Ethinylestradiol30μg-drospirenone and metformin: could this combination improve endothelial dysfunction in polycystic ovary syndrome?

Abstract

We are hereby investigating for the first time the effect of the association ethinylestradiol30μg-drospirenone 3mg (DRP/EE30μg) plus metformin and weight loss on endothelial status and C-reactive protein (hsCRP) levels in polycystic ovary syndrome (PCOS).
25 young women with PCOS (mean age 22.76 ± 0.83 years, body mass index (BMI): 28.44 ± 6.23) who completed the study were prospectively evaluated. The oral contraceptive- DRP/EE30μg (21 days/month) and metformin (1700 mg daily) were administered for 6 months to the PCOS group. Additionally, the 15 overweight and obese patients (BMI > 25 kg/m2) were instructed in a diet of no more than 1500 cal daily. Primary outcome measures were surrogate markers of cardiovascular disease and included endothelial function, i.e. flow-mediated dilatation (FMD) on the brachial artery and endothelin-1 levels, as well as hsCRP concentrations, body composition (measured by whole-body dual-energy X-ray-absorptiometry) and insulin resistance. Variables were assessed at baseline, as well as after our medical intervention.
The combination between DRP/EE30μg plus metformin combined with weight loss triggered a significant improvement in the FMD values (FMD-PCOSbasal 3.48 ± 1.00 vs FMD-PCOS6 months7.43 ± 1.04, p = 0.033), as well as body composition and insulin insensitivity (p < 0.05). Regarding hsCRP levels, there was no significant intragroup (PCOS6months - PCOSbasal) difference.
A 6-month course of metformin- DRP/EE30μg (associated with weight loss) improves the endothelial dysfunction in PCOS and shows neutral effects on hsCRP concentrations as an inflammation marker. These data demand for reevaluation of the medical therapy in PCOS, particularly in women with additional metabolic and cardiovascular risk factors (ClinicalTrials.gov Identifier: NCT01459445).

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RESEARCH ARTICLEOpen Access
Ethinylestradiol30μg-drospirenone and metformin:
could this combination improve endothelial
dysfunction in polycystic ovary syndrome?
Ioana Rada Ilie1*, Ioan Marian2, Teodora Mocan3, Razvan Ilie4, Lucian Mocan5, Ileana Duncea1and
Carmen Emanuela Pepene1
Abstract
Background: We are hereby investigating for the first time the effect of the association ethinylestradiol30μg
-drospirenone3mg(DRP/EE30μg) plus metformin and weight loss on endothelial status and C-reactive protein (hsCRP)
levels in polycystic ovary syndrome (PCOS).
Methods: 25 young women with PCOS (mean age 22.76±0.83 years, body mass index (BMI): 28.44±6.23) who
completed the study were prospectively evaluated. The oral contraceptive- DRP/EE30μg(21 days/month) and metformin
(1700 mg daily) were administered for 6 months to the PCOS group. Additionally, the 15 overweight and obese patients
(BMI>25 kg/m2) were instructed in a diet of no more than 1500 cal daily. Primary outcome measures were surrogate
markers of cardiovascular disease and included endothelial function, i.e. flow-mediated dilatation (FMD) on the brachial
artery and endothelin-1 levels, as well as hsCRP concentrations, body composition (measured by whole-body dual-energy
X-ray-absorptiometry) and insulin resistance. Variables were assessed at baseline, as well as after our medical intervention.
Results: The combination between DRP/EE30μgplus metformin combined with weight loss triggered a significant
improvement in the FMD values (FMD-PCOSbasal3.48±1.00 vs FMD-PCOS6 months7.43±1.04, p=0.033), as well as body
composition and insulin insensitivity (p<0.05). Regarding hsCRP levels, there was no significant intragroup
(PCOS6months– PCOSbasal) difference.
Conclusion: A 6-month course of metformin- DRP/EE30μg (associated with weight loss) improves the endothelial
dysfunction in PCOS and shows neutral effects on hsCRP concentrations as an inflammation marker. These data
demand for reevaluation of the medical therapy in PCOS, particularly in women with additional metabolic and
cardiovascular risk factors (ClinicalTrials.gov Identifier: NCT01459445).
Keywords: Ethinylestradiol30μg-drospirenone, Flow-mediated dilatation, Endothelial dysfunction, HsCRP, Metformin,
Polycystic ovary syndrome
Background
Women with polycystic ovary syndrome (PCOS) frequently
cluster several cardiovascular risk markers and early sub-
clinical atherosclerosis which seem to be in relation to their
unfavourable endocrine and metabolic milieu [1,2] repre-
sented mainly by insulin resistance, central obesity and
hyperandrogenemia [3]. Endothelial dysfunction and arter-
ial stiffness are considered early stages of cardiovascular
disease development [4]. Previous studies [3,5-7], have
shown that endothelial function is impaired in women with
PCOS. Moreover, low-grade chronic inflammation plays a
major role in inducing endothelial injury, atherosclerosis
development and progression in these patients.
Lifestyle modification, diet and weight loss are essential
for the cardiovascular disease and diabetes mellitus preven-
tion in overweight and obese PCOS women. In addition to
these, and aside from therapeutic strategies directed
towards fertility restoration, the current pharmacological
treatment of this disease is mainly based on two drug
* Correspondence: ioanamanaila@yahoo.com
1Department of Endocrinology, University of Medicine and Pharmacy, 3-5
Louis Pasteur, 400349, Cluj-Napoca, Romania
Full list of author information is available at the end of the article
© 2012 Ilie et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Ilie et al. BMC Endocrine Disorders 2012, 12:9
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categories: (combined) oral contraceptives ((C) OC) and in-
sulin sensitizers.
The insulin sensitizer metformin has been shown to
ameliorate insulin resistance, reduce hyperandrogenism
and triglyceride (TG) levels in PCOS [7-9]. Moreover,
some studies also reported endothelial structure and
function improvement [7,10] and a decrement of highly
sensitive C-reactive protein (hsCRP) concentrations in
PCOS after metformin [11,12].
On the other hand, COC have a long history of use in
these patients and are prescribed for obtaining a regular
menstrual cycle and for improving hyperandrogenism.
Because COCs might adversely influence insulin resist-
ance, glucose tolerance, lipid profile [9,11] or aggravate
chronic inflammation [11,13] the possibility of worsening
the already unfavorable cardiovascular risk profile of
PCOS subjects is of concern. However, the metabolic
effects of pills are extremely variable; for example estro-
gens impair insulin action dose-dependently and the
associated progestins may modify these effects [14]. It
was suggested that when a dose of ethinylestradiol (EE)
<50 μg/day is used, the effects of the COCs on the lipid
and glycoinsulinemic metabolism is related to the pro-
gestin used in the combination [15]. Drospirenone (DRP)
is a progestin with antiandrogenic and antimineralocorti-
coid activity. However, the studies assessing the effect of
the COC containing 30 μg EE+3mg DRP (DRP/EE30μg)
on surrogate markers of atherosclerosis are few and in-
conclusive [15,16]. All the hereinabove considered, the
present study aimed to assess the effects of DRP/EE30μg
combined with metformin and weight loss by means of
dietary intervention on indices of endothelial dysfunc-
tion, i.e. flow-mediated dilation (FMD) and serum
endothelin-1 (ET-1), serum hsCRP and lipids, and insulin
resistance in young women with PCOS.
Methods
Study populations
We prospectively studied 26 women with PCOS who pre-
sented to our clinic. The study protocol was conducted
with the approval of the local ethics committee (246/
09.02.2011), in accordance with the Helsinki Declaration.
Participants gave written informed consent before their in-
clusion in the study protocol. The diagnosis of PCOS was
based on Androgen Excess Society 2006 guidelines [17].
Hyperandrogenism was defined as hirsutism and/or as an
elevated total testosterone concentration. Menstrual irregu-
larities were defined as oligomenorrhea (eight or fewer
menses/ year) or amenorrhea (absence of menstruation for
3 consecutive months). Polycystic ovaries were defined by
the ultrasound appearance of 12 or more follicles in each
ovary measuring 2–9 mm in diameter and/or ovarian
volume greater than 10 ml. Secondary causes of hyper-
androgenism such as hyperprolactinemia, thyroid disease,
androgen-secreting tumours, Cushing’s syndrome and con-
genital adrenal hyperplasia were excluded in all patients.
The presence and extent of hirsutism were quantified using
the Ferriman-Gallwey (F-G) score. Exclusion criteria
included current or previous use (within 6 months) of oral
contraceptives, anti-androgens, ovulation induction medi-
cations, drugs known to affect carbohydrate-lipid metabol-
ism, or a personal history of diabetes.
Study design
At the first visit, subjects with PCOS and BMI<25 kg/m2
and those with BMI>25 kg/m2 were instructed in a diet
of no more than 2000- and respectively 1500 cal daily,
composed of 50% carbohydrates, 20% proteins, and 30 %
fat –with a polyunsaturated-saturate ratio of 2:1. Dietary
adherence was assessed by review with the same investiga-
tor (I.I.) at 3 and 6 months after inclusion in the study.
The treatment with the monophasic COC- DRP/EE30μg
(21 days/month) and metformin, given as 850 mg twice
per day, was started the fifth day of a spontaneous or
progestin-induced menstrual cycle. To reduce metformin
gastrointestinal side effects, doses were titrated up over a
20-day period starting at 425 mg up to a final dose of
1700 mg /day (two tablets daily, one before lunch and one
before dinner). All women received their medications for
6 months.
A complete history and physical examination, including
BMI and waist hip ratio (WHR) were determined by the
same physician. Systolic blood pressure (SBP) and diastolic
blood pressure (DBP) were measured in the right arm, with
the subjects in a seated position, using a mercury sphygmo-
manometer. More than 5 cigarettes/ day was considered
active smoking, while a sustained physical activity (fitness,
swimming, jogging etc.) of at least 1h per day, at least 3
times a week, for at least 3 consecutive months was consi-
dered significant. None of the study subjects performed sig-
nificant physical activity before or during the study. All
patients were instructed to not modify their physical acti-
vity throughout the trial.
PCOS subjects were investigated at baseline, during
early follicular phase (days 2–5) of a spontaneous or
progestin-induced menstrual cycle, and after six cycles
of treatment-during days 5–7 of week 4 (week of placebo
pills, no exogenous hormones). Evaluation included an-
thropometric, laboratory, flow-mediated dilation (FMD)
and body composition measurements. In seventeen
PCOS subjects (68%) menstruation was induced using
dydrogesterone, 20 mg/day for 5 days, a progestin which
appears to exert neutral effects on nitric oxide release, as
recently demonstrated [18].
Assay methods
All blood samples were obtained between 08.00 and 10.00 h
in the morning after overnight fasting. Blood samples were
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immediately centrifuged and the serum obtained was stored
at −80°C until the time of assay. Serum fasting glucose
(GLU, mg/dl) was measured by the glucose oxidase colour
method (Glucose GOD/PAP; Diagnosticum Zrt, Budapest,
Hungary). Total cholesterol (TC, mg/dl) and TG (mg/dl)
were measured by an enzymatic, colorimetric method
(Diagnosticum Zrt, Budapest, Hungary).
All other measurements were performed using the
ELISA TECAN auto-analyzer. Insulin (INS, μU/ml), total
testosterone (TT, ng/ml), sex hormone-binding globulin
(SHBG, nmol/l), endothelin-1 (ET-1, pg/ml) were all
measured using commercial enzyme–linked immunosor-
bent assay kits from DRG Instruments, Marburg, Germany.
The intra-assay coefficients of variation for low and high
values for INS, TT, SHBG and ET-1 respectively were as
follows: 2.6% and 1.8%, 4.1% and 3.3%, 8.6% and 5.3%, 8.8%
and 6.7% respectively. The intra-assay coefficients of va-
riance for hsCRP were 6. 9% for CRP values <1.0 mg/l and
4.1% for CRP values >3.0 mg/l, respectively. The minimal
detectable concentrations for ET-1 and hsCRP were 0.41
pg/ml and 0.02 μg/ml.
The free androgen index (FAI) was calculated according
to the equation: FAI (%)=TT (ng/ml) x 3.47 X 100/SHBG
(nmol/l). Insulin resistance was estimated by the homeosta-
sis model assessment of insulin resistance (HOMA-IR)
defined fasting glucose (mg/dl) x insulin (μU/ml)/405 and
by the quantitative insulin sensitivity check index (QUICKI)
defined as 1/ [log (fasting insulin)+log (fasting glucose)].
Body composition
Body composition was assessed by whole-body dual-
energy X-ray-absorptiometry (DXA) with a DPX-NT (GE,
Madison, USA) device. Fat-free mass was automatically
determined as the difference between total body weight
and bone mineral content and fat mass. Both total and seg-
mental fat mass and fat-free mass were expressed as weight
percentage. The coefficient of variance, evaluated at 3% for
total fat mass, was determined by measurements on 10
patients, each one evaluated 3 times.
Hemodynamic studies
FMD was measured for all subjects by the same cardio-
logist, who was blinded to PCOS status, using a colour
Doppler (AGILENT SONOS 4500) with a high-resolution
10-Mhz linear probe. Each patient was taken into a quiet,
temperature-controlled room at 20-25°C. After resting in a
supine position for 15 minutes, the right brachial artery
was identified and its position marked at about 5 cm above
the elbow joint. Diameter (mm) of the artery was measured
at end-diastole. After the resting measurement, limb flow
occlusion was produced by inflating a standard sphygmo-
manometer cuff on the upper arm to 50 mmHg above sys-
tolic pressure for 5 minutes. This caused ischemia and
consequently, dilatation of downstream resistance vessels.
Subsequent cuff deflation induced a brief high-flow state
through the brachial artery (reactive hyperemia) for endo-
thelial nitric oxide release, to accommodate the dilated
resistance vessels. The brachial artery was scanned conti-
nuously for 90 seconds after cuff deflation and the mea-
surements were performed during the 30–90 seconds
interval. The vessel’s diameter was measured at the same
point with resting measurement at least twice and the
maximal diameter was again defined (diameter during
reactive hyperemia). FMD was calculated as the percentage
maximum change in vessel size from baseline. The coeffi-
cient of variation for repeated measurements of resting
arterial diameter was 2.3%.
Statistical analysis
Prospective sample size estimation was performed based
on a pilot study data (n=10 for each group) which
showed FMD levels for before and after treatment mea-
surements of 3.37±6.6 and 7.97±4.77, respectively. For
alpha=0.05 and beta=0.20, we have calculated a sample
size of n=24 patients to be evaluated before and after
treatment. An additional estimative 8% was added to the
calculated sample size in order to cover patient loss,
resulting in a sample size of n=26. Analysis was per-
formed on the 25 subjects who completed the study.
Results are reported as mean values±SEM. The distribu-
tion of continuous variables was tested with Kolmogorov-
Smirnov test. Differences among continuous variables be-
fore and after treatment were assessed by Wilcoxon test.
For this type of analysis additional adjustments for dicho-
tomous weight loss were done using weight loss thresholds
of 5% and 10% respectively. Bivariate correlations were per-
formed calculating the Spearman rho coefficient. P values
of <0.05 were considered statistically significant. The ana-
lysis was performed using SPSS version 17.0 (Chicago, Il,
USA) and MedCalc 8.3.1.1.
Results
The study initially included all 26 subjects who were eli-
gible after screening. During the follow-up period, 1 pa-
tient withdrew from the study because of some
moderate gastrointestinal side effects related to metfor-
min and was, therefore, excluded. This resulted in a final
number of n=25 patients (ages: 15–30, mean age
22.76±0.83 years) completing the study.
Treatment and diet effects on measured parameters
Table 1 illustrates the anthropometric characteristics,
the main metabolic and hormonal pattern as well as the
endothelial and inflammatory profile of these 25 PCOS
patients over the 6 months.
The BMI, total fat mass %, lean mass, fasting insulin
and HOMA-IR, the F-G score and both TT and FAI were
significantly decreased by the pharmacologic approach
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and diet in the total PCOS group, while WHR and trunk
fat mass % declined only borderline. We also noted a sig-
nificant increment of QUICKI and SHBG concentrations
as well as of TC and TG levels over the study.
The combination of diet with metformin+DRP/EE30μg
was associated with a higher FMD (Table 1). A trend to-
wards lower ET-1 levels was found after 6-month DRP/
EE30μg and metformin therapy. However, the difference
failed to reach statistical significance. Moreover, there was
no change in hsCRP levels at the end of the follow-up
period as compared to baseline ones (Table 1).
HsCRP levels were significantly correlated with BMI
(r=0.554, p=0.004), TC (r=0.600, p=0.002), total fat mass
% (r=0.484, p=0.014) and trunk fat mass (r=0.404,
p=0.045) at the beginning of the research whereas at 6
months they significantly correlated with BMI (r=0.550,
p=0.004), trunk fat mass (r=0.431, p=0.032), insulin
(r=0.418, p=0.038), HOMA-IR (r=0.411, p=0.041),
QUICKI (r=−0.412, p=0.041), and TC concentrations
(r=0.403, p=0.046) but only borderline with total fat mass
(r=0.353, p=0.091). No other significant correlation be-
tween the studied parameters was found.
Influence of obesity and smoking habits on post-
therapeutic results
Overall the PCOS women achieved a 6.61% (−5.04±
5.31kg) moderate weight loss, whereas the obese- over-
weight and non-obese PCOS subgroups undergone a
weight loss of 8.78% (−7.53±4.92 kg) and 2.1 % (−1.3 ±3.41
kg), respectively.
Compared with their non-obese counterparts, obese
and overweightPCOSwomen
(p=0.02), DBP (p=0.017), FAI levels (p=0.035), fasting
glucose (p=0.009), fasting insulin (p=0.003), HOMA-IR
(p=0.001), total and trunk fat mass % (p<0.001), hsCRP
concentrations (p=0.045) and a lower QUICKI (p=0.001)
and SHBG concentrations (p=0.031). Considering non-
obese and obese PCOS subjects separately, the insulin re-
sistance indices, total fat mass and lean mass showed a sig-
nificant improvement after 6 months of therapy associated
with diet, compared to baseline in the sub-group of obese
and overweight women. However, none of the endothelial
dysfunction or inflammatory markers seemed to have sig-
nificantly improved (Table 2). In contrast, non-obese PCOS
patients only noted significant changes in markers of hyper-
androgenemia and a borderline increase in QUICK index.
Regarding the lipid profile, TG levels significantly rose in
the obese sub-group while TC concentrations were signifi-
cantly increased at the end of the 6-month period in the
non-obese subgroup and borderline in the obese one
(Table 2).
Even after adjusting for a decline of >5% in BMI, there
was still a significant difference between the basal values
of HOMA-IR (p=0.014), QUICKI (p=0.026), INS
(p=0.016),SHBG(p=0.003),
(p=0.007), trunk fat mass % (p=0.033), lean mass
(p=0.016) and a borderline one with respect to FMD
(p=0.093) and their values at 6 months. The adjustment
for>10% decrease in body weight was still accompanied
by significant differences in pre-treatment vs post treat-
ment values of total fat mass % (p=0.043) and SHBG
(p=0.028), respectively. Additionally, the unfavorable
increments in TG levels over the 6- month treatment
observed in our research were significant after applying
the same adjustments (p=0.012, p=0.027 respectively).
Eight (32%) PCOS women were currently light smo-
kers (i.e. no more than 10 cigarettes per day, <5 years).
There were however no statistically significant differ-
ences in any of the studied parameters between PCOS
smokers and PCOS non-smokers at baseline. Regarding
the smoking influence, the fasting glucose (p=0.021),
both HOMA-IR (p=0.003) and QUICKI (p=0.006),
SHBG levels (p<0.001) and FAI (p=0.003) improved
significantly in non-smokers during the study and they
had higher WHR
total fatmass%
Table 1 PCOS subjects (n=25) characteristics at baseline
and at the sixth cycles of treatment. Data as means ±
SEM
VariablesBaseline 6 months p-value
BMI (kg/m2) 28.44 ± 1.2426.51 ± 1.00
<0.001{
WHR0.83 ± 0.070.82 ± 0.01 0.071
Glucose (mg/dl)85.36 ± 1.43 83.08 ± 1.29 0.192
<0.001{
0.001†
0.001†
0.005†
HOMA-IR3.67 ± 0.322.82 ± 0.23
QUICKI
Insulin (μU/ml)
0.31 ± 0.0030.33 ± 0.004
17.26 ± 1.3713.74 ± 1.11
TC (mg/dl)198.00±4.87 215.28 ± 6.42
TG (mg/dl) 100.56 ± 6.82125.40 ± 12.88 0.04*
Total fat mass (%) 44.08 ± 1.5442.56 ± 1.470.018*
Trunk fat mass (%) 45.44 ± 1.8144.02 ± 1.81 0.065
0.009†
0.007†
<0.001{
<0.001{
Lean mass (kg)39.03 ± 1.38 37.84 ± 1.32
TT (ng/ml) 0.84 ± 0.06 0.68 ± 0.02
FAI (%)9.70 ± 1.723.55 ±1.02
SHBG (nmol/l)44.41 ± 5.34148.44 ± 16.22
F-G score9.72 ± 1.068.20 ± 0.920.02*
SBP (mmHg)106.20 ± 2.43104.00 ±2.390.184
DBP (mmHg)70.80 ± 1.9572.60 ± 1.680.318
hsCRP (mg/l)5.02 ± 0.725.20 ± 0.870.518
FMD (%)3.48 ± 1.007.43 ± 1.040.033*
Baseline artery diameter (mm)3.14 ± 0.073.03 ± 0.060.039*
ET-1 (pg/ml)
PCOS, polycystic ovary syndrome; BMI, body mass index; WHR, waist-to-hip
ratio; HOMA-IR, homeostasis model assessment of insulin resistance; QUICKI,
quantitative insulin sensitivity check index; TC, total cholesterol; TG,
triglycerides; TT, total testosterone; FAI, free androgen index; SHBG, sex-
hormone binding protein; SBP, systolic blood pressure; DBP, diastolic blood
pressure; FMD, flow-mediated dilatation; ET-1, endothelin-1
*P<0.05;†p<0.01;{p<0.001.
2.10 ± 0.591.60 ± 0.260.179
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showed no changes or only borderline modifications to-
wards a better status in smoker PCOS subjects. BMI, in-
sulin levels and F-G score decreased significantly
regardless of the smoking status (p<0.05). Neither
FMD nor ET-1 or hsCRP significantly changed during
the study as a function of smoking.
Discussions
We report for the first time that the 6-month treatment
with metformin 1700mg/day and the antiandrogenic
COC-DRP/EE 30μg combined with diet has an overall
beneficial effect on endothelial function and shows a
neutral effect on hsCRP levels in obese and non-obese
women with PCOS.
A novel finding here was the significant improvement
of FMD with the medical intervention. However, it is dif-
ficult to determine which of the three interventions are
responsible for this improvement, especially because when
individually assessed, the weight loss, metformin and par-
ticularly DRP/EE30μgeffects on the endothelial function are
not settled. Likewise, several recent studies have demon-
strated either the favourable impact of lifestyle modifica-
tions such as caloric restriction and increased physical
activity in improving endothelial function [19-21] or, on the
contrary, no improvement of FMD with weight loss [22,23].
However, in PCOS, metformin seems to have improved
endothelial function in both obese and non-obese subjects
[7,8,10,11,24,25], although not constantly [9]. FMD appears
to increase when circulating levels of estrogens augment
naturally or synthetically [26-29] and this beneficial vaso-
dilator effect of estrogens on the arterial function might be
antagonized by some certain types of progestins [30,31],
but not by DRP [32]. Regarding the DRP/EE30μg, there are
very few researches investigating its effect on endothelial
function and none evaluating the effect in combination
with metformin. Therefore, in PCOS the DRP/EE30μgcom-
bination administered for 6 months has been shown not to
modify the levels of ET-1 which, however, were found to be
similar to those of controls and within normal limits [15]
or the normal basal vascular reactivity of these women [16].
Others concluded that, while in lean patients with PCOS,
the DRP/EE30μgdoes not seem to affect endothelial func-
tion, in overweight PCOS women it does not counteract
the loss of weight due to healthier lifestyle changes, which
is associated with an improvement of insulin sensitivity and
FMD [15]. Of note, FMD did not report a significant
Table 2 Hormonal, metabolic, inflammatory and functional endothelial status in obese and non-obese PCOS women
pre- and post-medical intervention. Data as means ± SEM
VariablesObese PCOSp-
value
Non-Obese PCOS p-
value
Baseline6 monthsBaseline6 months
BMI (kg/m2) 32.56±1.1329.71±0.940.001†
22.26±0.55 21.72±0.660.263
WHR 0.87±0.010.86±0.010.4400.77±0.010.75±0.01 0.056
Glucose (mg/dl)88.40±1.62 84.60±3.96 0.117
0.002†
0.003†
0.002†
80.80±1.9180.80±2.18 0.859
HOMA-IR 4.48±0.42 3.29±0.30
2.46±0.172.10±0.23 0.139
QUICKI
Insulin (μU/ml)
0.31±0.004 0.32±0.005
0.33±0.0030.34±0.005 0.074
20.48±1.79 15.85±1.49
12.43±0.8510.57±1.11 0.169
TC (mg/dl) 202±20.65220±37.840.057
0.004†
192±29.24 208.20 ±20.770.047*
TG (mg/dl) 103.20±35.41135±72.67
96.60±33.48 111±49.69 1.00
Total fat mass (%)49.51±3.3047.43±4.360.016*35.94±4.3735.26±3.710.508
Trunk fat mass (%) 51.82±3.5650.39±4.000.173
0.006†
0.009†
35.86±5.4434.46±5.000.203
Lean mass (kg)40.54±8.52 38.72±8.27
36.75±2.38 36.51±2.560.721
TT (ng/ml)0.86±0.08 0.68±0.03
0.80±0.09 0.68±0.010.241
0.007†
FAI (%) 12.33±2.644.56±1.64 0.011*
0.007†
0.001†
5.74±0.82 2.04±0.41
F-G score10.40±1.428.67 ±1.21 8.70±1.647.50±1.450.016*
SHBG (nmol/l)35.98±6.10144.03±23.03
57.05±8.59155.06±22.530.013*
SBP (mmHg)109.67±3.02 104.67 ±3.29 0.042*101.00±3.63 103.00±3.590.732
DBP (mmHg)74.67±2.41 74.67±1.91 0.88665.00±2.35 69.50±2.930.098
HsCRP (mg/l) 6.10±0.916.88±1.14 0.3073.40±1.03 2.69±0.950.646
FMD (%)4.57±1.31 8.58±1.290.133 1.65±1.44 5.55±1.650.161
Baseline artery diameter (mm)3.15±0.113.06±0.09 0.4023.12±0.082.99±0.09 0.779
ET-1 (pg/ml)
PCOS, polycystic ovary syndrome; BMI, body mass index; WHR, waist-to-hip ratio; HOMA-IR, homeostasis model assessment of insulin resistance; QUICKI,
quantitative insulin sensitivity check index; TC, total cholesterol; TG, triglycerides; TT, total testosterone; FAI, free androgen index; SHBG, sex-hormone binding
protein; SBP, systolic blood pressure; DBP, diastolic blood pressure; FMD, flow-mediated dilatation; ET-1, endothelin-1;
*P<0.05;†p<0.01.
2.36±0.921.80±0.42 0.1981.71±0.55 1.32±0.20 0.508
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improvement when evaluating non-obese and obese PCOS
subjects separately and this can be accounted for by the
small number of subjects in the two groups.
The present results also show that the combination of
metformin+DRP/EE30μgassociated with lifestyle changes
has overall beneficial effects on body composition and
carbohydrate metabolism in the whole study population
and in the obese subjects. The milder metabolic abnormal-
ities in non-obese PCOS women (Table 2) and the small
number of subjects may explain the lack of significant effect
of medical intervention on body composition and the only
borderline improvement of insulin sensitivity in these sub-
jects. However, our results are similar to those of Ibanez
et al. who demonstrated that a 3-month treatment with
metformin 850 mg/day+DRP/EE30μg does not influence
the insulin resistance in another group of young non-obese
patients with PCOS but with increased adiposity [33].
Nevertheless, the decrease in body weight explains part of
our results as a modest weight reduction of 5-10% was
demonstrated to be enough to improve insulin resistance
[34]. However, we have shown that differences in several
parameters persisted after the adjustment for a decrease of
>5% or >10% of body weight thus suggesting that the
drug combination used displays an improvement in the in-
sulin sensitivity and can attenuate the central and total
body adiposity of young PCOS women. Previous results
showed that the increased adiposity found in non-obese
adolescents with PCOS diverged further from the norm in
subjects on DRP/EE30μgalone [35,36] and that the same
OC increased TG levels in both overweight and non-obese
women with this disease as well as in young, non-obese but
hyperinsulinemic PCOS patients [36-38]. Moreover, in a
subsequent research by the same authors, the addition of
small doses of metformin- 850mg/day to the treatment
with DRP/EE30μgfor 3 months failed to attenuate body adi-
posity of PCOS subjects and was accompanied by a further
deterioration of TG and interleukin-6 (IL-6) levels from the
norm [33]. In contrast, others observed that DRP/EE20μg+
metformin 1500mg/dayimproved
increased HDL-cholesterol concentrations and did not sig-
nificantly change TG levels in a group of young but non-
obese and non-insulin resistant women with PCOS [39].
However, a valid comparison between the study findings of
Ibanez et al. and Fruzzetti et al., as well as a further one be-
tween their results and ours cannot be performed since the
doses of metformin and EE used as well as the basal meta-
bolic profile were different between the populations of these
reports. Regarding lipid metabolism, although TG levels
rose significantly over the study period in the whole study
population as well as in the obese subgroup, most probably
as a result of the medical therapy, they remained within the
normal range. In other words, it can be hypothesized that
metformin clearly does not outperform DRP/EE30μgin in-
creasing TG concentrations. Unfortunately, we did not
insulinsensitivity,
undertake the analysis of cholesterol fraction in our study.
Likewise, we do not know if the increase in the total
cholesterol is due to a rise in HDL-cholesterol fraction, or
to an increase in LDL-cholesterol or both.
Most published data, yet not all [6,40,41], demon-
strate increased levels of hsCRP in women with
PCOS [5,12,42,43], which may be associated with
increased central fat excess rather than PCOS status
per se [41,44]. The change in hsCRP levels in our
study was not significant by the end of the 6-month
follow-up. However, the dosage of metformin and
the duration of our study assured the detection of
the effects of the medical intervention on hsCRP
levels. Therefore, we can conclude that even though
moderate weight loss (6–8 %) definitely conferred
significant metabolic benefits, it was not sufficient to
improve the low-grade chronic inflammation in the
total PCOS group and obese subgroup. The follow-
ing results at the end of the study are noteworthy:
both insulin resistance and body adiposity were still
abnormal, particularly in the total PCOS group and
the obese sub-group, on the one hand and the
hsCRP concentrations correlated with trunk fat mass,
BMI, TC and insulin resistance indices on the other
hand. Therefore, it appears that a more aggressive
therapeutic approach and a greater degree of weight
loss may be required to achieve metabolic benefits,
such as reductions in insulin resistance and body
adiposity andconsequent
chronic inflammation. Sustaining our results, it was
shown that a 4–5% weight loss improved lipid, glu-
cose, and insulin profiles in women with and with-
out PCOS, but was not effective in lowering CRP
concentrations in PCOS women [45] whereas a 15%
weight loss in a 2-yr dietary and exercise interven-
tion study was associated with hsCRP reduction [46].
The results obtained in the non-obese subgroup of
PCOS women and also those resulted after the ad-
justment for a decrease >5 % and 10% in body
weight showed that the drug combination used in
this study did not affect hsCRP levels. It could be
hypothesized that metformin and DRP/EE30μg may
have either different or even opposing effects on
chronic inflammation that may balance the risk out
and neutralize it overall or may both have neutral
effects on hsCRP. Our hypothesis is based on previ-
ous results showing that metformin either decreased
[11-13] or, used in lower doses (1000-1500mg/day)
and for shorter period of time (3 months), caused
no change in hsCRP [8,47], as well as on the fact that
the effect of DRP/EE30μgon chronic inflammation and es-
pecially on hsCRP has not been settled. As far as we know,
there are no reports evaluating the influence of DRP/EE30μg
monotherapy on hsCRP levels in PCOS or in other
decreaseof low-grade
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populations.
though, is that, DRP/EE30μgfurther increased the ab-
normal levels of IL-6 found in young women with
hyperinsulinemichyperandrogenism
reports suggested that metformin may have direct
actions on vascular cells [10,48]. Additionally, metfor-
min might decrease angiogenesis via nuclear factor-κB
and Erk1/Erk5 pathways by increasing the antiangio-
genic trombospondin-1, an adipokine, preferentially
produced by visceral adipose tissue and highly expressed
in obese insulin-resistant subjects [49]. However, it is
uncertain whether metformin exerts direct effects on
hsCRP levels or whether its beneficial changes on the
concentration of this inflammation marker are only the
result of improved glycemia, insulin resistance, abdom-
inal fat excess and weight loss. Complex interactions
and mechanisms might be implied. On the contrary, the
serum CRP levels have been shown to increase after a 6-
month treatment with COC in young overweight and
obese women with PCOS, even if the COC contains an
anti-androgen [11,13]. Both estrogens and progestin
content and dosage appear to be implicated in CRP
regulation [50-52], even though the role of oestrogen
might be more important than that of progestin [52].
As far as the mechanism of action is concerned, the litera-
ture has been sustaining the direct role of COCs in hsCRP
determination by affecting the latter’s metabolic and gen-
etic regulation [52]. Hence, a direct estrogen action on the
liver was proposed since it is the oral estrogen, and not
transdermal estradiol (the latter avoiding the first pass liver
effect), which leads to increased serum CRP levels [50,53].
Moreover, COCs have been demonstrated to increment
CRP concentrations, without increasing IL-6 ones, suggest-
ing that COCs stimulate hepatocytes to synthesize CRP in
a direct way and not via IL-6 mediated inflammation [52].
Regarding anti-androgens, even though their mechanisms
of action are not settled, they also appear to modulate and
decrease inflammation in PCOS. Hence, low-dose fluta-
mide added to metformin and a fourth-generation OC has
been identified to attenuate the hypoadiponectinemia, lean
mass deficit as well as central adiposity in young women
with PCOS [33]. Up-regulated in states of insulin resis-
tance, IL-6 differentially regulates androgen receptor trans-
activationviathree distinct
pathways, the overall effect depending on the balance
among these pathways and the androgen concentrations.
For instance, at androgen concentration above normal
female range and below normal male range, IL-6 and
androgens could act synergistically on the androgen recep-
tor [54,33], hereby increasing androgen action. On the
other hand, androgen excess in women favors an adipose
body composition, including in the abdominal region. A
vicious circle amplifying chronic inflammation is thus
established. Moreover, adiponectin, which exerts insulin-
What hasbeen previouslyobserved,
[36].Previous
signaling transduction
sensitizing effects, is reversibly down-regulated by andro-
gens and IL-6, but not by estrogens [55]. Therefore, the
mechanism of action of flutamide might be at least partly
explained by restoration of the androgen receptor transac-
tivation balance and counteraction of the androgen – and
IL-6- induced down-regulation of adiponectinemia [33].
Additionally, the mineralocorticoid and the androgen
receptor antagonist spironolactone has been shown to in-
hibit the production of proinflammatory cytokines in
patients with congestive heart failure, acting at the tran-
scriptional level and independent of its antimineralocorti-
coid and antiandrogen activities [56]. We found no data
on the effect of the combination metformin-DRP/EE30μg
on hsCRP, only that Ibanez et al. showed that the treat-
ment with flutamide-metformin plus DRP/ EE30μgis asso-
ciated with a consistent fall in CRP and TNF-α levels,
especially in patients with the most abnormal values [57].
In conclusion, our results suggest that the association
metformin- DRP/EE30μgwith lifestyle changes has posi-
tive impact on clinical and biochemical hyperandrogen-
emia, carbohydrate metabolism, body composition and
endothelial function-increasing FMD and neutral effects
on hsCRP levels. ET-1 levels were not significantly
altered by DRP/EE30μgand metformin in this follow-up
study; however, analyses for ET-1 were significantly
underpowered in our set of data. Future revaluation of
ET-1 on larger sample size groups is needed for a proper
characterization of this marker in relationship with the as-
sociation metformin- DRP/EE30μgand weight loss in PCOS.
Although our study is limited by the lack of a weight loss
control group, the results obtained, after adjusting for body
weight loss, further suggest that the medical treatment used
might have neutral/mild positive effects on hsCRP levels,
body adiposity and endothelial function, but it might
worsen TG levels. Hence, a thorough assessment of the
benefits of metformin- DRP/EE30μg therapy on vascular
function require further trials that should include a weight
loss group as a control arm. Concerning the subgroup of
non-obese PCOS women, the findings of our study suggest
that the use of the association metformin- DRP/EE30μgin
these women does not seem to affect endothelial function.
It can also be considered safe, as no further deterioration of
cardiovascular risk factors was noted in this population.
Furthermore, the specific effects of the individual compo-
nents of therapy: metformin and EE/DRP as well as those
of the individual COC components on cardiovascular risk
markers cannot be ascertained from this study. Neverthe-
less, these are only cardiovascular risk surrogate markers
and we do not know the clinical relevance of our results,
which do not provide comprehensive explanations but ra-
ther encourage further investigations.
Abbreviations
(BMI),: Body mass index; ((C) OC): (Combined) oral contraceptives;
(DBP): Diastolic blood pressure; (DRP): Drospirenone; (ET-1): Endothelin-1;
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(EE): Ethinylestradiol; (F-G): scoreFerriman-Gallwey; (FMD): Flow-mediated
dilation; (FAI): Free androgen index; (HOMA-IR): Homeostasis model
assessment of insulin resistance; (IL-6): Interleukin-6; (PCOS): Polycystic ovary
syndrome; (QUICKI): Quantitative insulin sensitivity check index; (SHBG): Sex
hormone-binding globulin; (SBP): Systolic blood pressure; (TC): Total
cholesterol; (TG): Triglycerides; (TT): Total testosterone; (WHR): Waist hip ratio.
Competing interests
There is no conflict of interest that would prejudice the impartiality of this
scientific work. The authors alone are responsible for the content and writing
of the article.
Acknowledgements
Funding:
This work was supported by academic grants CNCSIS 552/2007, PN-II-RU-PD-
2011-3-0287 and PNCDI II 41_068/2007.
Author details
1Department of Endocrinology, University of Medicine and Pharmacy, 3-5
Louis Pasteur, 400349, Cluj-Napoca, Romania.2Department of Internal
Medicine-Cardiology, University of Medicine and Pharmacy, Cluj-Napoca,
Romania.3Departmen of Physiology, University of Medicine and Pharmacy,
Cluj-Napoca, Romania.4Department of Obstetrics and Gynecology, University
of Medicine and Pharmacy, Cluj-Napoca, Romania.53-rd Department of
Surgery, University of Medicine and Pharmacy, Cluj-Napoca, Romania.
Authors' contributions
IRI has contributed to the design of this study; she was directly implicated in
conducting the research, in collecting, analyzing and interpreting data as
well as in editing the manuscript and critically revising it. IM has contributed
to the design of this study, the measurement of FMD and the data analysis
and interpretation. TM participated in the design of the study and performed
the statistical analysis. RI has contributed to the design of this study; data
collection and interpretation, gynecological ultrasound examination. LM
analyzed the data and acquired funding. ID participated in the design of the
study and helped to draft the manuscript. CEP has contributed to the design
of this study; she was directly implicated in conducting the research, in
interpreting data and editing the manuscript as well as in acquiring funding.
All authors read and approved the final manuscript.
Received: 15 February 2012 Accepted: 19 June 2012
Published: 19 June 2012
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doi:10.1186/1472-6823-12-9
Cite this article as: Ilie et al.: Ethinylestradiol30μg-drospirenone and
metformin: could this combination improve endothelial dysfunction in
polycystic ovary syndrome?. BMC Endocrine Disorders 2012 12:9.
Ilie et al. BMC Endocrine Disorders 2012, 12:9
http://www.biomedcentral.com/1472-6823/12/9
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