RED CELLS, IRON,AND ERYTHROPOIESIS
Sylvia T. Singer,1Elliott P. Vichinsky,1Ginny Gildengorin,2Jereon van Disseldorp,3Mitchell Rosen,4and Marcelle I. Cedars4
1Department of Hematology/Oncology and2Clinical Research Center, Children’s Hospital and Research Center, Oakland, CA;3Department of Reproductive
Medicine and Gynaecology, University Medical Center Utrecht, Utrecht, The Netherlands; and4Reproductive Endocrinology and Infertility, University of
California at San Francisco, San Francisco, CA
The pathophysiology of iron-induced
semia major (TM) was evaluated in
26 adult TM females. Low gonadotropin
secretion resulted in reduced ovarian an-
tral follicle count and ovarian volume, but
levels of anti-mu ¨llerian hormone (AMH), a
sensitive marker for ovarian reserve inde-
pendent of gonadotropin effect, were
mostly normal. AMH correlated with non–
transferrin-bound iron (NTBI), suggesting
a role of labile iron in the pathogenesis of
decreased reproductive capacity, possi-
bly occurring in parallel to cardiac iron
toxicity, as cardiac iron was associated
with the presence of amenorrhea and
with NTBI levels. AMH emerges as an
important biomarker for assessment of
reproductive capacity in TM, demonstrat-
ing that fertility is preserved in the major-
ity of those younger than 30 to 35 years.
AMH can be useful in future studies aim-
ervation, whereas NTBI and labile plasma
iron may be valuable for monitoring iron
Despite progress in chelation regimens, the deleterious effect of
excess iron to the reproductive system of women with thalassemia
major (TM) is still common.1,2Iron toxicity to the anterior pituitary
results in declining synthesis of lutenizing hormone (LH) and
follicle-stimulating hormone (FSH).3The effect of low gonadotro-
pin secretion on the ovarian oocyte maturation has not been
explored, and a possible direct effect of iron, in particular that of
plasma iron (LPI), on the ovaries is unknown. Some have
suggested iron-induced ovarian dysfunction1; but with reports of
successful ovulation-induction and pregnancies, it has been pro-
posed that ovarian function is preserved even in women with
Ovarian antral follicle pool can predict fertility capacity, and
reproductive aging is directly related to the decline in this pool.
Low ovarian reserve is associated with low chances for spontane-
ous pregnancy and poor response to hormonal stimulation.4-6
Antral follicle count (AFC), visualized by ultrasound, and anti-
mu ¨llerian hormone (AMH), which corresponds with AFC, can
accurately assess ovarian follicle pool and are used for ovarian
reserve testing, identifying women at risk for early ovarian
failure.7,8The usefulness of ovarian reserve testing for the care of
thalassemia females has not been evaluated. Reproductive capacity
in TM women cannot be well predicted by means of age, menstrual
status, or transfusion and chelation parameters.1,9Yet, as overall
survival continues to improve,10,11attainment of reproductive
capacity is crucial for many patients, a concern often brought up to
We evaluated the effect of iron burden on hypogonadism and
ovarian reserve inTM women, to better understand the pathophysi-
ology of reduced fertility and evaluate the predictive ability of
ovarian reserve testing in this unique patient-population.
Thalassemia women (? 17 years of age) who were treated with regular
transfusion therapy for at least 10 years before enrollment in the study were
eligible. Data from a multiethnic population of normo-ovulatory women
(n ? 769)12were used as the normal comparator. Children’s Hospital and
Research Center Oakland and University of California at San Francisco
Institutional Review Board approvals were obtained, and all the participants
provided written informed consent in accordance with the Declaration of
Annual liver iron concentration (LIC) was quantitated by superconduct-
ing quantum interference device biosusceptometer system; and cardiac iron
concentration by magnetic resonance imaging (1.5 Tesla; Philips Intera).
NTBI and LPI were measured at a standardized research laboratory
(University College London, London, United Kingdom) and vitamin C in a
commercial clinical laboratory. Hormone levels were determined in a
standardized research laboratory (CLASS Laboratory, University of Michi-
gan). AFC and ovarian volume measurements were performed all by the
same examiner, using a Shimadzu SDU-450XL ultrasound machine, with a
Statistical analysis was performed using SAS Version 9.2 software.
Bivariate Pearson correlations were computed, and linear regression of age
on theAMH values was performed.Asignificance level of .05 was used for
all statistical tests.
Results and discussion
Twenty-six females (transfused for 25.4 ? 7.4 years) were studied.
Mean LIC was 15.7 ? 12.9 mg/g dry weight. NTBI (n ? 23) was
4.2 ? 2.4?M, and LPI 0.8 ? 1.0?M, correlating with each other
(r ? 0.66;P ? .01).CardiacT2*ironestimationwas19.4 ? 9.4 ms
and inversely correlated with LPI (r ? ?0.5; P ? .04) and with
NTBI (r ? ?0.4; P ? .059).
Submitted June 10, 2011; accepted June 17, 2011. Prepublished online as
Blood First Edition paper, July 14, 2011; DOI 10.1182/blood-2011-06-360271.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2011 by TheAmerican Society of Hematology
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LH and FSH, inhibin B, and estradiol levels were low in 15 to
18 patients (? 60%), all amenorrheic (Table 1). AMH inversely
correlated with age (r ? ?0.48, P ? .01) and was lower in
amenorrheic women. AMH was within the range of the normal
controls (20.6 ? 14pM and 25.6 ? 19pM, respectively; 95% confi-
dence interval [CI], 13.4-26.8; Figure 1A). Mean follicle count
(AFC) was 7.4 ? 5 (range, 0-20), lower than that in normo-
ovulatory women (Figure 1B). Both AMH and AFC were reduced
in 5 women older than 30 years (mean, 34 ? 5 years), suggesting
combined pituitary and ovarian dysfunction. Ovarian volume was
low (1.25 ? 1.2 cm3; range, 0.3-6.8 cm3). FSH, LH, and estradiol
levels had no significant correlations with iron overload measures,
whereasAMHinverselycorrelatedwithNTBI(r ? ?0.5,P ? .01).
Mean vitamin C levels were 0.6 ? 0.5 mg/dL(normal, 0.4-2.0 mg/
dL) but very low in 11 patients (0.1 ? 0.2 mg/dL), inversely
correlating with LIC (r ? ?0.4, P ? .05).
Our findings imply a decrease in ovarian follicle development,
as reflected by low inhibin B, AFC, and ovarian volume, probably
because of lack of FSH stimulation. However, AMH levels are
overall normal, as they represent early and preantral follicles,
which are not affected by the low gonadotropin synthesis. This
explains successful cases of induction of follicle growth and
ovulation when TM women are stimulated with exogenous gonad-
otropins. Still, TM women seem to have a premature ovarian aging
compared with normo-ovulatory women, mostly in women in their
early-mid-30s, raising a concern of the optimal age for best
likelihood of successful response to hormonal stimulation. Indeed,
case series of successful pregnancy induction report a mean age of
24.5 and 29.5 years.2,13The rate of decline in ovarian reserve varies
considerably between individual women and is probably even more
variable among women with TM. Therefore, a test that can provide
information regarding the status of a woman’s remaining oocytes is
of great importance. Recent studies have shown that AMH is the
serum marker that best reflects the gradual reduced reproductive
capacity with increasing age.14-16Our results imply that AMH
shows significant promise to serve as such a marker in thalassemia
women with iron overload and seems a better marker than AFC,
which in many cases is lower despite a normal AMH. Thalassemia
women had a considerably lower ovarian volume compared with
reported normal controls (1.25 ? 1.2 cm3vs 6-6.6 cm3),17also
representing impaired ovarian reserve, probably a result of a halt in
follicle maturation because of lack of gonadotropin stimulation.
However, there may also be direct iron toxicity to ovarian tissue.
Ovarian tissue iron overload is supported by the finding of
inverse correlation of AMH levels, secreted exclusively by the
ovaries, and NTBI. Elevated NTBI, through its labile component,
is capable of permeating into organs and compromising organ
function.18An increase in reactive oxygen species and lower
enzymatic antioxidant defense mechanisms have been shown to
accelerate follicle aging.19High redox activity in the ovarian
follicular fluid of a TM woman was reported, signifying that
redox-active iron ions mediate free radical production, inducing
ovarian tissue injury.20A low antioxidant capacity is demonstrated
in our patients by having low ascorbic acid levels, which also has a
role in oocyte development.21Further study addressing iron-
induced oxidative stress and premature ovarian aging in TM is
The presence of amenorrhea did not correlate with LIC or “free”
thies, and length of time receiving regular transfusions. Interest-
ingly, we found an association of cardiac iron with NTBI and LPI.
Lack of correlation of cardiac iron and LIC, but an association with
NTBI, was shown before.22It is probable that labile, potentially
toxic, iron plays a major role in causing myocardial as well as
reproductive tissue damage,23-25suggesting a role for “free” iron in
the pathogenesis of impaired fertility.
In conclusion, our data suggest that ovarian reserve is preserved
in the majority of TM, ? 30-35 years old, despite a low follicle
Table 1. Clinical and endocrine characteristics
Amenorrhea (primary or secondary)Normal cycleP
No. (%) of patients
Mean age of secondary amenorrhea, y
Years receiving transfusions
LIC, mg Fe/g dry
Inhibin B, pg/mL
Mean ovarian volume, cm3*
Cardiac T2* MRI, ms
29 ? 5.7
19.2 ? 3
28 ? 6
13 ? 10
3.7 ? 1.6
2.7 ? 2.4
2.2 ? 2.7
17 ? 22
15 ? 7.1
7.5 ? 5
1.3 ? 1.4
1 in 1 woman
16.3 ? 8 (n ? 14)
27 ? 8
20 ? 6
17 ? 13
5.3 ? 3
8 ? 6
7.3 ? 9
63 ? 60
35 ? 15
20.7 ? 14
7 ? 1
1.3 ? 1
5 in 2 women
24.4 ? 10 (n ? 8)
Patients’baseline findings are shown based on the presence or absence of amenorrhea. Hormonal replacement therapy was discontinued for 1 month before obtaining all
reproductive measures. Iron chelation was halted until after obtaining NTBI and LPI levels. A factor of 6.0 was applied to convert in vivo LIC (wet weight) into dry weight,
comparable to paraffin-embedded biopsies, expressed in mg/g dry weight. Average LIC and results of the measure closest to the time of fertility evaluation were used for
analysis.Average previously obtained LIC of entire the group (3.7 ? 2/patient) was 14.5 ? 11 mg/g dry weight.
— indicates not applicable; E2, estradiol; OV, ovarian volume; and NS, not significant.
*Follicles with a mean diameter (of 2 dimensions) between 2 and 10 mm were counted. The volume of each ovary was calculated by applying the formula for an ellipsoid:
(length ? height ? width ? ?/6), the average of both ovaries was then applied.
†Other endocrinopathies were noted in 10 women, who had amenorrhea. All 5 patients older than 30 years who had markers of ovarian dysfunction were included in this
REPRODUCTIVE CAPACITY IN THALASSEMIAMAJOR WOMEN 2879BLOOD, 8 SEPTEMBER 2011?VOLUME 118, NUMBER 10
For personal use only. on November 5, 2015. by guest
count and reduced ovarian volume. AFC is affected by the low
gonadotropin secretion and cannot accurately reflect ovarian re-
serve, making AMH the better marker for determination of
reproductive stage. NTBI and LPI may have a significant contribu-
tion to the reproductive tissue damage, implying a need to better
define the appropriate chelation regimens, the role of antioxidant
supplementation, and the markers for improved monitoring of
treatment effects on the reproductive capacity.
and Dru Foote for their assistance in coordination of patients’
studies and data collection.
This work was supported by the Cooley’s Anemia Foundation
award on Translational Research in Adult Thalassemia, the Na-
tional Center for Research Resources (grant UL1RR024131-01),
and the National Institutes of Health/National Center for Research
This article’s contents are solely the responsibility of the
authors and do not necessarily represent the official views of the
National Institutes of Health.
Contribution: S.T.S. designed and performed research, analyzed
data, and wrote the manuscript; J.v.D. and M.R. assisted with data
analysis; M.I.C. performed all ovarian studies, analyzed data, and
assisted in writing the manuscript; E.P.V. assisted in writing and
critiquing the manuscript; and G.G. performed statistical analysis
and critically reviewed the manuscript.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Sylvia T. Singer, Department of Hematology/
Oncology and Clinical Research Center Oakland, Children’s
Hospital and Research Center, 747 52nd St, Oakland, CA 94609;
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Figure 1. AMH levels and AFC in TM women and normo-ovulatory controls.
(A) AMH levels in the thalassemia women, 25 years and older (n ? 23, red circles)
were compared with normal controls (F; n ? 759), showing that the slopes of the
regression lines against age were not statistically different (P ? .56). The slope was
significant for the normal controls (P ? .0001; 95% CI, ?1.867 to ?1.406) and for the
thalassemia patients (P ? .03; 95% CI, ?2.323 to ?0.1142), implying an association
with age. There was a 5.0pM (95% CI, 13.4 to 26.8) difference between the group
means. The levels in the thalassemia women were in the low-normal range of normal
and dropped to lower levels in women older than 30 years. (B) Age-dependent
AFC in thalassemia women and normal controls. AFC number includes all counted
follicles 2-10 mm in size, in thalassemia women, and in the cohort of normal controls
(n ? 769).
2880 SINGER et al BLOOD, 8 SEPTEMBER 2011?VOLUME 118, NUMBER 10
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Marcelle I. Cedars
Sylvia T. Singer, Elliott P. Vichinsky, Ginny Gildengorin, Jereon van Disseldorp, Mitchell Rosen and
Reproductive capacity in iron overloaded women with thalassemia
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