
Luteal phase excretion of E1C was also lower in ALL cycles than controls (P = 0.01) (Figure 4cGo). Reduced excretion of E1C was detected in both normal and short ALL cycles compared with controls (P = 0.04 and P = 0.03 respectively), but there was no significant difference between normal and short ALL cycles. Luteal phase P3G was slightly but not significantly lower in ALL cycles than controls, both when analysed as a 3-day peak or total luteal phase excretion (Figure 4dGo).

Plasma hormones
Blood samples taken during the early follicular phase of the cycle (days 3–5) were analysed for LH, FSH, oestradiol, inhibin A and inhibin B concentrations. LH and FSH concentrations were similar in ALL patients to controls (Figure 5aGo), but oestradiol was lower in ALL patients than controls (P < 0.05, Figure 5bGo). Inhibin A and B were similar in ALL patients and controls (Figure 5c and dGo). IGF1 and IGFBPs (not shown) were compared with published reference ranges (Blum, 1996Go) and were not significantly different from the reference range.



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Figure 5. Early follicular phase serum hormones in control and ALL subjects. Controls, open bars, n = 16; ALL subjects, filled bars, n = 12. *P < 0.05 versus control. Mean ± SEM.

 
Ovarian volumes
Mean ovarian volume in the ALL group was 4.8 ± 0.54 ml, which was not significantly different from that in controls (5.4 ± 0.57 ml). Ovarian morphology was normal in all subjects.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The treatment of childhood cancers frequently involves administration of drugs and radiation with potential adverse effects on reproductive function. There are few published data regarding long-term follow-up of fertility after standard treatment for ALL (Nygaard et al., 1991Go; Wallace et al., 1993Go). While fertility and successful pregnancies have been reported in patients after the treatment of childhood ALL with standard MRC regimens (and indeed three of the 30 patients identified but not studied here had had successful pregnancies), this does not rule out a significant and progressive effect on reproductive function. In a study of reproduction following treatment for childhood ALL in the Scandinavian countries, women who had received prophylactic radiation of the central nervous system had a significantly lower first birth rate than those without radiation, indicating that doses of 18–24 Gy to the brain may be a possible risk factor (Nygaard et al., 1991Go). Detailed analysis of endocrine function at long-term follow-up has not been previously reported.

We postulated that chemotherapy might have reduced the number of primordial/primary follicles in the ovary of patients with ALL. This was assessed by early follicular phase hormone assay and by ultrasound measurement of ovarian volume. While there are few direct data on changes in these markers of ovarian reserve in the context of chemotherapy, there is considerable evidence for their value in detecting the changes associated with normal ageing, and as predictors of the ovarian response to superovulation which is believed to be an index of the total follicular pool (Cruz and Gindoff, 1999Go). Ageing is accompanied by a progressive decline in the number of follicles within the ovary (Gougeon et al., 1994Go), which cannot be replaced. A rise in early follicular phase FSH with maintained oestrogen production is well recognized to be a feature of the perimenopause (Sherman et al., 1976Go; Reyes et al., 1977Go), and is detectable from ~20 years prior to the menopause (Ahmed Ebbiary et al., 1994Go). The pattern of secretion of inhibin B across the menstrual cycle is consistent with secretion by the developing cohort of follicles, whereas inhibin A is predominantly secreted by the dominant follicle (Groome et al., 1994Go, 1996Go; Welt et al., 1997Go). Inhibin B concentrations are reduced in the early follicular phase in older women (Klein et al., 1996Go; Welt et al., 1999Go), and are correlated with the ovarian response to exogenous gonadotrophin stimulation (Seifer et al., 1997Go; Hall et al., 1999Go; Eldar-Geva et al., 2000Go). Inhibin B appears to be the earliest endocrine marker of the perimenopause (Burger et al., 1998Go). Similarly, ovarian volume predicts the number of recruitable follicles during superovulation (Syrop et al., 1999Go). ALL patients did not show higher FSH concentrations or lower inhibin A and B concentrations than controls, or have smaller ovaries. These results therefore appear to be reassuring as to the number of primordial follicles remaining in the ovaries of these young girls after chemotherapy. The lack of sensitivity of the available markers and very limited longitudinal data (Welt et al., 1999Go) suggests, however, that such reassurance should be limited to stating that there was no evidence of any subjects being in the perimenopause. Further longitudinal data are required to assess the predictive value of these apparently normal results in this context.

By contrast, daily analysis of urinary hormone excretion provided clear evidence of abnormal reproductive function in some ALL patients. The great majority of monitored cycles in ALL patients were ovulatory, based on the presence of an LH surge and a rise in P3G excretion. This was consistent with the regular menses reported by these patients. However, LH excretion was reduced in ALL patients throughout the cycle, most markedly during the LH surge. There was also a high prevalence of cycles with short luteal phases in the ALL patients. While we have no direct evidence that this is related to reduced LH secretion in these patients, the LH surge was most deficient in cycles with short luteal phases. It is well-recognized that the function of the corpus luteum is dependent on LH secretion (Hutchison and Zeleznik, 1984Go) and in particular the magnitude and duration of the LH surge (Zelinski-Wooten et al., 1991Go). The apparent variability in the prevalence of short luteal phases between and within subjects (although based on small numbers) is consistent with the observed partial reduction in the magnitude of the LH surge rather than a more complete inability to mount the surge. Furthermore, the interval between treatment and investigation was longer in those patients showing short luteal defects, consistent with a progressive effect. Pituitary function shows progressive compromise following cranial irradiation in higher dose than received by the patients described here (Littley et al., 1989Go). Low dose central nervous system directed radiotherapy, as part of treatment for ALL, has been previously associated with perturbations in growth hormone secretion (Crowne et al., 1992Go; Brennan et al., 1998Go) but there are no previous reports of an effect on other pituitary hormone secretion in adulthood (Birkebaek et al., 1998Go).

Growth hormone (GH) is the primary stimulator of the synthesis of IGF-1. GH insufficiency has been associated with decreased fertility (Pellicer et al., 1994Go). IGF-1 may amplify the effects of gonadotrophins on ovarian tissue (Barreca et al., 1993Go) and has been postulated to have an effect on uterine receptivity (Potashnik et al., 1995Go). In this study, concentrations of IGF-1 were within the normal range and did not show a correlation with time since treatment. These data do not suggest a significant GH deficiency in these patients. However, subtle defects in GH secretion may not be detected by IGF-1 analysis.

Excretion of oestrone conjugates was significantly lower in the follicular phase in ALL cycles. Early follicular serum oestradiol concentrations were also lower in ALL patients. As oestradiol production requires both LH and FSH (The European Recombinant LH Study Group, 1998Go), this may reflect a reduction in gonadotrophic stimulus that was detected in the reduced urinary LH excretion in ALL patients. There was also evidence for reduced steroid excretion during the luteal phase, particularly in cycles with short luteal phases. This reached statistical significance only for E1C excretion, reflecting the greater intercycle variability in P3G excretion.

Current fertility prospects for this group of survivors appear to be good, as evidenced by successful pregnancies in three of the 30 long-term survivors identified (Nicholson and Byrne, 1993Go). However, this cohort is still young, with the majority not having tested their fertility. While none of the women showed evidence of overt ovarian damage, this may reflect our inability to detect this until relatively late when a woman has entered the perimenopause. The risk of premature menopause following childhood chemotherapy remains to be clarified (Byrne et al., 1992Go). Ovarian dysfunction has been reported following chemotherapy for standard risk ALL, but the chemotherapy protocols included greater doses of alkylating agents (Quigley et al., 1989Go). The currents trends in treatment of ALL are to increase the intensity of chemotherapy to improve survival (Hann et al., 2000Go). The late effects of the current treatment on reproductive function can only be measured many years after completion of therapy and the risk for the children currently undergoing chemotherapy without cranial irradiation may be greater than the cohort we are currently following, whose chemotherapy schedules were less intense.

These data demonstrate that low dose cranial irradiation has an adverse effect on the hypothalamic–pituitary–ovarian axis that may be progressive over time. Apparently minor disturbances in LH secretion may have an effect on reproductive potential: conception in normal women is more likely in cycles with greater LH surges and higher luteal phase progesterone and oestradiol (Baird et al., 1999Go). Short luteal phases are associated with reduced fertility and early miscarriage (Soules et al., 1989Go). These data therefore indicate the importance of continuing assessment of reproductive function in this cohort of survivors to detect effects of treatment that may only become apparent many years later.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Thanks to Dr Sarah Chambers and Dr Jane Walker for their assistance with the ultrasound scanning, to Dr Angela Thomas for allowing us to recruit her patients and to Mrs Martha Urquart for help with the urinary steroid analysis.


    Notes
 
4 To whom correspondence should be addressed at: MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, 37 Chalmers St., Edinburgh EH3 9ET, UK. E-mail: r.a.anderson{at}hrsu.mrc.ac.uk Back


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 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on February 5, 2001; accepted on June 5, 2001.