The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 1 14-16
Copyright © 1998 by The Endocrine Society
Autoantibody against Testosterone in a Woman with Hypergonadotropic Hypogonadism
Akira Kuwahara,
Masaharu Kamada,
Minoru Irahara,
Osamu Naka,
Toshiyuki Yamashita and
Toshihiro Aono
Department of Obstetrics and Gynecology, University of Tokushima
School of Medicine (A.K., M.K., M.I., T.A), 3–18-15 Kuramoto,
Tokushima 770; and the Departments of Obstetrics and Gynecology (O.N.)
and Urology (T.Y.), Miyoshi Prefectural Hospital, Ikeda 779, Japan
Address all correspondence and requests for reprints to: Akira Kuwahara, M.D., Ph.D., Department of Obstetrics and Gynecology, University of Tokushima School of Medicine, 3–18-15 Kuramoto, Tokushima 770, Japan.
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Abstract
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We demonstrate that antitestosterone autoantibody is produced in a
24-yr-old woman with hypergonadotropic hypogonadism. The serum
testosterone level measured by RIA was extremely elevated (5.80 ng/mL);
after elution, serum testosterone had returned to a normal female value
(0.21 ng/mL). The clinical features were suggestive of no androgen
activity. Primary follicles were present in the patent’s ovary. After
gonadotropin treatment, conception was achieved, and a normal female
infant was delivered.
A gel filtration study showed that the testosterone-binding activity
was eluted at the position of 150,000-kDa Ig. Scatchard analysis
revealed a low affinity antibody; the association constant was
0.034 x 103 mol-1, and the maximal
binding capacity was 162 µmol/mL. An immunoprecipitation study using
the chain-specific antibodies showed that the antitestosterone
autoantibody belonged to 
-type IgG. This subject is the first
reported case with an endocrine disorder who possessed autoantibodies
against testosterone.
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Introduction
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THE PRESENCE of autoantibodies against
circulating hormones have been reported in many cases with endocrine
disorders (1, 2, 3, 4). Nevertheless, only a few cases with endocrine
dysfunction possessing autoantibodies against steroid hormones have
been reported (5, 6).In this paper, we demonstrate that
antitestosterone autoantibody is produced in a woman with
hypergonadotropic hypogonadism. This subject is the first reported case
with an endocrine disorder who possessed autoantibodies against
testosterone.
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Case Report
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A 24-yr-old woman visited our hospital. She complained of an
irregular menstrual cycle since her menarche at 13 yr of age and
infertility. Menstruation occurred every 1–6 months. Hormonal analysis
revealed high serum concentrations of LH and FSH (LH, 40.7 IU/L; FSH,
97.9 IU/L), and estradiol was decreased to an undetectable level.
However, serum testosterone was elevated (5.80 ng/mL), comparable to
that in a normal adult man. Physical examination revealed no
virilization. Her karyotype was 46,XX. No tumor was detected in ovarian
or adrenal regions. Histological examination of ovarian tissue obtained
during laparotomy revealed the presence of a primary follicle, and no
lesions were observed, including signs of ovarian damage, inflammation,
and infiltration by immune cells; developing follicles were absent.
Various autoantibodies, i.e. antinuclear antibody, anti-DNA
antibody, anti-ribonucleoprotein antibodies, and anti-Sm antibody, were
not detected in the patient’s serum. Administration of clomiphene
citrate did not induce ovulation. Administration of human menopausal
gonadotropin at a dose of 225 IU for 5 days lead to ovulation and
conception. After 10 months, the patient was delivered of a female
infant. The infant showed a normal female phenotype and karyotype
(46,XX).
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Materials and Methods
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RIA for testosterone
Serum testosterone was measured by a solid phase RIA kit (DPC
Coat-A-Count total testosterone kit, Nippon DPC Corp., Chiba, Japan).
Serum testosterone was measured directory or after methanol
precipitation to remove high mol wt protein.
Binding activity of serum to 125I-labeled
testosterone
A serum sample (10 µL) and [125I]testosterone
solution (300 µL) were incubated with or without unlabeled
testosterone, androstenedione, dehydroxyepiandrosterone, estradiol, or
progesterone (Wako Pure Chemical Co., Tokyo, Japan; 0–1280 ng/tube) at
37 C for 3 h. Then, 250 µL 0.5% dextran-coated charcoal in
phosphate-buffered saline were added to each tube, and the mixture was
centrifuged at 3000 x g for 10 min. The
radioactivities of the supernatants were counted.
Binding activity to [125I]testosterone was examined in
the patient’s serum and sera from 10 women with normal menstrual
periods, who were used as age-matched controls. Furthermore, to
determine the incidence of development of testosterone-binding molecule
in the women with anovulation, 55 serum samples from anovulatory
patients with hypergonadotropic or normogonadotropic hypogonadism were
also examined.
Sephadex G-75 column chromatography
Serum from the patient (300 µL) was incubated with
[125I]testosterone solution (300 µL) at 37 C for 3
h. The mixture was applied to a Sephadex G-75 column (Pharmacia
Biotech, Uppsala, Sweden). The elute was collected in 1-mL fractions,
and the radioactivity of each fraction was counted directly.
Immunoprecipitation with antihuman Ig antibodies
Patient’s serum was incubated with 10 µL 0.1 N
HCl for 5 min to dissociate antibodies from the immune complex.
[125I]Testosterone solution (300 µL) was added. After
further incubation at 4 C overnight, 250 µL goat antihuman IgG, IgM,
or - or 
-chain antiserum (Sigma Chemical Co., St. Louis, MO) was
added. The mixture was incubated at 4 C overnight. After centrifugation
at 3000 x g for 30 min, radioactivity in the sediment
was counted.
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Results
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The serum testosterone level determined directly by RIA was high
(5.80 ng/mL). The testosterone level in resupernatant after treatment
of the same serum with methanol precipitation was 0.21 ng/mL.
The binding activities of the subject sera ranged from 59.4–75.78% of
the total serum [125I]testosterone added.
Testosterone-binding activities of patient’s sera during the ovulatory
phase and pregnancy showed no comparable differences from those during
the anovulatory phase. The mean
[125I]testosterone-binding activity of sera from 10
control women was very low (mean ± SD, 2.8 ±
0.6%). Sera obtained from subjects with anovulatory cycles did not
bind to [125I]testosterone (3.5 ± 0.4%).
Figure 1
shows a representative
displacement curve of the [125I]testosterone-binding
activity in the patient’s serum when incubated in the presence of
various concentrations of androgens. Androstenedione or
dehydroepiandrosterone had no inhibitory effect on serum
testosterone-binding activity. Testosterone inhibited serum
testosterone-binding activity, and Scatchard plot analysis revealed
that the association constant was 0.034 x 103
mol-1, and the maximal binding capacity was 162 µmol/mL.
Estradiol and progesterone also had no inhibitory effect (data not
shown)
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Figure 1. Inhibition of
[125I]testosterone-binding activities of patient’s serum
by various concentrations of unlabeled steroids. Testosterone inhibited
serum testosterone-binding activity. Androstenedione or
dehydroepiandrosterone had no inhibitory effect on testosterone-binding
activity.
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The elution profiles of the patient’s serum incubated with
[125I]testosterone on a Sephadex G-75 column are shown
in Fig. 2. Radioactive
testosterone was eluted as a single peak at the estimated mol
wt of approximately 150 kDa. The elution volume of the peak
was identical to that of rabbit antitestosterone antiserum
(IgG) incubated with [125I]testosterone.
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Figure 2. Elution profiles of the
[125I]testosterone-binding proteins in the patient’s
serum and in rabbit antiserum against testosterone. The
arrow indicates that radioactive testosterone was eluted
as a single peak at the estimated molecular mass of approximately 150
kDa, and elution volume of the peak was identical to that of rabbit
antitestosterone antiserum (IgG) incubated with
[125I]testosterone.
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Figure 3 showed the
[125I]testosterone-binding activities of mixtures of
subjects’ serum proteins and antisera against human IgG, IgM,
-chain, and -chain, respectively. Over 80% of serum
testosterone-binding protein was precipitated with antihuman IgG or
anti--chain antiserum.
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Discussion
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We describe here the first case of an endocrine disorder due to
the production of an antitestosterone autoantibody. The presence of
antitestosterone autoantibody was suspected based on the observation of
a discrepancy between the high serum testosterone level and the absence
of virilization. This was supported by the finding that the
testosterone level of the patient’s serum decreased dramatically to
the normal female level upon removal of the high mol wt proteins by
methanol precipitation. The binding molecules present in the patient’s
serum appeared to belong to the IgG class of Ig with -chain, based
on the findings that the estimated molecular mass was 150 kDa and it
was precipitated on incubation with antibodies to human IgG and
-chain.
Most antihormone autoantibodies are against peptide hormones. Steroid
hormones are considered to have low potential as immunogens because of
their small molecular size. Therefore, antisteroid hormone
autoantibodies are rarely reported. Antiestrogen antibody was detected
in sera of patients with systemic lupus erythematosus and women who had
a history of oral contraceptive use (5, 6). Autoantibodies against
thyroid hormones, which are as small as steroid hormones, are also
found only in cases of autoimmune thyroid disease. However, in the
present case, there was no evidence suggestive of a systemic autoimmune
disease or a history of taking oral contraceptives and androgenic
steroids. The cause of the production of antitestosterone autoantibody
is unclear. Bucala et al. (7) demonstrated that steroids
containing a vicinal hydroxyketone moiety in their structures can react
with some proteins covalently to form a steroid-protein conjugate that
may induce the production of autoantibody against the steroid
conjugate. Testosterone or its derivatives and some protein may form
the hapten-carrier complex and acquire antigenic potential to elicit an
immune response.
It is interesting that the present subject had experienced mild ovarian
dysfunction. The relationship between ovarian failure and autoimmune
disease has been shown in some patients with hypergonadotropic
hypogonadism (8). Destruction of steroid-producing cells by the
corresponding antibody has been considered to be involved in the
pathogenesis of ovarian failure. However, in this case, histological
examination of the ovarian tissue indicates no direct detrimental
effect of the antitestosterone antibody on steroid-producing cells.
Antibody to testosterone may interfere with the functional
aromatization pathway from testosterone to estradiol.
At present, the relationship of antitestosterone antibody to the
clinical syndrome remains unclear. To find additional cases of ovarian
dysfunction associated with the production of antitestosterone
antibodies would support the idea that these antibodies are involved in
the pathogenesis of ovarian failure.
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Acknowledgments
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We thank Mr. S. Umeda of Sumitomo Metal Bio-Science for
performing the steroid hormone assay and the column chromatography.
Received February 10, 1997.
Revised August 18, 1997.
Accepted September 8, 1997.
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Abstract
Introduction
