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Original Studies |
Departments of Obstetrics and Gynaecology (A.M., E.L.Y.) and Paediatrics (S.R.), National University of Singapore, Singapore 119074, Republic of Singapore
Address correspondence and requests for reprints to: Dr. E. L. Yong, Department Obstetrics and Gynaecology, National University Hospital, Level 2, Lower Kent Ridge Road, Singapore 119074, Republic of Singapore. E-mail: obgyel{at}nus.edu.sg
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Androgens act through the AR. The N-terminal portion of the X-linked
AR gene contains a highly polymorphic region with a variable
number of CAG repeats, encoding a polyglutamine tract. Expansion of the
CAG tract to beyond the polymorphic range (>40 CAGs) results in a
fatal neuromuscular disorder, spinal bulbal muscular atrophy (10).
In -vitro reporter gene assays indicate that changes in
AR-CAG length have an inverse relationship with
receptor activity, wherein ARs with short polyglutamine tracts have
high intrinsic receptor activity and, conversely, long polyglutamine
tracts are associated with reduced ability to activate
androgen-responsive genes (11, 12). Recently, variations of the
AR-CAG tract, while still within the normal
polymorphic range (11–38 CAGs), have been related to various disorders
associated with low or high androgenic activity. Short
AR-CAG tracts have been linked to an increase in
the severity (13) and an earlier age of onset (14) of the
androgen-driven tumor prostate cancer. On the other hand, long CAG
tracts (
28 CAGs) are associated with hypoandrogenicity (15) and
increased risk of male infertility due to impaired sperm production in
Chinese (12) and Caucasian (16) populations. Women who inherited
germ line BRCA1 mutations were at a significantly increased
risk of early onset breast cancer if they carried at least one
AR allele with 28 or more CAG repeats (17), suggesting that
the normal inhibitory action of androgens on breast tissue
proliferation have been reduced. Because AR-CAG length can
be correlated to androgenicity and androgen-regulated diseases, it is
possible that variations in its length may have a role in the etiology
of anovulatory infertility and polycystic ovaries. To test this
hypothesis, we performed a case-control study to evaluate the
association between AR-CAG length and the clinical diagnosis
of anovulatory infertility associated with polycystic ovaries.
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Subjects and Methods |
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Patients with of anovulatory infertility and polycystic ovaries were recruited from the Subfertility and Reproductive Endocrinology clinics of the National University Hospital in Singapore. The women must not have taken any hormonal medication for at least 2 months before the study. Inclusion criteria for patients were the presence of polycystic ovaries, infrequent periods with intermenstrual interval of more than 35 days, and involuntary infertility. An ovary with the ultrasound appearance of more than 10 subcapsular follicles (<10 mm in diameter) in the presence of prominent ovarian stroma was deemed polycystic. Patients with hyperprolactinemia, thyroid and adrenal diseases, 21-hydroxylase deficiency, and androgen-secreting tumors were excluded. The weight and height of all subjects were recorded. Hirsutism was defined as a Ferriman-Galwey score of more than 5. Hormonal parameters measured include LH, FSH, PRL, and testosterone (T). Normal controls of proven fertility, no history of subfertility treatment, and with normal menstrual cycles every 25–32 days were recruited from the contraceptive clinic. Ethical committee approval was received, and informed consent was obtained from all subjects and controls.
DNA amplification and Genescan analyses (PE Biosystems Asia, Singapore)
DNA was extracted from the peripheral blood of patients and control subjects using standard techniques (18). The CAG repeat segment was amplified using the sense (5'-TCCAGAATCTGTTCCAGAGCGTGC) and antisense (5'-GCTGTGAAGGTTGCTGTTCCTC) primers. The 30-µL reaction mix contained 500 ng genomic DNA, 50 µm dNTPs, 0.25 µM of each primer, the fluorescent-labeled R6G, and 0.5 U Taq polymerase. A two-step 30-cycle amplification protocol was used in which the denaturing temperature was 95C for 45 sec and the combined annealing and extension temperature was 68 C for 1.5 min. In the first cycle, the sample was denatured for 5 min. Amplified samples were mixed with formamide, loading buffer, and the Rox 500 size standard in a ratio 1:10:2:2, respectively. This mixture was denatured for 5 min at 95 C for 3 min and resolved on a 4% denaturing polyacrylamide gel. The sizes of the samples were analyzed on a 377 DNA Sequencer running Genescan 672 software. Some samples were sequenced to gain further accuracy in size determination. These samples of known length were subsequently inserted in every gel as controls.
Hormonal analysis
RIAs for serum LH, FSH, PRL, and T were performed using standard reagents supplied by the World Health Organization matched-reagent program (19). Free T was measured with the Coat-A-Count free T solid phase 125I RIA according to manufacturer’s instructions (Diagnostic Products, Los Angeles, CA). The inter- and intra-assay coefficients of variation were less than 15%.
Data analyses
The lengths of AR-CAG alleles of cases and the controls were compared using the two-sample independent t test. Because two X-linked AR alleles are present in every subject, comparisons between subject groups were performed by first considering the mean of the two AR-CAG alleles (biallelic mean) in each person and, second, the short and long alleles of each subject separately. To investigate whether CAG repeats are associated with any subset of anovulatory subjects, patients were categorized into "high T" and "low T" cases using a cut-off T value of 1.73 nmol/L (mean for normal patients). Pearson’s correlation coefficient was calculated to assess the relationship between CAG length and T. The mean AR-CAG lengths of patients with levels of FSH and LH above or below the respective means for normal subjects were similarly subjected to the t test. Finally, the contribution of ethnic differences in AR-CAG alleles was evaluated by analyzing the distribution of AR-CAG alleles among subjects of the two ethnic groups in our study population. Statistical analyses were performed using the SPSS, Inc. version 9.01 (SPSS, Inc., Chicago, IL) computer program. Statistical significance was defined as a two-sided P value less than 0.05, and data were reported as mean ± SE.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A total of 203 subjects, comprising 91 patients (83% Chinese and 17% Indians) and 112 healthy controls (92% Chinese and 8% Indians), were recruited. Patients had a higher mean body mass index of 26.9 ± 0.73 kg/m2 compared with 22.08 ± 0.43 kg/m2 for control subjects. Hirsutism was not common, occurring in 21% of our patients.
Distribution of AR-CAG alleles
There were 28 AR-CAG alleles in our population, ranging from 11–33 CAGs in patients and 14–38 in controls. There were no differences in the mean of the biallelic means of patients and controls, being 22.97 ± 0.24 and 23.09 ± 0.23, respectively. Because all females have two AR-CAG alleles, analyses were performed on the short and long alleles separately. The mean short-allele length of patients was no different from controls, being 21.38 ± 0.27 and 21.49 ± 0.25, respectively. Similarly, there were no significant differences in the long allelle between patients and controls, their mean lengths being 24.53 ± 0.25 and 24.7 ± 0.67, respectively.
T levels divide patients with polycystic ovaries into two subsets of AR-CAG length
Because there is a subset of PCOS patients who are
nonhyperandrogenemic (20), we investigated the relationship between
AR-CAG length and serum androgen levels (Fig. 1
). Total T was used in our analysis
because there was a close correlation between total and free T in our
patients (r = 0.82, P < 0.0001) and because free
T assays are also less readily available than total T. Furthermore,
there is evidence in the rabbit (21) and human (22) that at least some
of the albumin and sex-hormone globulin-bound T fractions may be
bioavailable and that measurement of non-SHBG-bound T and the
calculation of the free androgen index may provide no further
information in the diagnosis of PCOS beyond that provided by total T
(23).
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). The mean length of the shorter allele
for low T patients was significantly lower than for patients with high
T, being 20.38 ± 0.51 vs. 21.98 ± 0.29,
respectively (P = 0.004) (Table 1). A cut-off value of around 1.73 nmol/L
resulted in the most significant differences in CAG length. When the
cut-off was increased to 1.91 nmol/L, we noted that the P
value decreased to 0.058, of borderline significance, which may be a
result of the reduction in power with the reduced sample size in the
high T group. Bivariate correlation analysis between CAG repeats and
the T levels for each allele did not show any correlation when each of
the alleles was analyzed separately, values for the shorter and longer
allele being r = 0.063, P = 0.551 and r =
-0.113, P = 0.285, respectively. This suggests that
the relationship is not a dose-response or linear one, but is more
closely approximated by a threshold response. In comparison, there were
no differences in the long AR-CAG allele of patients with
low or high T. No differences were detected when those with low or high
FSH and LH were similarly analyzed (Table 1), suggesting that the
differences observed above were not likely to be due to chance.
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The average biallellic mean CAG length in Chinese subjects (patients and controls) was longer than for Indians, being 23.16 ± 0.17 and 22.08 ± 0.5, respectively (P = 0.035). The mean length of the short allele was also different between these two races, being 21.5 ± 0.19 and 20.4 ± 0.68 for Chinese and Indians, respectively (P = 0.045). There was also a difference between low T Chinese and Indians when the short allele alone was considered, being 20.7 ± 053 and 19.1 ± 1.42, respectively, although this did not reach statistical significance because of the small numbers.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Average biallelic mean AR-CAG length did not differ between
control and patients. There were also no significant differences when
the long and short alleles of the subjects were analyzed separately,
although AR-CAG length was slightly shorter in the patients.
Thus, it seems that the AR-CAG tract did not have any
important etiological role in the majority of patients. On the other
hand, we found a highly significant relationship between short
AR-CAG alleles and the subset of patients with low androgen
levels. About one third of our patients have serum T levels below the
normal laboratory mean, and these cases have a significantly lower mean
CAG length compared with patients whose T levels were above the
laboratory mean. This association between short AR-CAG
alleles and patients with low T was independent of ethnic origin, being
significant in both Chinese and Indian subjects. In contrast, no
significant relationships were observed when patients with different
serum levels of LH and FSH were analyzed. Our study is consistent with
the findings of a recent study, which did not find any difference in
the number of CAG repeats between hirsute patients and controls (26).
Although involving a relatively small number of patients, this study
intriguingly suggested that significant skewing of X-chromosome
inactivation occurred, allowing the shorter of the two
AR-CAG alleles to be expressed in cases of
nonhyperandrogenic hirsutism. If so, the effects of the short
AR allele would be accentuated, increasing the chances of
hirsutism in those with both long and short alleles in their
X-chromosomes. Our study shows that more than half of low T patients
have at least one AR-CAG allele of 21 or more (Fig. 2
) and
could potentially be at risk of hyperandrogenism due to the greater
intrinsic activity of the short AR-CAG allele. Our data
imply that excessive androgen action could have a greater role in PCOS
than expected from hyperandrogenemia alone.
The AR-CAG allele has well-established population differences. The most frequent allele in black Americans is 18 CAGs compared with 21 for white Americans (27). The most common allele length for our Asian subjects was 22, similar to the Asian population described by Edwards et al. (27). Ethnic differences were evident in our data, with Indian subjects having a shorter biallelic mean AR-CAG length compared with the Chinese. This ethnic difference was also observed when only the shorter AR allele was analyzed, providing, at least in part, a physiological basis for the observation that the prevalence of polycystic ovaries in Indian subcontinent Asian women is very high, about 52% compared to less than 10% in other populations (28). Interestingly, Indians have a higher age-standardized incidence rate of the androgen-driven tumor prostate cancer compared with Chinese (11.0 vs. 7.6 per 100,000/yr) (29). Strikingly, this same difference in prostate cancer rates is evident in black Americans compared with white Americans.
Short AR-CAG length resulting in a short AR polyglutamine tract is associated with high intrinsic AR activity in reporter gene assays (11, 12, 15). A possible molecular mechanism as to how a change in polyglutamine length can affect the activity of the receptor has been suggested by Hsiao and Chang (30). They have identified a novel nuclear G-protein, Ras-related nuclear protein/ARA24 that acts as a coactivator with the AR and can bind differentially with different lengths of polyglutamines within AR. AR-CAG/ARA24 interactions become stronger as the number of glutamines decreases, thereby increasing coactivation capability. Although the likely increase in AR intrinsic activity with each reduction in AR-CAG length is relatively small, these effects are genetically determined and, therefore, exert its effects over the entire lifetime of the individual. Small changes can, over time, have significant cumulative pathological effects.
Thus, our analysis of over 200 subjects suggest an association between
AR hypersensitivity and nonhyperandrogenemic patients with anovulatory
infertility and polycystic ovaries. An ethnic difference in
AR-CAG distribution was also observed. These data add to a
growing list (Table 2) of conditions that
are related to variations of AR-CAG tract and provide
evidence that the AR-CAG polymorphism has an essential
regulatory function in controlling intrinsic AR activity.
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Acknowledgments |
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Received November 22, 1999.
Revised April 21, 2000.
Accepted May 24, 2000.
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