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and Hyperandrogenism: A Clinical, Biochemical, and Molecular Genetic Study
Departments of Endocrinology (H.F.E.-M., R.M.C., J.S.) and Molecular Genetics (J.L.S.M.), Hospital Ramón y Cajal, 28034 Madrid, Spain
Address all correspondence and requests for reprints to: Héctor F. Escobar-Morreale, M.D., Ph.D., Department of Endocrinology, Hospital Ramón y Cajal, Carretera de Colmenar km. 9,100, 28034 Madrid, Spain. E-mail: hector.escobar{at}uam.es
Abstract
To evaluate the role of TNF-
in the pathogenesis of
hyperandrogenism, we have evaluated the serum TNF- levels, as well
as several polymorphisms in the promoter region of the TNF- gene, in
a group of 60 hyperandrogenic patients and 27 healthy controls matched
for body mass index.
Hyperandrogenic patients presented with mildly increased serum TNF-
levels as compared with controls (mean[median] ± SD:
7.2[7.0] ± 3.3 pg/ml vs. 5.6[4.4] ± 4.0 pg/ml,
P < 0.02). Although no differences in body mass
index and insulin resistance indexes were observed between patients and
controls, when subjects were classified by body weight, serum TNF-
was increased only in lean patients as compared with lean controls, but
this difference was not statistically significant when comparing obese
patients with obese controls.
The TNF- gene polymorphisms studied here (-1196C/T, -1125G/C,
-1031T/C, -863C/A, -857C/T, -316G/A, -308G/A, -238G/A, and
-163G/A) were equally distributed in hyperandrogenic patients and
controls. However, carriers of the -308A variant presented with
increased basal and leuprolide-stimulated serum androgens and
17-hydroxyprogesterone levels when considering patients and controls as
a group. No differences were observed in serum TNF- levels, body
mass index, and insulin resistance indexes, depending on the presence
or absence of these variants.
In conclusion, our present results suggest that the TNF- system
might contribute to the pathogenesis of hyperandrogenism, independent
of obesity and insulin resistance. However, elucidation of the precise
mechanisms underlying the relationship between the TNF- system and
androgen excess is needed before considering TNF- as a significant
contributing factor to the development of hyperandrogenism.
HYPERANDROGENISM, OR ANDROGEN excess, is possibly the most frequent endocrine disorder in women of reproductive age. Hirsutism is a clinical manifestation of androgen excess and is present in 7.1% of Spanish women in this age range (1). The most common cause of hirsutism is polycystic ovary syndrome (PCOS), as defined by endocrine criteria (2), showing a 6.5% prevalence in Spanish women (1).
The increasing evidence that hyperandrogenism and PCOS have a genetic basis (3, 4) has stimulated research into the genes involved in the pathogenesis of these disorders. Despite significant efforts, the precise genetic mechanisms leading to hyperandrogenism remain unknown, suggesting that this disorder is a complex trait in terms of inheritance.
TNF- influences the reproductive axis, inducing changes that closely
resemble those found in patients with PCOS and hyperandrogenism. TNF
stimulates proliferation and steroidogenesis in rat theca cells
in vitro (5, 6), facilitating the effects of
insulin and IGF-I in a dose-dependent and additive fashion
(6). Moreover, TNF may be involved in apoptosis and
anovulation in the rat ovary (7). In humans, increased
serum TNF levels have been found in lean PCOS patients
(8), but no differences in the follicular TNF content
have been found between normal and polycystic ovaries
(9).
TNF has several metabolic activities. Hyperexpression of TNF- in
adipose and muscle tissue has been proposed to play a key role in
the development of insulin resistance in humans (10, 11) by decreasing the tyrosine kinase activity of the insulin
receptor (12). This effect is mediated by the
insulin-receptor substrate-1 (12), is associated with
an increased expression of TNF receptors in adipose tissue
(13, 14), and can be antagonized by
troglitazone in experimental animals (15).
Although TNF acts through autocrine-paracrine mechanisms in adipose
and muscle tissue, increased serum levels of TNF- have been found in
several conditions associated with insulin resistance, such as type 2
diabetes mellitus (16) and obesity (17).
TNF is also a significant source of genetic variability. There are
several single nucleotide polymorphisms in the regulatory region of the
TNF gene, and some of them have been proposed to play a role in the
pathogenesis of insulin resistance, type 2 diabetes mellitus, and
obesity (18, 19, 20), among other disorders.
Hyperandrogenism, including PCOS and hirsutism with normal ovulation, is present in as many as 50% of women diagnosed with type 2 diabetes mellitus (21); conversely, impaired glucose tolerance is a frequent finding in PCOS and non-PCOS hyperandrogenic patients (22, 23). The association of obesity with hyperandrogenism is also well known (24). Therefore, the genes related to type 2 diabetes mellitus and obesity are considered candidate genes for the inheritance of hyperandrogenism (25).
The present study was undertaken to further delimitate the influence of
TNF on the pathogenesis of female hyperandrogenism, using a
clinical, biochemical, and molecular genetic approach.
Materials and Methods
Subjects
Sixty consecutive hyperandrogenic patients, aged 24.3 ± (SD) 6.6 yr with a body mass index (BMI) 29.0 ± (SD) 8.4 kg/m2, were studied. Hirsutism, as defined by the presence of excessive body hair distributed in an androgen-dependent pattern, with a modified Ferriman-Gallwey score (26) of 8 or more, was present in 57 of the patients with a score of 14.6 ± (SD) 5.3.
Menstrual cycle intervals were evaluated on recall for every patient. Oligomenorrhea was defined by the presence of 6 or more cycles of more than 36 days in the previous year, and amenorrhea by lack of vaginal bleeding for 3 consecutive months (27). Oligomenorrhea and amenorrhea were considered as indicative of ovulatory dysfunction, but no further effort was made to demonstrate oligo-ovulation. Oligomenorrhea or amenorrhea were present in 21 of the 60 patients, including 18 of the 57 hirsute patients and 3 women who did not have hirsutism (their hirsutism scores were 6, 7, and 7) but presented with oligomenorrhea and increased serum total T levels.
The reference values for the analytical procedures were obtained from a control group of 27 normal menstruating women, aged 30.2 ± (SD) 8.7 yr, matched for BMI (28.9 ± (SD) 7.7 kg/m2), who did not have signs and symptoms of hyperandrogenism or family history of endocrine diseases.
None of the patients had hypertension, features of Cushing’s disease, or drug-induced hirsutism. Hyperprolactinemia and congenital adrenal hyperplasia were ruled out because all the patients presented basal serum PRL levels <24 µg/liter, together with ACTH-stimulated 17-hydroxyprogesterone (17-OHP) levels <30 nmol/liter and ACTH-stimulated 11-deoxycortisol levels (S) <26 nmol/liter (which is the mean + 2 SD of the values obtained from the control group).
Data from some patients and controls, regarding different aspects of the pathophysiology of hirsutism, have been previously published (28). The patients and controls had not taken hormonal medications, including contraceptive pills, for the last 6 months. All the patients and controls were Caucasian. The ethics committee of the Hospital Ramón y Cajal approved the study, and informed consent was obtained from each patient and control.
Study protocol, hormone profiles, and diagnostic categories of hyperandrogenism
Studies were performed between days 5 and 10 of the menstrual
cycle or during amenorrhea, after excluding pregnancy by proper
testing. The patients reported to the Endocrine-Metabolic testing room
between 0800 and 0900 h after a 12-h overnight fast. An indwelling
iv line was placed in a forearm vein, and after 15–30 min, basal blood
samples were obtained for the measurement of TNF, total T,
4-androstenedione
(4-A), 17-OHP, dehydroepiandrosterone-sulfate
(DHEAS), cortisol (F), LH, FSH, E2, SHBG, glucose, and insulin.
Immediately after taking basal samples, a 250-µg iv bolus of 1–24
ACTH (Synacthen, Ciba-Geigy, Basel, Switzerland) was
injected and blood samples were obtained at 0 and 60 min for the
measurement of S, 17-OHP, and 4-A.
Finally, in all the patients and 19 controls, a 10-µg/kg body weight
sc dose of leuprolide was injected (29), and samples were
obtained at 0900 h on the next day for measurement of LH, FSH, E2,
F, T, SHBG, 17-OHP, and 4-A. Serum LH, FSH,
and E2 served to confirm the stimulatory effect of leuprolide on
gonadotropin and ovarian steroid secretion, and F levels were measured
in all the subjects to rule out an interference of the stimulatory
effect of the ACTH test performed the day before, on serum 17-OHP and
4-A levels. Samples were immediately
centrifuged and serum was separated and frozen at -20 C until
assayed.
Serum TNF levels were measured within a single assay by solid-phase,
two-site chemiluminescent enzyme immunometric technique (Immulite,
Diagnostic Products Corp., Los Angeles, CA) with a
sensitivity of 1.7 pg/ml and a mean intra-assay coefficient of
variation of 3.2%. The technical characteristics of the assays
employed for hormone measurements have been reported previously
(30, 31). The free testosterone (FT) concentration was
calculated from T and SHBG concentrations, assuming a serum albumin
concentration of 43 g/liter, and taking a value of 1 x
109 liter/mol for the association constant of SHBG for
T and a value of 3.6 x 104 liter/mol for that of
albumin for T (32).
Insulin resistance was estimated from fasting glucose and insulin levels using the homeostatic model assessment (HOMAIR) (33) and using the quantitative insulin sensitivity check index (QUICKI) (34).
Patients were classified clinically as follows: Women presenting with
hirsutism or hyperandrogenemia and menstrual dysfunction (n = 21)
were diagnosed with PCOS according to the criteria derived from the
National Institute for Child Health and Human Development 1990s
conference (2). Hirsute patients with increased
circulating concentrations of T, FT, DHEAS, and/or
4-A and regular menstrual cycles were
considered to have hyperandrogenemic hirsutism (n = 28). Hirsute
patients with normal serum T, FT, DHEAS, and
4-A levels and regular menstrual cycles were
diagnosed with idiopathic hirsutism (n = 11). Independently, the
presence or absence of functional ovarian hyperandrogenism (FOH), as
defined by increased 17-OHP levels after leuprolide administration, was
also considered.
The 95th percentile upper limits of normality for basal serum
androgens, derived from the control group of 27 healthy women, were
2.15 nmol/liter, 35 pmol/liter, 9.5 µmol/liter, and 15.7 nmol/liter
for T, FT, DHEAS, and 4-A, respectively. The
95th percentile upper limit of normality for the leuprolide test,
derived from 19 women in the control group, was a serum 17-OHP level of
7.6 nmol/liter.
DNA extraction and genotype analysis
Genomic DNA was extracted from leukocytes obtained from whole
blood samples, using commercial DNA purification kits (Wizard Genomic
DNA purification kit, Promega Corp., Madison, WI, and
Nucleon BAC C3, Amersham Pharmacia Biotech,
Buckinghamshire, United Kingdom). PCR primers were designed to amplify
2 fragments of the promoter of TNF gene. The first fragment,
spanning from -1232 to -732 relative to the TNF gene transcription
start site, comprises a DNA region that contains the -1196C/T,
-1125G/C, -1031T/C, -863C/A, and -857C/T polymorphisms described
previously (35). Primer sequences were 5'-TCT GCT TGT GTG
TGT GTG TCT G-3' (sense) and 5'-ATG AAG CTC TCA CTT CTC AGG G-3'
(antisense). A second fragment, spanning from -444 to -88 and
containing the -316G/A, -308G/A, -238G/A, and -163G/A polymorphisms
(36), was also amplified. Primer sequences were 5'-CAA CGG
ACT CAG CTT TCT GAA G-3' (sense) and 5'-TGG AGA AAC CCA TGA GCT CAT
C-3' (antisense).
DNA sequences were amplified in an automated thermocycler (GeneAmp PCR System 2400, PE Applied Biosystems, Foster City, CA), using a 25-µl reaction mix containing 1 U of AmpliTaq polymerase (PE Applied Biosystems). PCR conditions for both fragments included an initial denaturating step at 94 C for 1 min, 30 cycles at 94 C for 1 min, at 60 C for 1 min, and at 72 C for 1 min, and a final extension at 72 C for 10 min.
After PCR amplification, alleles for each polymorphism were identified
by direct sequence as follows. PCR products were purified using
QIAquick purification kit (QIAGEN GmbH, Hilden, Germany).
Sequence analysis was carried out using the dRhodamine Terminator Cycle
DNA Sequencing Ready Reaction Kit (PE Biosystems, Warrinton, UK) using
the antisense primer for the first fragment of the TNF gene promoter
and the sense primer for the second fragment. The cycle-sequencing
products were precipitated and analyzed by an ABI 310 automated
sequencer (PE Applied Biosystems) according to the
manufacturer’s instructions.
Statistical analysis
Results are expressed as mean [median] ± SD in
the text and tables, unless otherwise stated. The Kolmogorov-Smirnov
statistic, with a Lilliefors significance level for testing normality,
was applied to continuous variables. Because most of the variables did
not follow a normal distribution, nonparametric tests were applied. The
Mann-Whitney UWilcoxon rank sum
W test or the Kruskall-Wallis test were used to compare
variables among the groups of subjects, as appropriate. For variables
showing significant differences between the groups by the
Kruskall-Wallis test, repeated Mann-Whitney U -Wilcoxon rank
sum W tests were used to identify the differences between
each pair of groups, applying an a priori downward
correction to the level of significance to compensate for multiple
comparisons (i.e. P < 0.0125 is needed to
reach statistically significant differences when comparisons involve
four groups) (37). A 
2 test was
used for discontinuous variables, and Spearman’s nonparametric
correlation analysis was also used. P < 0.05 was
considered statistically significant, with the exception stated above.
Power analysis was performed using the G•Power software
(38). Because power analysis requires parametric tests
instead of the nonparametric tests used here, sample sizes for 0.80
statistical power were calculated for the equivalent parametric tests
(i.e., t test or one-way ANOVA), and the results
were corrected for the asymptotic relative efficiency of the
nonparametric tests relative to their parametric equivalents
(39).
Results
Serum TNF levels and hormone profiles
When considered as a whole, the group of 60 hyperandrogenic
patients presented with mildly increased serum TNF levels as
compared with the healthy controls, although there was a considerable
overlap among these groups (Fig.
1).
Patients presented increased serum T, FT, basal and ACTH-stimulated
4-A and 17-OHP, and DHEAS concentrations, and
decreased SHBG levels, as compared with controls (Table 1
). No differences were observed in basal
LH, FSH, E2, and insulin resistance as measured by HOMAIR and QUICKI
indexes (Table 1). Serum TNF levels showed weak but statistically
significant correlations, with basal and ACTH-stimulated serum
4-A concentrations (r = 0.286,
P < 0.01; and r = 0.222, P <
0.05, respectively), when considering patients and controls as a whole.
No other statistically significant correlations were observed (data not
shown).
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levels among controls
and patients with PCOS (n = 21), hyperandrogenemic hirsutism
(n = 28), and idiopathic hirsutism (n = 11) did not reach
statistical significance (controls, 5.6[4.4] ± 4.0 pg/ml; PCOS,
7.3[6.7] ± 3.7 pg/ml; hyperandrogenemic hirsutism 6.8[7.0] ± 2.7
pg/ml; and idiopathic hirsutism, 8.1[7.6] ± 4.1 pg/ml;
P = 0.099). However, the statistical power was not
enough to rule out such a difference (a total sample size of 209
subjects is needed for 0.80 power).
Fourteen patients (8 from the PCOS group and 6 from the group with
hyperandrogenic hirsutism) were diagnosed with FOH (40)
according to increased 17-OHP levels after the leuprolide challenge
(2 = 3.936, P < 0.05 by
Fisher’s exact test for the association between FOH and PCOS in
patients). Serum TNF levels were increased, compared with controls
only in the group of non-FOH patients, and FOH patients showed
intermediate values that were not different with respect to those of
controls and non-FOH patients (controls, 5.6[4.4] ± 4.0 pg/ml; FOH,
6.1[4.9] ± 3.2 pg/ml, non-FOH, 7.6[7.2] ± 3.3 pg/ml,
P < 0.05). No differences were observed in HOMAIR or
QUICKI indexes of insulin resistance or in the BMI when considering the
diagnostic categories of hyperandrogenism or the presence or absence of
FOH (data not shown).
Single nucleotide polymorphisms in the TNF gene
None of the polymorphisms in the promoter of the TNF gene
studied here was associated with patient or control status (Table 2), with the diagnostic category of
hyperandrogenism (data not shown) or with the presence or absence of
FOH (data not shown).
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. However, because of the
limited sample size, our study does not have enough statistical power
to detect true but small differences in the proportions of affected and
nonaffected individuals among patients and controls (Table 2).
Nevertheless, none of the subjects presented the -163A and the -1196T
variants, and only one patient presented the -1125C variant.
We then studied the influence of these polymorphisms on the serum
TNF levels and on the hormonal profiles. When considering
hyperandrogenic patients and controls as a whole, carriers of the
-308A variant presented with increased basal FT, 17-OHP,
4-A, and DHEAS levels and increased serum T,
FT, 17-OHP, and 4-A levels after leuprolide
administration, compared with subjects with wild-type (-308G) alleles
(Table 3). No differences were observed
in serum TNF, basal T, ACTH-stimulated 17-OHP, and
4-A and basal and leuprolide-stimulated LH,
FSH, and E2 levels or in insulin resistance as measured by HOMAIR and
QUICKI indexes (Table 3). All these parameters showed no statistically
significant differences when studying patients and controls
separately.
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Influence of obesity in serum TNF levels and association with
TNF gene polymorphisms
As stated above, the BMI was used to match patients and controls,
explaining why the study groups showed no statistically significant
differences for this variable. For comparisons between lean and obese
subjects (obesity was defined by a BMI 
25
kg/m2), patients and controls were studied as a
whole. Obese subjects presented higher serum TNF concentrations
(7.4[7.1] ± 4.0 vs. 5.6[5.0] ± 2.6 pg/ml,
P < 0.05), higher basal and leuprolide-stimulated FT
levels (basal: 38[35] ± 22 vs. 25[23] ± 13 pmol/liter,
P < 0.010; leuprolide-stimulated: 42[41] ± 23
vs. 29[27] ±12 pmol/liter, P < 0.010),
lower SHBG levels (39[30] ± 28 vs. 60[57] ± 29
nmol/liter, P < 0.010), higher HOMAIR (3.4[3.4] ±
1.6 vs. 2.1[2.0] ± 1.1, P < 0.001), and
lower QUICKI (0.32[0.32] ± 0.02 vs. 0.36[0.35] ± 0.06,
P < 0.001), compared with lean individuals. No
differences were observed in other variables.
These results persisted when studying patients alone, but when the analysis was restricted to healthy controls, only the differences in HOMAIR and QUICKI remained statistically significant (data not shown).
We then compared serum TNF levels between patients and controls in
obese and in lean subjects separately. Serum TNF levels were higher
in the 23 lean patients, compared with the 10 lean controls (6.2[5.1]
± 2.2 vs. 4.3[3.1] ± 3.0; U = 63; W = 15;
P < 0.05). On the contrary, serum TNF levels were
not different between the 37 obese patients and the 17 obese controls
(7.8[7.9] ± 3.8 vs. 6.4[6.5] ± 4.5; U = 227.5;
W = 380.5, P = 0.105). However, the statistical
power of the latter comparison was not enough to rule out such a
difference (a total sample size of 216 subjects is needed for 0.80
power).
Finally, none of the polymorphisms in the promoter of the TNF gene
were associated with obesity (data not shown).
Discussion
The possible involvement of TNF in the pathogenesis of
hyperandrogenism is based on several recent findings. TNF might be
related to increased ovarian steroid secretion, anovulation, and
ovarian apoptosis in animals (5, 6, 7), features that
resemble those of hyperandrogenism in humans.
Also, as reviewed by Hotamisligil (41), virtually all
animal and human models of obesity and insulin resistance are
associated with TNF messenger RNA and protein hyperexpression. As
stated above, obesity and insulin resistance are frequent findings in
hyperandrogenic women (42).
Our present results suggest that TNF might play a role in the
etiology of hyperandrogenism. Serum TNF levels were increased,
although with significant overlap, in patients, compared with healthy
controls. Because both groups were matched for BMI, this increase is
apparently independent of obesity. Moreover, in our series the indexes
of insulin resistance were similar in patients and controls. Therefore,
the mildly increased serum TNF levels found in our hyperandrogenic
patients appear to be also independent of insulin resistance.
Obesity alone modulates serum TNF levels, which, as has been
described by others (16, 17), were higher in obese
subjects, compared with lean individuals. Obese subjects also presented
a higher degree of insulin resistance, lower SHBG, and increased FT,
compared with lean subjects. When subjects were classified by body
weight, serum TNF levels were increased only in lean patients,
compared with lean controls, but this difference was not maintained
when comparing obese patients with obese controls. Gonzalez et
al. (8) recently reported similar results in PCOS
patients.
Therefore, our present results confirm that serum TNF levels
increase mainly because of obesity both in controls and hyperandrogenic
women but also point to a different mechanism in relation to
hyperandrogenism. Interestingly, the higher serum TNF levels were
found in the less severe hyperandrogenic women according to the
leuprolide test, and serum TNF levels tended to be higher in women
with idiopathic hirsutism.
Whether these tendencies reflect a pathogenic mechanism cannot be
solved by our present results but, because TNF acts mostly if not
completely by autocrine or paracrine mechanisms (43), it
is also possible that the fluctuations in serum TNF levels may have
no pathogenic significance, representing only secondary events not
actually related to the development of hyperandrogenism.
Nevertheless, in our series the TNF system also influenced
hyperandrogenism from a genetic perspective. Although none of the
polymorphisms studied here was more frequent in hyperandrogenic
patients, the -308A variant in the promoter of the TNF gene clearly
influenced the phenotype, resulting in increased basal and
leuprolide-stimulated androgen concentrations in the group of carriers
of this variant.
The -308G/A polymorphism in the promoter of the TNF gene has been
studied extensively. The -308A variant, which is associated with human
leukocyte antigens A1, B8, and DR3 alleles (44), is a much
more powerful transcription activator, compared with the -308G allele
(45, 46), explaining the increased TNF production found
in these individuals (47).
Based on these previous studies, we hypothesize that carriers of the
-308A allele might have increased TNF production in several
tissues, including the ovary. In such a case, the increased TNF
levels may stimulate ovarian 4-A secretion in
theca cells as occurs in experimental animals (5).
However, definite proof would require in vitro studies that
are far beyond the methodology of our present study.
Because the increased androgen secretion in -308A carriers was
observed when considering patients and controls as a whole, the -308A
variant in the promoter of the TNF gene should be considered only as
a contributing factor to the development of hyperandrogenism instead of
the main etiologic factor for this condition. Interestingly, serum
TNF levels were similar in -308A carriers and in subjects
presenting wild-type alleles, pointing to a local effect of TNF on
androgen secreting tissue rather than to an endocrine effect mediated
by circulating TNF.
On the contrary, neither the -308G/A polymorphism nor any of the other
TNF gene polymorphisms studied here were associated with patient or
control status. Milner et al. (48) recently
reported similar results, failing to demonstrate an association of the
-308G/A polymorphism with PCOS in their series. However, Milner
et al. (48) did not observe differences in
serum androgens depending on the -308G/A polymorphism, but they
measured only T and 4-A.
The association of variants in the promoter of the TNF gene with
obesity and insulin resistance is controversial, and there have been
negative (36, 49, 50, 51, 52) and positive (18, 19, 20)
reports for these associations over the last years. In our series, none
of the polymorphisms in the promoter of the TNF gene were associated
with obesity, and these polymorphisms did not influence serum TNF
levels, BMI, HOMAIR, or QUICKI, which were not different among the
carriers of these variants and subjects presenting wild-type alleles.
Albeit we have assessed insulin resistance by relatively insensitive
methods, our present results cannot demonstrate any association between
obesity and insulin resistance with the polymorphisms in the promoter
of the TNF gene studied here.
Sample size and statistical power merits an explanation. Statistical
power, as the complement of type II error, reflects the ability of a
study to detect a true difference (37). Therefore, low
power indicates an elevated probability of concluding erroneously that
there are no differences or associations in a study. As a rule, a 0.80
power is considered adequate (37). Our present study lacks
enough power to accurately rule out that the proportions of affected
and nonaffected individuals for the polymorphisms in the TNF gene
were actually different among hyperandrogenic patients and
controls.
Yet even in the case that these differences actually exist, they are too small (the higher was a 10-point difference in the percentage of affected individuals among patients and controls) to represent a major mechanism in the pathogenesis of hyperandrogenism. Moreover, as stated above, the study by Milner et al. (48) ruled out an association of the -308A variant with PCOS, and the sample size in this study provided adequate statistical power.
In conclusion, our present results suggest that the TNF system might
play a role in the pathogenesis of hyperandrogenism, independent of
obesity and insulin resistance. Not only serum TNF levels were
increased in patients, compared with controls, but also the -308G/A
polymorphism in the promoter of the TNF gene modulated ovarian
function, resulting in increased serum androgen levels in carriers of
the -308A variant. However, elucidation of the precise mechanisms
underlying the relationship between the TNF system and androgen
excess is needed before considering TNF as a significant
contributing factor to the development of hyperandrogenism.
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We thank Ms. Genoveva González for technical assistance.
Footnotes
This work was supported by a grant (Proyecto 08.6/0022/1998 to H.F.E.-M.) from the Consejería de Investigación y Cultura, Comunidad de Madrid, Spain, and by grants (Proyecto FIS 00/0414 to H.F.E.-M.; Contrato de Investigador FIS 98/3044 to R.M.C.) from the Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo, Spain. This work was presented at the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, June 21–24, 2000.
Abbreviations: BMI, Body mass index; 4-A,
4-androstenedione; F, cortisol; DHEAS,
dehydroepiandrosterone-sulfate; FT, free testosterone; FOH, functional
ovarian hyperandrogenism; 17-OHP, 17-hydroxyprogesterone; PCOS,
polycystic ovary syndrome.
Received December 5, 2000.
Accepted April 20, 2001.
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  H. F Escobar-Morreale, J. I Botella-Carretero, M. A. Martinez-Garcia, M. Luque-Ramirez, F. Alvarez-Blasco, and J. L S. Millan Serum osteoprotegerin concentrations are decreased in women with the polycystic ovary syndrome Eur. J. Endocrinol., September 1, 2008; 159(3): 225 - 232. [Abstract] [Full Text] [PDF]
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 T. Persico, V. Savasi, E. Ferrazzi, M. Oneta, A.E. Semprini, and G. Simoni Detection of human immunodeficiency virus-1 RNA and DNA by extractive and in situ PCR in unprocessed semen and seminal fractions isolated by semen-washing procedure Hum. Reprod., June 1, 2006; 21(6): 1525 - 1530. [Abstract] [Full Text] [PDF]
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E. Diamanti-Kandarakis, T. Paterakis, K. Alexandraki, C. Piperi, A. Aessopos, I. Katsikis, N. Katsilambros, G. Kreatsas, and D. Panidis Indices of low-grade chronic inflammation in polycystic ovary syndrome and the beneficial effect of metformin Hum. Reprod., June 1, 2006; 21(6): 1426 - 1431. [Abstract] [Full Text] [PDF]
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 E. Diamanti-Kandarakis and C. Piperi Genetics of polycystic ovary syndrome: searching for the way out of the labyrinth Hum. Reprod. Update, November 1, 2005; 11(6): 631 - 643. [Abstract] [Full Text] [PDF]
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 H. F. Escobar-Morreale, M. Luque-Ramirez, and J. L. San Millan The Molecular-Genetic Basis of Functional Hyperandrogenism and the Polycystic Ovary Syndrome Endocr. Rev., April 1, 2005; 26(2): 251 - 282. [Abstract] [Full Text] [PDF]
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T. Asghar, S. Yoshida, S. Kennedy, K. Negoro, W. Zhuo, S. Hamana, S. Motoyama, S. Nakago, D. Barlow, and T. Maruo The tumor necrosis factor-{alpha} promoter -1031C polymorphism is associated with decreased risk of endometriosis in a Japanese population Hum. Reprod., November 1, 2004; 19(11): 2509 - 2514. [Abstract] [Full Text] [PDF]
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 H. F. Escobar-Morreale, J. I. Botella-Carretero, G. Villuendas, J. Sancho, and J. L. San Millan Serum Interleukin-18 Concentrations Are Increased in the Polycystic Ovary Syndrome: Relationship to Insulin Resistance and to Obesity J. Clin. Endocrinol. Metab., February 1, 2004; 89(2): 806 - 811. [Abstract] [Full Text] [PDF]
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V. De Leo, A. la Marca, and F. Petraglia Insulin-Lowering Agents in the Management of Polycystic Ovary Syndrome Endocr. Rev., October 1, 2003; 24(5): 633 - 667. [Abstract] [Full Text] [PDF]
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 H. Chen, G. Sullivan, L. Q. Yue, A. Katz, and M. J. Quon QUICKI is a useful index of insulin sensitivity in subjects with hypertension Am J Physiol Endocrinol Metab, April 1, 2003; 284(4): E804 - E812. [Abstract] [Full Text] [PDF]
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B. Peral, J. L. San Millan, R. Castello, P. Moghetti, and H. F. Escobar-Morreale The Methionine 196 Arginine Polymorphism in Exon 6 of the TNF Receptor 2 Gene (TNFRSF1B) Is Associated with the Polycystic Ovary Syndrome and Hyperandrogenism J. Clin. Endocrinol. Metab., August 1, 2002; 87(8): 3977 - 3983. [Abstract] [Full Text] [PDF]
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A. Katsuki, Y. Sumida, E. C. Gabazza, S. Murashima, H. Urakawa, K. Morioka, N. Kitagawa, T. Tanaka, R. Araki-Sasaki, Y. Hori, et al. QUICKI Is Useful for Following Improvements in Insulin Sensitivity after Therapy in Patients with Type 2 Diabetes Mellitus J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2906 - 2908. [Abstract] [Full Text] [PDF]
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G. Villuendas, J. L. San Millan, J. Sancho, and H. F. Escobar-Morreale The -597 G->A and -174 G->C Polymorphisms in the Promoter of the IL-6 Gene Are Associated with Hyperandrogenism J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1134 - 1141. [Abstract] [Full Text] [PDF]
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M. J. Quon QUICKI Is a Useful and Accurate Index of Insulin Sensitivity J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 949 - 950. [Full Text]
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G. E. Duncan, A. D. Hutson, and P. W. Stacpoole QUICKI Is Not a Useful and Accurate Index of Insulin Sensitivity following Exercise Training J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 950 - 951. [Full Text]
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