ORIGINAL ARTICLE

Defining Hyperandrogenism in Women

With Polycystic Ovary Syndrome:
A Challenging Perspective

Renato Pasquali, Laura Zanotti, Flaminia Fanelli, Marco Mezzullo, Alessia Fazzini,
Antonio Maria Morselli Labate, Andrea Repaci, Danilo Ribichini,
and Alessandra Gambineri
Division of Endocrinology (R.P., L.Z., F.F., M.M., A.F., A.M.M.L., A.R., D.R., A.G.), Department of Medical
and Surgical Sciences, and Centre for Applied Biomedical Sciences (F.F., M.M., A.F.), University Alma
Mater Studiorum, Sant’Orsola-Malpighi Hospital, Bologna, 40138 Italy

Objective: This study was designed to assess the steroid profiling by liquid chromatography coupled
with tandem mass spectrometry in PCOS women with different phenotypes.

Design: Cross-sectional study.

Setting: University hospital of Bologna, Italy.

Patients and Methods: A total of 156 PCOS women and 141 controls comparable for age were
investigated. All underwent a steroid profiling by liquid chromatography coupled with tandem
mass spectrometry. Metabolic parameters were also investigated and hirsutism was measured by
the modified Ferriman-Gallwey (mF-G) score.

Results: Three distinct phenotypes were initially defined according to the combination of hirsutism
(mF-G ⱖ 8) and/or high testosterone (T) (HA), oligo-amenorrhea (OA), and polycystic ovarian
morphology (PCOm); OA ⫹ PCOm (n ⫽ 43), HA ⫹ OA (n ⫽ 65), and HA ⫹ OA ⫹ PCOm (n ⫽ 45). T,
androstenedione (A), and free androgen index (FAI) levels progressively increased in the 3 PCOS
phenotypes with respect to the controls, with the highest values in the HA ⫹ OA ⫹ PCOm phe-
notype. The various combinations of hirsutism, high T, high A, and high FAI made it possible to
categorize the 3 original phenotypes into 8 hyperandrogenic subgroups, characterized by diver-
gent additional steroid profile and metabolic pattern. A total of 90% of patients with PCOS thus
proved hyperandrogenic. Interestingly, half the PCOS women originally classified as having the
OA-PCOm phenotype were categorized in a hyperandrogenic subgroup. No significant correlation
was found between T, A, and the mF-G score. In contrast, significant correlation was found be-
tween A and both T and FAI.

Conclusions: This study provides evidence that, by including a steroid profile in the definition of
hyperandrogenemia, the majority of women with PCOS are hyperandrogenic, although a clinical
and biochemical heterogeneity exists. In addition, these data demonstrate that hirsutism and high
androgen levels cannot be used indifferently to define hyperandrogenism. (J Clin Endocrinol
Metab 101: 2013–2022, 2016)

ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: A, androstenedione; ANCOVA, analysis of covariance; BMI, body mass
Printed in USA index; DHEA, dehydroepiandrosterone; FAI, free androgen index; HA, hirsutism and/or
Copyright © 2016 by the Endocrine Society high T; HDL, high density lipoprotein; HOMA-IR, homeostasis model assessment for insulin
Received November 18, 2015. Accepted March 4, 2016. resistance; IR, ion ratio; LC-MS/MS, liquid chromatography coupled with tandem mass
First Published Online March 10, 2016 spectrometry; mF-G, modified Ferriman-Gallwey; 17OHP, 17hydroxiprogesterone; OA,
oligo-amenorrhea; P, progesterone; PCOm, polycystic ovarian morphology; PCOS, poly-
cystic ovary syndrome; QUICKI, quantitative insulin sensitivity check index; ROC, receiver
operating characteristics; SHBG, sex hormone-binding globulin; T, testosterone.

doi: 10.1210/jc.2015-4009 J Clin Endocrinol Metab, May 2016, 101(5):2013–2022 press.endocrine.org/journal/jcem 2013

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2014 Pasquali et al Hyperandrogenism in PCOS
yperandrogenism is the main criterion in the diag-
H nostic work-up of polycystic ovary syndrome
(PCOS). According to the current criteria, it can be indis-
tinctly defined by either hirsutism and/or excess of blood
testosterone (T) levels (1–3). However, most but not all
PCOS women are hirsute (3), and no more than 50% have
increased T levels measured by commonly used immuno-
assay methods (3). This implies that the population of
PCOS is rather heterogeneous in displaying its hyperan-
drogenic phenotype, which may be related to the relative
unreliability of the diagnostic criteria used to define hy-
perandrogenism. The current definition of hirsutism by
the modified Ferriman-Gallwey (mF-G) score is in fact
largely unsatisfactory, due to poor interobserver agree-
ment and he intrinsic variability of the scoring system (4).
In addition, although most women with hirsutism have
high T levels (5), and some correlation is found between
hirsutism scores and T levels in women with PCOS (6, 7),
this relationship becomes only marginally significant
when T is measured by more appropriate techniques (8).
The recent use of these techniques has increased, partic-
ularly in females, partly because most of the available im-
munoassays for T measurement are decidedly unsatisfac-
tory (9 –12). Recent studies have emphasized that a steroid
profile (also including androstenedione [A] and free T)
might be more appropriate than T alone in the diagnostic
work-up of PCOS (13–16) and might optimize the diag-
nosis of hyperandrogemia in women with PCOS, therefore
improving our understanding on the heterogeneity of the
phenotypes of the disorder. We therefore planned this
study to assess the steroid profiling by liquid chromatog-
raphy coupled with tandem mass spectrometry (LC-MS/
MS) in PCOS women with different phenotypes.
Materials and Methods
Subjects
We recruited 156 consecutive Caucasian PCOS women in
reproductive age attending the Endocrinology Unit of the
Sant’Orsola-Malpighi Hospital of Bologna. The diagnosis of
PCOS was performed according to the Rotterdam criteria (2).
Oligo-anovulation was defined by the association between oligo-
amenorrhea (OA) and progesterone (P) levels less than or equal
to 2 ng/mL in the luteal phase of the last cycle (17). Clinical
hyperandrogenism was defined by the mF-G score more than or
equal to 8 (18), whereas biochemical hyperandrogenism was
defined by a total T measured by LC-MS/MS more than or equal
to 0.454 ng/mL (HA) (19). This cut-off value was derived from
a previous study, in which we did not find any significant dif-
ference between normal-weight (0.245 ⫾ 0.087 ng/mL) and
overweight/obese (0.268 ⫾ 0.088 ng/mL) non-PCOS healthy
women (19). The other causes of hyperandrogenism, including
Cushing’s syndrome, nonclassic congenital adrenal hyperplasia,
androgen-secreting tumors, hyperprolactinemia, and drugs,
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J Clin Endocrinol Metab, May 2016, 101(5):2013–2022
were excluded by clinical and biochemical assessment (5). The
polycystic ovarian morphology (PCOm) was evaluated through
an ultrasound endovaginal examination (20). No subjects had
thyroid dysfunction, diabetes, cardiovascular, renal, or liver dys-
function, based on clinical and laboratory findings, or had taken
any medication in the previous quarter.
A total of 141 Caucasian healthy women of similar age were
also included. They derived from 2 population-based studies
performed by our group with the aim of evaluating the preva-
lence of hyperandrogenic disorders in the general population
(21, 22). All controls had normal menses and were ovulatory
(luteal phase P levels ⱖ3 ng/mL) (17), and no signs of
hyperandrogenism.
The study protocol was approved by the local Ethics Com-
mittee and written informed consent was obtained from the
participants.
Experimental protocol
Each subject underwent medical examination and blood sam-
pling. The same sample used to determine total T for the diag-
nosis of PCOS was also used to measure other steroids by LC-
MS/MS, gonadotropins and sex hormone-binding globulin
(SHBG). Blood samples were obtained after on overnight fast (d
3– 8 of the menstrual cycle) in subjects with eumenorrhea or
mild-to-moderate oligomenorrhea. In those with severe oligom-
enorrhea or amenorrhea, samples were taken after withdrawal
bleeding induced by a course of oral medroxyprogesterone ac-
etate, pregnancy having been excluded by the measurement of
human chorionic gonadotropin. All subjects had followed a
3-day diet containing at least 200-g carbohydrates daily before
blood sampling.
Anthropometry included the measurement of height, weight,
and waist circumference, and body mass index (BMI), according
to the World Health Organization guidelines (23).
The population of PCOS was categorized in 3 distinct phe-
notypes according to the combination of the classic criteria, in-
cluding hirsutism and/or high T (HA), OA, and PCOm (2).
Thereafter, we included A and free androgen index (FAI) in the
diagnosis of biochemical hyperandrogenism. A single cut-off
value of 1.602 ng/mL was used to define A excess, because no
significant difference in the reference intervals between normal-
weight and overweight/obese healthy women was found, as pre-
viously described (19). At variance, the cut-off values to define
high FAI were more than or equal to 1.80 in normal-weight and
more than or equal to 0.622 in overweight obese women (19).
Therefore, we further categorized the original phenotypes of
PCOS into different hyperandrogenic subgroups identified by
various combinations of hirsutism, high T, high A, and high FAI.
Assays
Gonadotropins (LH, FSH), SHBG, glucose, insulin, and lipids
were measured. Intra- and interassay coefficient of variation for
each analyte have been previously reported (20). Steroids (T, A,
dehydroepiandrosterone [DHEA], 17hydroxiprogesterone (17OHP),
and P), cortisol, corticosterone, and 11-deoxycortisol were mea-
sured by LC-MS/MS as detailed elsewhere (19). Briefly, serum
was purified by protein precipitation followed by solid phase
extraction. Extracts were then injected into a 2-dimensional liq-
uid chromatography system consisting of an on line purification
followed by separation on a Luna RP-C8 100x4.6 mm, 5 ␮m
(Phenomenex) in 21-minute runtime. Afterward, analytes un-
doi: 10.1210/jc.2015-4009 press.endocrine.org/journal/jcem 2015

derwent atmospheric pressure chemical ionization and multiple SE and the 95% confidence intervals of the ROC curves were
reaction monitoring detection by an API4000 QTrap mass spec- also evaluated. The sensitivities and the specificities were eval-
trometer (AB-Sciex). Specific quantifier and qualifier transitions uated by using the best cut-off assessed according to a max-
were monitored for each analyte and the ion ratios (IRs) were imum likelihood method (28). Data analyses were performed
monitored in each sample: data were excluded whenever the IR by using the IBM SPSS Statistics package (version 21; IBM
value exceeded ⫾20% of the expected IR. Isotopic dilution Co). Two-tailed P ⬍ .05 was considered significant.
quantitation was performed by using d4-cortisol as internal stan-
dard for cortisol, d8-corticosterone for corticosterone, d2-11-
deoxycortisol for 11-deoxycortisol, and 13C2-T (for T, A, and Results
DHEA; d8-17OHP for 17OHP, and 13C2-P for P). Data pro-
cessing and quantitation were performed by Analyst 1.4.2 AB- Comparisons among the 3 PCOS phenotypes and
Sciex. Intra- and interassay coefficient of variation were less than
controls
10% and less than 11%, respectively, and accuracy ranged be-
tween 83.7% and 106.2%, for all the analytes. The sensitivity in All PCOS women were initially categorized into 3 phe-
serum matrix was 0.244 ng/mL for cortisol, 0.078 ng/mL for notypes according to the combination of the 3 major Rot-
11-deoxycortisol and 17OHP, 0.019 ng/mL for T, 0.039 ng/mL terdam’s criteria: 1) patients with all the criteria (named
for A, 0.78 ng/mL for DHEA, and 0.049 ng/mL for P. HA ⫹ OA ⫹ PCOm; n ⫽ 48, 31%), 2) patients with HA
The FAI was calculated according to the formula total T ⫻ and OA (named HA ⫹ OA; n ⫽ 65, 42%), and 3) patients
100/SHBG blood levels (24). To investigate insulin sensiti-
with OA and PCOm (named OA ⫹ PCOm; n ⫽ 43, 28%).
vity, the homeostasis model assessment for insulin resistance
(HOMA-IR) (25) and the quantitative insulin sensitivity check
No subject had the phenotype characterized by HA plus
index (QUICKI) (26) were also calculated. PCOm.
Anthropometry, metabolic parameters and steroids
Statistics and data analysis were compared among the 3 PCOS phenotypes and be-
Data are reported as mean ⫾ SD and absolute and relative tween each PCOS phenotype and controls (Table 1). All
frequencies. All the source variables but QUICKI had signif- PCOS phenotypes were characterized by BMI, waist cir-
icantly different distribution from normal (P values ranging cumference and fasting glucose similarly higher compared
between ⬍0.001 and 0.003) at the Kolmogorov-Smirnov test
with controls, whereas no difference in fasting insulin,
(27) and showed a significant positive skewness coefficient.
Hence, these variables were transformed according to the for- HOMA-IR, QUICKI, and lipids between PCOS groups
mula log10 or to the formula log10 (x ⫹ k) if the constant was and controls was found.
needed in order to reach a nonsignificant value. The test of As expected, unlike the OA ⫹ PCOm phenotype, T
normal distribution showed P values ranging from 0.065 to levels were significantly higher in the HA ⫹ OA
1.000 after variable transformation. The data were compared (P ⬍ .01) and OA ⫹ HA ⫹ PCOm (P ⬍ .001) phenotypes
between the groups by means of the general linear models:
compared with controls, with an increasing trend ac-
ANOVA was used in order to test age and BMI, whereas the
analysis of covariance (ANCOVA) with BMI as covariate was cording to phenotype severity (P ⬍ .001). At variance,
used in order to test the other variables. The linear contrast A and FAI levels progressively increased in the 3 PCOS
was applied in order to test the relationship of the various phenotypes with respect to the controls, with the high-
parameters with increasing phenotype severity, whereas the est values in the HA ⫹ OA ⫹ PCOm phenotype
simple contrast was used in testing the different groups vs the (P ⬍ .001). No difference with respect to controls and
reference one (controls or OA ⫹ PCOm) in order to avoid
among the PCOS phenotypes was found in 17OHP and
multiple comparisons. A four-way ANOVA was applied in
order to evaluate the independent effects of hirsutism, high T, 11-deoxycortisol levels. By contrast, there was some
high A, and high FAI, as well as of their combination, on heterogeneity among the PCOS phenotypes in DHEA
hormones, anthropometry, and metabolic parameters accord- levels (P ⬍ .05) without any difference in comparison
ing to a hierarchical model: only the effects of the single factors with the controls. Finally, no significant difference was
were considered in the analysis when all the combinations detected in cortisol and corticosterone levels among the
between factors (ie, the interactions of second order) were not
PCOS phenotypes, but the HA ⫹ OA phenotype had
significant. The effects estimated by the ANOVA were anti-
transformed in order to obtain values showing the fractions of significantly higher values than controls (P ⬍ .05 and
the nontransformed values of the metabolic parameters in P ⬍ .01, respectively). SHBG levels were significantly
patients with presence of the factor vs the values in patients lower in all PCOS phenotypes compared with controls,
without the factor as far as the single factors were concerned, without any difference among them.
and in order to obtain values showing the additional fraction
attributable to the association of the contemporary presence Prevalence of high A and high FAI, other than
of both factors as far as the interactions were concerned. The
high T and hirsutism, alone or in combination,
relationship between variables was tested by means of the
linear regression. The accuracy of A and FAI in discriminating within the different phenotypes of PCOS
PCOS and control women was estimated by means of the area We found that 87 out of the 156 PCOS women (56%)
under the receiver operating characteristics (ROC) curve. The had high A; 35 (73%) within the HA ⫹ OA ⫹ PCOm

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Table 1. Comparison of Anthropometry, Metabolic Parameters, and Steroids Between the 3 PCOS Phenotypes and
Controls
PCOS Phenotypes
P for
HA ⴙ OA ⴙ Trend
Controls OA ⴙ PCOm HA ⴙ OA PCOm in
(n ⴝ 141) (n ⴝ 43) (n ⴝ 65) (n ⴝ 48) PCOS
Age (y) 28.8 ⫾ 10.3 29.6 ⫾ 7.4 30.2 ⫾ 8.3 27.7 ⫾ 6.9 .23d
BMI (kg/m2) 25.2 ⫾ 5.8 27.6 ⫾ 6.9a 28.9 ⫾ 7.4b 29.7 ⫾ 6.6c .19d
Waist circumference (cm) 84.7 ⫾ 14.1 89.1 ⫾ 15.8b 90.2 ⫾ 17.9b 91.7 ⫾ 13.8c .64e
Glucose, fasting (mg/dL) 81.7 ⫾ 13.6 86.7 ⫾ 9.9a 87.0 ⫾ 12.6a 89.9 ⫾ 20.9a .77e
Insulin, fasting (␮U/mL) 8.3 ⫾ 4.8 11.2 ⫾ 8.1 10.5 ⫾ 6.2 11.6 ⫾ 6.2 .92e
HOMA-IR 1.75 ⫾ 1.16 2.50 ⫾ 2.06 2.30 ⫾ 1.48 2.53 ⫾ 1.43 .86e
QUICKI 0.158 ⫾ 0.014 0.152 ⫾ 0.017 0.152 ⫾ 0.152 0.149 ⫾ 0.013 .92e
Total cholesterol (mg/dL) 176 ⫾ 31 174 ⫾ 44 173 ⫾ 36 175 ⫾ 27 .84e
HDL-cholesterol (mg/dL) 59.7 ⫾ 13.9 54.2 ⫾ 12.8 53.9 ⫾ 11.2 52.3 ⫾ 12.8 .83e
Triglycerides (mg/dL) 74.7 ⫾ 55.0 86.3 ⫾ 45.5 79.0 ⫾ 42.9 92.2 ⫾ 52.1 .98e
T (ng/mL) 0.259 ⫾ 0.085 0.276 ⫾ 0.084 0.323 ⫾ 0.129b 0.412 ⫾ 0.178c ⬍.001e
A (ng/mL) 0.82 ⫾ 0.34 1.44 ⫾ 0.41c 1.79 ⫾ 0.74c 2.05 ⫾ 0.67c ⬍.001e
17OHP (ng/mL) 0.620 ⫾ 0.454 0.589 ⫾ 0.349 0.592 ⫾ 0.379 0.618 ⫾ 0.332 .43e
DHEA (ng/mL) 7.39 ⫾ 4.83 6.22 ⫾ 4.39 8.79 ⫾ 5.86 7.64 ⫾ 3.50 .04e
SHBG (mmol/L) 51.0 ⫾ 32.8 39.2 ⫾ 23.0a 33.7 ⫾ 24.3c 30.4 ⫾ 14.1c .43e
Cortisol (ng/mL) 106.9 ⫾ 44.1 100.7 ⫾ 35.8 116.9 ⫾ 47.9a 105.2 ⫾ 44.3 .52e
Corticosterone (ng/mL) 3.37 ⫾ 2.91 3.74 ⫾ 3.95 5.74 ⫾ 6.47b 4.48 ⫾ 5.37 .40e
11-deoxycortisol (ng/mL) 0.334 ⫾ 0.241 0.283 ⫾ 0.199 0.321 ⫾ 0.189 0.322 ⫾ 0.258 .32e
FAI 0.68 ⫾ 0.45 1.06 ⫾ 0.87b 1.22 ⫾ 0.71c 1.55 ⫾ 0.78c ⬍.001e
Data are presented as mean ⫾ SD.
a
P ⬍ .05 vs controls (ANOVA for age and BMI; ANCOVA with BMI as covariate for the other variables).
b
P ⬍ .01 vs controls (ANOVA for age and BMI; ANCOVA with BMI as covariate for the other variables).
c
P ⬍ .001 vs controls (ANOVA for age and BMI; ANCOVA with BMI as covariate for the other variables).
d
ANOVA for trend.
e
ANCOVA for trend with BMI as covariate.

phenotype, 38 (58%) within the HA ⫹ OA phenotype notype. Consequently, the inclusion of FAI, isolated or
and, interestingly, 14 (33%) within the OA ⫹ PCOm combined with high T and/or high A, further increased
phenotype (Table 2). Collectively, the inclusion of A in the prevalence of hyperandrogenic women from 81% to
the diagnosis of biochemical hyperandrogenism led to a 90%.
prevalence of hyperandrogenic PCOS of 81%. In both the OA ⫹ HA ⫹ PCOm and the HA ⫹ OA
In addition, we found that 79 out of the 156 PCOS phenotypes, we were able to identify 8 hyperandrogenic
women (51%) had high FAI; 31 (65%) within the HA ⫹ subgroups, characterized by different combinations of
OA ⫹ PCOm phenotype, 35 (54%) within the HA ⫹ OA high T, high A, and high FAI with or without hirsutism
phenotype, and 13 (30%) within the OA ⫹ PCOm phe- (Table 2). Intriguingly, the original OA-PCOm phenotype

Table 2. Distribution of Hirsutism, High T, High A, and High FAI, Alone or in Combination, According to the 3
PCOS Phenotypes
Prevalence Frequency of Combination of High A and High FAI

High High Normal A- Normal A- High A- High A-

Phenotypes A FAI Normal FAI High FAI Normal FAI High FAI
Overall (n ⫽ 156) 87 (56%) 79 (51%) 52 (33%) 17 (11%) 31 (20%) 56 (36%)
HA ⫹ OA ⫹ PCOm (n ⫽ 48) 35 (73%) 31 (65%) 8 (17%) 5 (10%) 9 (19%) 26 (54%)
No hirsutism-high T (n ⫽ 14) 13 (93%) 12 (86%) 0 1 (7%) 2 (14%) 11 (79%)
Hirsutism-normal T (n ⫽ 29) 17 (59%) 15 (52%) 8 (28%) 4 (14%) 6 (21%) 11 (38%)
Hirsutism-high T (n ⫽ 5) 5 (100%) 4 (80%) 0 0 1 (20%) 4 (80%)
HA ⫹ OA (n ⫽ 65) 38 (58%) 35 (54%) 15 (23%) 12 (18%) 15 (23%) 23 (35%)
No hirsutism-high T (n ⫽ 3) 3 (100%) 2 (67%) 0 0 1 (33%) 2 (67%)
Hirsutism-normal T (n ⫽ 55) 28 (51%) 30 (54%) 15 (27%) 12 (22%) 10 (18%) 18 (33%)
Hirsutism-high T (n ⫽ 7) 7 (100%) 3 (43%) 0 0 4 (57%) 3 (43%)
OA ⫹ PCOm (n ⫽ 43) 14 (33%) 13 (30%) 16 (37%) 13 (30%) 14 (33%) 0

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doi: 10.1210/jc.2015-4009 press.endocrine.org/journal/jcem 2017

Table 3. Comparison of Hormonal Parameters Between the 8 Hyperandrogenic PCOS Subgroups, Defined
According to the Different Combinations of Hirsutism, High T, High A, and High FAI
Hyperandrogenic PCOS Subgroups

Hirsutism ⴙ
High T ⴙ Hirsutism ⴙ High T ⴙ
High A ⴙ Hirsutism ⴙ Hirsutism ⴙ High A ⴙ High A ⴙ
OA ⴙ PCOm Hirsutism HighA High FAI High FAI High FAI HighA High FAI High FAI
Parameters (n ⴝ 16) (n ⴝ 23) (n ⴝ 14) (n ⴝ 13) (n ⴝ 16)d (n ⴝ 16) (n ⴝ 16) (n ⴝ 29) (n ⴝ 12)e

DHEA (ng/mL) 4.59 ⫾ 2.55 5.69 ⫾ 2.68 8.58 ⫾ 5.59b 5.26 ⫾ 3.50 8.83 ⫾ 4.52b 5.66 ⫾ 2.96 8.68 ⫾ 6.09b 11.1 ⫾ 5.6c 9.14 ⫾ 4.76b
SHBG (mmol/L) 58.0 ⫾ 17.5 45.1 ⫾ 31.1a 34.4 ⫾ 27.1c 28.5 ⫾ 12.7c 39.4 ⫾ 18.9a 24.1 ⫾ 13.1c 35.5 ⫾ 6.9a 21.9 ⫾ 9.7 c 32.8 ⫾ 18.6b
Cortisol (ng/mL) 103.5 ⫾ 40.1 103.8 ⫾ 46.0 102.9 ⫾ 31.4 93.8 ⫾ 35.8 99.9 ⫾ 36.1 89.0 ⫾ 41.0 139.7 ⫾ 45.3a 126.6 ⫾ 52.4 103.9 ⫾ 32.6
Corticosterone 4.17 ⫾ 4.78 3.39 ⫾ 3.38 3.87 ⫾ 4.27 2.86 ⫾ 2.08 5.41 ⫾ 6.26 3.66 ⫾ 6.60 7.32 ⫾ 6.16a 6.97 ⫾ 7.66 3.71 ⫾ 2.22
(ng/mL)
11-deoxycortisol 0.304 ⫾ 0.229 0.221 ⫾ 0.114 0.316 ⫾ 0.227 0.205 ⫾ 0.088 0.335 ⫾ 0.219 0.235 ⫾ 0.161 0.385 ⫾ 0.160 0.422 ⫾ 0.308 0.301 ⫾ 0.142
(ng/mL)
LH (␮U/mL) 8.58 ⫾ 8.52 5.47 ⫾ 2.99 9.81 ⫾ 6.86 5.69 ⫾ 2.14 13.80 ⫾ 4.00b 5.51 ⫾ 2.42 7.71 ⫾ 3.88 7.72 ⫾ 4.57 11.48 ⫾ 6.51
LH/FSH 2.36 ⫾ 2.23 1.19 ⫾ 0.63a 2.13 ⫾ 1.19 1.90 ⫾ 0.89 2.67 ⫾ 0.81a 0.98 ⫾ 0.35a 1.61 ⫾ 0.83 2.01 ⫾ 1.10 2.19 ⫾ 1.28

The nonhyperandrogenic OA-PCOm subgroup was used as reference. Data are presented as mean ⫾ SD.
a
P ⬍ .05 vs the phenotype OA ⫹ PCOm (by ANOVA).
b
P ⬍ .01 vs the phenotype OA ⫹ PCOm (by ANOVA).
c
P ⬍ .001 vs the phenotype OA ⫹ PCOm (by ANOVA).
d
A total of 13 out of 16 patients included in this subgroup had high FAI.
e
A total of 7 out of 12 patients included in this subgroup had high FAI.

without any androgen excess decreased from 43 to 16 perandrogenic subgroups except those with isolated hir-
patients; ie, from 28% to 10% of the entire population of sutism or combined with high A had higher BMI and waist
PCOS women (Table 2). values with respect to the nonhyperandrogenic OA ⫹
PCOm subgroup. All the subgroups except the hirsut-
Additional steroids and gonadotropins in the 8 ism ⫹ high A one had significantly higher fasting insulin
hyperandrogenic PCOS subgroups with respect to
and HOMA-IR, and lower QUICKI. Fasting glucose was
the nonhyperandrogenic subgroup (OA ⴙ PCOm)
higher in all the subgroups with high FAI alone or com-
We investigated the pattern of other steroids and of
bined with hirsutism, high T, and/or high A. Finally, high
gonadotropin profile (Table 3) in the 8 hyperandrogenic
subgroups obtained by the combination of high hirsutism, density lipoprotein (HDL)-cholesterol levels were lower
high T, high A, and high FAI. The residual nonhyperan- only in the subgroups with hirsutism plus high FAI with or
drogenic OA ⫹ PCOm subgroup (n ⫽ 16) served as the without high A, whereas triglycerides were higher in the
reference group. subgroup with hirsutism ⫹ high A ⫹ high FAI only.
LH blood levels and the LH to FSH ratio were signif-
icantly higher only in the nonhirsute subgroup with high Effect of hirsutism, high T, high A, high FAI,
T ⫹ high A ⫹ high FAI. The subgroups with isolated hir- and of their combination on hormones,
sutism and hirsutism ⫹ high FAI were characterized by a anthropometry, and metabolic parameters
significantly lower LH to FSH ratio with respect to the within the population of PCOS
reference group. DHEA was higher in all subgroups with Finally, we measured the independent effect of hirsut-
high A, regardless of the coexistence of hirsutism, high T, ism, high T, high A, and high FAI, as well as of their
and high FAI. Notably, the subgroup with hirsutism ⫹ combination on hormones, anthropometry, and meta-
high A had significantly higher cortisol and corticosterone bolic parameters. Only the effects that were statistically
circulating levels. All the hyperandrogenic subgroups had significant are shown in Table 5.
lower SHBG levels with respect to the nonhyperandro-
We confirmed that hirsutism is associated with lower
genic OA ⫹ PCOm subgroup.
levels of LH and of LH to FSH ratio, with higher DHEA
Anthropometric and metabolic parameters in the 8 circulating levels, and also with lower levels of SHBG.
hyperandrogenic PCOS subgroups with respect to In addition, we found that hirsutism is associated with
the nonhyperandrogenic subgroup (OA ⴙ PCOm) higher fasting insulin and a worse insulin resistance
Accordingly, we extended the analysis of anthropomet- state only when it is combined with high T. Interestingly
ric and metabolic parameters in the 8 hyperandrogenic high A was associated with higher levels of adrenal ste-
PCOS subgroups described above (Table 4). All the hy- roids (DHEA, cortisol, cortisone, and 11-deoxycorti-

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Table 4. Comparison of Anthropometric and Metabolic Parameters Between the 8 Hyperandrogenic PCOS
Subgroups, Defined According to the Different Combinations of Hirsutism, High T, High A, and High FAI
Hyperandrogenic PCOS Subgroups

Hirsutism ⴙ
High T ⴙ Hirsutism ⴙ High T ⴙ
High A ⴙ Hirsutism ⴙ Hirsutism ⴙ High A ⴙ High A ⴙ
OA ⴙ PCOm Hirsutism HighA High FAI High FAI High FAI HighA High FAI High FAI
Parameters (n ⴝ 16) (n ⴝ 23) (n ⴝ 14) (n ⴝ 13) (n ⴝ 16)d (n ⴝ 16) (n ⴝ 16) (n ⴝ 29) (n ⴝ 12)e

Age (y) 30.6 ⫾ 8.5 29.7 ⫾ 7.9 25.7 ⫾ 5.3 32.3 ⫾ 6.8 27.6 ⫾ 6.2 34.3 ⫾ 9.3 30.7 ⫾ 6.9 26.2 ⫾ 6.7 28.6 ⫾ 8.3
BMI (kg/m2) 23.4 ⫾ 5.8 25.1 ⫾ 6.7 27.7 ⫾ 6.8a 33.3 ⫾ 4.7c 29.5 ⫾ 6.3b 35.3 ⫾ 5.4c 22.1 ⫾ 1.9 32.2 ⫾ 5.8c 30.7 ⫾ 6.2c
Waist circumference 80.0 ⫾ 15.7 80.0 ⫾ 14.9 86.5 ⫾ 15.1a 100.5 ⫾ 9.4c 92.2 ⫾ 11.9b 104.8 ⫾ 16.2c 74.6 ⫾ 6.4 95.5 ⫾ 15.7c 91.8 ⫾ 16.7a
(cm)
Glucose, fasting 81.8 ⫾ 7.2 85.4 ⫾ 7.8 86.1 ⫾ 10.1 92.1 ⫾ 10.7a 93.6 ⫾ 33.2a 94.4 ⫾ 18.6b 84.4 ⫾ 8.6 87.5 ⫾ 10.3 86.2 ⫾ 12.4
(mg/dL)
Insulin, fasting 5.8 ⫾ 2.6 9.6 ⫾ 5.8a 13.2 ⫾ 6.1c 16.3 ⫾ 10.8c 10.6 ⫾ 5.2b 12.8 ⫾ 5.6c 5.7 ⫾ 1.9 12.7 ⫾ 6.7c 14.0 ⫾ 6.5c
(mg/dL)
HOMA-IR 1.18 ⫾ 0.59 2.02 ⫾ 1.26a 2.83 ⫾ 1.32c 3.82 ⫾ 2.85c 2.28 ⫾ 1.23b 3.05 ⫾ 1.62c 1.19 ⫾ 0.46 2.79 ⫾ 1.58c 2.99 ⫾ 1.47c
QUICKI 0.166 ⫾ 0.013 0.154 ⫾ 0.014b 0.146 ⫾ 0.014c 0.142 ⫾ 0.015c 0.151 ⫾ 0.014b 0.144 ⫾ 0.012c 0.164 ⫾ 0.011 0.147 ⫾ 0.014c 0.144 ⫾ 0.012c
Total cholesterol 175 ⫾ 33 166 ⫾ 33 181 ⫾ 61 166 ⫾ 29 179 ⫾ 31 172 ⫾ 41 166 ⫾ 26 184 ⫾ 34 167 ⫾ 18
(mg/dL)
HDL-cholesterol 59.0 ⫾ 13.3 55.9 ⫾ 8.8 52.3 ⫾ 13.1 51.5 ⫾ 11.4 55.2 ⫾ 10.2 48.0 ⫾ 9.3a 60.4 ⫾ 14.4 50.0 ⫾ 13.3a 52.9 ⫾ 11.3
(mg/dL)
Triglycerides 69.9 ⫾ 33.9 75.7 ⫾ 55.1 99.7 ⫾ 55.9 90.2 ⫾ 39.7 79.1 ⫾ 32.2 103.1 ⫾ 69.4 53.7 ⫾ 19.4 98.3 ⫾ 39.1 a
78.5 ⫾ 26.8
(mg/dL)

The nonhyperandrogenic OA-PCOm subgroup was used as reference. Data are presented as mean ⫾ SD.
a
P ⬍ .05 vs the phenotype OA ⫹ PCOm (by ANOVA).
b
P ⬍ .01 vs the phenotype OA ⫹ PCOm (by ANOVA).
c
P ⬍ .001 vs the phenotype OA ⫹ PCOm (by ANOVA).
d
A total of 13 out of 16 patients included in this subgroup had high FAI.
e
A total of 7 out of 12 patients included in this subgroup had high FAI.

sol), higher levels of LH and of LH to FSH ratio, a lower istence of high T. Finally, high FAI was associated with
BMI and waist circumference but higher fasting glu- lower SHBG levels, higher fasting glucose and higher
cose. The increasing effect of high A on fasting glucose fasting insulin, a worse insulin resistance state and
was accentuated in those PCOS women with the coex- lower HDL-cholesterol. In addition, high FAI was as-

Table 5. Effect of Hirsutism, High T, High A, High FAI, and of Their Combination on Hormones, Anthropometry,
and Metabolic Parameters Within the #2.1 Population of PCOS
Effect

Hirsutism ⴙ High A ⴙ High A ⴙ High FAI ⴙ High FAI ⴙ

Parameters Hirsutisma High Ta High Aa High FAIa High Tb Hirsutismb High Tb High Ab High Tab

DHEA 1.22 (P ⫽ .035) 1.84 (P ⬍ .001)

SHBG (mmol/L) 0.59 (P ⫽ .004) 2.91 (P ⫽ .003) 0.17 (P ⬍ .001) 0.45 (P ⫽ .049) 1.61 (P ⫽ .049)
Cortisol 1.25 (P ⫽ .003)
Corticosterone 1.73 (P ⫽ .003)
11-deoxycortisol 1.72 (P ⬍ .001)
LH 0.79 (P ⫽ .024) 1.43 (P ⫽ .019) 1.46 (P ⫽ .001)
LH/FSH 0.71 (P ⫽ .005) 1.52 (P ⫽ .051)
BMI 0.93 (P ⫽ .035) 1.46 (P ⬍ .001)
Waist 0.94 (P ⫽ .028) 1.32 (P ⬍ .001)
circumference
Glucose, fasting 0.79 (P ⫽ .023) 1.19 (P ⫽ .043) 1.16 (P ⫽ .018) 1.40 (P ⫽ .047) 0.86 (P ⫽ .019)
Insulin, fasting 2.21 (P ⬍ .001) 1.94 (P ⫽ .031) 0.61 (P ⫽ .024)
HOMA-IR 2.46 (P ⬍ .001) 2.05 (P ⫽ .032) 0.60 (P ⫽ .032)
QUICKI 0.87 (P ⬍ .001) 0.90 (P ⫽ .038) 1.08 (P ⫽ .021)
Total cholesterol 1.26 (P ⫽ .014)
HDL-cholesterol 0.83 (P ⬍ .001)
Triglycerides 1.73 (P ⫽ .008) 0.51 (P ⫽ .040)

Only the statistically significant effects are shown.

a
The effect of each factor estimates the fraction of the values of the parameters in patients with presence of the factor vs the values in patients
without the factor.
b
The effect of the association between factors (interaction between factors) estimates the additional fraction of the values of the parameters
attributable to the association of the contemporary presence of both factors.

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doi: 10.1210/jc.2015-4009 press.endocrine.org/journal/jcem 2019

sociated with higher total cholesterol and triglycerides

when combined with high A.

Relationship between hirsutism score and T, A,

and FAI in all women with PCOS
No significant correlation was found between the
mF-G score and T (r ⫽ ⫺0.088; P ⫽ .28) or A (r ⫽ 0.066;
P ⫽ .41). By contrast, the mF-G score showed a modest,
but significant, positive correlation with FAI (r ⫽ 0.182;
P ⫽ .033) and an inverse correlation with the T to A ratio
(r ⫽ ⫺0.228; P ⫽ .004). Notably, highly significant cor-
relation coefficients were found between A and both T
(r ⫽ 0.772; P ⬍ .001) and FAI (r ⫽ 0.653; P ⬍ .001).

ROC analysis of A and FAI in discriminating PCOS

and control women
A high accuracy for A (91.1%; P ⬍ .001) in differen-
tiating the overall PCOS women from controls, whereas
FAI showed less, although significant, accuracy (75.0%;
P ⬍ .001) (Supplemental Table 1). Specifically, A showed
high values of sensitivity, whereas FAI showed high values
of specificity. The accuracy of both androgens increased
with the severity of the PCOS phenotypes, reaching values
of 94.4% and 85.0% in HA ⫹ OA ⫹ PCOm for A and FAI,
respectively (see ROC curves in Figure 1).

Discussion
This study shows that the clinical and biochemical pre-
sentation of androgen excess in PCOS women is hetero-
geneous, as suggested in previous studies in the field (8,
13–16), confirming that an androgen profile approach
may provide an advantage in defining the different hy-
perandrogenic phenotypes of PCOS. This is expected to
potentially favor an improvement of both preventive and
therapeutic individual strategies, as previously suggested
(5, 11). This is partly due to the adoption of modern an-
alytical technologies, such as LC-MS/MS, and their intrin- Figure 1. ROC curves of A (A) and FAI (B) in discriminating PCOS and
sic high accuracy and precision in the measurement of control women. The ROC curves discriminating each of the 3
steroids, particularly in women (5), provided that ade- phenotypes of PCOS and controls are also shown. The best cut off
points are highlighted.
quate reference ranges according to age and ethnicity is
available (17, 19, 29).
We confirm here that the combined use of a triad of also found that half patients with the OA ⫹ PCOm
parameters, including total T, A, and FAI, can accurately phenotype displayed a specific pattern of steroid ab-
define hyperandrogenemia in the majority (90%) of PCOS normality, characterized by high A or high FAI, which
patients and that their blood concentrations tend to in- supports the appropriateness of defining these patients
crease with the severity of the phenotype. In addition, this as hyperandrogenic (30), as suggested years ago by De-
steroid profile has been proved to help in defining the wailly et al (31).
different phenotypes, independently of the association The results of the ROC curves clearly support that A
with hirsutism, with high sensitivity in categorizing the levels displayed a high accuracy in differentiating the
heterogeneous presentation of hyperandrogenemia in PCOS status, as previously suggested by others (12),
PCOS, as previously suggested (12–14). Unexpectedly, we whereas FAI showed less accuracy in discriminating PCOS

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2020 Pasquali et al Hyperandrogenism in PCOS J Clin Endocrinol Metab, May 2016, 101(5):2013–2022

from controls, although demonstrating a high specificity, and these findings have been partly replicated in other
particularly in patients with the classic phenotype. studies (14, 16). Here we confirm that the combination of
Gonadotropin blood levels are not strictly required for high A with high total or free T is associated with a wors-
diagnosis of PCOS (2), nonetheless it is well known that an ened metabolic profile. Moreover, our data support that
increase of LH levels and altered LH to FSH ratio fre- hyperinsulinemia and insulin resistance are associated
quently occurs in these women, particularly in normal- with specific PCOS subgroups. This represents an intrigu-
weight individuals. In fact, obesity tends to decrease go- ing area for further research, aimed at more properly
nadotropin levels and LH responsiveness to GnRH identifying patients in whom insulin excess may play a
stimulation (32). Accordingly, we have found that those potential pathophysiological role in determining the hy-
PCOS women characterized by high A alone, had lower perandrogenic phenotype.
BMI, and a higher LH levels and LH to FSH ratio. By Finally, our study shows that the mF-G score did not
contrast, PCOS women with hirsutism presented with sig- significantly correlate with both total T and A blood levels,
nificantly lower LH to FSH ratio and normal LH levels. which implies that not only circulating androgens are re-
These findings may imply different dysregulated neuroen- sponsible for the development of hirsutism, which may
docrine mechanisms responsible for androgen excess in also reflect local hormone concentrations at the piloseba-
PCOS. ceous unit, where androgen effects also depend on the
Intriguingly, the present and previous data (12, 14, 16) expression of the androgen receptor (39). In addition, it
strongly support that the mechanisms responsible for an- should be considered that dihydrotestosterone is formed
drogen excess in PCOS women may differ among individ- directly from T by the 5␣-reductase type 1, which is abun-
uals. In fact, we found a significant heterogeneity on dant in the skin tissues. The same enzyme can also convert
DHEA blood levels among the different subgroups; those A into the 5␣-androstanedione which, in turn, can be con-
subgroups characterized only by hirsutism or high A had verted into dihydrotestosterone, through the action of
higher circulating DHEA levels. In addition, high A was 17␤-hydroxysteroid dehydrogenase3,5 (40). An increased
associated with higher levels of cortisol, corticosterone, cutaneous 5␣-reductase type 1 activity has been found in
and 11-deoxycortisol concentrations. These data support hirsute women with PCOS (41); however, studies on the
that a cluster of hyperandrogenic PCOS women may have role of 17␤-hydroxysteroid dehydrogenase3,5 in the skin
a dysregulation of steroidogenetic enzymes in the adrenals are still inconclusive (42). Therefore skin cells may pro-
or in peripheral tissues. Accordingly, increased 3␤-hy- duce biologically active androgens and express crucial
droxysteroid-dehydrogenase, which converts DHEA to genes of steroidogenesis (43). The potential role of cuta-
androgens, or reduced 17␤-hydroxysteroid-dehydroge- neous androgens in the pathophysiology of hirsutism in
nase, may represent potential targets for further research specific PCOS phenotypes should therefore be considered.
in explaining high A, particularly when T blood levels are In conclusion, we have provided evidence that the
normal (33). High A levels have also been reported in some triad defined by the combination of total T, A, and FAI
PCOS women with enhanced peripheral 5␣-reductase ac- may provide an excellent accuracy in defining the PCOS
tivity (34). Intriguingly, we found that combined high A status and its different hyperandrogenemic subpheno-
and DHEA has been found to be significantly more fre- types. Moreover we identified a subset of women pre-
quent in normal-weight than in overweight or obese PCOS senting with the OA ⫹ PCOm phenotype, who should
women (35). Overall, these data support an important be considered as hyperandrogenic. Metabolic issues do
impact of excess body weight on androgen blood levels not seem to correlate with the severity of hyperandro-
and metabolism. genism, rather they seem to characterize specific sub-
The additional contribution of FAI in increasing the groups of PCOS women with different androgen pro-
power of the hyperandrogenemic subgroups cannot be files. Finally, we suppose that hirsutism by itself and
totally related to T, but also to SHBG circulating levels, hyperandrogenemia should be used separately to define
which in turn are negatively regulated by insulin and pro- hyperandrogenism in PCOS women.
inflammatory cytokines (36). Intriguingly, because SHBG
levels are subjected to an inherited trait (37), it should be
considered that genetic variations in the regulatory se- Acknowledgments
quences of SHBG may influence its production and func-
We thank Susan West for assistance in the language revision of
tion (36, 38).
the manuscript.
The pioneering study by O’Reilly et al (13) showed that
the combination of high T and high A may predict insulin Address all correspondence and requests for reprints to:
resistance and a dysmetabolic milieu in women with PCOS Renato Pasquali, Division of Endocrinology, Department of

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doi: 10.1210/jc.2015-4009
Medical and Surgical Sciences, University Alma Mater Studio-
rum, Sant’Orsola-Malpighi Hospital, Via Massarenti 9, 40138
Bologna, Italy. E-mail: renato.pasquali@unibo.it.
Author contributions: R.P. and A.G. conceived, designed,
and wrote the study and wrote the manuscript; F.F., M.M., and
A.F. performed all laboratory analyses using LC-MS/MS; L.Z.,
D.R., and R.A. contributed in preparing the database and tables
and in discussing the data and the major findings of the study;
and A.M.M.L. performed statistical analysis.
This work was supported by Programma Regione Emilia-
Romagna Università 2007–2009, Programma Regione Emilia-
Romagna Università 2010 –2012, Bando Giovani Ricercatori
Alessandro Liberati, and the Programma Regione Università
area 1 Giovani Ricercatori (PRUa1GR-2012-004).
Disclosure Summary: The authors have nothing to disclose.
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