1 Department of Molecular and Clinical Endocrinology and Oncology, 2 Division of Haematology, 3 Department of Gynaecology and Obstetrics, Federico II University of Naples, via S.Pansini 5, 80131 Naples and 4 Chair of Obstetrics and Gynecology, University of Cantazaro, Magna Graecia, Italy
5 To whom correspondence should be addressed at: Department of Molecular and Clinical Endocrinology & Oncology, Federico II University of Naples, Italy. e-mail: colao{at}unina.it
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Abstract |
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Key words: busulphan/cyclophosphamide/graft-versus-host disease/ovarian failure/stem cell transplantation
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Introduction |
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Ovarian failure associated with SCT has been generally ascribed to total body irradiation (TBI) and antiblastic alkylating agents, both widely used for the treatment of haematological malignancies and in conditioning regimens to transplant (Schubert et al., 1990; Shalet et al., 1995
; Couto-Silva et al., 2001
). Ovarian damage results in immediate menopause in most women; recovery is rare, being less frequent at older age (Schubert et al., 1990
; Shalet et al., 1995
). The conditioning regimen aims at eradicating the underlying malignant disease and, in the allogeneic setting, at suppressing the host immune system. Conditioning regimens avoiding TBI, such as the combination of busulphan and cyclophosphamide, have been employed more frequently in recent years: such a combination, however, still confers a consistent risk for developing ovarian failure, by altering germ cell viability and gonadal hormone production (Sanders et al., 1988
; Afify et al., 2000
; Sklar et al., 2001
). Additionally, a cytokine storm is released in the peri-transplantation period (Jadus et al., 1992
), and a prolonged immune deficiency usually occurs due to delayed and incomplete recovery of the immune system in both allogeneic and autologous SCT recipients.
Allogeneic graft compromises the host immune system more severely than the autologous one due to a prolonged treatment by immunosuppressive drugs needed to avoid the reverse rejection. Indeed, acute or chronic graft-versus-host disease (aGVHD, cGVHD)a complication affecting >50% of patients after allograftinginduces additional relevant alteration in the immune system (Sullivan et al., 1991).
We have recently demonstrated that cGVHD profoundly impairs endocrine functions in allo-STC recipients, even in the absence of TBI (Tauchmanovà et al., 2002). Moreover, cGVHD was associated with lower sperm count in male long-term survivors after busulphan/cyclophosphamide conditioning (Grigg et al., 2000
). It should be emphasized that most previous studies focused on the etiopathogenetic role of chemotherapy and TBI in inducing ovarian failure and infertility and considered allo- and auto-SCT patients as one group (Sanders et al., 1988
; Chatterjee and Goldstone, 1996
; Schimmer et al., 1998
; Afify et al., 2000
; Grigg et al., 2000
; Couto-Silva et al., 2001
; Tauchmanovà et al., 2002
). Conversely, we observed more frequent recovery of menstrual cycles in women after auto-SCT than in those all-transplanted.
To investigate whether differences in ovarian residual function exist in the allogeneic and autologous setting treated with similar conditioning regimen without TBI, we designed this observational, analytical, prospective, controlled study.
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Materials and methods |
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Five women were excluded because of hyperthyroidism (n = 1), delayed puberty (n = 1), history of oligomenorrhoea prior to the disease (n = 2) and radiation treatment (n = 1). In the remaining 45 patients, primary diseases were acute or chronic myelogenous leukaemia (AML, n = 19 and CML, n = 7 respectively), acute lymphoblastic leukaemia (ALL, n = 4), chronic lymphocytic leukaemia (CLL, n = 3), Hodgkin disease (HD, n = 8) and non-Hodgkin lymphoma (NHL, n = 4). Allo- and auto-transplanted patients with AML received SCT in their first complete remission, patients with ALL, HD, NHL and CLL were transplanted in second complete remission. Profile at study entry of the patients is summarized in Table I.
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Informed consent was given by all patients and the study was designed in accordance with the Helsinki II Declaration.
Treatments
Both allo- and auto-transplanted patients for AML, CML and ALL received the same conditioning regimen BUCY2, including busulphan (16 mg/kg in 4 days) and cyclophosphamide (120 mg/kg in 2 days). Patients with HD, NHL and CLL were conditioned with the BEAM protocol, consisting of carmustine (300 mg/m2 in 1 day), etoposide (200 mg/m2 in 4 days), cytarabine (400 mg/m2 in 4 days) and melphalan (140 mg/m2 in 1 day). Allo-SCT recipients received bone marrow-derived stem cells from HLA-identical siblings. After allografting, aGVHD prophylaxis was performed with cyclosporin A (1 mg/kg i.v. from day 1 to +21, then 10 mg/kg p.o. for 6 months) and short course methotrexate (10 mg/kg for four doses). Acute GvHD was treated by high-dose methylprednisolone (210 mg/kg for 10 days), followed by slow dose tapering in the following 6 months, monitoring patients clinical conditions. Chronic GVHD was treated by prednisolone at doses of 12 mg/kg, associated with CsA at doses ranging from 1 to 8 mg/kg/day. Previous treatment history included alkylating agents for patients with ALL, CLL, HD and NHL (Table I) and consisted in a mean 6 month duration of chemotherapy for all diagnoses. Acute GVHD was considered according to the Glucksberg et al. (1974) grading system, and cGVHD was graded as either limited or extensive, based upon clinical severity and target organ involvement (Shulman et al., 1980
). Nine women had been affected by aGVHD of global grade 13 and 13 women were affected by cGVHD (six limited and seven extensive form). Three women with the limited form had liver involvement and the other three skin manifestation. Liver, skin, intestine and eyes were the most common cGVHD sites in the extensive form with variable combination [liver and gastrointestinal (n = 2), liver and eyes (n = 2), skin, gut and eyes (n = 2), skin, gut and kidney (n = 1)]. Liver manifestation included an increase of liver enzymes in all women (ALT: 501100 U/l; AST: 82761 U/l;
-glutamyltransferase: 90350 U/l) and reversible cholestasis with alkaline phosphatase (487785 U/l) and bilirubin increase (59 mg/dl) associated with icterus in two. Skin lesions included lichen planus and focal epidermal atrophy. Ocular dryness (Sjogren-like syndrome) was the major ophthalmological manifestation, while oral mucosa dryness and lichenoid lesions were the upper gastrointestinal tract symptoms. Diarrhoea and malabsorption were present in two women. One woman developed reversible membranous glomerulonephritis with nephrotic syndrome. All these patients were treated as stated above for a period ranging 624 months (Table I). After evaluation, hormone substitution treatment with estradiol and medroxyprogesterone was initiated in all women with amenorrhoea unless contraindicated or refused.
Design
Previous medical records were reviewed to obtain complete information on patients outcome, including data on menstrual history pre-transplantation. Post-transplantation data were recorded prospectively. BMI (kg/m2) was determined in all subjects. In the control group the endocrine and ultrasonography was performed in the early follicular phase of the menstrual cycle (23 day).
Endocrine evaluation
In all subjects, blood samples were obtained between 08:00 and 10:00 a.m. Circulating FSH, LH, prolactin, 17-estradiol, testosterone and
4-androstenedione levels were assayed to assess the hypothalamic pituitaryovarian function. Dehydroepiandrosterone sulphate (DHEAS) was also measured as the major androgen of adrenal origin. All measurements were performed by commercially available kits: 17
-estradiol by radioimmunoassay (RIA) (Nichols Institute Diagnostics, USA), total testosterone and DHEAS using Immulite, solid phase chemiluminescent enzyme immunoassay (DPC, USA); FSH and LH with RIA Biodata S,p.A. kits (Rimini, Italy); androstenedione using RIA Diagnostic Systems Laboratories kit (Webster, USA); at least two different samples were taken in all subjects and the average value was calculated for each hormone. Detection limits were 3 pg/ml for 17
-estradiol, 5 ng/dl for testosterone, 10 ng/dl for
4-androstenedione and 2 µg/dl for DHEAS. Intra-assay and inter-assay coefficients of variation were <7 and <15% respectively, for all endocrine determinations.
Ultrasonographic evaluation
Pelvic ultrasonography was performed in all patients and controls using a 7.5 MHz transvaginal transducer. The study was performed by a single well-trained operator (S.P.), blind in respect to patient or control examination. Ovarian and uterine volumes were estimated using the ellipsoid formula (longitudinalxantero-posteriorxtransversal diameterx0.523) (Pavlik et al., 2001). The mean ovarian volume for each woman was calculated. Number of follicles and endometrium thickness were also recorded.
Statistical analysis
Students t-test for paired and unpaired data was used to compare the group of patients and controls, and different subgroups of patients respectively. The non-parametric MannWhitney U-test was used to compare hormone levels in patients and controls as well as in allo- versus auto-SCT patient group, when WilkShapiros test was not consistent with the Gaussian distribution of the data. To assess the dependence between endocrine and ultrasonographic features of the patients, the Pearsons correlation coefficient was calculated. As possible predictor variables of menstrual cycle recovery, age at transplant, BMI, time from SCT, previous use of alkylating agents and type of transplant were considered. Univariate and regression analysis was performed using the cycle recovery as a binary outcome variable. Two-sided P < 0.05 was taken as statistically significant. Data are expressed as mean ± SEM.
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Results |
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Endocrine evaluation
Endocrine parameters of the two patients groups and controls are shown in Table IV. Serum gonadotrophin levels were significantly higher (P < 0.001) while 17-estradiol,
4-androstenedione, testosterone (P < 0.001) and DHEAS (P < 0.05) were lower in allo-SCT patients than in controls. The auto-SCT women had significantly higher (P < 0.001) gonadotrophin values and lower 17
-estradiol (P < 0.001) than controls while
4-androstenedione and testosterone levels were similar. Serum gonadotrophin levels were significantly higher (P < 0.001) and 17
-estradiol,
4-androstenedione and testosterone significantly lower (P < 0.05) in the allo-SCT compared with the auto-SCT group. Prolactin levels were within the normal range in all subjects, without any difference between patients and controls. Similar results with the same statistical significance were obtained when only women affected by AML were compared (10 auto- and 9 allo-transplanted, data not shown). When allo-SCT patients were divided according to presence of cGVHD, ovarian failure was found to be slightly more severe in women with cGVHD (Table V).
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In the patient group, the mean number of follicles was significantly (P < 0.001) lower than in the controls; and allo-transplanted women had fewer follicles than those auto-transplanted (P < 0.001). In particular, four to 12 small and larger follicles (diameter, <12 mm) per ovary were observed in the control group, several small ovarian follicles (diameter <6 mm) were found in 12 women after auto-SCT and in only four after allo-SCT. In the allo- and auto-SCT groups, without difference between groups, the mean endometrial thickness was significantly (P < 0.05) lower in comparison with controls (Table IV). In fact, endometrial thickness was very low or undetectable in most SCT patients.
With regard to the presence of cGVHD, ovarian and uterine volumes were significantly lower in women with than in those without GVHD (P < 0.001; Table V).
As expected, in the patient group considered as a whole, 17-estradiol (r = 0.51; P < 0.05) and Ä4-androstenedione (r = 0.55; P < 0.05) values correlated with ovarian volume; 17
-estradiol was also inversely correlated with FSH levels (r = 0.67; P < 0.05). Uterine volume correlated with ovarian volumes (r = 0.77; P < 0.01). On the other hand, no correlation was found between age, BMI, time elapsed from transplantation and endocrine or ultrasonographic parameters.
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Discussion |
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Ovarian failure is reported to be a rather frequent event after myelo-ablative therapy followed by SCT in postpubertal patients (Sanders et al., 1988; Schubert et al., 1990
; Shalet et al., 1995
; Chatterjee and Goldstone, 1996
; Grigg et al., 2000
). However, whether it is due to high-dose alkylating agents and/or TBI used in the conditioning regimens, to conventional chemotherapy before myelo-ablative treatment, to the combination of all three treatment approaches (Keilholtz et al., 1997
; Couto-Silva et al., 2001
) or to other unknown factors is still to be fully elucidated. Most previous studies (Sanders et al., 1988
; 1996; Chatterjee and Goldstone, 1996
; Schimmer et al., 1998
; Afify et al., 2000
; Grigg et al., 2000
; Couto-Silva et al., 2001
; Tauchmanovà et al., 2002
), having as their endpoints spontaneous recovery of ovarian function, infertility or pregnancy rates, have focused on the role of different treatments, such as TBI and chemotherapy, in inducing ovarian failure. Additionally, in previous studies no distinction between allo- and auto-SCT subjects has been performed, and different conditioning regimens, also including TBI, were considered altogether. In an attempt to overcome these limitations, we included patients receiving a similar conditioning treatment, excluding TBI, and analysed auto- and allo-SCT patients separately.
One of the relevant finding of the current study, is that allo-transplanted recipients had lower 17-estradiol and androgen circulating levels and lower ovarian volume than in the auto-transplanted ones. Importantly, among allo-transplanted women those who developed cGVHD had lower 17
-estradiol,
4-androstenedione, testosterone and DHEAS and higher FSH and LH levels than those who did not develop GVHD. The potential role of a lower BMI (indicating less fat mass after cGVHD) in inducing androgen aromatization to estrogens seems to be negligible, considering the lower circulating androgen levels and the absence of correlation between BMI and endocrine parameters. It should also be noted that auto-transplanted women had an ovarian volume similar to that observed in women after a corresponding interval from physiological menopause; in contrast the ovarian volume of allo-SCT was lower (Wehba et al., 1996
; Flaws et al., 2000
; Pavlik et al., 2001
).
The differences in endocrine and ultrasound parameters between auto- and allo-transplanted patients were similar in the subgroup including only women with AML, indicating that results did not depend on the underlying disease, but likely on different transplant procedures. Chemotherapy is known to induce ovarian failure by apoptotic changes in pre-granulosa cells causing follicle loss (Warne et al., 1973; Chabner et al., 1996
). In particular, alkylating drugs have been shown to alter base pairs, leading to DNA cross-links and single-strand breaks (Epstein, 1990
). The combination of high-dose cyclophosphamide and busulphan is one of the most potent conditioning regimens to induce ovarian failure (Brennan and Shalet, 2002
). Primordial follicles were absent in some patients at ovarian biopsy, likely due to damage of oocytes and proliferating as well as resting follicles supporting granulosa cells (Warne et al., 1973
).
An additional finding in our cohort of patients was that, besides having lower 17-estradiol levels, allo-SCT women had lower testosterone levels than both auto-SCT and control women. The ovarian secretion of estrogens is reported to decline faster than that of androgens after physiological menopause, likely due to progressive follicular atresia with persistent androgen production from stromal ovarian tissue (Lindgren et al., 2000
). Consequently, the ovaries were hypothesized to become primarily androgen-producing glands, capable of maintaining gonadotrophin responsiveness for many years (Lindgren et al., 2000
). As a matter of fact, different studies had shown that ovarian testosterone production remains relatively constant, thereby increasing the relative ovarian contribution to the global testosterone production and androgen:estrogen ratio (Judd et al., 1974
; Adashi, 1994
). Conversely, absent gonadotrophin receptors and insignificant 17
-estradiol and androgen production have been recently shown in post-menopausal women after cessation of menstrual cycles for
5 years (Couzinet et al., 2001
). The different period of time elapsing from menopause onset to evaluation could explain the difference between these apparently contrasting findings. Serum 17
-estradiol,
4-androstenedione and testosterone levels in our cohort of allo-SCT patients were significantly decreased, indicating both follicles and stromal endocrine cell damage, with signs of more severe damage in women affected by cGVHD. Conversely, in auto-SCT patients only serum 17
-estradiol levels were decreased, while
4-androstenedione and testosterone levels were normal 1224 months after transplantation. The ovarian contribution to lower serum androgens is confirmed by the correlation between ovarian volume and 17
-estradiol and androgen levels. However, since lower DHEA-S values were found in allo-SCT patients, particularly in those with cGVHD, effects of longer and more recent corticosteroid use on adrenal androgen secretion cannot be ruled out. The degree of immune system involvement might be hypothesized as a major difference between the two groups.
While the toxic effects of antiblastic agents have been widely described as detrimental, the impact of immune system damage on endocrine function in transplanted women is not clear. Allogeneic SCT is an exceptional condition in that a massive number of donor immunological cells are infused into a host, which can recognize the host as non-self. The best-known target organs for such a graft versus host reaction are skin, liver, gut and lung, but any other organ can be targeted including gonads, and the neoplastic tissue itself. The prophylaxis and treatment of GVHD require immunosuppressive drugs, which further compromise immune system function and whose effects cannot be separate from those of the immune system dysregulation itself. Azoospermia related to cGVHD (Grigg et al., 2000) was also reported in allo-SCT recipients, which is in line with our hypothesis on a possible involvement of the immune system derangement in the gonadal damage.
In the current study, we did not investigate circulating autoantibodies against ovary since the existence of a correlation between anti-ovarian antibody titre, cellular immune dysfunction and histological evidence of inflammation in women with autoimmune ovarian failure is still undefined. Circulating anti-ovary autoantibodies are not correlated with clinical activity of autoimmune oophoritis and their pathogenic role remains questionable (Kinch et al., 1965; Hoek et al., 1997
).
In conclusion, ovaries after auto-SCT show features similar to those found after physiological menopause, whereas allo- SCT seems to be associated with major injury, including lower estradiol and androgen production and smaller ovarian size. Chronic GVHD following allografting further worsens this condition. Ovarian failure in SCT recipients seems to be related principally to the myelo-ablative conditioning regiments; however, ovarian steroid levels and size can be further influenced in the allogeneic setting, likely by a major deregulation of the immune system and its treatments. The impact of a lower residual estrogen and androgen steroid secretion after allo-transplantation on womens health, especially in terms of osteoporosis, cardiovascular risks and quality of life remains to be established as well as any future potential therapeutic implication.
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Submitted on November 13, 2002; resubmitted on February 17, 2003; accepted on April 3, 2003.