Iron overload is associated with low anti-müllerian hormone in women with transfusion-dependent b-thalassaemia

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1 DOI: /j x Fertility and assisted reproduction Iron overload is associated with low anti-müllerian hormone in women with transfusion-dependent b-thalassaemia H-H Chang, a,b M-J Chen, c M-Y Lu, a JPS Chern, d C-Y Lu, a Y-L Yang, a,e S-T Jou, a D-T Lin, a,e Y-S Yang, c K-H Lin a a Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan b Graduate Institute of Clinical Medicine, National Taiwan University, College of Medicine, Taipei, Taiwan c Department of Obstetrics and Gynaecology, National Taiwan University Hospital, Taipei, Taiwan d Department of Family Medicine, Tao-Yuan General Hospital, Department of Health, Taiwan e Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan Correspondence: Dr M-J Chen, Department of Obstetrics and Gynaecology, National Taiwan University Hospital, No. 7 Chung-Shan South Road, 10016, Taipei, Taiwan. mjchen04@ntu.edu.tw Accepted 27 January Published Online 15 March Objective To investigate anti-müllerian hormone (AMH) as a best test of ovarian reserve in women with transfusion-dependent b-thalassaemia, and the relationship between AMH and iron overload. Design and setting A case control study in a tertiary medical centre. Population Twenty-nine women with transfusion-dependent b-thalassaemia and 29 healthy controls of a similar age were recruited. Methods Blood sampling, questionnaires and medical record reviews were used. Main outcome measures The history of iron overload-related morbidities, haematological phenotypes, serum levels of AMH and ferritin, and hormonal profiles were analysed. Results The serum levels of AMH, luteinising hormone, and estradiol were lower in women with transfusion-dependent b-thalassaemia than in age-matched normal controls. In women with transfusion-dependent b-thalassaemia, the serum AMH level was significantly inversely related to the ferritin level, but not related to the presence of hypogonadotrophic hypogonadism, diabetes and haematological phenotypes. The serum ferritin level was positively associated with advanced age and the presence of hypogonadotrophic hypogonadism in the study participants. However, the inverse relationship between AMH and ferritin still exists after further adjustment for advanced age in women with transfusion-dependent b-thalassaemia. Conclusions The present study indicates that the serum AMH levels in women with transfusion-dependent b-thalassaemia are lower when compared with normal healthy women of a similar age, and are significantly negatively correlated with their serum ferritin levels. This implies that ovarian function might be impaired by the chronic iron overload status in women with transfusion-dependent b-thalassaemia. Keywords Anti-müllerian hormone, ferritin, hypogonadotrophic hypogonadism, iron overload, b-thalassaemia. Please cite this paper as: Chang H, Chen M, Lu M, Chern J, Lu C, Yang Y, Jou S, Lin D, Yang Y, Lin K. Iron overload is associated with low anti-müllerian hormone in women with transfusion-dependent b-thalassaemia. BJOG 2011;118: Introduction Beta-thalassaemia is a disorder of hereditary chronic anaemia that is derived from a defect in b-globin chain production. 1 Transfusion-dependent b-thalassaemia, also known as b-thalassaemia major, most often results from homozygosity or compound heterozygosity of a mutant b-thalassaemia allele. Women with transfusion-dependent b-thalassaemia generally need to receive regular red blood cell transfusions from their first year of life as a life-saving measure. 2,3 However, repeated red blood cell transfusions may lead to iron deposition in various organs and tissues, such as the liver, heart and most endocrine glands, hence causing tissue damage and organ dysfunction and failure. 1 Therefore, iron overload after repeated red blood cell transfusions causes morbidity and mortality in women with transfusion-dependent b-thalassaemia. Because transfusions and advances in iron-chelating therapy have significantly improved the long-term survival and quality of life for women with transfusion-dependent b-thalassaemia, 4,5 ª 2011 The Authors BJOG An International Journal of Obstetrics and Gynaecology ª 2011 RCOG 825

2 Chang et al. evaluation of reproductive function in such women has became an emerging clinical issue. 6,7 Women with thalassaemia major and chronic iron overload are known to have a high prevalence of hypogonadotrophic hypogonadism 8,9 because of the predilection for iron deposition in the pituitary gonadotrophs and hypothalamus. 10 However, whether or not gonadal function is intact has always been a matter of controversy in previous studies. 7,11,12 The follicle-stimulating hormone (FSH) and estradiol (E 2 ) levels in the follicular phase in women with hypogonadotrophic hypogonadism are not reliable as markers to evaluate gonadal function. Some studies have measured the response to gonadotrophin-releasing hormone and gonadotrophin stimulation tests by measuring serum E 2 levels 7,11,12 to evaluate gonadal function. However, such indirect methods have produced discrepant results. 7,11,12 Therefore, although ovarian function has been reported to be unaffected in women with transfusiondependent b-thalassaemia major and hypogonadotrophic hypogonadism, 7 there are some studies that have reported impaired gonadal function in these women. 11,12 Because iron deposition has been detected in the ovaries of adolescent women with transfusion-dependent b-thalassaemia on histological evaluation, 13 it is reasonable to speculate that iron overload after repeated red blood cell transfusions might possibly impair gonadal function. After a review of the literature, there is only one study 14 available currently to suggest such an association. Anti-müllerian hormone (AMH), a member of the transforming growth factor-b superfamily, is primarily secreted by the granulosa cells of growing follicles. 15 The number of growing follicles may indirectly reflect the size of the primordial follicle pool in the ovary, 16 which constitutes the ovarian reserve. 17 Anti-müllerian hormone can be reliably detected in the serum, 18 and AMH concentrations are constant during the menstrual cycle. 19 Serum AMH levels also have an excellent correlation with the antral follicle count, as determined by vaginal sonography 17 and serum AMH levels significantly decline with age. 20 Therefore, serum AMH levels can be used as a marker of ovarian reserve. 17 The aim of this study was to determine the serum AMH levels in a cohort of women with transfusion-dependent b-thalassaemia in comparison with healthy women of a similar age. We also determined the effect of iron overload on serum AMH levels in women with transfusiondependent b-thalassaemia. Methods Participants and data collection The study protocol was approved by the Institutional Review Board of the National Taiwan University Hospital. Written informed consent was obtained from all of the women or their parents or legal guardian before blood sampling. Fifty-eight women were enrolled in this study, 29 of whom had transfusion-dependent b-thalassaemia and the other 29 were healthy female volunteers of a similar age who served as the control group. The control women were all regularly menstruating healthy volunteers and were recruited in the outpatient clinics of either paediatrics or gynaecological departments, where they were attending for infectious disease screening before receiving vaccination. Each study participant was matched with the control with the principle of not more than 1 year difference in age. b-thalassaemia major was diagnosed on the basis of clinical and laboratory findings, as in a previous study. 9 All study women were recruited and received thalassaemia care, including regular red blood cells transfusion (10 15 ml packed erythrocytes/kg of body weight every 2 3 weeks to maintain a haemoglobin level of at least 10 g/dl before each transfusion) and iron-chelating therapy, in the National Taiwan University Hospital. The history of menstrual regularity and menarche were obtained by selfreport. The clinical data, including blood transfusion and iron overload-related morbidities (hepatitis C virus infection, diabetes and hypogonadotrophic hypogonadism) of the 29 women with transfusion-dependent b-thalassaemia at the time of blood sampling was obtained by reviewing the medical records. None of the women enrolled in this study had poorly controlled systemic disease or had ever received pelvic surgery before. We defined the clinical diagnosis of diabetes mellitus as a fasting plasma glucose 126 mg/dl. Hypogonadotrophic hypogonadism was defined as FSH and luteinising hormone (LH) levels 2 iu/l, with accompanying E 2 levels 20 pg/ml. Blood sampling of all participants was obtained during the early follicular phase of the menstrual cycle wherever possible, whereas for women without regular menstrual cycles, the blood samples were collected randomly. For those women with elevated levels of progesterone (>2 ng/ml) or E 2 ( 150 pg/ml), FSH and LH were not analysed because of the asynchronous phase of the menstrual cycle. Blood sampling of the study participants was performed before red blood cell transfusions. Blood was processed within 30 minutes of collection. Haemoglobin and ferritin levels were analysed on the day of sampling. Serum and plasma were split into aliquots and frozen at )70 C until assayed. The genomic DNA was also extracted to determine the haematological phenotypes (b 0 /b 0 or b 0 /b + ) of the participants by mutation analysis. Assay methods The serum concentrations of FSH, LH, E 2 and progesterone were measured in all participants as described previously. 21 Serum AMH levels were assessed as in a previous study 22 using a second-generation enzyme immunoassay 826 ª 2011 The Authors BJOG An International Journal of Obstetrics and Gynaecology ª 2011 RCOG

3 Iron overload and low AMH in b-thalassaemia major (AMH-EIA kit; reference number: A16507; Immunotech A Beckman Coulter Company, Marseilles, France), according to the supplier s instructions. The intra-assay and interassay coefficients of variation of all the assays were <10%. Analysis of b-thalassaemia mutations and haematological phenotypes Mutations for b-thalassaemia were detected by direct sequencing of the amplified genomic DNA, as described previously. 9 The b 0 -thalassaemia mutation alleles were IVS II-654 (C-T), codons 41/42 (-TCTT), codons 27/28 (+C), and codons 17 (A-T) and the b + -thalassaemia mutation alleles detected were )28 (A-G) and Hb E (codons 26 [GAG-AAG]). Statistical analysis The numeric variables are presented as the untransformed mean ± SD and the categorical variables are presented as the number with the percentage in parentheses, unless indicated otherwise. The Shapiro Wilk W test was used to identify whether or not all the variables were normally distributed. Log transformation was performed on variables with significant deviation from a normal distribution before further analysis. Student s t test was applied for comparisons of age between study and control groups. Non-parametric analysis with Wilcoxon signed rank test was applied for comparisons of AMH, FSH, LH and E 2 between study and control women after log transformation to correct the heterogeneity of variance. Fisher s exact test, binominal test and logistic regression analysis were used for proportional data comparison. The Spearman s rank correlation analyses and univariate linear regression analyses were performed to assess the relationship between AMH, ferritin and other variables as indicated. For comparisons of variables between four categories of AMH levels, the Kruskall Wallis test for age and ferritin levels was performed where appropriate. A statistically significant difference was defined as a P < Results Table 1. Basic demographic data of women with transfusiondependent b-thalassaemia and control women Variable Study group (n = 29), % Control group (n = 29), % Age (years) (52) 13 (45) (41) 14 (48) (7) 2 (7) Menarche 24 (83) 29 (100) Hypogonadotrophic 15 (52) 0 hypogonadism Hepatitis C 14 (48) 0 Diabetes 4 (14) 0 Haematological phenotype b 0 /b 0 22 (76) 0 b 0 /b + 7 (24) 0 Table 2. Comparison of clinical characteristics between women with transfusion-dependent b-thalassaemia and healthy control women Variable Study group (n = 29) Mean (SD) Control group (n = 29) Mean (SD) P value Age (years) (5.86) (6.27) 0.98 AMH (pmol/l) (21.16) (20.00) 0.06** Ferritin (ng/dl) (4262.2) NA FSH* (miu/ml) 6.56 (9.60) LH* (miu/ml) 2.49 (2.11) E2* (pg/ml) (20.46) 5.27 (1.69) 6.44 (4.68) (30.46) 0.46** 0.004** 0.023** NA, not available. *Only 20 age-matched pairs of control and study women whose blood samples were all collected in early follicular phase were enrolled in the final analyses. **P values by Wilcoxon signed rank tests after log transformation of variables. The basic and clinical characteristics of the 29 women with transfusion-dependent b-thalassaemia and 29 healthy control women of a similar age recruited in this study are depicted in Table 1. The median age of all participants enrolled in the study was 21 years and ranged from 12 to 33 years. The serum AMH levels of the study group were lower, although not significantly so, than the control group (24.8 ± 21.2 and 31.0 ± 20.0 pmol/l, respectively, P = 0.06). The age and FSH levels were no different between the study and control women. In addition, participants with transfusion-dependent b-thalassaemia were also noted to have significantly lower levels of LH and E 2 in comparison with those in the controls, as shown in Table 2, because 15 participants (52%) with transfusion-dependent b-thalassaemia had concurrent hypogonadotrophic hypogonadism (Table 1) and at the same time impaired ovarian reserve. The serum ferritin level was significantly higher in study participants with hypogonadotrophic hypogonadism than in those without ( ± and ± ng/dl, respectively, P = 0.003), whereas the serum AMH levels were comparable between study participants with and without hypogonadotrophic hypogonadism. The ª 2011 The Authors BJOG An International Journal of Obstetrics and Gynaecology ª 2011 RCOG 827

4 Chang et al. serum AMH levels also showed no significant differences between participants with and without other blood transfusion and iron overload-related morbidities such as the status of menarche, hepatitis C virus infection and diabetes. In addition, the women with b-thalassaemia major with the b 0 /b + haematological phenotype had a significantly lower risk of hypogonadotrophic hypogonadism than women with the b 0 /b 0 haematological phenotype (odds ratio 0.095, exact 95% CI , two-sided exact P = 0.001). Univariate regression analyses were applied to determine the explanatory factors that may possibly correlate with the serum AMH levels and ferritin concentrations in the 29 women with transfusion-dependent b-thalassaemia (Table 3). When using AMH as the dependent variable, only the serum ferritin levels of all the explanatory variables were noted to be significantly and inversely correlated with the serum AMH levels in these women (b = )0.929, P = 0.036; Table 3).The other clinical conditions, including age, status of menarche, hypogonadotrophic hypogonadism, diabetes and the haematological phenotypes, did not correlate significantly with the AMH levels in these study participants. However, when using ferritin as the dependent variable, the serum AMH levels were still negatively related to the ferritin level, whereas advanced age and the presence of hypogonadotrophic hypogonadism were noted to be positively related to the elevated ferritin levels (Table 3). Serum levels of FSH, LH, and E 2, were also found not to correlate with the serum AMH and ferritin concentrations. Table 4 illustrates that the study participants with extremely low AMH concentrations tended to be older and had higher ferritin levels. As the serum AMH level may decline and the ferritin level may increase with aging, we then used an age-adjusted Spearman s rank partial correlation analysis 23 to assess the relationship between AMH and ferritin levels in women with transfusion-dependent b-thalassae- Table 3. Univariate regression analyses between serum AMH and ferritin and other clinical factors in 29 women with transfusion-dependent b-thalassaemia Log (AMH) Log (Ferritin) b SE P value b SE P value Age ) Log (AMH) ) Log (Ferritin) ) Hypogonadotrophic hypogonadism Diabetes ) Haematological phenotype (b 0 /b 0 vs b 0 /b + ) ) ) Log (FSH)* ) ) Log (LH)* ) ) Log (E 2 )* ) *Only 26 participants left in the final analyses after exclusion of three women whose blood samples were not taken during the early follicular phase. Table 4. The age and ferritin levels of different clinical categories of AMH levels AMH (pm) Control group (n = 29) Study group (n = 29) Ferritin (ng/dl) mean (SD) n (%) Age (years) mean (SD) n (%) Age (years) mean (SD) (0) 4 (13.79) (8.06) (7621) (20.69) (7.99) 8 (27.59) (7.30) 5846 (3295) (41.38) (6.43) 5 (17.24) (5.18) 4669 (2823) (20.69) (6.95) 5 (17.24) (3.19) 3453 (1578) >48 5 (17.24) (4.16) 7 (24.14) (4.93) 3591 (1900) P value Comparisons between five groups were performed with Kruskall Wallis test for age and ferritin levels. 828 ª 2011 The Authors BJOG An International Journal of Obstetrics and Gynaecology ª 2011 RCOG

5 Iron overload and low AMH in b-thalassaemia major Figure 1. Scatter plot with fit line of the untransformed serum ferritin and AMH levels in 29 women with transfusion-dependent b-thalassaemia. Age-adjusted Spearman correlation coefficient is shown. mia, and found that an inverse relationship of serum AMH and ferritin levels still significantly existed (c = )0.393, P = 0.039); the untransformed data of scatter plot with fit line is shown in Figure 1. Discussion There is debate concerning the integrity of ovarian function in women with b-thalassaemia major receiving chronic repeated red blood cell transfusions and iron-chelating therapy. 7,11,12 A previous study of female adolescents implied that gonadal function may not be intact, as illustrated by histological findings of iron deposition and a reduced number of primordial follicles. 13 Therefore, it is reasonable to hypothesise that long-term red blood cell transfusions in these girls may lead to iron deposition in the ovaries and further reduce the ovarian reserve. In the present study, we demonstrated that serum AMH levels, as a marker of ovarian reserve, were lower in women with transfusion-dependent b-thalassaemia when compared with healthy women of a similar age. In addition, serum ferritin levels in women with transfusion-dependent b-thalassaemia were also noted to be significantly and inversely related to the AMH concentrations. Although the other iron overload-related morbidities and risks for women with transfusion-dependent b-thalassaemia, like advanced age, haematological phenotypes, diabetes and the presence of hypogonadotrophic hypogonadism, were not related to the AMH levels in this study, it is possible that the small study population and the lower vulnerability of the gonads were related. In addition, the decreased ovarian reserve of women with transfusion-dependent b-thalassaemia may lead to a compensatory increase of FSH levels, resulting in the lack of a difference in FSH levels compared with controls because of the high prevalence of hypogonadotrophic hypogonadism with significantly lower LH levels in the study group. The hormone AMH is exclusively secreted from the granulosa cells of the ovary after menarche and has high reproducibility and consistency over the menstrual cycle. It has been suggested that AMH is an excellent marker for predicting ovarian reserve 17,18,24,25 and AMH is used clinically as a predictor of ovarian response before starting gonadotrophin treatment for women who undergo assisted reproductive treatments 26 as well as being considered the most applicable predictor in general practice compared with other commonly used ovarian reserve tests. 25 Our study is the first to investigate circulating AMH levels and their relationship with serum ferritin levels in women with transfusion-dependent b-thalassaemia. The endocrinopathies caused by iron overload in women with transfusion-dependent b-thalassaemia have been extensively investigated in previous studies, 9,13,27 29 some of which have reported that different genotypes of b-globin gene mutations (b 0 or b + ) may result in different amounts of blood transfused (haematological phenotypes, b 0 /b 0 or b 0 /b + ) and/or vulnerability to free radical damage. 9,28 However, while we have shown that the risk of hypogonadotrophic hypogonadism is also significantly related to the haematological phenotype of women with transfusiondependent b-thalassaemia, as has been shown previously, 9 the risk of depletion of the ovarian reserve, as indicated by low serum AMH levels, appears not to be associated with the haematological phenotypes. This discrepancy may derive from the small study population included in this study or may be explained by the fact that the mechanisms by which iron overload causes damage to ovarian reserve differ from those in the pituitary gland or hypothalamus. Women with transfusion-dependent b-thalassaemia major are a small population because pregnant women who conceived a fetus with diagnosed transfusion-dependent b-thalassaemia major mostly decide to terminate the pregnancy. 30 Women with transfusion-dependent b-thalassaemia major who receive life-long red blood cell transfusions now live longer because of advances in the current medical care systems and in iron-chelating agents. Therefore the reproductive endocrinology and fertility requirements for such women are becoming more important and should be considered. The results of our study may provide additional evidence to suggest that chronic transfusion and heavy metal overload are at high risk of being associated with impaired gonadal function. Such findings might also be applied to other diseases that involve repeated transfusions, iron-chelating therapy and heavy metal overload. Furthermore, elective cryopreservation of ovarian tissue or oocytes should possibly be discussed to preserve future fertility. ª 2011 The Authors BJOG An International Journal of Obstetrics and Gynaecology ª 2011 RCOG 829

6 Chang et al. In conclusion, we have demonstrated that women with transfusion-dependent b-thalassaemia were found to have lower serum AMH levels than control women of a similar age. The serum AMH levels in women with transfusiondependent b-thalassaemia were inversely related to the serum ferritin levels, independent of the effect of advancing age. The results of our study are consistent with the hypothesis that the ovarian reserve might be impaired in women with transfusion-dependent b-thalassaemia because of iron overload. However, further studies with larger study populations are required to substantiate our findings. Disclosure of interests There are no conflicts of interest from the authors. Contribution to authorship H-HC contributed to the study on data analysis and to drafting of the manuscript. M-YL, J-PC, Y-LY, S-TJ, D-TL and K-HL contributed to enrolment of study participants and data collection. C-YL and Y-SY contributed to enrolment of control women and data collection. 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