1. health and fitness
2. disease
3. infertility

D 1299
OULU 2015
UNIVERSITY OF OUL U P.O. Box 8000 FI-90014 UNIVERSITY OF OULU FINLA ND
U N I V E R S I TAT I S
O U L U E N S I S
ACTA
A C TA
D 1299
ACTA
U N I V E R S I T AT I S O U L U E N S I S
Johanna Puurunen
University Lecturer Santeri Palviainen
Postdoctoral research fellow Sanna Taskila
Professor Olli Vuolteenaho
University Lecturer Veli-Matti Ulvinen
Johanna Puurunen
Professor Esa Hohtola
ANDROGEN SECRETION
AND CARDIOVASCULAR RISK
FACTORS IN WOMEN WITH
AND WITHOUT PCOS
STUDIES ON AGE-RELATED CHANGES AND
MEDICAL INTERVENTION
Director Sinikka Eskelinen
Professor Jari Juga
University Lecturer Anu Soikkeli
Professor Olli Vuolteenaho
Publications Editor Kirsti Nurkkala
ISBN 978-952-62-0808-4 (Paperback)
ISBN 978-952-62-0809-1 (PDF)
ISSN 0355-3221 (Print)
ISSN 1796-2234 (Online)
UNIVERSITY OF OULU GRADUATE SCHOOL;
UNIVERSITY OF OULU,
FACULTY OF MEDICINE;
MEDICAL RESEARCH CENTER OULU;
OULU UNIVERSITY HOSPITAL;
NATIONAL GRADUATE SCHOOL OF CLINICAL INVESTIGATION
D
MEDICA
ACTA UNIVERSITATIS OULUENSIS
D Medica 1299
JOHANNA PUURUNEN
ANDROGEN SECRETION AND
CARDIOVASCULAR RISK FACTORS IN
WOMEN WITH AND WITHOUT PCOS
Studies on age-related changes and medical intervention
Academic Dissertation to be presented with the assent
of the Doctoral Training Committee of Health and
Biosciences of the University of Oulu for public defence
in Auditorium 4 of Oulu University Hospital, on 5 June
2015, at 12 noon
U N I VE R S I T Y O F O U L U , O U L U 2 0 1 5
Copyright © 2015
Acta Univ. Oul. D 1299, 2015
Supervised by
Professor Juha Tapanainen
Doctor Terhi Piltonen
Reviewed by
Docent Maritta Hippeläinen
Professor Risto Kaaja
Opponent
Professor Antoni Duleba
ISBN 978-952-62-0808-4 (Paperback)
ISBN 978-952-62-0809-1 (PDF)
ISSN 0355-3221 (Printed)
ISSN 1796-2234 (Online)
Cover Design
Raimo Ahonen
JUVENES PRINT
TAMPERE 2015
Puurunen, Johanna, Androgen secretion and cardiovascular risk factors in women
with and without PCOS. Studies on age-related changes and medical intervention
University of Oulu Graduate School; University of Oulu, Faculty of Medicine; Medical
Research Center Oulu; Oulu University Hospital; National Graduate School of Clinical
Investigation
Acta Univ. Oul. D 1299, 2015
University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland
Abstract
Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of
reproductive age. The main features of the syndrome include menstrual irregularities and
hyperandrogenism. In addition to symptoms related to fertility, some women also suffer from an
unfavourable metabolic profile including impaired glucose tolerance, dyslipidaemia and lowgrade chronic inflammation.
In the present studies we aimed to investigate the role of age on adrenal and ovarian androgen
secretion in 79 women with PCOS and 98 healthy women, with special focus on the menopause.
Furthermore, we studied the effects of combined hormonal contraceptives (CHCs) administered
orally, transdermally and vaginally (n=42, healthy women, 9 weeks) and atorvastatin treatment
(n=28, women with PCOS, 6 months) on androgen levels and metabolic factors. Androgen
secretion capacity was analysed by using adrenal and ovarian stimulation tests and glucose
tolerance by using oral and intravenous glucose tolerance tests. Furthermore, chronic
inflammation was assessed via assay of C-reactive protein and pentraxin-3.
Basal and stimulated adrenal and ovarian androgen production was elevated and levels
remained higher in women with PCOS compared with healthy women even after the menopause.
Furthermore, women with PCOS presented with enhanced insulin resistance and chronic
inflammation, which persisted beyond menopausal transition. During CHC treatment, the route of
administration was insignificant, and all treatments impaired insulin sensitivity and increased
chronic inflammation. In women with PCOS, treatment with atorvastatin improved chronic
inflammation and the lipid profile as expected, but worsened glucose tolerance and did not affect
testosterone levels.
Regardless of strict exclusion criteria, where only relatively healthy women with PCOS were
recruited, the results showed that enhanced androgen secretion and unfavourable metabolic
alterations associated with PCOS persist through menopausal transition. The findings emphasize
the importance of monitoring glucose metabolism during the use of CHCs, especially in women
with known risks of type 2 diabetes. Atorvastatin treatment exacerbates insulin resistance in
women with PCOS and therefore the treatment should only be considered after individual risk
assessment of cardiovascular disease and not just because of PCOS.
Keywords: aging, androgens, cardiovascular diseases, combined hormonal
contraceptives, CRP, cutaneous administration, hyperandrogenism, inflammation,
insulin resistance, intravaginal administration, menopause, oral administration,
polycystic ovary syndrome, statins
Puurunen, Johanna, Miessukuhormonieritys ja sydän- ja verisuonitautien riskitekijät
monirakkulaisessa munasarjaoireyhtymässä ja terveillä naisilla. Ikääntymisen ja
lääkehoidon vaikutukset
Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Medical Research
Center Oulu; Oulun yliopistollinen sairaala; Valtakunnallinen kliininen tutkijakoulu
Acta Univ. Oul. D 1299, 2015
Oulun yliopisto, PL 8000, 90014 Oulun yliopisto
Tiivistelmä
Monirakkulainen munasarjaoireyhtymä (PCOS) on hedelmällisessä iässä olevien naisten yleisin
hormonaalinen ongelma. Tyypillisiä PCOS:n oireita ovat munarakkuloiden epäsäännöllisestä
kypsymisestä johtuvat kuukautiskierron häiriöt ja miessukuhormonien eli androgeenien liikatuotanto. Hedelmällisyyttä heikentävien oireiden lisäksi PCOS:än liittyy aineenvaihdunnan ongelmia, kuten heikentynyttä sokerinsietoa sekä taipumus rasva-aineenvaihdunnan häiriöihin ja
krooniseen tulehdukseen.
Tutkimuksessa selvitettiin ikääntymisen ja vaihdevuosien vaikutuksia lisämunuais- ja munasarjaperäiseen androgeenieritykseen 79 PCOS-naisella ja 98 terveellä naisella. Lisäksi tutkittiin
eri yhdistelmäehkäisyvalmisteiden antoreittien (suu, iho, emätin) (n=42, terveet naiset, 9 viikkoa) ja atorvastatiinihoidon (n=28, PCOS-naiset, 6 kuukautta) vaikutuksia androgeenitasoihin ja
aineenvaihdunnallisiin muuttujiin. Androgeenieritystä tutkittiin lisämunuaisten ja munasarjojen
stimulaatiotesteillä ja sokeriaineenvaihdunnan muutoksia suun kautta ja suonensisäisesti tehtävillä sokerirasituskokeilla. Tulehduksellista tilaa mitattiin määrittämällä C-reaktiivisen proteiinin
ja pentraksiini-3:n pitoisuuksia.
Lisämunuaisten ja munasarjojen androgeenieritys oli PCOS-naisilla lisääntynyt terveisiin
naisiin verrattuna, ja ero säilyi vaihdevuosi-iän jälkeen. PCOS-naisilla esiintyi myös enemmän
heikentynyttä sokerinsietoa ja kroonista tulehdusta vielä vaihdevuosi-iän jälkeenkin. Hormonaalinen yhdistelmäehkäisy heikensi insuliiniherkkyyttä sekä pahensi pitkäaikaista tulehdusta
annostelureitistä riippumatta. Atorvastatiinihoito puolestaan paransi pitkäaikaista tulehdusta
sekä rasva-aineenvaihduntaa PCOS-naisilla, mutta huononsi sokerinsietoa ja insuliiniherkkyyttä
eikä sillä ollut vaikutusta testosteronitasoihin.
Koska poissulkukriteerit olivat tiukat, tutkimuksiin valikoitui varsin terveitä PCOS-naisia.
Siitä huolimatta osoittautui, että PCOS:än liittyvä lisääntynyt androgeenituotanto sekä epäedulliset aineenvaihdunnan muutokset jatkuvat vielä vaihdevuosi-iän jälkeen. Hormonaalisen yhdistelmäehkäisyn käytön aikana olisi hyvä seurata sokeriaineenvaihdunnan muutoksia erityisesti
niillä naisilla, joilla on kohonnut riski sairastua aikuistyypin diabetekseen. Atorvastatiinihoito
huonontaa PCOS-naisilla insuliiniherkkyyttä, minkä vuoksi hoito tulisi aloittaa vain yksilöllisen
riskiarvion perusteella.
Asiasanat: androgeenit, CRP, emättimen kautta annostelu, hormonaalinen
yhdistelmäehkäisy, hyperandrogenismi, ihon kautta annostelu, ikääntyminen,
insuliiniresistenssi, monirakkulainen munasarjaoireyhtymä, statiinit, suun kautta
annostelu, sydän- ja verisuonitaudit, tulehdus, vaihdevuodet
There are only two ways to live your life. One is as though nothing is a
miracle. The other is as though everything is a miracle.
–Albert Einstein
To Olli and our lovely sons
8
Acknowledgements
The present work was carried out at the Department of Obstetrics and
Gynecology, University of Oulu, and the Medical Research Center Oulu, Oulu
University Hospital, during the years 2004–2015.
First and foremost, I wish to express my deepest gratitude to my supervisor
professor Juha Tapanainen for introducing the fascinating field of medical
research to me. I am grateful for his expert guidance, encouraging attitude and
support, and also for his patience during my periods of parental leave. I have been
privileged to work as a member of his research group in an excellent scientific
environment.
I owe warm and grateful acknowledgement to my supervisor Terhi Piltonen,
M.D., Ph.D., who has had an enormous role in this project. Her excellent
guidance ever since I started as a medical student has been invaluable. I also want
to thank Terhi for her warm and close friendship and those numerous hours
around her dining table.
I wish to express my sincerest gratitude to docent Laure Morin-Papunen for
her energetic and supportive attitude and her helpfulness in any difficulties I have
had. Of course, I also thank her for many summer parties arranged in her lovely
home. I thank professor Hannu Martikainen, docent Ilkka Järvelä and docent
Tommi Vaskivuo for their valuable advice and supportive comments in meetings
and elsewhere.
I wish to thank professor Aimo Ruokonen, professor Markku Savolainen,
professor Karl-Heinz Herzig, docent Antti Perheentupa, Antti Nissinen,
Shivaprakash Mutt, Katri Puukka and Pirjo Hedberg for their help and valuable
contributions during the course of this project. I also want to thank Risto Bloigu
for his guidance in the field of statistics.
I have had the privilege to work in an inspiring and cheerful atmosphere and I
want to thank my colleagues Mervi Haapsamo, Minna Jääskeläinen, Sanna
Koskela, Kristiina Mäkelä, Katriina Niemelä, Pekka Pinola, Anni Rantala, Reeta
Törmälä, Outi Uimari and Zdravka Veleva for their support, encouragement and
excellent company in the office and at evening gatherings. I also want to thank
the new members of our research group, Anna-Maaria Auvinen, Marika
Kangasniemi, Salla Karjula, Masuma Khatun, Shilpa Lingaiah, Maija Ollila,
Henna Rossi and Saara Vuontisjärvi for many memorable moments.
9
I wish to thank Mirja Ahvensalmi and Anu Ojala for their friendly and skilful
care of the subjects. I also want to thank our new research nurse Elina Huikari for
her energetic and helpful attitude and pleasant conversations during coffee breaks.
I am grateful to laboratory manager Tiina Hurskainen for her help and for
arranging a quiet room to write this thesis. I wish to thank all the members in
other MRC Oulu research groups for the warm atmosphere in the coffee room.
Special thanks are owed to my roommate Johanna Huusko for all the help.
The official reviewers of my thesis, docent Maritta Hippeläinen and professor
Risto Kaaja, are gratefully acknowledged for their constructive comments and
suggestions, which improved the quality of the thesis. I thank Nicholas Bolton for
his skilful linguistic revision of the thesis and my manuscripts over the years.
I want to thank my dear friends and colleagues Marjo Dahlbacka, Minna
Kivenmäki, Sanna Koskela, Marianne Rasi, Kati Retsu-Heikkilä, Saara
Vuontisjärvi, Anne Vuontissalmi and Laura Ylikauma for their support in the field
of medicine and in life in general. I also want to thank Päivi Haaranen, Anne
Kivilahti, Jenni Krankka and Anna Mäkivuoti for their warm friendship and
support. I wish to thank Jenni Rask for deep conversations and numerous
evenings in the swimming pool. I have spent several enjoyable times with you all
and I am so lucky to have you in my life.
I am grateful to my parents Eeva-Liisa and Kari Kesti for our lovely family
and for their support and encouragement in life. I want to thank my father for
those thousands of kilometres driven due to sailing, and my mother for those
desperate hours teaching me English. I thank my little sisters Jutta Kesti and Jenni
Lämsä for close friendship and unforgettable moments spent together. I have
always felt loved. I also thank my brother-in-law Mikko Lämsä, my godson Eino
and a newborn nephew for providing such enjoyable company in our family.
Above all, my deepest thanks are owed to my beloved husband Olli for
believing in me. I am grateful for all his help with computers and for
encouragement during the deepest moments of desperation. I thank my dear and
precious sons, Joona and Peetu, for reminding me of the importance of life apart
from work. You three are my everything and I am deeply grateful for having you
beside me.
I want to express my sincere gratitude to all the study subjects who
volunteered to participate in this project. Without those ladies my thesis would
never have been possible.
This study was financially supported by the Academy of Finland, the Sigrid
Jusélius Foundation, Oulu University Hospital, the National Graduate School of
10
Clinical Investigation, the North Ostrobothnia Regional fund of the Finnish
Cultural Foundation, Tyyni Tani Foundation of the University of Oulu, the
Finnish-Norwegian Medical Foundation, the Finnish Society of Obstetrics and
Gynecology, Oulu Medical Research Foundation, the Finnish Medical Foundation
and the Paulo Foundation, who are gratefully acknowledged.
Oulu, April 2015
Johanna Puurunen
11
12
Abbreviations
17-OHP
A
ACTH
AMH
BMI
CHC
CHD
CRH
CRP
CVD
DHEA
DHEAS
DHT
E2
EE
ET-1
FAI
FSH
GDM
GnRH
hCG
HDL
HOMA
IFG
IGF
IGT
IMT
IR
IVGTT
LDL
LH
OGTT
OR
PCO
PCOS
17-hydroxyprogesterone
androstenedione
adrenocorticotrophic hormone
anti-Müllerian hormone
body mass index
combined hormonal contraceptive
coronary heart disease
corticotrophin-releasing hormone
C-reactive protein
cardiovascular disease
dehydroepiandrosterone
dehydroepiandrosterone sulphate
dihydrotestosterone
estradiol
ethinyl estradiol
endothelin-1
Free Androgen Index
follicle-stimulating hormone
gestational diabetes mellitus
gonadotrophin-releasing hormone
human chorionic gonadotrophin
high-density lipoprotein
homeostatic model assessment
impaired fasting glucose
insulin-like growth factor
impaired glucose tolerance
intima-media thickness
insulin receptor
intravenous glucose tolerance test
low-density lipoprotein
luteinising hormone
oral glucose tolerance test
odds ratio
polycystic ovary
polycystic ovary syndrome
13
PTX-3
SHBG
StAR
T
T2DM
VLDL
WHR
14
pentraxin 3
sex hormone-binding globulin
steroidogenic acute regulatory protein
testosterone
type 2 diabetes mellitus
very low density lipoprotein
waist to hip ratio
List of original publications
This thesis is based on the following articles, which are referred to in the text by
their Roman numerals:
I
Puurunen J, Piltonen T, Jaakkola P, Ruokonen A, Morin-Papunen L & Tapanainen JS
(2009) Adrenal androgen production capacity remains high up to menopause in
women with polycystic ovary syndrome. J Clin Endocrinol Metab 94(6):1973–8.
II Puurunen J, Piltonen T, Morin-Papunen L, Perheentupa A, Järvelä I, Ruokonen A &
Tapanainen JS (2011) Unfavorable hormonal, metabolic, and inflammatory alterations
persist after menopause in women with PCOS. J Clin Endocrinol Metab 96(6):1827–
34.
III Puurunen J, Piltonen T, Puukka K, Ruokonen A, Savolainen MJ, Bloigu R, MorinPapunen L & Tapanainen JS (2013) Statin therapy worsens insulin sensitivity in
women with polycystic ovary syndrome (PCOS): a prospective, randomized, doubleblind, placebo-controlled study. J Clin Endocrinol Metab 98(12):4798–807.
IV Piltonen T*, Puurunen J*, Hedberg P, Ruokonen A, Mutt SJ, Herzig KH, Nissinen A,
Morin-Papunen L & Tapanainen JS (2012) Oral, transdermal and vaginal combined
contraceptives induce an increase in markers of chronic inflammation and impair
insulin sensitivity in young healthy normal-weight women: a randomized study. Hum
Reprod 27(10):3046–56.
*equal contribution
15
16
Contents
Abstract
Tiivistelmä
Acknowledgements
9
Abbreviations
13
List of original publications
15
Contents
17
1 Introduction
19
2 Review of the literature
21
2.1 Polycystic ovary syndrome (PCOS)........................................................ 21
2.1.1 Criteria for PCOS ......................................................................... 21
2.1.2 Prevalence of PCOS ..................................................................... 24
2.2 Androgen synthesis and ovulatory dysfunction in PCOS ....................... 24
2.2.1 Hyperandrogenism ....................................................................... 24
2.2.2 Oligoamenorrhoea ........................................................................ 34
2.2.3 Polycystic ovaries (PCO) ............................................................. 34
2.3 Metabolic alterations and health consequences of PCOS ....................... 35
2.3.1 Obesity ......................................................................................... 36
2.3.2 Insulin sensitivity and glucose tolerance ...................................... 38
2.3.3 Cardiovascular risk factors and outcomes .................................... 44
2.4 Treatment of PCOS-related disorders ..................................................... 51
2.4.1 Combined hormonal contraceptives ............................................. 51
2.4.2 Insulin sensitizers ......................................................................... 53
2.4.3 Statins ........................................................................................... 55
3 Purpose of the present study
59
4 Subjects and methods
61
4.1 Subjects ................................................................................................... 61
4.1.1 Women with PCOS (Studies I, II and III)..................................... 62
4.1.2 Control (healthy) subjects (Studies I, II and IV) .......................... 62
4.1.3 Exclusion criteria and medication ................................................ 63
4.2 Methods................................................................................................... 63
4.2.1 Study visits ................................................................................... 63
4.2.2 Intervention measures ................................................................... 63
4.2.3 Laboratory analyses ...................................................................... 66
4.2.4 Power and statistical analyses ...................................................... 67
17
5 Results and Discussion
69
5.1 Hyperandrogenism .................................................................................. 69
5.1.1 Androgen secretion ....................................................................... 69
5.1.2 The effects of combined contraceptives and statin therapy
on androgen secretion ................................................................... 72
5.2 Metabolic alterations in women with and without PCOS ....................... 74
5.2.1 Glucose tolerance ......................................................................... 74
5.2.2 Lipid profile .................................................................................. 79
5.2.3 Chronic inflammation ................................................................... 81
5.3 Limitations of the study .......................................................................... 84
6 Conclusions
85
References
87
Original publications
113
18
1
Introduction
Polycystic ovary syndrome, PCOS, is the most common endocrine disorder in
fertile-aged women, with a prevalence of 6–20% depending on the diagnostic
criteria used (Yildiz et al. 2012). The main symptoms of the syndrome include
irregularities in menstrual cycles and hyperandrogenism. Hyperandrogenism can
be detected by clinical findings of male-type hair growth, hirsutism, or by the
measurement of elevated androgen levels or their bioactivity. One of the typical
signs of PCOS is anovulation, and often it leads to a finding of polycystic ovaries
(PCO) in ultrasonography. Furthermore, anovulation is the most common cause of
infertility in these women.
Hyperandogenism has a prominent role in PCOS, and it can be of both
ovarian and adrenal origin. Furthermore, especially in the presence of obesity,
decreased concentrations of sex hormone-binding globulin (SHBG) also play a
role in hyperandrogenism, resulting in increased levels of bioactive hormones.
How androgen secretion changes with ageing in PCOS is not well described. It is
an important question, as hyperandrogenism has been associated with increased
risks of several adverse health effects such as cardiovascular diseases.
Women with PCOS present with increased risks of insulin resistance,
metabolic disorders and development of glucose intolerance and type 2 diabetes
mellitus (T2DM). Furthermore, obesity is common among these women. For a
long time it was unclear if women with PCOS also had an increased risk of
cardiovascular disease. However, today there are several studies supporting this
perception.
As women with PCOS present with several health risks, different types of
medication for the symptoms have been studied extensively. Insulin-sensitizing
drugs have been shown to be useful for the treatment of glucose intolerance and
in specific cases of menstrual irregularities and infertility. There has also been a
growing interest in statin therapy, as it could potentially affect both dyslipidaemia
and hyperandrogenism. However, up to now the most common and widely used
forms of medication for menstrual irregularities and hyperandrogenism are
combined contraceptives.
The aim of this study was to explore hyperandrogenism of adrenal and
ovarian origin, especially age-related changes and the role of menopausal
transition in PCOS. Secondly, we aimed to compare the metabolic effects of three
different administration routes of combined contraceptives, which are commonly
used to treat irregular cycles and hyperandrogenism in women with PCOS.
19
Thirdly, given that PCOS has a strong metabolic component, there was an interest
to investigate the effects of atorvastatin treatment on metabolic factors in women
with PCOS.
20
2
Review of the literature
2.1
Polycystic ovary syndrome (PCOS)
Polycystic ovary syndrome was first described as early as 1935, when doctors
Stein and Leventhal discovered an association between excessive hair growth,
irregular menstrual cycles and polycystic ovaries (Stein & Leventhal 1935).
Today it is widely accepted that PCOS is a hyperandrogenic anovulatory disorder
combined with an unfavourable metabolic and inflammatory profile where the
first two characteristics are considered as hallmarks of the diagnosis. PCOS is
considered to be the most common endocrine dysfunction among fertile-aged
women and one of the most common causes of female infertility worldwide.
Despite the efforts of the scientific community to define the cause of PCOS, its
aetiology and pathogenesis remain unclear. It might involve a genetic
predisposition influenced by gestational environment, lifestyle factors or both
(Norman et al. 2007). As the syndrome is related to infertility and several adverse
health consequences, understanding the mechanisms that cause PCOS is an
important goal in medical research. It has been estimated that more than 4 million
women suffer from PCOS in the U.S. and the estimated costs for the treatment of
different symptoms exceed 4 billion dollars annually, the largest proportion of the
money consumed being connected to the treatment of PCOS-associated diabetes
(Azziz et al. 2005).
2.1.1 Criteria for PCOS
There are three different diagnostic criteria for PCOS available today, and the
definitions underline some findings in cases of PCOS differently. In 1990 the
expert conference on PCOS was held in the U.S., sponsored by the National
Institutes of Health (NIH). The diagnostic criteria for PCOS were defined by
consensus among the participants and were published by Zawadski and Dunaif as
follows: “the criteria for PCOS should include (in order of importance): 1)
clinical or biochemical signs of hyperandrogenism, 2) chronic anovulation and 3)
exclusion of other possible pathologies, such as hyperprolactinemia, thyroid
disorders, and nonclassic adrenal hyperplasia” (Zawadzki & Dunaif 1992). The
NIH criteria represented an important step as regards standardization of the
21
diagnosis of PCOS, as those in the research field were able to publish studies on
women fulfilling standardized diagnostic criteria for PCOS.
However, there was a need for broader diagnostic criteria, as it had been
recognised that some women with PCOS displayed ovarian dysfunction without
evidence of androgen excess. Therefore, revised diagnostic criteria were defined
in 2003 at an expert conference in Rotterdam sponsored by the European Society
for Human Reproduction and Embryology and the American Society for
Reproductive Medicine (Rotterdam ESHRE/ASRM-Sponsored PCOS consensus
workshop group 2004). The Rotterdam criteria were defined as follows: “the
diagnosis of PCOS can be established if at least two of the following criteria are
met: 1) oligo- and/or anovulation, 2) clinical and/or biochemical
hyperandrogenism, or 3) PCO. Hyperandrogenism should be evaluated by
assessing the degree of hirsutism or by biochemical measurement of free
testosterone (T) or the free androgen index (FAI). Polycystic ovaries were defined
as the presence of 12 or more follicles in one ovary measuring 2–9 mm in
diameter, and/or increased ovarian volume (> 10 ml)” (Balen et al. 2003). In
addition, other aetiologies such as congenital adrenal hyperplasia, androgensecreting tumours, Cushing’s syndrome and hyperprolactinaemia should be
excluded. The use of Rotterdam criteria has increased the prevalence of PCOS, as
the criteria have broadened the spectrum of the clinical symptoms, thereby
allowing inclusion of women with milder forms of the syndrome. Notably,
recently it has been suggested that a follicle number threshold of more than 25 per
ovary could be considered to be a more accurate measure of PCO when modern
ultrasonographic technology is used (Dewailly et al. 2014), although in clinical
practice ovarian volume measurements may be more useful.
A third set of diagnostic criteria was suggested by The Androgen Excess and
PCOS Society (AE-PCOS Society) in 2006 (Azziz et al. 2006). This forum
considered that hyperandrogenism was one of the main traits of PCOS and that
the Rotterdam criteria were leading to phenotypes of PCOS that were too diverse.
Thus, the AE-PCOS Society criteria were defined as follows: 1)
hyperandrogenism and 2) ovarian dysfunction (oligo-anovulation and/or PCO)
and 3) exclusion of other causes of androgen excess or related disorders.
22
Table 1. Different phenotypes of PCOS included in different sets of diagnostic criteria
Criteria
Hyperandrogenism
Oligo-anovulation
NIH 1990
+
+
+
+
Rotterdam 2003
+
AE-PCOS Society 2006
PCO
+
+
+
+
+
+
+
+
+
+
+
+
+
(Azziz et al. 2006, Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group 2004,
Zawadzki & Dunaif 1992)
The serum level of anti-Müllerian hormone (AMH) has been shown to correlate
well with ovarian antral follicle count, and naturally the levels are higher in
women with PCOS when compared with healthy women (Maas et al. 2015,
Piltonen et al. 2005). Today, the possibility to use assay of AMH produced by the
granulosa cells of ovarian follicles for the diagnosis of PCO has been of interest
and the results seem promising (Lauritsen et al. 2014). However, the variability of
the assays used for the measurement of AMH, has so far been an obstacle to
inclusion of AMH levels in the criteria of PCO/PCOS. After the assays become
more reliable, the diagnosis of PCO based on AMH measurement may spread into
general practice.
Women with PCOS diagnosed by means of NIH criteria (hyperandrogenism
with ovarian dysfunction) have a greater prevalence of menstrual irregularities
and a greater degree of hyperandrogenism, total and abdominal obesity and
insulin resistance and have more severe risk factors of T2DM and cardiovascular
disease (CVD) than women diagnosed using Rotterdam or AE-PCOS Society
criteria (Wild et al. 2010). Ovulatory women with PCOS have a lower body mass
index (BMI) and less abdominal obesity, lesser degrees of hyperandrogenism and
hyperinsulinaemia, a reduced prevalence of metabolic syndrome and milder forms
of dyslipidaemia, whereas normoandrogenic women with PCOS have the most
metabolically favourable profile, in some cases even comparable with that among
women without PCOS (Wild et al. 2010).
23
2.1.2 Prevalence of PCOS
The prevalence of PCOS varies according to the diagnostic criteria used, and it
has been estimated to be 0.9–10.7% based on NIH criteria (Asunción et al. 2000,
Azziz et al. 2004b, Diamanti-Kandarakis et al. 1999, Knochenhauer et al. 1998,
Lauritsen et al. 2014, March et al. 2010, Tehrani et al. 2011, Yildiz et al. 2012),
11.9–19.9% according to Rotterdam criteria (Lauritsen et al. 2014, March et al.
2010, Tehrani et al. 2011, Yildiz et al. 2012) and 10.2–15.3% with respect to AEPCOS Society criteria (Lauritsen et al. 2014, March et al. 2010, Tehrani et al.
2011, Yildiz et al. 2012). The prevalence of diagnostic features of PCOS has been
shown to decrease with age, as the prevalence of PCOS diagnosed by Rotterdam
criteria in women under 30 years of age has been found to be 33.3%, but only
10.2% in women older than 35 years. Similarly, the prevalence of PCOS
according to AE-PCOS Society criteria was 25.0% in women under 30 years and
9.0% in women more than 35 years (Lauritsen et al. 2014). Furthermore, the
prevalence of PCO also declines with age (Koivunen et al. 1999).
2.2
Androgen synthesis and ovulatory dysfunction in PCOS
2.2.1 Hyperandrogenism
In women, biochemical hyperandrogenism is considered when serum levels of
total T or free androgens are higher than the upper limit of the normal range.
Clinical hyperandrogenism, on the other hand, is diagnosed when a woman
presents with hirsutism, excessive male-type hair growth, alopecia or acne.
Hirsutism is the most commonly studied clinical marker of hyperandrogenism,
whereas acne has been shown to have a poor correlation with hyperandrogenism.
Hirsutism is usually evaluated by using the scoring system introduced by
Ferriman and Gallwey, where a score of 8 or more indicates hirsutism (Ferriman
& Gallwey 1961). Today in clinical practice, the assessment of hirsutism is
challenging, as women tend to shave their body-hair intensively.
The majority of women suffering from hyperandrogenism, even up to 82%,
are diagnosed as having PCOS (Azziz et al. 2004a), and approximately 60% of
women with PCOS have hirsutism, when the diagnosis has been defined by NIH
criteria (Azziz et al. 2006, Legro et al. 2006). Hyperandrogenism in women with
PCOS originates from both the ovaries and adrenals. In addition, low circulating
levels of SHBG in women with PCOS increase the concentration of free
24
androgens (biologically active forms) and thereby worsen the level of
hyperandrogenism. Lower SHBG levels are seen especially in overweight
women, as obesity-induced hyperinsulinaemia inhibits the production of SHBG
(Lim et al. 2013). Interestingly, hyperandrogenism in women with PCOS is at
least partly of genetic origin, as mothers and sisters of women with PCOS present
with elevated androgen levels when compared with control women (Yildiz et al.
2003).
Even though circulating levels of androgens are significantly lower in women
than in men, androgens are also essential for women, as they serve as substrates
for oestrogen biosynthesis. In women, the ovaries and adrenals are capable of
synthesising cholesterol de novo from acetate, which serves as a precursor of
androgen synthesis. Extracellular cholesterol is transported by circulating
lipoproteins, in humans mostly by low-density lipoproteins (LDLs) and less by
high-density lipoproteins (HDLs), representing the major source of substrate for
steroidogenesis in the ovaries and adrenals. LDL and HDL cholesterol are
transferred into cells via endocytosis, and the cholesterol is freed. If cholesterol
uptake exceeds the rate of steroidogenesis, free cholesterol is transferred to
storage directly as intracellular cholesteryl ester for future use, to ensure its
availability (Gwynne & Strauss 1982, Wood & Strauss 2002). In the cell,
cholesterol is trafficked to the outer membrane of the mitochondria and
translocated to the inner mitochondrial membrane by steroidogenic acute
regulatory protein (StAR). The movement of cholesterol from the outer to the
inner mitochondrial membrane by StAR is the rate-limiting step of
steroidogenesis (Jamnongjit & Hammes 2006). After cholesterol is transferred to
the inner mitochondrial membrane, the enzymatic chain of steroidogenesis can
begin (Gwynne & Strauss 1982, Wood & Strauss 2002).
Androstenedione (A), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), T and dihydrotestosterone (DHT) are the main
circulating androgens in women (Figure 1). DHT and T bind to nuclear androgen
receptors, and thereby these two steroid hormones induce most of the androgenic
effects, whereas the first three androgens are considered as pro-androgens, and
require conversion to T and DHT to present their androgenic potential (Burger
2002). It has been estimated that at fertile age 25% of A and T production is of
ovarian origin, 25% is of adrenal origin, and 50% is produced in peripheral
tissues (Baptiste et al. 2010). After menopause the role of peripheral tissues on
androgen secretion is emphasized, at adrenal and especially at ovarian expense.
25
In the circulation the majority of androgens are bound to various carrier
proteins, mostly SHBG (carrying 65% of the total amount of circulating T) and
albumin (carrying 33%) and only 1–2% of testosterone circulates unbound
(Catteau-Jonard,S., Dewailly,D. 2013). As only free androgens have biological
activity, calculation of the free androgen index (FAI, 100 × T/SHBG) is of
importance when estimating androgenic potential and effects (Burger 2002). Even
though the measurement of free androgens (mainly T) would be preferable to the
measurement of total T, there are considerable weaknesses in the method and
calculation of the FAI is considered to be a significantly more reliable
measurement as regards active androgens.
26
27
Fig. 1. Androgen synthesis in the ovaries and adrenals.
Ovarian androgen production
Pulsatile secretion of pituitary follicle-stimulating hormone (FSH) and luteinising
hormone (LH) are regulated by hypothalamic gonadotrophin releasing-hormone
(GnRH) (Figure 2). Luteinising hormone stimulates ovarian theca cell androgen
production by increasing the expression and activity of StAR (Jamnongjit &
Hammes 2006), and the conversion of cholesterol to pregnenolone and further to
androgens. This is mediated through an increase in synthesis of the steroidogenic
enzymes CYP11A and CYP17 activity (Figure 1) (Payne & Hales 2004, Wood &
Strauss 2002). Some of the androgens diffuse to granulosa cells, where they are
converted into oestrogens by aromatase enzyme, which is under the control of
FSH (Figures 1 and 2). As ovarian androgens are by-products of oestrogen
synthesis, their excess is not under specific negative feedback regulation by
pituitary LH secretion, and therefore the control of ovarian hyperandrogenism in
connection with normal oestrogen synthesis is weak.
Fig. 2. Ovarian steroid production.
In cases of PCOS the ovaries are known to hyper-secrete androgens, and several
mechanisms such as arrested follicular development have been suggested to play
a role. Women with PCOS present with altered hypothalamic-pituitary axis
28
function, shown as increased LH secretion (Cook et al. 2002, Gilling-Smith et al.
1997, McCartney et al. 2004, Morales et al. 1996, Morin-Papunen et al. 2000)
and elevated LH pulse frequency and amplitude when compared with healthy
women (Eagleson et al. 2000, Morales et al. 1996). Thus these abnormalities
promote androgen secretion in ovarian theca cells by enhancing the expression of
StAR, CYP11A and CYP17 (Wood & Strauss 2002). The excess ovarian
androgen secretion can be explained at least partly by greater LH and diminished
FSH responses to GnRH stimulation especially in lean women with PCOS
compared with healthy women (Barnes et al. 1989).
In addition to central mechanisms, the number of ovarian theca cells has been
shown to be increased in women with PCOS, and they seem to hyper-respond to
LH and human chorionic gonadotrophin (hCG) stimulation in vivo even at
physiological concentrations (Barnes et al. 1989, Gilling-Smith et al. 1997,
McCartney et al. 2004, Piltonen et al. 2004). In vitro, theca cells obtained from
women with PCOS have been shown to exhibit increased expression of LH
receptors when compared with theca cells obtained from healthy women
(Jakimiuk et al. 2001). Furthermore, polymorphism in the LH receptor gene has
been associated with an increased risk of PCOS (Mutharasan et al. 2013). These
features may partly account for the increased responsiveness to LH and
pronounced androgen secretion (Gilling-Smith et al. 1994, Gilling-Smith et al.
1994, McCartney et al. 2004, Nelson et al. 1999). Furthermore, theca cells have
been shown to have enhanced gene expression and/or activities of StAR,
CYP11A, CYP17, 3β-HSD and 17-HSD (Daneshmand et al. 2002, Jakimiuk et al.
2001, Nelson et al. 1999, Nelson et al. 2001, Nelson-Degrave et al. 2005,
Wickenheisser et al. 2000, Wickenheisser et al. 2004, Wickenheisser et al. 2005).
It has also been suggested that women with PCOS can be divided into those with
and without pronounced ovarian responses to hCG measured by 17hydroxyprogesterone (17-OHP) levels (Maas et al. 2015).
Women with PCOS are often insulin resistant. Concomitant
hyperinsulinaemia and increased insulin action mimics the gonadotrophic actions
of LH, having trophic effects on theca cells, promoting proliferation and
increased androgen secretion, as shown in in vitro studies (Nestler et al. 1998, Wu
et al. 2014). Furthermore, theca cells obtained from women with PCOS have been
suggested to be hyper-responsive and to have a lower stimulatory threshold to
insulin when compared with controls (Nestler et al. 1998). An animal study has
confirmed a causal pathway between hyperinsulinaemia and divergent ovarian
androgen secretion and function, as depletion of insulin receptors in ovarian theca
29
cells protects mice with diet-induced obesity from these disturbances (Wu et al.
2014). Moreover, several studies have demonstrated that improvement of insulin
resistance in women with PCOS results in lower androgen levels, improves
ovulatory function and ameliorates the exaggerated steroidogenic response to LH
stimulation (Baillargeon et al. 2004, Nestler & Jakubowicz 1996).
Women with PCOS have been found to have elevated serum levels of free
insulin-like growth factor I (IGF-I) (Thierry van Dessel et al. 1999). IGFs, which
exhibit high structural homology to insulin and signal through IGF-I receptors,
have also been shown to promote ovarian steroidogenesis by enhancing the
expression and activity of several steroidogenic enzymes such as CYP17 and
CYP11A1 (Jamnongjit & Hammes 2006, Nahum et al. 1995, Nestler et al. 1998,
Wood & Strauss 2002). Furthermore, IGF signalling has been shown to increase
the expression of LH receptors, which in turn enhances LH-induced expression
and activity of StAR (Jamnongjit & Hammes 2006).
All in all, the mechanisms that lie behind enhanced ovarian androgen
secretion in women with PCOS are complex, with numerous abnormal actions
and interactions taking place.
Adrenal androgen production
The adrenal cortex is composed of three layers where each layer has separate
enzymatic cascades resulting in three different types of steroid hormones:
glucocorticoids (zona fasciculata and reticularis), mineralocorticoids (zona
glomerulosa) and androgens (zona fasciculata and reticularis). However, even
though the zona fasciculata and reticularis mostly express the same enzymes, they
differ in enzyme activities. The zona fasciculata produces mainly cortisol and the
zona reticularis androgens (Miller & Auchus 2011). The zona glomerulosa
synthesizes mineralocorticoids under regulation of the renin-angiotensin system.
Steroid production by the adrenal zona reticularis and fasciculata is regulated by
pituitary adrenocorticotrophic hormone (ACTH), and ACTH is controlled by
hypothalamic corticotrophin-releasing hormone (CRH) (Figure 3) (Miller &
Auchus 2011). Androgens are used as substrates for mineralocorticoid and
glucocorticoid synthesis (Figure 1). As previously mentioned, androgens do not
exert significant feedback on the pituitary gland, and therefore they do not have a
significant regulatory effect on ACTH production. DHEAS is almost uniquely
secreted by the adrenal zona reticularis as a result of its sulphate activity. Thus, in
clinical practice, DHEAS is often measured in order to evaluate the rate of
30
adrenal androgen production, even though the liver and small intestine also have
the capability of converting DHEA to DHEAS (Goodarzi et al. 2015).
Fig. 3. Adrenal steroid production.
Around 20–65% of women with PCOS have been shown to have increased
adrenal androgen production (Carmina et al. 1986, Carmina et al. 1992, Gallagher
et al. 1958, Gallagher et al. 1958, Hoffman et al. 1984, Kumar et al. 2005). It has
been postulated that women that are genetically prone to secrete more adrenal
androgens would have a greater predisposition to develop PCOS (Azziz et al.
2001).
The possible mechanisms behind enhanced adrenal androgen secretion in
PCOS have been of great interest. Interestingly, serum ACTH levels are
comparable in women with PCOS and healthy women (Chang et al. 1982,
Hoffman et al. 1984, Lanzone et al. 1995, Stewart et al. 1993). Furthermore, the
pituitary response to CRH stimulation seems to be similar in women with PCOS
with and without adrenal hyperandrogenism (Azziz et al. 1998), although
contrasting findings have also been reported (Lanzone et al. 1995). It is possible
that women with PCOS secrete above-normal levels of ACTH during stress or
31
other central nervous system stimulus, as these women have been shown to
present with increased sympathetic nervous system activity (Sverrisdottir et al.
2008). Furthermore, increased peripheral metabolism or impaired reactivation of
cortisol may result in decreased negative feedback suppression of ACTH
secretion (Tsilchorozidou et al. 2003).
Women with PCOS have an increased capacity to secrete androgens during
ACTH stimulation when compared with healthy controls (Azziz et al. 1998, Cinar
et al. 2012, Glintborg et al. 2005). In particular, hyperandrogenic women with
PCOS exhibit higher basal and ACTH-stimulated adrenal androgen secretion
compared with non-hyperandrogenic women with PCOS, whose adrenal
androgen secretion is comparable to that in controls (Cinar et al. 2012). Thus, the
aetiology of adrenal hyperandrogenism in women with PCOS appears to be
related to pronounced activity of the adrenal cortex and not to abnormal function
of the hypothalamic-pituitary-axis or increased sensitivity of the adrenal cortex to
ACTH stimulation. This is supported by the finding that women with PCOS and
increased adrenal androgen secretion have increased 17α-hydroxylase activity of
CYP17, although not all investigators agree (Azziz et al. 1995, Moran et al.
2004). Mutations in the CYP21 gene, which would increase steroid production
towards the CYP17 route, do not seem to play a role in adrenal hyperandrogenism
(Glintborg et al. 2005). In vivo, a strong correlation between the levels of adrenal
androgens and insulin has been found in blood samples taken from adrenal veins
in hyperandrogenic women (Martikainen et al. 1996). However, the relationship
is incompletely understood and studies with contrasting results have been
published (Goodarzi et al. 2015). Elevated levels of estradiol or increased
peripheral sulphatase activity may also have a role in the increase of serum
DHEAS levels (Carmina et al. 1999).
Elevated serum LH levels could participate in adrenal hyperandrogenism in
women with PCOS, as the presence of LH receptors has been found in
steroidogenic cells of the human zona reticularis and fasciculata (Pabon et al.
1996). It has been reported that transgenic female mice over-expressing LH
develop aberrant adrenal gland function entailing altered morphology of the
adrenals, increased LH receptor expression and elevated corticosterone
production (Kero et al. 2000). Furthermore, in women with PCOS salivary gland
cortisol levels positively correlate with LH levels (Tock et al. 2014), supporting a
link between LH and adrenal function. However, in a clinical setting adrenal
steroid secretion in healthy women does not seem to respond to short-term
hCG/LH exposure (Piltonen et al. 2002). Nevertheless, the length of time of
32
exposure to LH may play an important role in this issue. All in all, several extraadrenal factors may influence adrenal androgen secretion and participate in the
pathogenesis of adrenal hyperandrogenism in PCOS.
Peripheral tissues
The most important organs that take part in peripheral androgen conversion are
the skin, liver, lungs and adipose tissue. These tissues have 3β-HSD and 17β-HSD
activity, and thereby they can convert DHEA and DHEAS to A and subsequently
to T (Payne & Hales 2004). DHT, the biologically active form of T, is converted
in the target tissue by 5α-reductase. Furthermore, peripheral tissues have an
important role in regulating the levels of circulating androgens, as they have
aromatase activity, which converts androgens to oestrogens.
The effect of age on ovarian and adrenal androgen production
It is estimated that approximately 400 ovulations occur during a woman’s
lifespan. The rest of the follicles are depleted through apoptosis, programmed cell
death (Vaskivuo et al. 2001). The number of follicles decreases gradually and by
the age of 50 years a woman reaches the menopause, where the follicle pool has
become exhausted and only a low number of follicles remains. As a result of
follicle depletion, ovarian secretion of oestrogens decreases dramatically towards
menopause. The secretion of androgens has also been shown to decrease towards
menopause, and a change in circulating androgen levels can be detected as early
as after the age of 20 years. The decrease seems to be steepest in the early
reproductive years, with stabilisation before menopausal transition (Davison et al.
2005). In postmenopausal women only the adrenal cortex and ovarian stroma
produce some androgens (Couzinet et al. 2001). The availability of free
androgens remains stable or even increases as SHBG levels decrease during
menopausal transition (Burger et al. 2000).
Ovarian androgen production decreases towards menopause both in healthy
women and in women with PCOS. However, ovarian capacity to synthesise and
release androgens remains high through the reproductive years in women with
PCOS when compared with healthy women (Piltonen et al. 2003, Piltonen et al.
2004). Similarly, a cohort study performed in Sweden revealed significantly
elevated T levels in middle-aged women with PCOS, but the difference had
33
disappeared after 21 years of follow-up (mean ages 49 and 70 years) (Schmidt et
al. 2011).
Adrenal DHEAS levels increase markedly during the adolescent years in
girls; they peak in early adulthood and decrease linearly with age thereafter
(Orentreich et al. 1984). Similarly to ovarian aging, adrenal androgen production
in hyperandrogenic women and women with PCOS decreases with age (Kumar et
al. 2005, Moran et al. 1999). However, in a follow-up study of 3–5 years
duration, serum DHEAS levels remained stable in the majority of women with
PCOS (Yildiz et al. 2004).
2.2.2 Oligoamenorrhoea
Oligoamenorrhoea (cycle length more than 35 days) and amenorrhoea (absence of
menstrual bleeding for 3 months or more) are typical signs of anovulatory cycles
in PCOS. In fact, NIH criteria for PCOS require menstrual dysfunction for the
diagnosis of PCOS. However, Rotterdam and AE-PCOS criteria are broader, as
the diagnosis can be set without menstrual dysfunction. Among women diagnosed
by using Rotterdam criteria, approximately 90% have oligo- or amenorrhoea
(March et al. 2010). Interestingly, women with menstrual disturbances were
diagnosed with PCOS with up to 90% probability in a study carried out in Sri
Lanka (Kumarapeli et al. 2008), whereas the Northern Finland Birth Cohort
Study revealed only a 62% probability of being diagnosed with PCOS (Rotterdam
criteria), when the women reported having oligomenorrhoea (Taponen et al.
2004a). Interestingly only 30–40% of women with amenorrhoea have PCOS
(Sirmans & Pate 2013).
2.2.3 Polycystic ovaries (PCO)
The recruitment of resting follicles and follicular maturation is a continuous
process from fetal life throughout women’s reproductive years until menopause.
The recruitment, growth and development of primordial follicles until the
preantral stage takes 6 months or more and is gonadotrophin-independent, as
these changes can occur even with minimal circulating FSH levels (Gougeon
1996) and in women with inhibiting FSH receptor mutations (Aittomaki et al.
1996). Menstrual cyclicity is based on changes in specific hormone levels. The
increase in FSH levels as early as in the late luteal phase of the previous
menstrual cycle, due to a decrease of serum E2 levels from the atrophic corpus
34
luteum, induces the growth of follicles towards ovulation and stimulates the
production of E2. One of the preovulatory follicles grows faster than the others
and is selected as dominant follicle. During the late follicular phase granulosa
cells in the dominant follicle produce high amounts of E2, resulting in positive
feedback, sensitizing the pituitary to GnRH and consequently sharply increased
LH levels, leading to ovulation. Granulosa and theca cells from the ovulated
follicle form a corpus luteum, which secretes progesterone under the regulation of
LH. Regression of the corpus luteum in the late luteal phase enables follicle
maturation for the next menstrual cycle (Messinis et al. 2014).
The microscopic and macroscopic ovarian architecture in cases of PCOS is
abnormal, as these women have a greater density of primordial, primary, preantral
and antral follicles when compared with normal ovaries (Dewailly et al. 2010,
Hughesdon 1982, Maciel et al. 2004, Pigny et al. 2003, Webber et al. 2003). The
follicles in PCO stop growing and developing, typically when they reach 4–7 mm
in diameter (Franks et al. 1998), and hyperandrogenism seems to play a central
role in this divergent early follicular growth (Abbott et al. 2005, Jonard et al.
2003, Vendola et al. 1998). The cessation of folliculogenesis at this stage results
in the accumulation of multiple irregular-sized preantral and antral follicles
typically situated in the ovarian cortex, with pearl-shaped manifestation, thereby
generating the typical appearance of a PCO. However, it is important to notice
that PCO criteria do not define “typical” PCO morphology, as only the number of
follicles or ovarian volume are counted. Furthermore, a PCO finding in only one
ovary is sufficient. Even though PCO are related to anovulation, this distinctive
ovarian morphology is also observed in 6–16% of the female population with
normal menstrual cycles (Abdel Gadir et al. 1992, Koivunen et al. 1999, Polson
et al. 1988).
2.3
Metabolic alterations and health consequences of PCOS
Although infertility is common in cases of PCOS, irregular cycles, acne and
hirsutism may be the most common reasons for seeking medical care during
reproductive life. Obesity, dyslipidaemia, low-grade inflammation and
hypertension together with insulin resistance are the most important determinants
of long-term health in women with PCOS, and their roles increase with ageing.
35
2.3.1 Obesity
The World Health Organization defines overweight BMI as 25.0–29.9 kg/m2 and
obesity as BMI ≥ 30 kg/m2. Not only weight but also the distribution of the fat
tissue matters: abdominal obesity is considered more deleterious than female-type
obesity, and the measurement of waist circumference and waist to hip ratio
(WHR) are useful tools to assess the future risks of T2DM and CVD.
An overweight condition and obesity are common characteristics of women
with PCOS, and the prevalence of these features varies between different
countries (Table 2). It seems that in the U.S. women with PCOS are significantly
more obese than the general population, whereas in Europe this is not the case. In
one study, women with PCOS in the U.S. had a significantly higher BMI (35.1
kg/m2) than women in Italy (27.0 kg/m2). Despite the difference in BMI, WHRs
were similar in these women. The difference in BMI was not explained by total
calorie intake but by a higher amount of saturated fat in women living in the U.S.
(Carmina et al. 2003).
Table 2. The prevalence of obesity among women with PCOS
Study
Country
Criteria
N
PCOS women
Overweight
Diamanti-
Greece
NIH
13
General population
Obese
Overweight Obese
38%
Kandarakis 1999
Asunción 2000
Spain
NIH
10
Cibula 2000
Czech R
NIH
28/752
30%
10%
36%1
Azziz 2004
U.S.
NIH
11/400 24%
42%
BMI 25.9 kg/m2
40%1
24%
32%
Taponen 2004
Finland
Symptoms2
67/663
Lo 2006
U.S.
PCOS4
12734
67.0%
BMI 24.1 kg/m2
Yildiz 2012
Turkey
NIH
24
25.0%
10.2%
Rotterdam
78
15.4%
10.2%
AE-PCOS
60
31.4%
15.0%
10.2%
Joham 2014
Australia
Self-reported 478
25.7%
32.2%
22.4%
15.1%
Lauritsen 2014
Denmark
Rotterdam
16.2%
14.9%
13.1%
5.6%
74
1
2
The definition of obesity BMI > 28.9 kg/m
2
The symptoms of PCOS are self-reported signs of hyperandrogenism and/or irregular menstruation and
the finding of PCO in ultrasonography.
3
The number of women with PCOS / the number of control women
4
The diagnosis of PCOS was not further defined in the publication.
(Asunción et al. 2000, Azziz et al. 2004b, Balen et al. 1995, Cibula et al. 2000, Diamanti-Kandarakis et al.
1999, Joham et al. 2014, Lauritsen et al. 2014, Lo et al. 2006b, Taponen et al. 2004b, Yildiz et al. 2012)
36
BMI has been shown to correlate with an increased rate of hirsutism, serum T
concentrations, menstrual cycle disturbances and infertility in women with PCOS
(Lim et al. 2013, Liou et al. 2009). Obesity further increases the risks of T2DM
and CVD. Given that obesity significantly increases the severity of clinical
features of PCOS (Table 3), weight loss is important for these women. Even 5–
10% weight loss has been shown to improve PCOS symptoms and endocrine
profiles (Moran et al. 2009)
Table 3. Detrimental effects of obesity in PCOS
Endometrium & pregnancy
Androgenic features
Glucose metabolism
Menstrual irregularities ↑
Androgen secretion ↑
Insulin resistance ↑
Dysfunctional bleeding ↑
SHBG levels ↓
Hyperinsulinaemia ↑
Infertility ↑
Hirsutism ↑
T2DM ↑
Gestational diabetes ↑
Acne ↑
Dyslipidaemia ↑
Pre-eclampsia ↑
Preterm birth ↑
Birth-weight of singletons ↑
Caesarean sections ↑
(De Frène et al. 2014, Lim et al. 2013, Liou et al. 2009)
The association between obesity and the prevalence of PCOS has not been
demonstrated in all studies (Table 4). However, the observations support the
assumption that PCOS may be more prevalent among overweight and obese
women.
Table 4. The prevalence of PCOS in different BMI groups
Study
Country Criteria
Alvarez-Blasco Spain1
NIH
N
Underweight
Normal weight Overweight
113
Obese
40.0%
25.0%
9.9%
9.0%
2006
Yildiz 2008
U.S.
NIH
675
Yildiz 2012
Turkey
NIH
392
1
8.2%
9.8%
5.1%
15.0%
Rotterdam 392
18.8%
30.0%
AE-PCOS
14.5%
22.5%
392
The prevalence of PCOS in the general population in the same city is 6.5% (Asunción et al. 2000).
(Alvarez-Blasco et al. 2006, Yildiz et al. 2008, Yildiz et al. 2012)
Up to as many as 80–85% of women with PCOS diagnosed by NIH criteria have
abdominal obesity (Ehrmann et al. 2006, Glueck et al. 2003). When waist
circumference exceeds 88 cm, the risks of T2DM and CVD are significantly
37
increased. Most women with PCOS are thought to have an abdominal body fat
distribution regardless of BMI. This assumption was supported by studies
performed in Sweden and Ireland showing that women with PCOS have higher
WHRs compared with BMI-matched controls (Mannerås-Holm et al. 2011,
O'Connor et al. 2010). However, these results have not been confirmed in
magnetic resonance or computed tomography imaging studies concerning fat
distribution between visceral, abdominal, gluteal and mid-femoral subcutaneous
regions (Barber et al. 2008, Ezeh et al. 2013, Mannerås-Holm et al. 2011,
Yalamanchi et al. 2012). Abdominal subcutaneous and visceral fat accumulation
in cases of PCOS has been reported in three other studies involving
ultrasonography, total-body dual X-ray absorptiometry and subcutaneous adipose
tissue topography (Carmina et al. 2007, Horejsi et al. 2004, Yildirim et al. 2003).
Interestingly, a cohort study performed in Sweden revealed a significant
difference in WHR between middle-aged women with PCOS and controls (mean
age 49 years), but the difference had disappeared later in life (mean age 70 years)
(Schmidt et al. 2011).
Today, the metabolic abnormalities seen in PCOS are not primarily thought to
result from an increased amount of abdominal adipose tissue, but they may be
linked to abnormal adipocyte function and morphology. Adipocytes aspirated
from women with PCOS are significantly larger when compared with those from
control women with a similar BMI (Dunaif et al. 1992, Faulds et al. 2003,
Mannerås-Holm et al. 2011). This finding most likely contributes to altered
adipose tissue and metabolic functions and the increased risk of T2DM in these
women, as adipocyte size negatively correlates to adipose tissue insulin
sensitivity (Salans et al. 1968), and enlarged adipocyte size has been shown to be
a risk factor of T2DM (Lönn et al. 2010).
2.3.2 Insulin sensitivity and glucose tolerance
Insulin is secreted by the β-cells of the pancreatic islets of Langerhans in response
to increased circulating levels of glucose and amino acids after a meal (Pessin &
Saltiel 2000), and its role is to maintain whole body glucose homeostasis and
promote efficient glucose utilization. The human INS gene encodes preproinsulin,
which is converted to proinsulin. Insulin, a heterotetrameric protein consisting of
two α-subunits and two β-subunits, is produced by cleavage of C-peptide from
proinsulin.
38
In the liver, insulin inhibits the production of glucose by inhibiting
gluconeogenesis and glycogenolysis and also promotes glycogen storage. In
muscle and adipose tissue, insulin stimulates the uptake, storage and use of
glucose (Moller & Flier 1991, Pessin & Saltiel 2000). Insulin also affects lipid
metabolism by increasing lipid synthesis in the liver and adipocytes and it
attenuates fatty acid release by suppressing lipolysis from triglycerides in adipose
tissue and muscle, resulting in a decrease in circulating free fatty acid levels
(Diamanti-Kandarakis & Dunaif 2012, Muniyappa et al. 2008, Pessin & Saltiel
2000).
Obesity, body fat distribution and muscle mass all have important
independent effects on insulin sensitivity. Abnormally low, i.e. impaired insulin
sensitivity is called insulin resistance. It can be defined as a decreased ability of
insulin to mediate its metabolic actions on glucose uptake, glucose production and
lipolysis, resulting in a requirement for an increased amount of insulin to achieve
euglycaemia (Diamanti-Kandarakis & Dunaif 2012). This leads to an increased
release of insulin from the pancreas, and thereby compensatory
hyperinsulinaemia, which is a characteristic of insulin resistance. As a
consequence of insulin resistance, glucose concentrations rise slightly, although
remaining within the normal range before reaching the diagnostic criteria for
glucose intolerance (Smith 1994). The most important factors underlying insulin
resistance are obesity, physical inactivity and genetic factors. Furthermore, insulin
resistance is tightly associated with major public health problems – obesity,
hypertension, dyslipidaemias, coronary artery disease and metabolic syndrome
(Ginsberg 2000).
Impaired glucose regulation (impaired glucose tolerance [IGT] and impaired
fasting glucose [IFG]) refers to a metabolic state intermediate between normal
glucose homeostasis and diabetes. Furthermore, IGT and IFG are not clinical
entities but risk categories as regards future T2DM and/or CVD (World Health
Organization 2006). The development of T2DM, a state where euglycaemia is not
reached, originates from insulin resistance occurring in skeletal muscle and
adipose tissue, leading to failure in glucose uptake from the blood, with a
simultaneous insufficient compensatory increase in insulin production from βcells. Thus, it is a metabolic aetiology characterized by chronic hyperglycaemia
resulting from defects in insulin secretion or action, or both (Alberti & Zimmet
1998). Impaired glucose tolerance, IFG and T2DM can be diagnosed from fasting
blood samples or by way of oral glucose tolerance tests (OGTTs). In Table 5, the
specific limits for diagnosing these disorders are listed.
39
Table 5. The World Health Organization criteria for diagnosing diabetes mellitus,
impaired glucose tolerance and impaired fasting glycaemia
Diagnosis
Glucose concentration in plasma (mmol/l)
Diabetes mellitus
Fasting glucose
≥ 7.0
2-hour glucose
≥ 11.1
Impaired glucose tolerance
Fasting glucose
< 7.0
2-hour glucose
7.8 ≤ glucose ≤ 11.0
Impaired fasting glycaemia
Fasting glucose
6.1 ≤ glucose ≤ 6.9
2-hour glucose
< 7.8
(World Health Organization 2006)
Measuring glucose homeostasis
The gold standard for assessing insulin resistance in vivo is the
hyperinsulinaemic, euglycaemic glucose clamp technique, which directly
determines metabolic insulin sensitivity (Muniyappa et al. 2008). At steady state,
the amount of glucose that is infused equals the amount of glucose taken up by
the peripheral tissues, and it can be used as a measure of peripheral sensitivity to
insulin – insulin-mediated glucose disposal (Diamanti-Kandarakis & Dunaif
2012, Dunaif 1997). Insulin sensitivity can also be evaluated indirectly by way of
the intravenous glucose tolerance test (IVGTT). The minimal model provides a
measurement of insulin sensitivity on the basis of glucose and insulin data
obtained during IVGTTs (Muniyappa et al. 2008). This index reflects insulin
action in stimulating glucose uptake and suppressing glucose production
(Diamanti-Kandarakis & Dunaif 2012).
Because both the above-mentioned techniques are time-consuming,
expensive and experimentally demanding methods to accomplish in practical use,
the possibility to use fasting parameters as measurements of insulin resistance has
been studied. Fasting levels of glucose or insulin are not suitable for insulin
resistance measurements by themselves, as fasting glucose reflects endogenous
glucose production (an index of hepatic insulin action) and fasting insulin reflects
insulin secretion and metabolic clearance in addition to insulin sensitivity
(Hücking et al. 2008). The indexes better describing insulin resistance are
homeostatic model assessment data (Matthews et al. 1985), the fasting glucose to
insulin ratio, and the quantitative insulin sensitivity check index (Katz et al.
40
2000). However, these indexes are mathematically related and provide essentially
identical information.
The oral glucose tolerance test is widely used in clinical practice as a firstline measurement, and it reflects the efficiency of the body to dispose of glucose
after an oral glucose load (Muniyappa et al. 2008). As an advantage, the OGTT
mimics glucose and insulin dynamics under physiological conditions more
closely than conditions of the glucose clamp or the IVGTT (Hücking et al. 2008).
The OGTT provides useful information about glucose tolerance but not insulin
sensitivity or resistance per se.
Biochemical screening of glucose tolerance is indicated for at least obese
women with PCOS and those with increased visceral adiposity as measured by
waist circumference. However, a current statement from the AE-PCOS Society
recommends an OGTT with assessment of fasting and 2-hour glucose
concentrations for all women diagnosed with PCOS (Salley et al. 2007, Wild et
al. 2010), as fasting glucose levels are poorly associated with 2-h glucose
concentrations in women with PCOS (Ehrmann et al. 2005). The Endocrine
Society recommends rescreening by way of an OGTT every 3–5 years or even
more often if a woman is obese, for example (Legro et al. 2013). In addition, if an
OGTT is not possible, insulin resistance of women with PCOS may also be
evaluated by measurement of fasting glucose and insulin concentrations and
calculation of the glucose to insulin ratio. A ratio of < 4.5 is considered significant
for insulin resistance in obese Caucasian women with PCOS, and the ratio has
been shown to correlate well with insulin sensitivity obtained by way of IVGTTs
in women with PCOS (Legro et al. 1998). Assay of glycated haemoglobin,
HbA1c, the level of which reflects average plasma glucose concentrations over
the previous month, has not been found to be a useful tool, as in women with
PCOS it has low sensitivity to detect IGT and T2DM when compared with
OGTTs (Velling Magnussen et al. 2011).
Prevalence of glucose intolerance and related factors in PCOS
Insulin resistance and compensatory hyperinsulinemia play a prominent role in
the pathogenesis of PCOS, since as many as 60–80% of women with this
syndrome suffer from these conditions, independently of obesity (Stepto et al.
2013, Wild et al. 2010). Insulin resistance has been recognised as a major risk
factor as regards the development of T2DM (Lillioja et al. 1993), and PCOS has
been associated with higher prevalence rates of IGT, gestational diabetes mellitus
41
(GDM) and T2DM (Boomsma et al. 2006, Ehrmann et al. 1999, Kjerulff et al.
2011, Legro et al. 1999, Moran et al. 2010, Norman et al. 2001). Furthermore,
insulin resistance has been shown to correlate positively with FAI and triglyceride
and CRP levels, and negatively with SHBG concentrations (Karakas et al. 2010).
Table 6. Prevalence of IGT and T2DM in women with PCOS
Study
Country
Criteria
N
PCOS
IGT
Dahlgren 1992
Sweden
PCO2
33/1321
Ehrmann 1999
U.S.
NIH
122
35.2%
Legro 1999
U.S.
NIH
254/801
31.1%
Cibula 2000
Czech R
NIH
28/7521
Ehrmann 2005
U.S.
NIH
408
Lo 2006
U.S.
PCOS3
11035/551751
Hudecova 2011
Sweden
Rotterdam4
84/871
Control women
T2DM
IGT
15.0%
T2DM
2.3%
9.8%
7.5%
14.0%
32.1%
23.0%
3.9%
9.5%
8.3%
0%
8.0%
9.0%
1.9%
2.3%
1.1%
Joham 2014
Australia
Self-reported 478/81341
5.1%
0.3%
Sirmans 2014
U.S.
ICD-95
17.6%
4.7%
1689/50671
1
Number of women with PCOS / number of control women
2
Histopathologically confirmed PCO
3
Diagnoses of PCOS not further defined in the publication
4
All women presented with PCO
5
ICD-9 diagnosis code for PCOS or oligomenorrhoea/amenorrhoea and hyperandrogenism
(Cibula et al. 2000, Dahlgren et al. 1992, Ehrmann et al. 1999, Ehrmann et al. 2005, Hudecova et al.
2011, Joham et al. 2014, Legro et al. 1999, Lo et al. 2006b, Sirmans et al. 2014)
The prevalence of glucose intolerances are increased in women with PCOS (Table
6). A meta-analysis has revealed that the odds ratios (ORs) for IGT and T2DM are
significantly increased in women with PCOS compared with BMI-matched
controls, being 2.54 for IGT and 4.00 for T2DM (Moran et al. 2010). Therefore, it
has been recommended that all women with PCOS should be screened for glucose
intolerance by means of OGTTs (Legro et al. 2013, Salley et al. 2007).
Of women with PCOS, 15–67% are overweight or obese (Table 2). A recent
study revealed that the impact of BMI on insulin resistance is even greater in
cases of PCOS than in controls (Stepto et al. 2013). Even though the risk of
glucose intolerance is further amplified by obesity (Boudreaux et al. 2006, Dunaif
et al. 1992, Ehrmann et al. 1999, Legro et al. 1999), lean women with PCOS have
also been shown to have increased rates of insulin resistance and glucose
intolerance compared with BMI-matched healthy women (Dunaif et al. 1989,
Legro et al. 1999, Morales et al. 1996, Stepto et al. 2013). The risk of glucose
42
intolerance varies among women with PCOS, as it may be highest in women with
ovulatory dysfunction and hyperandrogenism, and lowest among women with
anovulation and PCO without hyperandrogenism (Barber et al. 2007, Barber et al.
2007, Dewailly et al. 2006). Interestingly, irregular menstrual cycles have been
shown to present as an independent risk factor of T2DM in women in general
(Solomon et al. 2001).
Besides having an increased risk of T2DM, women with PCOS are at high
risk of developing GDM. In a meta-analysis it was estimated that women with
PCOS have a significantly higher risk of developing GDM (OR 2.94) compared
with women without the syndrome (Boomsma et al. 2006). In addition, a large
population-based study revealed a 2.4-fold increased risk of GDM in women with
PCOS (Lo et al. 2006a). Moreover, women with previous GDM presented with
PCO more often than controls (Koivunen et al. 2001).
Hyperinsulinaemia and defects in insulin secretion in PCOS
Pancreatic β-cell dysfunction lies behind the development of T2DM, as
hyperglycaemia develops when insulin secretion is not sufficient for the increased
requirements of insulin resistance. Therefore, the ability to dispose of
carbohydrates depends on the responsiveness of pancreatic β-cells to glucose as
well as the sensitivity of the glucose-utilizing tissues to secreted insulin (Bergman
et al. 1981). The high prevalence of hyperglycaemia in women with PCOS
suggests the presence of defects in insulin secretion in addition to peripheral
insulin resistance.
It is well known that basal insulin secretion rates are elevated in women with
PCOS, which results from the greater degree of insulin resistance compared with
that in healthy women. However, previous studies have also suggested that
women with PCOS present with a greater degree of compensatory insulin
secretion for a given increment in insulin resistance, in a fasting state (Goodarzi et
al. 2005). Even though first-phase insulin secretion has been shown to be normal
in women with PCOS, decreased postprandial secretion of insulin in these women
has been reported independently of obesity, especially when insulin release is
proportioned to the degree of insulin resistance (Dunaif & Finegood 1996,
Ehrmann et al. 1995, Ehrmann et al. 2004, O'Meara et al. 1993). However,
studies with differing results have also been published (Ciampelli et al. 1997,
Holte et al. 1994). The decreased postprandial insulin secretion resembles
pancreatic β-cell dysfunction, which is also present in T2DM and may therefore
43
explain the high prevalence of glucose intolerance in women with PCOS.
Interestingly, women with PCOS who present with a first-degree relative with
T2DM are more likely to present these defects (Colilla et al. 2001, Ehrmann et al.
1995). Furthermore, daughters of women with PCOS as young as 8–12 years old
present with postprandial β-cell dysfunction (Torchen et al. 2014). These findings
suggest a genetic contribution to the disturbances in β-cell function in PCOS. One
possible explanation for alterations in insulin secretion may be changes in the
secretion of incretins, which augment insulin secretion after a meal. In lean
women with PCOS, increased total glucose-dependent insulinotropic polypeptide
and lower late-phase active glucagon-like peptide-1 concentrations during OGTTs
have been observed when compared with BMI- and age-matched control subjects
(Vrbikova et al. 2008). However, another study did not reveal an independent
effect of PCOS on the incretin response (Svendsen et al. 2009). Furthermore,
inflammation has been suggested to play a role in β-cell dysfunction in PCOS
(Malin et al. 2015).
2.3.3 Cardiovascular risk factors and outcomes
Women with PCOS present with several risk factors of CVD. Both the Endocrine
Society and The AE-PCOS Society have published recommendations that
adolescents and women with PCOS should be screened for cardiovascular disease
risk factors, as shown in Table 7 (Legro et al. 2013, Wild et al. 2010).
44
Table 7. Different cardiovascular risk categories of women with PCOS (Legro et al.
2013, Wild et al. 2010)
Risk category
Symptom / Diagnosis
At risk – women with any of the following
Obesity (especially increased abdominal obesity)
Cigarette smoking
Hypertension
Dyslipidaemia
Subclinical vascular disease
Impaired glucose tolerance
Family history of premature cardiovascular disease
At high risk – women with
Metabolic syndrome
Type 2 diabetes
Vascular or renal disease, cardiovascular diseases
Obstructive sleep apnoea
Dyslipidaemia
Disturbances in lipid and lipoprotein metabolism are fairly common metabolic
abnormalities in women with PCOS, diagnosed in approximately 70% of such
women in the U.S. (Diamanti-Kandarakis et al. 2007, Legro et al. 2001, Randeva
et al. 2012). Similarly, the incidence of dyslipidaemia in Brazil has been reported
to be as high as 76% (Rocha et al. 2011). The diagnostic limits of dyslipidaemia
can be seen in Table 8.
Table 8. Diagnostic limits of dyslipidaemia in women in Finland. An abnormal
concentration can be found in one or more variable.
Variable
Limit
LDL cholesterol
> 3.0 mmol/l
Triglycerides
> 1.7 mmol/l
HDL cholesterol
< 1.2 mmol/l
There are several studies in which lipid abnormalities in women with PCOS have
been explored. In general, decreased levels of HDL cholesterol together with
elevated levels of triglycerides and LDL cholesterol have been reported among
these women (Berneis et al. 2007, Conway et al. 1992, Ehrmann et al. 2006,
Glueck et al. 2003, Glueck et al. 2009, Legro et al. 2001, Rocha et al. 2011,
Yildirim et al. 2003). Although the results concerning the levels of LDL
cholesterol have been divergent, a meta-analysis revealed that women with PCOS
present with significantly higher serum LDL cholesterol concentrations, even
45
when BMI has been taken into account (Wild et al. 2011). Furthermore, women
with PCOS have elevated levels of small, dense LDL cholesterol particles
compared with age- and BMI-matched controls (Berneis et al. 2007, Berneis et al.
2009, Dejager et al. 2001, Pirwany et al. 2001), which further exposes these
women to CVD, as small dense LDL particles are taken up by arterial wall more
easily and have decreased oxidative susceptibility compared with larger LDL
particles (Rizzo & Berneis 2006). Furthermore, both normal-weight and obese
women with PCOS have been shown to present with increased levels of oxidised
LDL cholesterol when compared with age- and BMI-matched controls (Macut et
al. 2006). All in all, the most common lipid profile in PCOS seems to be an
atherogenic lipoprotein phenotype characterized by hypertriglyceridaemia,
increased levels of small, dense LDL cholesterol and decreased HDL cholesterol
levels, representing a risk factor of coronary heart disease (CHD) (National
Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation,
and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III)
2002).
In PCOS, the main pathophysiological mechanisms of dyslipidaemia are
probably central adiposity, hyperandrogenism and insulin resistance, but diet,
exercise and genetics have their roles as well. However, overweight conditions
and obesity have been suggested to be predominant factors affecting
dyslipidaemia in cases of PCOS (Joharatnam et al. 2011). This hypothesis was
supported by the results of a well-matched case-control study, which revealed
differences only in the composition of LDL particles (Phelan et al. 2010).
Chronic inflammation
Atherosclerosis has been shown to involve a chronic inflammatory process (Ross
1999). At present, C-reactive protein (CRP) appears to be the classic marker of
inflammation and it is commonly measured by a highly sensitive method able to
detect very low concentrations. However its use in clinical practice is still random.
CRP is released by the liver in response to interleukin-6 and it acts both as a marker
and a contributor to the vascular inflammatory process (Deanfield et al. 2005). Lowgrade chronic inflammation is characterized by persistent moderately elevated CRP
concentrations within the normal range. CRP independently predicts future coronary
events such as myocardial infarction, stroke, coronary revascularization processes and
cardiovascular death in asymptomatic individuals more strongly and more
significantly than other inflammatory markers (Ridker et al. 1998, Ridker et al. 2000,
46
Rifai et al. 2002) or even LDL (Ridker et al. 2002). Furthermore, there is growing
evidence that the lesions of atherosclerosis represent a series of highly specific
cellular and molecular responses that can be described as an inflammatory disease
(Ross 1999). In addition, inflammation plays a role in destabilizing the fibrous cap in
coronary plaques, predisposing individuals to plaque rupture and thereby enhancing
the risk of coronary thrombosis (van der Wal et al. 1994). Moreover, the results of an
in vitro study suggested that CRP may play a direct role in promoting the
inflammatory component of atherosclerosis (Pasceri et al. 2000). Obesity, metabolic
syndrome and cigarette smoking have been shown to be associated with increased
CRP levels (Tracy et al. 1997, Visser et al. 1999). Another marker of chronic
inflammation, pentraxin 3 (PTX-3), is produced by peripheral tissues such as vascular
endothelial cells, smooth muscle cells and leucocytes in response to proinflammatory
signals. Therefore its secretion is not influenced by drug-induced hepatic protein
synthesis (Mantovani et al. 2008).
It has been shown that women with PCOS present with increased levels of
CRP when compared with control women, even after adjustment for age and BMI
(Boulman et al. 2004, Diamanti-Kandarakis et al. 2006, Diamanti-Kandarakis et
al. 2006, Kelly et al. 2001). Positive relationships between CRP levels and insulin
resistance, body weight and adipose tissue mass have also been observed
(Diamanti-Kandarakis et al. 2006, Diamanti-Kandarakis et al. 2006, Kelly et al.
2001, Morin-Papunen et al. 2003). In addition, levels of other low-grade chronic
inflammation markers including cell adhesion molecules are increased in PCOS
(Diamanti-Kandarakis et al. 2006, Diamanti-Kandarakis et al. 2006).
Furthermore, levels of the proinflammatory cytokine interleukin-18, which
promotes the synthesis of interleukin-6, are elevated in women with PCOS
(Escobar-Morreale et al. 2004).
Endothelial dysfunction
The endothelium is a thin single-cell layer that covers the inner surface of the
blood vessels and the heart. The endothelium plays a key role in the maintenance
of a healthy vascular wall by regulating vascular tone and inhibiting several preatherogenic processes such as monocyte recruitment, platelet adhesion, LDL
oxidation, synthesis of pro-inflammatory cytokines and smooth muscle cell
proliferation (Cannon 1998). Endothelial dysfunction can be defined as a
reduction of the bioavailability of vasodilators, such as nitric oxide, and an
increase in vasoconstrictors, such as endothelin-1 (ET-1), produced by or acting
47
on endothelial cells (Deanfield et al. 2005), and it is an early marker of
atherosclerosis (Brunner et al. 2005). In addition to nitric oxide, insulin has a
physiological action in the vasodilation of skeletal muscle vasculature in humans
(Cleland et al. 1998).
When obese women with PCOS were compared with weight-matched
controls, women with PCOS exhibited significantly diminished endotheliumdependent vasodilation and ability of insulin to cause vasodilation (DiamantiKandarakis et al. 2006, Kelly et al. 2002, Paradisi et al. 2001). A diminished
vasodilatory response seems to be at least in part due to impaired production
and/or release of nitric oxide (Paradisi et al. 2001), and it can be normalized by
way of metformin treatment (Diamanti-Kandarakis et al. 2005). Therefore, one
could speculate that the insulin resistance mechanisms concerning impaired
vasodilatation are the same as seen in adipocytes and skeletal muscle, as
discussed above.
Increased serum levels of ET-1 have also been reported in women with PCOS
when compared with BMI-matched controls (Diamanti-Kandarakis et al. 2001,
Diamanti-Kandarakis et al. 2005, Diamanti-Kandarakis et al. 2006, Orio et al.
2004). Furthermore, the difference remained when BMI was used as a covariate
(Diamanti-Kandarakis et al. 2001). A putative cause of the increased ET-1 levels
could be insulin resistance, as insulin stimulates secretion of ET-1 in vivo and in
vitro (Ferri et al. 1995). In line with this, metformin treatment reduces the level of
ET-1 in PCOS (Diamanti-Kandarakis et al. 2001, Diamanti-Kandarakis et al.
2005).
In line with previous findings, reduced vascular compliance in the brachial
and carotid arteries of women with PCOS has been reported, versus controls
(Kelly et al. 2002, Lakhani et al. 2002). Decreased vascular compliance is a
feature of subclinical atherosclerosis, as arterial elasticity is reduced during fattystreak formation before other pathological changes occur (Hironaka et al. 1997),
and it has a strong correlation with cardiovascular and all-cause mortality
(Laurent et al. 2001).
The role of androgens as regards impaired endothelial function in cases of
PCOS is an interesting question. Endothelial function has been found to be
inversely correlated with serum free T levels (Paradisi et al. 2001). Furthermore,
androgens have induced endothelial dysfunction in animal models (Hutchison et
al. 1997). In line with this, in female to male transsexuals, androgen
administration has been associated with impaired vascular reactivity, consistent
with a deleterious effect of androgen excess on arterial physiology (McCredie et
48
al. 1998). The mechanism by which hyperandrogenism affects vascular reactivity
is unknown. It is possible that T could have direct effects on the vessel wall,
although an indirect effect via alteration of the lipoprotein profile cannot be
excluded.
Hypertension
Essential hypertension is a highly prevalent pathological condition that is
considered as one of the most relevant cardiovascular risk factors and is an
important cause of morbidity and mortality around the world. Hypertension is
considered, when systolic blood pressure is ≥ 140 mmHg or diastolic blood
pressure is ≥ 90 mmHg (National Cholesterol Education Program (NCEP) Expert
Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in
Adults (Adult Treatment Panel III) 2002).
Among women with PCOS who are of reproductive age, 21–48% develop
elevated blood pressure or hypertension (≥ 130/85 mmHg) (Apridonidze et al.
2005, Dokras et al. 2005, Ehrmann et al. 2006, Glueck et al. 2003). A study
performed in the U.S. revealed that 26.6% of women with PCOS aged from 15 to
44 years had diagnosed hypertension or elevated blood pressure compared with
11.7% of age-matched controls and women with PCOS were more likely to have
elevated blood pressure or hypertension even after adjustment for BMI, age,
diabetes and dyslipidaemia (Lo et al. 2006b). Furthermore, a recent cohort study
revealed that women with PCOS have an OR of 2.85 for hypertension when
compared with controls (Sirmans et al. 2014). Notably, a study performed in
Sweden revealed that 39% of women with PCOS aged from 40 to 59 years were
being treated for hypertension, whereas only 11% of age-matched control women
used medication for this condition (Dahlgren et al. 1992). Twenty-one years later,
69% of the women with PCOS and 41% of the controls had hypertension and the
difference was significant (Schmidt et al. 2011). However, some investigators
have not found an increased prevalence of hypertension in cases of PCOS (Wild
et al. 2000).
Obesity has been suggested as the primary cause of elevated blood pressure
in women with PCOS (Luque-Ramírez et al. 2007). Furthermore,
hyperandrogenism might have a role (Chen et al. 2007). In addition, increased
activity of the sympathetic nervous system in PCOS results in vasoconstriction,
the extent of which strongly correlates with hyperandrogenism (Sverrisdottir et al.
49
2008). In summary, the development of hypertension in PCOS is multifactorial
and the role of PCOS per se is not well understood.
Subclinical atherosclerosis
One of the first detectable stages of atherosclerosis is thickening of the arterial
wall. Carotid intima-media thickness (IMT) is the thickness of two layers (the
intima and media) of the walls of the carotid arteries, the largest conduits of blood
going to the brain (Roger et al. 2012). Ultrasonographic measurement of common
carotid IMT is a morphological marker used to assess precocious atherosclerosis
(van den Oord et al. 2013) and a recent meta-analysis revealed that increased IMT
is a predictor of future myocardial infarction and stroke (van den Oord et al.
2013). Increased IMT has been associated with general and abdominal obesity
and increasing insulin resistance (De Michele et al. 2002, Rajala et al. 2002). It
has also been reported in relatively young women with PCOS when compared
with control women (Orio et al. 2004), as well as in women with PCOS older
than 40 years (Guzick et al. 1996, Talbott et al. 2000). Furthermore, 7.2% of
women with PCOS and 0.7% of control women have been reported to have a
carotid plaque index of three or more (Talbott et al. 2000), indicating more
intense carotid atherosclerosis in these cases.
Coronary artery calcification is a measure of the extent of atherosclerosis in
the heart arteries. Detection of coronary calcium confirms the presence of
coronary atherosclerotic plaque independently of symptoms (Roger et al. 2012).
Women with PCOS have a significantly greater prevalence and extent of coronary
artery and aortic calcification than age-matched ovulatory control women
(Christian et al. 2003, Talbott et al. 2004). After adjustment for BMI, PCOS did
not predict the presence of coronary calcium in one study (Christian et al. 2003),
but it remained a significant predictor of coronary artery calcification in another
(Talbott et al. 2004).
Diastolic dysfunction is considered to be an early sign of coronary artery
disease. When women with PCOS were compared with BMI-matched but older
control women, they presented several unfavourable changes in diastolic function
and therefore they may be likely to develop diastolic dysfunction (Yarali et al.
2001).
50
Cardiovascular disease, cardiovascular events and mortality
As women with PCOS present with several risk factors of CVD, a question is
whether they also have greater prevalence rates of CVDs and events, or deaths
caused by CVD. It has been estimated that on the basis of specific risk factors,
women with PCOS aged 40–49 yr present with a fourfold increased risk and
women aged 50–60 yr even an 11-fold risk of developing CVD {{640
Dahlgren,E. 1992}}. However, divergent results concerning CVD diagnosis have
been published. Symptoms related to PCOS such as hirsutism, irregular
menstruation and PCO have been associated with coronary artery disease
(Azevedo et al. 2006, Birdsall et al. 1997, Wild et al. 1990), but not all studies
support this (Krentz et al. 2007).
Studies carried out to investigate the association between PCOS and CVD are
few and the diagnosis of PCOS varies. Furthermore, the subjects are relatively
young in some studies. Coronary artery disease has been reported to be more
prevalent in women with PCOS in some (Cibula et al. 2000, Shaw et al. 2008)
but not all (Wild et al. 2000) studies. Again, women with PCOS may have a
higher prevalence of cerebrovascular disease (Wild et al. 2000). Cumulative
cardiovascular event-free survival may be significantly shorter in women with
PCOS and these women might even have a 3.3-fold greater risk of cardiovascular
death or myocardial infarction compared with women without PCOS (Shaw et al.
2008), although differing results exist (Elting et al. 2001, Pierpoint et al. 1998,
Schmidt et al. 2011). One meta-analysis of five studies revealed a twofold
increased risk of CHD and stroke in women with PCOS when compared with
women without the syndrome. When studies with results adjusted for BMI were
investigated, the risk further increased by 55% (de Groot et al. 2011). In
summary, the conclusions drawn from the results of clinical studies vary. When
women with definitive diagnoses of PCOS are older, more data can be analysed
and the answers will be found in the future.
2.4
Treatment of PCOS-related disorders
2.4.1 Combined hormonal contraceptives
Combined hormonal contraceptives (CHCs), including both oestrogens and
progestogens, are the most widely used forms of contraception in the world. Besides
contraception, these agents can be used in the treatment of menstrual irregularities,
51
hirsutism and acne, including women with PCOS. Furthermore, CHCs are used in
connection with severe dysmenorrhoea, dysfunctional uterine bleeding, endometriosis
and scheduling the cycles for in vitro fertilization. Combined hormonal contraceptives
can be administered orally, transdermally or vaginally.
The effects of CHCs are based on the effects of both oestrogens and
progestogens. The most frequently used oestrogen is ethinyl estradiol (EE), but today
more physiological oestrogens are available such as 17β-estradiol and estradiol
valerate. Ethinyl estradiol exerts a stronger effect on hepatic metabolism than natural
oestrogens. There are several different progestins available with various chemical
structures, clinical effects and metabolic risks. The progestins are structurally related
either to testosterone or progesterone. New 4th generation progestins have been
designed to bind with high specificity to progesterone receptors and avoid interactions
with androgen, oestrogen and glucocorticoid receptors, and they are promising in the
treatment of hyperandrogenism. Unfortunately they are associated with a slightly
increased risk of venous thrombosis when compared with 2nd generation preparations
(Sitruk-Ware & Nath 2011). Progestins have been divided into different generations
based on the occasions when they have been introduced to the market.
In addition to contraceptive effects, CHCs modify surrogate markers such as
glucose tolerance, the lipid profile and coagulation factors, which have been
associated with cardiovascular and venous risks. A cross-sectional study revealed
7.1-fold increased risk of IGT in current users and a 2.1-fold risk in former users
of oral contraceptives compared with nonusers (Deleskog et al. 2011). However,
this has not been confirmed as regards T2DM (Chasan-Taber et al. 1997, Rimm et
al. 1992), and CHCs are not considered to cause clinically significant changes in
glucose tolerance (Lopez et al. 2014). The oestrogen component is primarily
responsible for the insulin resistance induced by CHCs, in a dose-dependent
matter (Godsland et al. 1992, Kojima et al. 1993). When male to female
transsexuals were treated with EE, peripheral glucose uptake was diminished,
reflecting impaired insulin sensitivity (Polderman et al. 1994). Progesterone
receptor expression has been observed in the pancreas (Pasanen et al. 1997), and
progestins seem to increase insulin half-life, to varying degrees (Godsland et al.
1992, Kojima et al. 1993). In addition, the androgenic properties of progestins
might play a role in the effects of CHCs on insulin sensitivity, as androgen
supplementation in female to male transsexuals decreases insulin sensitivity
(Polderman et al. 1994) and administration of anti-androgens improves it
(Moghetti et al. 1996).
52
The effect of different CHCs on the lipid profile reflects the balance of oestrogen
and progestin, both in terms of steroid dose and progestin type. Generally, oestrogen
administration results in a favourable lipid profile and the magnitude is related to the
potency of the oestrogen. On the other hand, progestins may counteract these effects
and the detrimental effects are related to the androgenic properties of progestins
(Sitruk-Ware & Nath 2013). Interestingly, CHCs may reduce LDL particle size
(Crook & Godsland 1998), and they have been associated with a denser LDL
subfraction pattern (de Graaf et al. 1993). Exposure to EE increases hepatic
secretion of particles that are triglyceride-rich such as very low density lipoprotein
(VLDL) and thereby the use of combined contraceptives leads to increased levels of
triglycerides.
The Endocrine Society recommends the use of CHCs as first-line treatment for
the menstrual irregularities, hirsutism and acne in women with PCOS. It does not
suggest one formulation or administration route over another, and, naturally, it
recommends screening for possible contraindications before the use of hormonal
contraceptives (Legro et al. 2013). In a recent review, individualized risk
stratification prior to prescribing combined hormonal contraceptives was
suggested (Yildiz 2015).
2.4.2 Insulin sensitizers
Insulin sensitizers are drugs that improve insulin sensitivity in target cells.
Insulin-lowering agents improve endocrine and reproductive abnormalities in
women with PCOS by reducing insulin resistance and hyperinsulinaemia as these
conditions are considered to be significant contributors to the pathophysiology of
PCOS.
Metformin
Metformin is an orally administered drug that has been used extensively in the
treatment of T2DM for decades. The beneficial effect of metformin on insulin
sensitivity is complex (Hardie 2013). Furthermore, a small decrease in body
weight might also contribute to its effects. The effects of metformin on glucose
metabolism include a reduction of hepatic glucose production by inhibition of
gluconeogenesis in the liver, an increase in glucose uptake by stimulation of
GLUT4 expression and intracellular glucose transport, acceleration of glucose
53
utilization by enhancement of glucose metabolism, and activation of insulin
receptors by stimulating their tyrosine kinase activity (De Leo et al. 2003).
Metformin is the most extensively studied insulin-lowering drug used in the
treatment of PCOS. It improves insulin resistance and hyperandrogenism and
increases the levels of SHBG in these women (Palomba et al. 2009)(Nestler &
Jakubowicz 1996, Velazquez et al. 1994). It has also been widely reported that
metformin improves menstrual irregularity and restores ovulatory function (De
Leo et al. 2003, Fleming et al. 2002). The results of some randomized placebocontrolled studies (Kjøtrød et al. 2011, Morin-Papunen et al. 2012), but not all
(Fleming et al. 2002), suggest that metformin treatment improves pregnancy and
live-birth rates in women with PCOS. A meta-analysis revealed an improvement
in clinical pregnancy rate but not in live-birth rate during metformin treatment
versus placebo (Tang et al. 2012). In line with this, similar results were reported
when metformin was used before or during assisted reproductive techniques (Tso
et al. 2014). Moreover, metformin improves metabolic cardiovascular risk
parameters in PCOS, such as dyslipidaemia (Fleming et al. 2002, Rautio et al.
2005) and chronic inflammation (Morin-Papunen et al. 2003). The advantage of
metformin is that it is considered to be non-teratogenic drug (Cassina et al. 2014).
The Endocrine Society gave recommendations concerning metformin use in
women with PCOS in 2013. Metformin use is recommended for women with
PCOS and IGT or T2DM who fail to modify their lifestyles. The Endocrine
Society recommends metformin as a second-line therapy for the treatment of
menstrual irregularities in women who cannot take or do not tolerate hormonal
contraceptives. However, in clinical practice, when treating obese women,
metformin can also be considered as first-line treatment. Furthermore, the
Endocrine Society suggests the use of metformin as adjuvant therapy during in
vitro fertilization in order to prevent ovarian hyperstimulation syndrome. Notably,
the Endocrine Society does not suggest the use of metformin in first-line
treatment of cutaneous manifestations, for prevention of pregnancy
complications, or for the treatment of obesity (Legro et al. 2013).
Thiazolidinediones
Thiazolidinediones, TZDs, were first introduced as promising drugs for the
treatment of insulin resistance, as they enhance insulin action in skeletal muscle,
liver and adipose tissue. However, the first TZD, troglitazone, was withdrawn
from the market by the U.S. Food and Drug administration in 2000 as a result of
54
its hepatotoxicity. In Europe, only one TZD, pioglitazone, is currently available,
as rosiglitazone has been withdrawn from the market because of an excess of
cardiovascular events.
Thiazolidinediones have been shown to improve menstrual cyclicity,
hyperandrogenism, insulin resistance and hyperinsulinaemia in women with PCOS
(Rautio et al. 2006). However, as TZDs have been suspected of retarding fetal
development in animal studies, their use is contraindicated for infertility treatment
and during pregnancy. The Endocrine Society does not recommend the use of
thiazolidinediones for the treatment of PCOS because of safety concerns (Legro
et al. 2013).
2.4.3 Statins
Statins are widely used to reduce plasma cholesterol levels and the risk of
cardiovascular events (Taylor et al. 2013). They reduce the conversion of 3hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) to L-mevalonate by
inhibiting the activity of HMG-CoA reductase, which plays a central role in the
production of cholesterol in the liver. The reduction in the intracellular levels of
cholesterol induces enhanced LDL receptor expression on hepatocyte cell
surfaces, resulting in increased extraction of LDL cholesterol from the blood and
thereby decreased circulating concentrations. In addition to decreased cholesterol
levels, this results in inhibition of the biosynthesis of isoprenoids such as
geranylgeranyl pyrophosphate and farnesyl pyrophosphate. These isoprenoid
metabolites participate in the maintenance of cell shape, motility, factor secretion,
differentiation and proliferation. Furthermore, cholesterol-independent beneficial
effects of statins include, for example, improvement of endothelial function,
decrease of vascular inflammation and oxidative stress, and stabilization of
atherosclerotic plaques (Gazzerro et al. 2012). According to their
pharmacokinetics, statins can be classified as lipophilic (atorvastatin, fluvastatin,
lovastatin and simvastatin) and hydrophilic (pravastatin, rosuvastatin) (Gazzerro
et al. 2012). Lipophilic statins show efficient activity at both hepatic and extrahepatic sites, whereas hydrophilic statins are more hepato-selective (Gazzerro et al.
2012). The most important adverse effects associated with statins are myopathy and
an asymptomatic increase in hepatic transaminases (Girardi 2014). It has been
suggested that the risk of myopathy may be lowest with hydrophilic statins as a result
of minor muscle penetration (Girardi 2014).
55
As up to 70% of women with PCOS present with dyslipidaemia and an increased
risk of CVD (Wild et al. 2010), the use of statins is often indicated. As cholesterol
serves as a substrate for androgen synthesis, the possible effects of statin use on
hyperandrogenism besides cardiovascular health have also been studied. However,
there is no good evidence available that statins would improve menstrual
irregularities, ovulation rate, hirsutism or acne in cases of PCOS (Raval et al. 2011).
Unfortunately, the use of statins cannot be extended to women who wish to become
pregnant, as statin treatment during pregnancy is considered to be teratogenic.
However, the results of some studies suggest that statins may not be major teratogens
and animal studies suggest that statins might even be a good therapeutic option for the
treatment of pre-eclampsia (Girardi 2014). The American Endocrine Society does not
recommend the use of statins for the treatment of hyperandrogenism or anovulation
until there are more studies that demonstrate more benefits than risks (Legro et al.
2013). The studies concerning statin therapy in women with PCOS are specified in
Table 9.
56
57
Randomised
Duleba 2006
RDBPC
Randomised
Randomised placebo-
Sathyapalan 2009
Kaya 2010
Kazerooni 2010
RDBPC
Sathyapalan 2012
40
9
42
32
32
19
simvastatin
metformin +
simvastatin
atorvastatin
atorvastatin
3 months
12 weeks
12 weeks
6 months
3 months
12 weeks
12 weeks
Duration
20 mg
40 mg
20 mg
3 months
6 weeks
500 mg 12 weeks
20 mg
20 mg
20 mg
20 mg
simvastatin
atorvastatin
20 mg
20 mg
20 mg
20 mg
20 mg
Dose
atorvastatin
simvastatin
simvastatin
/ OCP
simvastatin + OCP
simvastatin + OCP
Treatment
NIH and LDL>2.6 atorvastatin
Rotterdam
Rotterdam
NIH criteria
Rotterdam
Rotterdam
AE-PCOS
Rotterdam
Rotterdam
Criteria
Age
T
↓
↓
↓
→
→
↓
↓
→
↓
→
→
↑
↑
→
↑
→
↑
→
→
Raja-Khan et al. 2011, Sathyapalan et al. 2009, Sathyapalan et al. 2012)
(Banaszewska et al. 2007, Banaszewska et al. 2009, Banaszewska et al. 2011, Duleba et al. 2006, Kaya et al. 2009, Kaya et al. 2010, Kazerooni et al. 2010,
↓
↓
→
↓
↓
→
↓
→
↓
→
→
DHEAS f-insulin IS
40.1 33.8 → ↓
28.5 25.6 ↓
24.2 26.8 ↓
24.0 27.6 ↓
33.2 26.6 ↓
25.2
24.7 23.4 ↓
↓
23.1 26.1 ↓
22.3 24
21.7 24.0 ↓
BMI
No, Number of subjects; IS, insulin sensitivity; OCP, oral contraceptive pill; RDBPC, randomized double blind placebo-controlled
RDBPC
Raja-Khan 2011
controlled
Randomised
Kaya 2009
40
28
2011
Randomised
45
24
No
2009
Banaszewska
Banaszewska 2007 Randomised cross-over
Design
Trial
Table 9. Studies on statin therapy in women with PCOS.
58
3
Purpose of the present study
The pathogenesis of PCOS has been studied intensively and not just a single
factor but several mechanisms have been identified. Furthermore, for a long time
the focus in PCOS was on infertility and hyperandrogenic symptoms such as acne
and hirsutism and not much attention was paid to the long-term metabolic
consequences of the syndrome. However, as the age-related health risks became
obvious the diversity of the syndrome revealed itself.
There are only a few studies available on age-related androgen secretion in
women with PCOS and hardly any data on the effect of menopausal transition on
the hormonal and metabolic profiles in these women. As women with PCOS
usually have oligomenorrhoea, CHCs are commonly used to control the menstrual
cycle and improve androgenic symptoms. Moreover, these women often have an
atherogenic lipid profile and an increased risk of T2DM and therefore they could
benefit from statin therapy. At least theoretically, statins could also improve
hyperandrogenism by decreasing cholesterol, which is a proximal substrate of
androgens. However, both CHCs and statins have also been suggested to have
unbeneficial metabolic effects, and the use of statins in particular has been
associated with disturbed carbohydrate metabolism and even an increased risk of
T2DM. Thus, the specific aims were:
1. To evaluate age-related changes and the role of menopausal transition on
adrenal and ovarian androgen secretion, glucose metabolism and
inflammation in women with PCOS (Studies I and II)
2. To study the effect of different administration routes of CHCs on
hormonal, metabolic and inflammatory parameters (Study IV)
3. To evaluate whether or not atorvastatin is useful in improving
hyperandrogenism and metabolic and inflammatory alterations in women
with PCOS (Study III)
59
60
4
Subjects and methods
All study protocols were approved by the Ethics Committee of Oulu University
Hospital. Studies III and IV were also approved by the Finnish Medicines
Agency. Studies III and IV were registered at the Clinical Trials Register
(http://clinicaltrials.gov) with identifier codes NCT01072097 and NCT01087879
and the EU Clinical Trials Register (https://www.clinicaltrialsregister.eu) with
identifier codes 2006-003584-31 and 2007-004984-23.
4.1
Subjects
All subjects signed a written informed consent document prior to participating in
the study.
Table 10. Characteristics of the subjects in Studies I–IV.
Study details
Study II
Study III
Study IV
No. of subjects who 58 PCOS*
Study I
21 PCOS*
28 PCOS*
42 Controls
completed the
69 Controls
29 Controls
2003–2006
2003–2010
2007–2011
2008–2010
18–59 PCOS
43–59 PCOS
29–50
20–33
19–62 Controls
45–62 Controls
19.0–42.9 PCOS
25.9–36.9 PCOS
19.9–53.8
17.9–26.4
19.2–35.0 Controls
20.8–29.7 Controls
-
-
study
Time period of
subject recruitment
Age (yr)
BMI (kg/m2)
Medication
Duration of
-
-
Atorvastatin or
Oral, transdermal or
placebo
vaginal CHC
6 months
9 weeks
treatment
*PCOS according to Rotterdam criteria
61
4.1.1 Women with PCOS (Studies I, II and III)
Women with PCOS were recruited from the Department of Obstetrics and
Gynaecology, Oulu University Hospital. In addition, for Study III, subjects were
also recruited from the central hospital of Kemi and the district hospital of
Oulaskangas, and also by advertising in local newspapers and personnel handouts
at Oulu University Hospital.
A diagnosis of PCOS was made if at least two of the following criteria were
met: 1) oligomenorrhoea (intermenstrual interval > 35 d or irregular menstruation
[menstrual interval > 7 d different from one period to another]), 2)
hyperandrogenism (hirsutism score ≥ 8 [Ferriman-Gallwey score] (Ferriman &
Gallwey 1961) or serum testosterone ≥ 2.7 nmol/L), or 3) PCO (observed in
transvaginal ultrasonography; at least eight follicles of 3–8 mm in diameter in one
plane, in at least one ovary). Thus, the criteria were based on the Rotterdam
consensus (Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop
group 2004) except for the diagnosis of PCO which was determined according to
the old PCO criteria presented by Adams (Adams et al. 1985). In addition, other
disorders that can present with a PCOS-like phenotype, such as
hyperprolactinaemia, Cushing’s syndrome, androgen-secreting tumours and 21hydroxylase-deficient non-classic adrenal hyperplasia were excluded in the
clinical investigation and by using assay of 17-OHP.
The PCOS diagnoses were made for all participants during their reproductive
age and confirmed by the researchers from the medical records if the women were
pre- or postmenopausal when they entered the study. For postmenopausal women
with PCOS, the diagnosis was based on oligomenorrhoea or irregular
menstruation combined with hyperandrogenism reported in their medical records,
as ultrasonographic examinations were not available at the time of their diagnosis.
A woman was considered postmenopausal if she had not had menstrual bleeding
for at least one year or her serum FSH level was more than 30 U/l. In Study II,
one premenopausal and one postmenopausal woman with PCOS had undergone
hysterectomy. Subjects with previous ovarian drilling, wedge resection or
oophorectomy were excluded.
4.1.2 Control (healthy) subjects (Studies I, II and IV)
Control women were recruited by advertising in personnel handouts at Oulu
University Hospital and by sending advertisement emails to students at the
62
Faculty of Medicine, University of Oulu and the School of Health and Social
Care, Oulu University of Applied Sciences.
Control subjects had regular menstrual cycles at fertile age, no signs of
hyperandrogenism and normal-appearing ovaries in transvaginal ultrasonography
when entering the study. Postmenopausal women had not had menstrual bleeding
for at least a year and their serum FSH levels were over 30 U/l.
4.1.3 Exclusion criteria and medication
General exclusion criteria included pregnancy and lactation, cigarette smoking,
abuse of alcohol, previous history of cancer, and medication for glucose tolerance
or hyperlipidaemia. Further information on medication and wash-out periods are
clarified in original publications I–IV.
4.2
Methods
4.2.1 Study visits
All study visits were performed at the research unit of the Department of
Obstetrics and Gynaecology, Oulu University Hospital, with the exception of
three-month blood samplings in Study III, which were performed at the nearest
laboratory for subjects at a long distance from Oulu. In these cases, the blood
samples were sent to the Oulu research unit on the day of collection. All visits
were performed between 7:00–12:00h.
4.2.2 Intervention measures
Clinical measurements
Blood pressure was measured after a 15-minute rest in a sitting position. Weight
was measured with underwear and height was measured without shoes. Body
mass index was calculated as follows: weight (kg) / [height (m)]2. Waist
circumference was measured at the midpoint between the lower margin of the last
palpable rib and the top of the iliac crest and hip circumference at the maximum
circumference over the buttocks. Thus, the measurements were performed
according to the World Health Organization STEPS protocol (World 2005) with a
63
soft tape, and the waist to hip ratio was calculated as [waist circumference (cm) /
hip circumference (cm)].
Imaging examinations
Vaginal ultrasonography was performed at the first study visit in all Studies (I–
IV). The size of the ovaries was measured using three different dimensions
(length, width, thickness) and the number of follicles in each ovary was calculated
in one plane (Studies I–III) or in the whole ovary (Study IV).
Although the breasts of all subjects were clinically examined, one subject was
diagnosed with breast cancer after entering Study II. Thereafter, mammography
within one year before entering Study II was required. If this was not the case,
mammography was performed prior to the study examinations.
64
Table 11. Interventions used in Studies I–IV
Method / variable
Study I
Study II
Study III
Study IV
X
X
Tests
ACTH test
X
hCG test
X
OGTT
X
IVGTT
X
Hormonal parameters
LH
X
X
FSH
X
X
17-OHP
X
X
A
X
X
X
X
T
X
X
X
X
DHEA
X
DHEAS
X
Cortisol
X
X
X
E2
X
Inhibin-B
X
X
SHBG
X
X
X
FAI
X
X
X
Glucose tolerance
Glucose
X
X
X
Insulin
X
X
X
X
X
C-peptide
Lipids
Total cholesterol
X
X
LDL cholesterol
X
X
HDL cholesterol
X
X
Trigycerides
X
X
X
X
Inflammatory parameters
CRP
PTX-3
X
X
Atorvastatin
In Study III the subjects were randomized to use atorvastatin (20 mg/day;
Lipitor®, Pfizer Incorporated) or placebo for 6 months. The subjects were advised
to take the medication in the evening before going to bed.
Randomization was conducted using a computer-generated randomization list
in blocks of six. It was performed at the Pharmacy of Oulu University Hospital by
personnel not involved in the study. They also repacked the medication into
65
envelopes, which were sequentially numbered and then closed. The subjects were
advised not to change their physical activity or dietary habits during the study
period.
Combined contraceptives
In Study IV the subjects were randomized to use oral contraceptive pills (EE 20
μg and desogestrel 150 μg; Mercilon®; Organon Ltd., Dublin, Ireland), a
transdermal contraceptive patch (EE 20 μg/day and norelgestromin 150 μg/day;
Ortho Evra®; Janssen Pharmaceutica N.V., Beerse Belgium) or a contraceptive
vaginal ring (EE 15 μg/day and etonogestrel 120 μg/day; NuvaRing®; N.V.
Organon, Oss, the Netherlands) continuously for 9 weeks.
Randomization was carried out with an allocation ratio of 1:1:1 and a block
size of six. The randomization list was computer-generated and constructed by a
pharmacist at Oulu University Hospital. The research nurse controlled the
randomization list and assigned participants to their groups at their first study
visit.
The subjects started the treatment after the first study visit on cycle days 2–4,
and they were advised to use barrier contraception during the first 7 days. Advice
on use of the different contraceptive methods was given by the research nurse and
the researcher at study-entry. The subjects were told not to change their dietary
habits or physical activity during the study.
4.2.3 Laboratory analyses
Blood samples were centrifuged (3000 rpm, 10 minutes), and serum and EDTA
plasma were stored at -20 °C (Studies I and II) or at -80 °C (Studies III and IV)
for later analyses. However, serum glucose and plasma creatinine and ALAT
measurements were performed immediately. All assays were performed
according to the instructions of the reagent manufacturers, and details are given in
Table 12.
66
Table 12. Characteristics of the laboratory assays used in Studies I–IV.
Analyte
Method
Typical sensitivity Coefficient of Coefficient of
intra-assay
interassay
variation (%)
variation (%)
17-OHP
RIA
0.2 nmol/l
5.0
5.4
A
RIA/CIA
0.14/1.0 nmol/l
5.0/6.3
7.0/8.6
Reference
range
0.6–8.8 nmol/l
1.4–14.3
nmol/l
T
ACLS/LC-MS/MS
0.35/0.03 nmol/l
4.0/5.3
5.6/5.3
0.4–2.3 nmol/l
DHEA
RIA
0.1 nmol/l
7.9
11.9
9–38 nmol/l
DHEAS
RIA/CIA
0.03/0.08 µmol/l
5.3/6.5
7.0/9.3
1–14 µmol/l
Cortisol
ACLS
5.0 nmol/l
4.0
4.3
150–650
E2
RIA
5 pmol/l
4.6
5.8
nmol/l
0.07–0.53
nmol/l
LH
ACLS
0.07 U/l
2.9
3.8
FSH
ACLS
0.3 U/l
2.1
2.8
2–12 U/l
Inhibin-B
ELISA
<15 ng/l
<7
<7
30–90 ng/l
SHBG
TRFIA/CIA
0.02/0.5 nmol/l
3.9/2.7
4.6/5.2
20–140 nmol/l
CRP
immunonephelometry
0.18 mg/l
0.9
3.5
<3 mg/l
PTX-3
ELISA
0.025 ng/ml
3.8
6.1
-
Glucose
Advia 1800
1.2
2.2
2–10 U/l
4.2–6.3
mmol/l
Insulin
ACLS
0.5 mU/l
4.6
7.5
3–25 mU/l
C-peptide
ACLS
0.02 nmol/l
3.2
7.1
0.26–1.2
Cholesterol
Advia 1800
1.7
3.1
nmol/l
<5 mmol/l
(desirable)
LDL
Advia 1800
0.8
2.8
Advia 1800
2.3
2.6
Triglycerides Advia 1800
1.6
2.4
cholesterol
HDL
≤3 mmol/l
(desirable)
≥1.2 mmol/l
(desirable)
cholesterol
≤1.7 mmol/l
(desirable)
RIA = radiomimmunoassay, CIA = chemiluminometric immunoassay, ACLS = automated
chemiluminescence system, LC-MS/MS = liquid chromatography-tandem mass spectrometry, ELISA =
enzyme-linked immunosorbent assay, TRFIA = time-resolved fluoroimmunoassay
4.2.4 Power and statistical analyses
Power analyses were conducted for Studies III and IV, and specific justifications
are explained in original publications.
67
All variables with skewed distribution were logarithmically transformed prior
to statistical analysis. Statistical analyses were conducted by using SPSS (the
Statistical Package for the Social Sciences software, SPSS Inc. Released 2006.
SPSS for Windows, Version 15.0. Chicago, SPSS Inc.). The limit of statistical
significance was set at p ≤ 0.05.
To compare the results between two different study groups, the independentsamples t test was used for normally distributed variables and the Mann–Whitney
U test for variables with a skewed distribution (Studies I–IV). Pearson’s
correlation coefficient (r) was calculated to correlate age with the hormone levels
measured (Study I). One-way analysis of variance (ANOVA) was used to assess
the possible changes between different age or treatment groups (Studies I and IV).
Differences in hormone responses between two groups were analysed and the
impact of BMI was controlled by using multiple linear regression analysis (Study
I) and ANOVA (Study II). To compare changes during treatment at two different
time points, the paired samples t-test was used for normally distributed variables
and Wilcoxon’s non-parametric test for variables with skewed distribution
(Studies III and IV). To explore changes during treatment at three different time
points, repeated measures ANOVA was performed for normally distributed
variables and Friedman’s test for variables with a skewed distribution (Studies III
and IV). The baseline level of T (Study III) and the change in BMI (Study IV)
were used as covariates to adjust for changes in glucose metabolism in repeated
measures ANOVA.
68
5
Results and Discussion
Table 13. Main results of Studies I–IV
Study I
Study II
Study III
Study IV
Adrenal steroid
Insulin resistance,
Atorvastatin therapy
CHCs impair insulin
production remains
enhanced androgen
improves lipid profile and sensitivity and increase
enhanced up to
secretion and systemic
inflammation but worsens systemic inflammation in
menopause in women
inflammation persist after insulin sensitivity in
healthy women
with PCOS
menopausal transition in women with PCOS
regardless of
PCOS
administration route
5.1
Hyperandrogenism
Even though hyperandrogenism is a crucial feature of PCOS and the significance
of hyperandrogenism during fertile life in these women is well established, the
pattern of androgen secretion during ageing is not widely described. To clarify
this phenomenon further, ovarian and adrenal androgen secretion were
investigated in women with PCOS and in healthy women in Studies I and II, and
the changes in androgen levels were explored during the use of CHCs in healthy
women (Study IV) and atorvastatin in women with PCOS (Study III).
5.1.1 Androgen secretion
Women with PCOS had significantly elevated levels of A and/or T when
compared with control women younger than 31 and older than 41 years up to the
postmenopausal period (Figure 4) (Studies I and II).
69
Fig. 4. Mean basal serum testosterone levels in different age groups (Study I). MP,
menopausal; *significant difference between women with PCOS and control women in
the same age group.
Serum levels of DHEAS decreased significantly during ageing in healthy women.
In women with PCOS, a decrease was also observed, but it did not reach
statistical significance (Study I). The adrenal androgen response (A, T, DHEAS)
during ACTH tests was elevated in women with PCOS and the responses
remained unchanged up to menopausal transition, whereas adrenal androgen
secretion correlated negatively with age in control women (Study I). In Study II
we compared premenopausal and postmenopausal women and found that the
capacity of the ovaries to secrete 17-OHP and A in response to hCG decreased in
both controls and women with PCOS during menopausal transition. Notably, the
secretory capacity of these hormones remained significantly higher in women
with PCOS even after menopause (Figure 5).
70
Fig. 5. Basal and hCG-stimulated mean levels of androstenedione in pre- and
postmenopausal women (Study II). MP, menopausal; *significant difference between
women with PCOS and control women in the same age group.
Although women with PCOS presented with significantly elevated BMI in
Studies I and II, it did not explain the differences between the groups and proved
that PCOS per se was the determining factor.
In our previous studies we have found that the ovarian capacity to synthesise
and release androgens remains enhanced throughout the reproductive years in
women with PCOS when compared with healthy women (Piltonen et al. 2003,
Piltonen et al. 2004). In the present studies we further explored these observations
as regards adrenal androgen secretion and ovarian androgen activity at the time of
menopausal transition.
The enhanced androgen secretion in women with PCOS after menopause has
also been reported in other studies (Markopoulos et al. 2011). Despite the fact that
serum T levels were higher in women with PCOS compared with controls,
testosterone levels decreased among women with PCOS up to premenopausal age
(Study I), which is also supported by the results of a previous study (Winters et al.
2000). Furthermore, a cohort study revealed significantly elevated T levels in
middle-aged women with PCOS when compared with control women, but the
difference had disappeared by a mean age of 70 (Schmidt et al. 2011). The
finding shows that enhanced androgen secretion decreases with age in women
71
with PCOS and at some point it reaches the levels of control women. However, it
does not occur at menopause.
Approximately 20–30% of women with PCOS present with adrenal
hyperandrogenism (Goodarzi et al. 2015). The finding in Study I showing that
adrenal androgen secretion decreases with age in women with PCOS is supported
by the results of other studies in hyperandrogenic women and women with PCOS
(Kumar et al. 2005, Moran et al. 1999). On the other hand, in a follow-up study
of 3–5 years, serum DHEAS levels remained unchanged in the majority of
middle-aged women with PCOS, which reflects the fact that the decrease in
adrenal androgen secretion is a slow process (Yildiz et al. 2004).
In summary, enhanced androgen secretion in women with PCOS remains
after menopause. This finding is important, as increased testosterone levels in
older women have been found to be associated with an increase in metabolic
disturbances and cardiovascular diseases (Patel et al. 2009).
5.1.2 The effects of combined contraceptives and statin therapy on
androgen secretion
Combined contraceptives
According to the results of Study IV, orally, transdermally and vaginally
administered combined contraceptives equally decreased the levels of 17-OHP
and A. However, T concentrations remained unchanged in the pill and vaginal
ring study groups and even an increase was observed in the patch group. As a
result of a more than 200% increase in serum SHBG levels in all study groups,
the FAI significantly decreased. The increase in SHBG level was significantly
lower in women using a vaginal ring than in those using transdermal patches or
oral pills (Study IV).
The increase in SHBG concentrations during the use of combined contraceptives
originates from the hepatic impact of EE. It is related to the 17α-ethinyl group of the
molecule, which prevents the inactivation of EE and results in slow metabolism, long
tissue retention and a long half-life, and thereby even 500-fold more potent hepatic
effects when compared with estradiol (Sitruk-Ware & Nath 2013). The hepatic effects
of EE appear during both oral and vaginal administration (Goebelsmann et al. 1985).
In contrast to our results, two studies on oral (20 µg/30 µg EE) and vaginal (15 µg)
combined contraceptives have revealed that vaginal administration increased the
72
levels of SHBG more significantly than oral administration (Elkind-Hirsch et al.
2007, Tuppurainen et al. 2004) and thereby induced a significantly greater reduction
of the FAI (Elkind-Hirsch et al. 2007). This difference may result from the different
progestin components used in the pill (desogestrel vs. levonogestrel), as the progestins
modify the hepatic effect of EE depending on their dose and type (Odlind et al.
2002). The production of SHBG is highly oestrogen-sensitive and levonorgestrel,
which is less oestrogenic than desogestrel, does not enhance the synthesis of SHBG as
much (Hammond et al. 1984, Odlind et al. 2002, Wiegratz et al. 2003). Interestingly,
use of the patch (EE, 20 µg/day and norelgestromin 150 µg/day) has been reported to
result in a greater increase of SHBG than that associated with an oral combined
contraceptive containing 35 µg EE and 250 µg norgestimate, despite the fact that
norelgestromin is the metabolic product of norgestimate (White et al. 2005, White et
al. 2006). However, it has been shown that exposure to EE during patch use is 1.7
times higher when compared with an orally administered combined contraceptive
(van den Heuvel et al. 2005), which may account for the results. This finding may
also explain the differences in SHBG levels in Study IV, as the progestins used in the
preparations are fairly similar as regards their oestrogenic features.
Atorvastatin
In Study III, atorvastatin treatment decreased serum levels of DHEAS, but the
levels of LH, FSH, A, T and SHBG remained unchanged.
In line with our results, one earlier study revealed no change in serum
testosterone levels during atorvastatin treatment (Raja-Khan et al. 2011).
However, all other studies on atorvastatin have shown a decrease in serum levels
of T (Kaya et al. 2009, Kaya et al. 2010, Sathyapalan et al. 2009). Studies
exploring the effects of simvastatin in women with PCOS have also shown a
decrease in T levels (Banaszewska et al. 2007, Banaszewska et al. 2011, Duleba
et al. 2006, Kaya et al. 2009, Kaya et al. 2010). One significant difference
between our study and several other studies is the androgenic state of the study
subjects. In Study III, PCOS was diagnosed on the basis of Rotterdam criteria and
not on NIH criteria, in which hyperandrogenism is one required criterion.
Moreover, our subjects were older (late fertile age) and had lower androgen levels
as a result of an age-related decrease of both ovarian and adrenal androgen
secretion.
The idea that statins could theoretically affect androgen levels originated
from the fact that cholesterol acts as a substrate for androgen synthesis. Later,
73
statins were found to directly inhibit rat theca cell proliferation in vitro (Izquierdo
et al. 2004, Kwintkiewicz et al. 2006), and this has also been confirmed in human
theca cells (Sokalska et al. 2010). Furthermore, statins have been shown to inhibit
steroidogenesis (Izquierdo et al. 2004, Ortega et al. 2012, Sokalska et al. 2014),
where inhibition of CYP17A1 mRNA expression has been thought to be a
principal mechanism (Ortega et al. 2012). Simvastatin seems to be the most
effective statin in the inhibition of growth and steroidogenesis of rat theca cells,
whereas atorvastatin and pravastatin present with lower effectiveness and
lovastatin has an effect between simvastatin and atorvastatin (Sokalska et al.
2014). Interestingly, resveratrol, a type of natural phenol, potentiates the effects of
simvastatin on androgen secretion from the ovaries (Ortega et al. 2014).
Serum levels of DHEAS significantly decreased in Study III. This has also
been reported in other studies on atorvastatin (Raja-Khan et al. 2011, Sathyapalan
et al. 2012) and simvastatin (Banaszewska et al. 2009, Banaszewska et al. 2011,
Duleba et al. 2006, Kazerooni et al. 2010), although not all studies agree
(Banaszewska et al. 2007, Kaya et al. 2010). Because DHEAS serves as a
precursor for testosterone synthesis, its decrease during atorvastatin treatment
may reflect a compensatory mechanism to maintain prior testosterone levels.
However, potentially statins may have direct inhibitory effects on androgen
synthesis in the adrenal glands, similarly to the ovaries (Sathyapalan et al. 2012).
5.2
Metabolic alterations in women with and without PCOS
5.2.1 Glucose tolerance
In Study II, fasting glucose levels were comparable in women with PCOS and
controls in both premenopausal and postmenopausal study groups, but fasting
insulin levels were elevated twofold in cases of PCOS. However, the difference
reached statistical significance only between the postmenopausal groups, as did
the AUC of glucose. Fasting and 2-hour insulin sensitivity indexes were
significantly lower in postmenopausal women with PCOS when compared with
control women. In addition, HOMA-IR results were increased and Matsuda
indexes decreased significantly among both premenopausal and postmenopausal
women with PCOS when compared with control women. When the differences
between the groups were analysed using BMI as a covariate, higher BMI levels in
74
cases of PCOS explained differences in several glucose tolerance parameters, but
not the AUC of insulin or the insulin sensitivity index at 120 min.
It is well known that most women with PCOS present with insulin resistance.
However, the possible changes during menopausal transition are not well
understood. The results of Study II strongly suggest that impaired glucose
metabolism in women with PCOS persists after the menopause. There are only a
few studies available on metabolic parameters in older women with the syndrome.
However, premenopausal and postmenopausal women with a history of PCOS
have been found to present with T2DM more often than controls (Hudecova et al.
2011, Shaw et al. 2008). A study performed in Greece did not reveal any
differences in fasting insulin and glucose, or insulin sensitivity indexes between
postmenopausal women with PCOS and healthy women, but women with PCOS
had significantly higher insulin levels during OGTTs without changes in glucose
levels, reflecting increased insulin resistance (Markopoulos et al. 2012). These
findings are of importance, as impaired glucose metabolism and increased
prevalence of T2DM further predisposes these women to CVD.
The effects of combined contraceptives on glucose tolerance
Fasting glucose levels remained unchanged during the use of oral, transdermal
and vaginal contraceptives. However, the insulin sensitivity index during OGTTs
and the Matsuda index decreased significantly in all study groups, reflecting
deterioration of insulin sensitivity (Figure 6). This was accompanied by
compensatory increases of fasting and glucose-stimulated insulin levels. A
significant increase was also found in the levels of fasting C-peptide during use of
the transdermal preparation. All the parameters reflecting insulin sensitivity were
fairly similar among the study groups (Study IV).
75
Fig. 6. Changes in the Matsuda index (mean) before and after 9-week use of combined
contraceptives (Study IV). Open circles, the pill group; open squares, the patch group;
closed circles, the vaginal ring group.
Several previous investigators have reported similar results concerning alterations
in insulin sensitivity in healthy women in prospective (Cagnacci et al. 2009a,
Cagnacci et al. 2009b, Deleskog et al. 2011, Elkind-Hirsch et al. 2007) as well as
cross-sectional studies (Frempong et al. 2008, Morin-Papunen et al. 2008).
However, there are also data showing no change in glucose tolerance during
combined contraceptive use (Cagnacci et al. 2009a, Gaspard et al. 2003). In
women with PCOS, an oral contraceptive pill containing EE and drospirenone
caused deterioration of insulin sensitivity (Battaglia et al. 2010, Duijkers et al.
2004), whereas treatment with EE and cyproterone acetate did not affect glucose
tolerance (Gode et al. 2011). Interestingly, a new preparation containing estradiol
valerate with dienogest has been reported not to worsen insulin sensitivity but to
improve it (De Leo et al. 2013). Furthermore, favourable effects on glucose
tolerance have been reported among hirsute women after the use of EE and
desogestrel (Escobar-Morreale et al. 2000). The discrepancies between the studies
may be explained in part by different doses of oestrogen and different progestins
used in the formulations.
In Study IV, significant impairment in glucose metabolism was also found in
subjects using transdermal and vaginal preparations as increases in the AUC of
glucose and decreases in insulin sensitivity indexes were found in both groups.
However, earlier investigators have published divergent results. One study on
76
transdermal patches revealed a decrease in fasting glucose levels (Kiriwat &
Petyim 2010), whereas previous studies on vaginal rings have not shown any
changes in insulin sensitivity (Cagnacci et al. 2009b, Elkind-Hirsch et al. 2007).
Moreover, in women with type 1 diabetes, glycosylated haemoglobin
concentrations and average daily insulin requirements remained stable
(Grodnitskaya et al. 2010). In women with PCOS, the use of vaginal rings has
even been reported to result in improvement of variables reflecting glucose
tolerance (Battaglia et al. 2010). The discrepancies between our study and earlier
studies can be partly explained by the different dosing patterns, in other words
longer exposure to hormonal preparations due to continuous administration for
nine weeks in Study IV vs. traditional cyclic dosing.
The effect of atorvastatin on glucose tolerance
In Study III, atorvastatin treatment resulted in a significant increase in fasting
levels of insulin and AUCs of insulin and C-peptide, and a deterioration of insulin
sensitivity indexes (Figure 7). Serum levels of glucose remained unchanged,
reflecting a sufficient compensatory increase in insulin secretion (Figure 7).
77
Fig. 7. Glucose and insulin responses during OGTTs in the atorvastatin group (Study
III). *Specific time points during OGTTs where the concentration was significantly
higher after 6 months’ use of atorvastatin. Open circles, before treatment; closed
circles, after 6 months of treatment
The findings are interesting, as other studies concerning the use of atorvastatin in
women with PCOS have revealed a decrease in fasting insulin levels and
improvement of insulin resistance (Kaya et al. 2009, Kaya et al. 2010,
Sathyapalan et al. 2009), or no change (Raja-Khan et al. 2011). Simvastatin
treatment has not had any effect on values reflecting glucose tolerance
(Banaszewska et al. 2007, Banaszewska et al. 2011, Duleba et al. 2006, Kaya et
78
al. 2009, Kaya et al. 2010). The reported unaltered or improved glucose tolerance
during simvastatin and atorvastatin treatment in women with PCOS in other
studies may be explained by the fact that these studies have included women
presenting with biochemical hyperandrogenism and improvement of this feature
may result in improvement of insulin resistance by decreased release of free fatty
acids (Xu et al. 1991).
In the general population a slightly increased risk of the development of
diabetes during statin therapy has been observed (OR 1.09; 95% CI 1.02–1.17)
(Sattar et al. 2010). However, a recent study revealed an increased risk of T2DM
during atorvastatin and simvastatin use of up to 46% and the risk seemed to be
dose-dependent (Cederberg et al. 2015). Furthermore, on the basis of the results
of clinical trials, the mechanisms underlying statin-induced diabetes have been
suggested to include an increase in insulin resistance and a decrease in insulin
sensitivity-corrected insulin secretion (Cederberg et al. 2015). Similar alterations
have been found in in vitro studies, as statins have been suggested to inhibit the
uptake of glucose by human liver, adipose tissue and skeletal muscle cells by
inducing conformational changes in glucose transporters (Nowis et al. 2014) and
reducing glucose-induced insulin secretion by blocking Ca2+ channels (Yada et al.
1999). The divergent effects on glucose tolerance between different statins may
be explained by the degree of lipophilicity. Lipophilic statins may have more
adverse metabolic consequences than hydrophilic statins, such as impairment of
insulin secretion and promotion of insulin resistance, by affecting other tissues
besides the liver, while hydrophilic statins show greater hepatoselectivity
(Schachter 2005). This has also been seen in clinical trials, in which hydrophilic
pravastatin even improved insulin sensitivity and lipophilic simvastatin impaired
it (Baker et al. 2010).
5.2.2 Lipid profile
The effects of combined contraceptives on the lipid profile
In Study IV, the effects on the lipid profile were fairly similar in all study groups.
Levels of total cholesterol remained stable and levels of HDL cholesterol
significantly increased. However, the change in HDL cholesterol was not
significant in the oral contraceptive group when baseline and nine-week treatment
were compared. Serum levels of LDL cholesterol were significantly increased
79
temporarily at five weeks during the use of oral contraceptives but remained
unchanged in the other study groups. As expected, the levels of triglycerides
significantly increased in all study groups during the total treatment period (oral
group +78%, transdermal group +60% and vaginal group +53%) and the changes
did not differ between the groups.
The results concerning total cholesterol are in line with that of a crosssectional study showing comparable levels of total cholesterol between non-users
and oral, transdermal and vaginal contraceptive users (Palan et al. 2010).
However, an increase in the levels of total cholesterol has also been reported
among the users of oral (Cagnacci et al. 2009a, Cagnacci et al. 2009b, Cauci et al.
2008, Morin-Papunen et al. 2008), transdermal (Creasy et al. 2003, Kiriwat &
Petyim 2010) and vaginal (Barreiros et al. 2011) contraceptives. In line with our
results, there are also studies available that have not revealed any changes in total
cholesterol during the use of vaginal ring (Cagnacci et al. 2009b, Elkind-Hirsch et
al. 2007, Tuppurainen et al. 2004).
The improvement in HDL cholesterol might be of notable significance, as
HDL cholesterol plays a role in the prevention of atherosclerosis (National
Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation,
and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III)
2002). An increase in HDL cholesterol has also been found in other studies on
oral (Cagnacci et al. 2009a, Cagnacci et al. 2009b, Cauci et al. 2008, Frempong et
al. 2008), transdermal (Creasy et al. 2003, Kiriwat & Petyim 2010) and vaginal
(Barreiros et al. 2011) contraceptives.
The increment in triglyceride levels in Study IV is in line with the results of
other studies (Barreiros et al. 2011, Cagnacci et al. 2009a, Cagnacci et al. 2009b,
Cauci et al. 2008, Creasy et al. 2003, Frempong et al. 2008, Kiriwat & Petyim
2010, Krintus et al. 2010, Morin-Papunen et al. 2008). This may have a
significant impact, as elevated levels of triglycerides emerge as strong predictors
of coronary atherosclerosis and CHD (Harchaoui et al. 2009). On the other hand,
the increase in triglyceride levels may be caused by increased levels of larger
VLDL particles, which may be less atherogenic than small VLDL particles
(Crook & Godsland 1998).
80
The effects of atorvastatin on the lipid profile
As expected, levels of total and LDL cholesterol and triglycerides decreased
during atorvastatin treatment in Study III, while the level of HDL cholesterol
remained unchanged.
As LDL cholesterol is the major atherogenic lipoprotein, the beneficial effect
of atorvastatin medication in reducing the risk of cardiovascular diseases is
mediated through this effect. There are two other studies in which decreases in the
levels of triglycerides during the use of atorvastatin in women with PCOS have
been reported (Raja-Khan et al. 2011, Sathyapalan et al. 2009). This effect is also
of an importance, as triglyceride levels have been identified as a risk factor of
coronary atherosclerosis and CHD (Tenenbaum et al. 2014). However, one study
revealed no change in triglyceride levels (Kaya et al. 2009). Studies on
simvastatin have not shown any changes in triglyceride levels (Banaszewska et al.
2009, Banaszewska et al. 2011, Kaya et al. 2009). Decreases in total and LDL
cholesterol levels, however, are well documented (Banaszewska et al. 2009,
Banaszewska et al. 2011, Kaya et al. 2009, Raja-Khan et al. 2011, Sathyapalan et
al. 2009).
5.2.3 Chronic inflammation
In Study II, premenopausal women with PCOS presented with serum levels of
CRP almost twice as high as in premenopausal control women. In addition,
postmenopausal women with a history of PCOS had significantly increased levels
of CRP when compared with control women, and the differences between the
groups remained after adjustment for BMI. Interestingly, menopause did not
affect the level of chronic inflammation in either the PCOS or the control group.
Chronic inflammation (with high CRP levels) predisposes individuals to
CVD by taking part in atherosclerosis plaque formation (Ridker et al. 2000).
Notably, the detrimental effects of chronic inflammation are not limited to CVD,
as CRP levels have also been shown to predict the incidence of type II diabetes
(Pradhan et al. 2001). It is well known that women with PCOS present with
increased systemic inflammation when compared with control women of fertile
age (Diamanti-Kandarakis et al. 2006). This was also confirmed in Study II,
where it was shown that chronic inflammation persists in women with PCOS after
menopause and predisposes these women to unbeneficial health consequences
after their reproductive years.
81
Inflammatory changes during combined contraceptive treatment
Serum concentrations of CRP rose significantly in all three groups in Study IV
during combined contraceptive treatment (Figure 8). Even though subjects with
CRP concentrations of more than 10 mg/l were excluded from the analysis, the
increase remained significant in all study groups. Plasma levels of another marker
of inflammation, PTX-3, increased significantly during the use of oral and
transdermal contraceptives, and a similar trend was seen in the vaginal ring group
(Study IV).
Fig. 8. Changes in serum CRP levels during the use of combined contraceptives
(Study IV). Open circles, vaginal ring group; open squares, patch group; solid circles,
pill group. Values of p show the significance of changes within each study group.
Increased levels of CRP have been reported previously during the use of
transdermal (White et al. 2006) and oral combined contraceptives (MorinPapunen et al. 2008, White et al. 2006). Study IV was the first one in which an
increase was also found during the use of vaginal contraception.
As combined contraceptives stimulate the synthesis of proteins such as CRP
in hepatocytes through a mechanism which is not known, we wanted to examine
another inflammation marker to investigate whether or not the elevated levels of
CRP also represented extrahepatic systemic inflammation. We analysed the levels
of PTX-3, which is produced by peripheral tissues such as vascular endothelial
cells, smooth muscle cells and leucocytes in response to proinflammatory signals,
82
and thereby the secretion of PTX-3 is not influenced by drug-induced hepatic
protein synthesis (Mantovani et al. 2008). Hence its assay gives more precise
information on the actual inflammation. The finding of increased PTX-3 levels in
the pill and patch groups supports the idea that the increase in CRP levels may not
be only a consequence of liver induction but also a reflection of a true increase in
systemic inflammation. However, the increase in the levels of CRP regardless of
the exact mechanism might be of significance, as the presence of CRP molecules
might lead to increased foam-cell formation in atherosclerotic plaques (Zwaka et
al. 2001). Furthermore, CRP can accelerate atherosclerotic processes, because it
can induce the expression of adhesion molecules (Pasceri et al. 2000) and inhibit
nitric oxide expression in endothelial cells, causing endothelial dysfunction
(Verma et al. 2002). Interestingly, progestin-only contraceptives do not stimulate
the production of CRP, whether administered orally or in utero (Morin-Papunen
et al. 2008).
As there seems to be a pronounced inflammatory milieu in users of combined
contraceptives, it may be of some clinical significance in the long term through
worsening endothelial function and promoting accumulation of asymptomatic
atherosclerotic changes in women. However, the clinical importance warrants
further investigations.
Inflammatory changes during atorvastatin treatment
Atorvastatin therapy significantly decreased serum levels of CRP in women with
PCOS after excluding women with infection (CRP > 10 mg/l) (Study III).
Several studies have shown that statin therapy reduces chronic inflammation,
usually assessed by the measurement of CRP levels (Girardi 2014). Similar
findings have also been reported in women with PCOS by other investigators
exploring the effects of atorvastatin (Kaya et al. 2010, Raja-Khan et al. 2011,
Sathyapalan et al. 2009) and simvastatin (Banaszewska et al. 2007, Banaszewska
et al. 2009, Banaszewska et al. 2011, Kaya et al. 2010). As CRP levels
independently predict the future risk of coronary events (Ridker et al. 2000),
statin therapy in women with PCOS probably decreases the risk of CVD. The
reduction of inflammation by way of statin therapy is of great importance, as
statin therapy is able to reduce the risk of CVD in men and women without
hyperlipidaemia only by reducing inflammation (Ridker et al. 2008).
83
5.3
Limitations of the study
The limitations of Studies I–IV are related to the limited numbers of study
subjects, although for the clinical trials (Studies III and IV) appropriate power
calculations were performed. In Studies I and II, women with PCOS had a
significantly higher BMI when compared with control women. However, this
difference was controlled by using BMI as a covariate in statistical analyses. As
the study subjects were healthy in Study IV, the changes observed during the use
of combined contraceptives may not be generalised to women with PCOS.
Healthy women were selected for the study population, since the purpose was to
explore the changes in metabolic parameters without confounding factors such as
insulin resistance, which is commonly found in PCOS.
Further limitations are related to the methods used. During the ACTH test, for
example, tetracosactide hexacetate might have had a more powerful effect on the
adrenals if it had been injected intravenously. Furthermore, the IVGTTs may have
benefitted from more frequent blood sampling. In principle, Rotterdam criteria
were used for the diagnosis of PCOS, although the diagnosis of PCO was based
on the older criteria, where eight or more follicles in one plane are needed for the
diagnosis (Adams et al. 1985). The Rotterdam criteria require 12 follicles in the
whole ovary (2004). Nevertheless, if there are eight follicles in one plane it is
very obvious that there are twelve or more follicles in the whole ovary, and
therefore the risk of over-diagnosing PCOS is small.
84
6
Conclusions
The present data support the fact that unfavourable changes in androgen secretion,
glucose metabolism and chronic inflammation associated with PCOS persist
beyond menopause, when the prevalent symptoms/findings related to PCOS itself
have already disappeared, and this potentially has long-term health effects
(Studies I and II). In Study IV we found that the use of combined contraceptives,
often used to treat hyperandrogenism in women with PCOS, leads to impairment
of insulin sensitivity in young healthy women regardless of administration route.
As a result of the disadvantageous health consequences in PCOS, statin therapy
has been suggested in order to improve lipid and androgen profiles in these
women. However, on the basis of the results of Study III, despite the
improvement in lipid profile, atorvastatin treatment did not have an effect on
androgen secretion and it seemed to worsen glucose tolerance. Therefore, the
treatment of women with PCOS should be based on a comprehensive individual
risk evaluation in order to avoid adverse health effects related to treatments
themselves.
Fig. 9. The timeline of PCOS
85
86
References
Abdel Gadir A, Khatim MS, Mowafi RS, Alnaser HM, Muharib NS & Shaw RW (1992)
Implications of ultrasonically diagnosed polycystic ovaries. I. Correlations with basal
hormonal profiles. Hum Reprod 7(4): 453–457.
Adams J, Franks S, Polson DW, Mason HD, Abdulwahid N, Tucker M, Morris DV, Price J
& Jacobs HS (1985) Multifollicular ovaries: clinical and endocrine features and
response to pulsatile gonadotropin releasing hormone. Lancet 2(8469–70): 1375–1379.
Aittomaki K, Herva R, Stenman UH, Juntunen K, Ylostalo P, Hovatta O & de la Chapelle
A (1996) Clinical features of primary ovarian failure caused by a point mutation in the
follicle-stimulating hormone receptor gene. J Clin Endocrinol Metab 81(10): 3722–
3726.
Alberti KG & Zimmet PZ (1998) Definition, diagnosis and classification of diabetes
mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus
provisional report of a WHO consultation. Diabet Med 15(7): 539–553.
Alvarez-Blasco F, Botella-Carretero JI, San Millan JL & Escobar-Morreale HF (2006)
Prevalence and characteristics of the polycystic ovary syndrome in overweight and
obese women. Arch Intern Med 166(19): 2081–2086.
Apridonidze T, Essah PA, Iuorno MJ & Nestler JE (2005) Prevalence and characteristics
of the metabolic syndrome in women with polycystic ovary syndrome. J Clin
Endocrinol Metab 90(4): 1929–1935.
Asunción M, Calvo RM, San Millan JL, Sancho J, Avila S & Escobar-Morreale HF (2000)
A prospective study of the prevalence of the polycystic ovary syndrome in unselected
Caucasian women from Spain. J Clin Endocrinol Metab 85(7): 2434–2438.
Azevedo GD, Duarte JM, Souza MO, Costa-E-Silva TD, Soares EM & Maranhao TM
(2006) Menstrual cycle irregularity as a marker of cardiovascular risk factors at
postmenopausal years. Arq Bras Endocrinol Metabol 50(5): 876–883.
Azziz R, Black V, Hines GA, Fox LM & Boots LR (1998) Adrenal androgen excess in the
polycystic ovary syndrome: sensitivity and responsivity of the hypothalamic-pituitaryadrenal axis. J Clin Endocrinol Metab 83(7): 2317–2323.
Azziz R, Bradley EL,Jr, Potter HD & Boots LR (1995) Adrenal androgen excess in women:
lack of a role for 17-hydroxylase and 17,20-lyase dysregulation. J Clin Endocrinol
Metab 80(2): 400–405.
Azziz R, Carmina E, Dewailly D, Diamanti-Kandarakis E, Escobar-Morreale HF,
Futterweit W, Janssen OE, Legro RS, Norman RJ, Taylor AE, Witchel SF &
Androgen Excess Society (2006) Positions statement: criteria for defining polycystic
ovary syndrome as a predominantly hyperandrogenic syndrome: an Androgen Excess
Society guideline. J Clin Endocrinol Metab 91(11): 4237–4245.
Azziz R, Fox LM, Zacur HA, Parker CR,Jr & Boots LR (2001) Adrenocortical secretion of
dehydroepiandrosterone in healthy women: highly variable response to
adrenocorticotropin. J Clin Endocrinol Metab 86(6): 2513–2517.
87
Azziz R, Marin C, Hoq L, Badamgarav E & Song P (2005) Health care-related economic
burden of the polycystic ovary syndrome during the reproductive life span. J Clin
Endocrinol Metab 90(8): 4650–4658.
Azziz R, Sanchez LA, Knochenhauer ES, Moran C, Lazenby J, Stephens KC, Taylor K &
Boots LR (2004a) Androgen excess in women: experience with over 1000 consecutive
patients. J Clin Endocrinol Metab 89(2): 453–462.
Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES & Yildiz BO (2004b) The
prevalence and features of the polycystic ovary syndrome in an unselected population.
J Clin Endocrinol Metab 89(6): 2745–2749.
Baillargeon JP, Jakubowicz DJ, Iuorno MJ, Jakubowicz S & Nestler JE (2004) Effects of
metformin and rosiglitazone, alone and in combination, in nonobese women with
polycystic ovary syndrome and normal indices of insulin sensitivity. Fertil Steril 82(4):
893–902.
Baker WL, Talati R, White CM & Coleman CI (2010) Differing effect of statins on insulin
sensitivity in non-diabetics: a systematic review and meta-analysis. Diabetes Res Clin
Pract 87(1): 98–107.
Balen AH, Conway GS, Kaltsas G, Techatrasak K, Manning PJ, West C & Jacobs HS
(1995) Polycystic ovary syndrome: the spectrum of the disorder in 1741 patients. Hum
Reprod 10(8): 2107–2111.
Balen AH, Laven JS, Tan SL & Dewailly D (2003) Ultrasound assessment of the
polycystic ovary: international consensus definitions. Hum Reprod Update 9(6): 505–
514.
Banaszewska B, Pawelczyk L, Spaczynski RZ & Duleba AJ (2009) Comparison of
simvastatin and metformin in treatment of polycystic ovary syndrome: prospective
randomized trial. J Clin Endocrinol Metab 94(12): 4938–4945.
Banaszewska B, Pawelczyk L, Spaczynski RZ & Duleba AJ (2011) Effects of simvastatin
and metformin on polycystic ovary syndrome after six months of treatment. J Clin
Endocrinol Metab 96(11): 3493–3501.
Banaszewska B, Pawelczyk L, Spaczynski RZ, Dziura J & Duleba AJ (2007) Effects of
simvastatin and oral contraceptive agent on polycystic ovary syndrome: prospective,
randomized, crossover trial. J Clin Endocrinol Metab 92(2): 456–461.
Baptiste CG, Battista MC, Trottier A & Baillargeon JP (2010) Insulin and
hyperandrogenism in women with polycystic ovary syndrome. J Steroid Biochem Mol
Biol 122(1–3): 42–52.
Barber TM, Golding SJ, Alvey C, Wass JA, Karpe F, Franks S & McCarthy MI (2008)
Global adiposity rather than abnormal regional fat distribution characterizes women
with polycystic ovary syndrome. J Clin Endocrinol Metab 93(3): 999–1004.
Barber TM, Wass JA, McCarthy MI & Franks S (2007) Metabolic characteristics of
women with polycystic ovaries and oligo-amenorrhoea but normal androgen levels:
implications for the management of polycystic ovary syndrome. Clin Endocrinol (Oxf)
66(4): 513–517.
Barnes RB, Rosenfield RL, Burstein S & Ehrmann DA (1989) Pituitary-ovarian responses
to nafarelin testing in the polycystic ovary syndrome. N Engl J Med 320(9): 559–565.
88
Barreiros FA, Guazzelli CA, Barbosa R, Torloni MR, Barbieri M & Araujo FF (2011)
Extended regimens of the combined contraceptive vaginal ring containing
etonogestrel and ethinyl estradiol: effects on lipid metabolism. Contraception 84(2):
155–159.
Battaglia C, Mancini F, Fabbri R, Persico N, Busacchi P, Facchinetti F & Venturoli S
(2010) Polycystic ovary syndrome and cardiovascular risk in young patients treated
with drospirenone-ethinylestradiol or contraceptive vaginal ring. A prospective,
randomized, pilot study. Fertil Steril 94(4): 1417–1425.
Bergman RN, Phillips LS & Cobelli C (1981) Physiologic evaluation of factors controlling
glucose tolerance in man: measurement of insulin sensitivity and beta-cell glucose
sensitivity from the response to intravenous glucose. J Clin Invest 68(6): 1456–1467.
Berneis K, Rizzo M, Hersberger M, Rini GB, Di Fede G, Pepe I, Spinas GA & Carmina E
(2009) Atherogenic forms of dyslipidaemia in women with polycystic ovary
syndrome. Int J Clin Pract 63(1): 56–62.
Berneis K, Rizzo M, Lazzarini V, Fruzzetti F & Carmina E (2007) Atherogenic lipoprotein
phenotype and low-density lipoproteins size and subclasses in women with polycystic
ovary syndrome. J Clin Endocrinol Metab 92(1): 186–189.
Birdsall MA, Farquhar CM & White HD (1997) Association between polycystic ovaries
and extent of coronary artery disease in women having cardiac catheterization. Ann
Intern Med 126(1): 32–35.
Boomsma CM, Eijkemans MJ, Hughes EG, Visser GH, Fauser BC & Macklon NS (2006)
A meta-analysis of pregnancy outcomes in women with polycystic ovary syndrome.
Hum Reprod Update 12(6): 673–683.
Boudreaux MY, Talbott EO, Kip KE, Brooks MM & Witchel SF (2006) Risk of T2DM
and impaired fasting glucose among PCOS subjects: results of an 8-year follow-up.
Curr Diab Rep 6(1): 77–83.
Boulman N, Levy Y, Leiba R, Shachar S, Linn R, Zinder O & Blumenfeld Z (2004)
Increased C-reactive protein levels in the polycystic ovary syndrome: a marker of
cardiovascular disease. J Clin Endocrinol Metab 89(5): 2160–2165.
Brunner H, Cockcroft JR, Deanfield J, Donald A, Ferrannini E, Halcox J, Kiowski W,
Luscher TF, Mancia G, Natali A, Oliver JJ, Pessina AC, Rizzoni D, Rossi GP, Salvetti
A, Spieker LE, Taddei S, Webb DJ & Working Group on Endothelins and Endothelial
Factors of the European Society of Hypertension (2005) Endothelial function and
dysfunction. Part II: Association with cardiovascular risk factors and diseases. A
statement by the Working Group on Endothelins and Endothelial Factors of the
European Society of Hypertension. J Hypertens 23(2): 233–246.
Burger HG (2002) Androgen production in women. Fertil Steril 77 Suppl 4: S3–5.
Burger HG, Dudley EC, Cui J, Dennerstein L & Hopper JL (2000) A prospective
longitudinal study of serum testosterone, dehydroepiandrosterone sulfate, and sex
hormone-binding globulin levels through the menopause transition. J Clin Endocrinol
Metab 85(8): 2832–2838.
89
Cagnacci A, Ferrari S, Tirelli A, Zanin R & Volpe A (2009a) Insulin sensitivity and lipid
metabolism with oral contraceptives containing chlormadinone acetate or desogestrel:
a randomized trial. Contraception 79(2): 111–116.
Cagnacci A, Ferrari S, Tirelli A, Zanin R & Volpe A (2009b) Route of administration of
contraceptives containing desogestrel/etonorgestrel and insulin sensitivity: a
prospective randomized study. Contraception 80(1): 34–39.
Cannon RO,3rd (1998) Role of nitric oxide in cardiovascular disease: focus on the
endothelium. Clin Chem 44(8 Pt 2): 1809–1819.
Carmina E, Bucchieri S, Esposito A, Del Puente A, Mansueto P, Orio F, Di Fede G & Rini
G (2007) Abdominal fat quantity and distribution in women with polycystic ovary
syndrome and extent of its relation to insulin resistance. J Clin Endocrinol Metab
92(7): 2500–2505.
Carmina E, Gonzalez F, Vidali A, Stanczyk FZ, Ferin M & Lobo RA (1999) The
contributions of oestrogen and growth factors to increased adrenal androgen secretion
in polycystic ovary syndrome. Hum Reprod 14(2): 307–311.
Carmina E, Koyama T, Chang L, Stanczyk FZ & Lobo RA (1992) Does ethnicity influence
the prevalence of adrenal hyperandrogenism and insulin resistance in polycystic ovary
syndrome? Am J Obstet Gynecol 167(6): 1807–1812.
Carmina E, Legro RS, Stamets K, Lowell J & Lobo RA (2003) Difference in body weight
between American and Italian women with polycystic ovary syndrome: influence of
the diet. Hum Reprod 18(11): 2289–2293.
Carmina E, Rosato F & Janni A (1986) Increased DHEAs levels in PCO syndrome:
evidence for the existence of two subgroups of patients. J Endocrinol Invest 9(1): 5–9.
Cassina M, Dona M, Di Gianantonio E, Litta P & Clementi M (2014) First-trimester
exposure to metformin and risk of birth defects: a systematic review and metaanalysis. Hum Reprod Update 20(5): 656–669.
Catteau-Jonard,S., Dewailly,D. (2013) Pathophysiology of Polycystic Ovary Syndrome:
The Role of Hyperandrogenism. In: Grossman A (ed) Polycystic Ovary Syndrome Novel Insights into Causes and Therapy. Switzerland, Karger: 6–7.
Cauci S, Di Santolo M, Culhane JF, Stel G, Gonano F & Guaschino S (2008) Effects of
third-generation oral contraceptives on high-sensitivity C-reactive protein and
homocysteine in young women. Obstet Gynecol 111(4): 857–864.
Cederberg H, Stancakova A, Yaluri N, Modi S, Kuusisto J & Laakso M (2015) Increased
risk of diabetes with statin treatment is associated with impaired insulin sensitivity
and insulin secretion: a 6 year follow-up study of the METSIM cohort. Diabetologia .
Chang RJ, Mandel FP, Wolfsen AR & Judd HL (1982) Circulating levels of plasma
adrenocorticotropin in polycystic ovary disease. J Clin Endocrinol Metab 54(6):
1265–1267.
Chasan-Taber L, Willett WC, Stampfer MJ, Hunter DJ, Colditz GA, Spiegelman D &
Manson JE (1997) A prospective study of oral contraceptives and NIDDM among U.S.
women. Diabetes Care 20(3): 330–335.
90
Chen MJ, Yang WS, Yang JH, Chen CL, Ho HN & Yang YS (2007) Relationship between
androgen levels and blood pressure in young women with polycystic ovary syndrome.
Hypertension 49(6): 1442–1447.
Christian RC, Dumesic DA, Behrenbeck T, Oberg AL, Sheedy PF,2nd & Fitzpatrick LA
(2003) Prevalence and predictors of coronary artery calcification in women with
polycystic ovary syndrome. J Clin Endocrinol Metab 88(6): 2562–2568.
Cibula D, Cifkova R, Fanta M, Poledne R, Zivny J & Skibova J (2000) Increased risk of
non-insulin dependent diabetes mellitus, arterial hypertension and coronary artery
disease in perimenopausal women with a history of the polycystic ovary syndrome.
Hum Reprod 15(4): 785–789.
Cinar N, Harmanci A, Aksoy DY, Aydin K & Yildiz BO (2012) Adrenocortical steroid
response to ACTH in different phenotypes of non-obese polycystic ovary syndrome. J
Ovarian Res 5(1): 42-2215-5-42.
Cleland SJ, Petrie JR, Ueda S, Elliott HL & Connell JM (1998) Insulin as a vascular
hormone: implications for the pathophysiology of cardiovascular disease. Clin Exp
Pharmacol Physiol 25(3–4): 175–184.
Colilla S, Cox NJ & Ehrmann DA (2001) Heritability of insulin secretion and insulin
action in women with polycystic ovary syndrome and their first degree relatives. J
Clin Endocrinol Metab 86(5): 2027–2031.
Conway GS, Agrawal R, Betteridge DJ & Jacobs HS (1992) Risk factors for coronary
artery disease in lean and obese women with the polycystic ovary syndrome. Clin
Endocrinol (Oxf) 37(2): 119–125.
Cook CL, Siow Y, Brenner AG & Fallat ME (2002) Relationship between serum
mullerian-inhibiting substance and other reproductive hormones in untreated women
with polycystic ovary syndrome and normal women. Fertil Steril 77(1): 141–146.
Couzinet B, Meduri G, Lecce MG, Young J, Brailly S, Loosfelt H, Milgrom E & Schaison
G (2001) The postmenopausal ovary is not a major androgen-producing gland. J Clin
Endocrinol Metab 86(10): 5060–5066.
Creasy GW, Fisher AC, Hall N & Shangold GA (2003) Transdermal contraceptive patch
delivering norelgestromin and ethinyl estradiol. Effects on the lipid profile. J Reprod
Med 48(3): 179–186.
Crook D & Godsland I (1998) Safety evaluation of modern oral contraceptives. Effects on
lipoprotein and carbohydrate metabolism. Contraception 57(3): 189–201.
Dahlgren E, Johansson S, Lindstedt G, Knutsson F, Oden A, Janson PO, Mattson LA,
Crona N & Lundberg PA (1992) Women with polycystic ovary syndrome wedge
resected in 1956 to 1965: a long-term follow-up focusing on natural history and
circulating hormones. Fertil Steril 57(3): 505–513.
Daneshmand S, Weitsman SR, Navab A, Jakimiuk AJ & Magoffin DA (2002)
Overexpression of theca-cell messenger RNA in polycystic ovary syndrome does not
correlate with polymorphisms in the cholesterol side-chain cleavage and 17alphahydroxylase/C(17-20) lyase promoters. Fertil Steril 77(2): 274–280.
91
Davison SL, Bell R, Donath S, Montalto JG & Davis SR (2005) Androgen levels in adult
females: changes with age, menopause, and oophorectomy. J Clin Endocrinol Metab
90(7): 3847–3853.
De Frène V, Vansteelandt S, T'Sjoen G, Gerris J, Somers S, Vercruysse L & De Sutter P
(2014) A retrospective study of the pregnancy, delivery and neonatal outcome in
overweight versus normal weight women with polycystic ovary syndrome. Hum
Reprod 29(10): 2333–2338.
de Graaf J, Swinkels DW, Demacker PN, de Haan AF & Stalenhoef AF (1993) Differences
in the low density lipoprotein subfraction profile between oral contraceptive users and
controls. J Clin Endocrinol Metab 76(1): 197–202.
de Groot PC, Dekkers OM, Romijn JA, Dieben SW & Helmerhorst FM (2011) PCOS,
coronary heart disease, stroke and the influence of obesity: a systematic review and
meta-analysis. Hum Reprod Update 17(4): 495–500.
De Leo V, Fruzzetti F, Musacchio MC, Scolaro V, Di Sabatino A & Morgante G (2013)
Effect of a new oral contraceptive with estradiol valerate/dienogest on carbohydrate
metabolism. Contraception 88(3): 364–368.
De Leo V, la Marca A & Petraglia F (2003) Insulin-lowering agents in the management of
polycystic ovary syndrome. Endocr Rev 24(5): 633–667.
De Michele M, Panico S, Iannuzzi A, Celentano E, Ciardullo AV, Galasso R, Sacchetti L,
Zarrilli F, Bond MG & Rubba P (2002) Association of obesity and central fat
distribution with carotid artery wall thickening in middle-aged women. Stroke 33(12):
2923–2928.
Deanfield J, Donald A, Ferri C, Giannattasio C, Halcox J, Halligan S, Lerman A, Mancia
G, Oliver JJ, Pessina AC, Rizzoni D, Rossi GP, Salvetti A, Schiffrin EL, Taddei S,
Webb DJ & Working Group on Endothelin and Endothelial Factors of the European
Society of Hypertension (2005) Endothelial function and dysfunction. Part I:
Methodological issues for assessment in the different vascular beds: a statement by
the Working Group on Endothelin and Endothelial Factors of the European Society of
Hypertension. J Hypertens 23(1): 7–17.
Dejager S, Pichard C, Giral P, Bruckert E, Federspield MC, Beucler I & Turpin G (2001)
Smaller LDL particle size in women with polycystic ovary syndrome compared to
controls. Clin Endocrinol (Oxf) 54(4): 455–462.
Deleskog A, Hilding A & Ostenson CG (2011) Oral contraceptive use and abnormal
glucose regulation in Swedish middle aged women. Diabetes Res Clin Pract 92(2):
288–292.
Dewailly D, Catteau-Jonard S, Reyss AC, Leroy M & Pigny P (2006) Oligoanovulation
with polycystic ovaries but not overt hyperandrogenism. J Clin Endocrinol Metab
91(10): 3922–3927.
Dewailly D, Lujan ME, Carmina E, Cedars MI, Laven J, Norman RJ & Escobar-Morreale
HF (2014) Definition and significance of polycystic ovarian morphology: a task force
report from the Androgen Excess and Polycystic Ovary Syndrome Society. Hum
Reprod Update 20(3): 334–352.
92
Dewailly D, Pigny P, Soudan B, Catteau-Jonard S, Decanter C, Poncelet E & Duhamel A
(2010) Reconciling the definitions of polycystic ovary syndrome: the ovarian follicle
number and serum anti-Mullerian hormone concentrations aggregate with the markers
of hyperandrogenism. J Clin Endocrinol Metab 95(9): 4399–4405.
Diamanti-Kandarakis E, Alexandraki K, Piperi C, Protogerou A, Katsikis I, Paterakis T,
Lekakis J & Panidis D (2006) Inflammatory and endothelial markers in women with
polycystic ovary syndrome. Eur J Clin Invest 36(10): 691–697.
Diamanti-Kandarakis E, Alexandraki K, Protogerou A, Piperi C, Papamichael C, Aessopos
A, Lekakis J & Mavrikakis M (2005) Metformin administration improves endothelial
function in women with polycystic ovary syndrome. Eur J Endocrinol 152(5): 749–
756.
Diamanti-Kandarakis E & Dunaif A (2012) Insulin resistance and the polycystic ovary
syndrome revisited: an update on mechanisms and implications. Endocr Rev 33(6):
981–1030.
Diamanti-Kandarakis E, Kouli CR, Bergiele AT, Filandra FA, Tsianateli TC, Spina GG,
Zapanti ED & Bartzis MI (1999) A survey of the polycystic ovary syndrome in the
Greek island of Lesbos: hormonal and metabolic profile. J Clin Endocrinol Metab
84(11): 4006–4011.
Diamanti-Kandarakis E, Papavassiliou AG, Kandarakis SA & Chrousos GP (2007)
Pathophysiology and types of dyslipidemia in PCOS. Trends Endocrinol Metab 18(7):
280–285.
Diamanti-Kandarakis E, Paterakis T, Alexandraki K, Piperi C, Aessopos A, Katsikis I,
Katsilambros N, Kreatsas G & Panidis D (2006) Indices of low-grade chronic
inflammation in polycystic ovary syndrome and the beneficial effect of metformin.
Hum Reprod 21(6): 1426–1431.
Diamanti-Kandarakis E, Spina G, Kouli C & Migdalis I (2001) Increased endothelin-1
levels in women with polycystic ovary syndrome and the beneficial effect of
metformin therapy. J Clin Endocrinol Metab 86(10): 4666–4673.
Dokras A, Bochner M, Hollinrake E, Markham S, Vanvoorhis B & Jagasia DH (2005)
Screening women with polycystic ovary syndrome for metabolic syndrome. Obstet
Gynecol 106(1): 131–137.
Duijkers I, Killick S, Bigrigg A & Dieben TO (2004) A comparative study on the effects
of a contraceptive vaginal ring NuvaRing and an oral contraceptive on carbohydrate
metabolism and adrenal and thyroid function. Eur J Contracept Reprod Health Care
9(3): 131–140.
Duleba AJ, Banaszewska B, Spaczynski RZ & Pawelczyk L (2006) Simvastatin improves
biochemical parameters in women with polycystic ovary syndrome: results of a
prospective, randomized trial. Fertil Steril 85(4): 996–1001.
Dunaif A (1997) Insulin resistance and the polycystic ovary syndrome: mechanism and
implications for pathogenesis. Endocr Rev 18(6): 774–800.
Dunaif A & Finegood DT (1996) Beta-cell dysfunction independent of obesity and glucose
intolerance in the polycystic ovary syndrome. J Clin Endocrinol Metab 81(3): 942–
947.
93
Dunaif A, Segal KR, Futterweit W & Dobrjansky A (1989) Profound peripheral insulin
resistance, independent of obesity, in polycystic ovary syndrome. Diabetes 38(9):
1165–1174.
Dunaif A, Segal KR, Shelley DR, Green G, Dobrjansky A & Licholai T (1992) Evidence
for distinctive and intrinsic defects in insulin action in polycystic ovary syndrome.
Diabetes 41(10): 1257–1266.
Eagleson CA, Gingrich MB, Pastor CL, Arora TK, Burt CM, Evans WS & Marshall JC
(2000) Polycystic ovarian syndrome: evidence that flutamide restores sensitivity of
the gonadotropin-releasing hormone pulse generator to inhibition by estradiol and
progesterone. J Clin Endocrinol Metab 85(11): 4047–4052.
Ehrmann DA, Barnes RB, Rosenfield RL, Cavaghan MK & Imperial J (1999) Prevalence
of impaired glucose tolerance and diabetes in women with polycystic ovary syndrome.
Diabetes Care 22(1): 141–146.
Ehrmann DA, Breda E, Corcoran MC, Cavaghan MK, Imperial J, Toffolo G, Cobelli C &
Polonsky KS (2004) Impaired beta-cell compensation to dexamethasone-induced
hyperglycemia in women with polycystic ovary syndrome. Am J Physiol Endocrinol
Metab 287(2): E241–6.
Ehrmann DA, Kasza K, Azziz R, Legro RS, Ghazzi MN & PCOS/Troglitazone Study
Group (2005) Effects of race and family history of type 2 diabetes on metabolic status
of women with polycystic ovary syndrome. J Clin Endocrinol Metab 90(1): 66–71.
Ehrmann DA, Liljenquist DR, Kasza K, Azziz R, Legro RS, Ghazzi MN &
PCOS/Troglitazone Study Group (2006) Prevalence and predictors of the metabolic
syndrome in women with polycystic ovary syndrome. J Clin Endocrinol Metab 91(1):
48–53.
Ehrmann DA, Sturis J, Byrne MM, Karrison T, Rosenfield RL & Polonsky KS (1995)
Insulin secretory defects in polycystic ovary syndrome. Relationship to insulin
sensitivity and family history of non-insulin-dependent diabetes mellitus. J Clin Invest
96(1): 520–527.
Elkind-Hirsch KE, Darensbourg C, Ogden B, Ogden LF & Hindelang P (2007)
Contraceptive vaginal ring use for women has less adverse metabolic effects than an
oral contraceptive. Contraception 76(5): 348–356.
Elting MW, Korsen TJ, Bezemer PD & Schoemaker J (2001) Prevalence of diabetes
mellitus, hypertension and cardiac complaints in a follow-up study of a Dutch PCOS
population. Hum Reprod 16(3): 556–560.
Escobar-Morreale HF, Botella-Carretero JI, Villuendas G, Sancho J & San Millan JL
(2004) Serum interleukin-18 concentrations are increased in the polycystic ovary
syndrome: relationship to insulin resistance and to obesity. J Clin Endocrinol Metab
89(2): 806–811.
Escobar-Morreale HF, Lasuncion MA & Sancho J (2000) Treatment of hirsutism with
ethinyl estradiol-desogestrel contraceptive pills has beneficial effects on the lipid
profile and improves insulin sensitivity. Fertil Steril 74(4): 816–819.
94
Ezeh U, Pall M, Mathur R, Dey D, Berman D, Chen IY, Dumesic DA & Azziz R (2013)
Effects of endogenous androgens and abdominal fat distribution on the
interrelationship between insulin and non-insulin-mediated glucose uptake in females.
J Clin Endocrinol Metab 98(4): 1541–1548.
Faulds G, Ryden M, Ek I, Wahrenberg H & Arner P (2003) Mechanisms behind lipolytic
catecholamine resistance of subcutaneous fat cells in the polycystic ovarian syndrome.
J Clin Endocrinol Metab 88(5): 2269–2273.
Ferri C, Pittoni V, Piccoli A, Laurenti O, Cassone MR, Bellini C, Properzi G, Valesini G,
De Mattia G & Santucci A (1995) Insulin stimulates endothelin-1 secretion from
human endothelial cells and modulates its circulating levels in vivo. J Clin Endocrinol
Metab 80(3): 829–835.
Ferriman D & Gallwey JD (1961) Clinical assessment of body hair growth in women. J
Clin Endocrinol Metab 21: 1440–1447.
Fleming R, Hopkinson ZE, Wallace AM, Greer IA & Sattar N (2002) Ovarian function and
metabolic factors in women with oligomenorrhea treated with metformin in a
randomized double blind placebo-controlled trial. J Clin Endocrinol Metab 87(2):
569–574.
Franks S, Mason H, White D & Willis D (1998) Etiology of anovulation in polycystic
ovary syndrome. Steroids 63(5–6): 306–307.
Frempong BA, Ricks M, Sen S & Sumner AE (2008) Effect of low-dose oral
contraceptives on metabolic risk factors in African-American women. J Clin
Endocrinol Metab 93(6): 2097–2103.
Gallagher TF, Kappas A, Hellman L, Lipsett MB, Pearson OH & West CD (1958)
Adrenocortical hyperfunction in idiopathic hirsutism and the Stein-Leventhal
syndrome. J Clin Invest 37(6): 794–799.
Gaspard U, Scheen A, Endrikat J, Buicu C, Lefebvre P, Gerlinger C & Heithecker R (2003)
A randomized study over 13 cycles to assess the influence of oral contraceptives
containing ethinylestradiol combined with drospirenone or desogestrel on
carbohydrate metabolism. Contraception 67(6): 423–429.
Gazzerro P, Proto MC, Gangemi G, Malfitano AM, Ciaglia E, Pisanti S, Santoro A, Laezza
C & Bifulco M (2012) Pharmacological actions of statins: a critical appraisal in the
management of cancer. Pharmacol Rev 64(1): 102–146.
Gilling-Smith C, Story H, Rogers V & Franks S (1997) Evidence for a primary
abnormality of thecal cell steroidogenesis in the polycystic ovary syndrome. Clin
Endocrinol (Oxf) 47(1): 93–99.
Gilling-Smith C, Willis DS, Beard RW & Franks S (1994) Hypersecretion of
androstenedione by isolated thecal cells from polycystic ovaries. J Clin Endocrinol
Metab 79(4): 1158–1165.
Ginsberg HN (2000) Insulin resistance and cardiovascular disease. J Clin Invest 106(4):
453–458.
Girardi G (2014) Can statins prevent pregnancy complications? J Reprod Immunol 101102: 161–167.
95
Glintborg D, Hermann AP, Brusgaard K, Hangaard J, Hagen C & Andersen M (2005)
Significantly
higher
adrenocorticotropin-stimulated
cortisol
and
17hydroxyprogesterone levels in 337 consecutive, premenopausal, caucasian, hirsute
patients compared with healthy controls. J Clin Endocrinol Metab 90(3): 1347–1353.
Glueck CJ, Morrison JA, Goldenberg N & Wang P (2009) Coronary heart disease risk
factors in adult premenopausal white women with polycystic ovary syndrome
compared with a healthy female population. Metabolism 58(5): 714–721.
Glueck CJ, Papanna R, Wang P, Goldenberg N & Sieve-Smith L (2003) Incidence and
treatment of metabolic syndrome in newly referred women with confirmed polycystic
ovarian syndrome. Metabolism 52(7): 908–915.
Gode F, Karagoz C, Posaci C, Saatli B, Uysal D, Secil M & Akdeniz B (2011) Alteration
of cardiovascular risk parameters in women with polycystic ovary syndrome who
were prescribed to ethinyl estradiol-cyproterone acetate. Arch Gynecol Obstet 284(4):
923–929.
Godsland IF, Walton C, Felton C, Proudler A, Patel A & Wynn V (1992) Insulin resistance,
secretion, and metabolism in users of oral contraceptives. J Clin Endocrinol Metab
74(1): 64–70.
Goebelsmann U, Mashchak CA & Mishell DR,Jr (1985) Comparison of hepatic impact of
oral and vaginal administration of ethinyl estradiol. Am J Obstet Gynecol 151(7):
868–877.
Goodarzi MO, Carmina E & Azziz R (2015) DHEA, DHEAS and PCOS. J Steroid
Biochem Mol Biol 145: 213–225.
Goodarzi MO, Erickson S, Port SC, Jennrich RI & Korenman SG (2005) beta-Cell
function: a key pathological determinant in polycystic ovary syndrome. J Clin
Endocrinol Metab 90(1): 310–315.
Gougeon A (1996) Regulation of ovarian follicular development in primates: facts and
hypotheses. Endocr Rev 17(2): 121–155.
Grodnitskaya EE, Grigoryan OR, Klinyshkova EV, Andreeva EN, Melnichenko GA &
Dedov II (2010) Effect on carbohydrate metabolism and analysis of acceptability
(menstrual cycle control) of extended regimens of the vaginally inserted hormonereleasing system 'NuvaRing' as compared with the standard 21/7 regime in
reproductive-age women with type 1 diabetes mellitus. Gynecol Endocrinol 26(9):
663–668.
Guzick DS, Talbott EO, Sutton-Tyrrell K, Herzog HC, Kuller LH & Wolfson SK,Jr (1996)
Carotid atherosclerosis in women with polycystic ovary syndrome: initial results from
a case-control study. Am J Obstet Gynecol 174(4): 1224–9; discussion 1229–32.
Gwynne JT & Strauss JF,3rd (1982) The role of lipoproteins in steroidogenesis and
cholesterol metabolism in steroidogenic glands. Endocr Rev 3(3): 299–329.
Hammond GL, Langley MS, Robinson PA, Nummi S & Lund L (1984) Serum steroid
binding protein concentrations, distribution of progestogens, and bioavailability of
testosterone during treatment with contraceptives containing desogestrel or
levonorgestrel. Fertil Steril 42(1): 44–51.
96
Harchaoui KE, Visser ME, Kastelein JJ, Stroes ES & Dallinga-Thie GM (2009)
Triglycerides and cardiovascular risk. Curr Cardiol Rev 5(3): 216–222.
Hardie DG (2013) AMPK: a target for drugs and natural products with effects on both
diabetes and cancer. Diabetes 62(7): 2164–2172.
Hironaka K, Yano M, Kohno M, Tanigawa T, Obayashi M, Konishi M, Umemoto S &
Matsuzaki M (1997) In vivo aortic wall characteristics at the early stage of
atherosclerosis in rabbits. Am J Physiol 273(3 Pt 2): H1142–7.
Hoffman DI, Klove K & Lobo RA (1984) The prevalence and significance of elevated
dehydroepiandrosterone sulfate levels in anovulatory women. Fertil Steril 42(1): 76–
81.
Horejsi R, Moller R, Rackl S, Giuliani A, Freytag U, Crailsheim K, Sudi K & Tafeit E
(2004) Android subcutaneous adipose tissue topography in lean and obese women
suffering from PCOS: comparison with type 2 diabetic women. Am J Phys Anthropol
124(3): 275–281.
Hücking K, Watanabe RM, Stefanovski D & Bergman RN (2008) OGTT-derived
measures of insulin sensitivity are confounded by factors other than insulin sensitivity
itself. Obesity (Silver Spring) 16(8): 1938–1945.
Hudecova M, Holte J, Olovsson M, Larsson A, Berne C & Poromaa IS (2011) Diabetes
and impaired glucose tolerance in patients with polycystic ovary syndrome – a long
term follow-up. Hum Reprod 26(6): 1462–1468.
Hughesdon PE (1982) Morphology and morphogenesis of the Stein-Leventhal ovary and
of so-called "hyperthecosis". Obstet Gynecol Surv 37(2): 59–77.
Hutchison SJ, Sudhir K, Chou TM, Sievers RE, Zhu BQ, Sun YP, Deedwania PC, Glantz
SA, Parmley WW & Chatterjee K (1997) Testosterone worsens endothelial
dysfunction associated with hypercholesterolemia and environmental tobacco smoke
exposure in male rabbit aorta. J Am Coll Cardiol 29(4): 800–807.
Izquierdo D, Foyouzi N, Kwintkiewicz J & Duleba AJ (2004) Mevastatin inhibits ovarian
theca-interstitial cell proliferation and steroidogenesis. Fertil Steril 82 Suppl 3: 1193–
1197.
Jakimiuk AJ, Weitsman SR, Navab A & Magoffin DA (2001) Luteinizing hormone
receptor, steroidogenesis acute regulatory protein, and steroidogenic enzyme
messenger ribonucleic acids are overexpressed in thecal and granulosa cells from
polycystic ovaries. J Clin Endocrinol Metab 86(3): 1318–1323.
Jamnongjit M & Hammes SR (2006) Ovarian steroids: the good, the bad, and the signals
that raise them. Cell Cycle 5(11): 1178–1183.
Joham AE, Ranasinha S, Zoungas S, Moran L & Teede HJ (2014) Gestational diabetes and
type 2 diabetes in reproductive-aged women with polycystic ovary syndrome. J Clin
Endocrinol Metab 99(3): E447–52.
Joharatnam J, Barber TM, Webber L, Conway GS, McCarthy MI & Franks S (2011)
Determinants of dyslipidaemia in probands with polycystic ovary syndrome and their
sisters. Clin Endocrinol (Oxf) 74(6): 714–719.
Karakas SE, Kim K & Duleba AJ (2010) Determinants of impaired fasting glucose versus
glucose intolerance in polycystic ovary syndrome. Diabetes Care 33(4): 887–893.
97
Katz A, Nambi SS, Mather K, Baron AD, Follmann DA, Sullivan G & Quon MJ (2000)
Quantitative insulin sensitivity check index: a simple, accurate method for assessing
insulin sensitivity in humans. J Clin Endocrinol Metab 85(7): 2402–2410.
Kaya C, Cengiz SD, Berker B, Demirtas S, Cesur M & Erdogan G (2009) Comparative
effects of atorvastatin and simvastatin on the plasma total homocysteine levels in
women with polycystic ovary syndrome: a prospective randomized study. Fertil Steril
92(2): 635–642.
Kaya C, Pabuccu R, Cengiz SD & Dunder I (2010) Comparison of the effects of
atorvastatin and simvastatin in women with polycystic ovary syndrome: A prospective,
randomized study. Exp Clin Endocrinol Diabetes 118(3): 161–166.
Kazerooni T, Shojaei-Baghini A, Dehbashi S, Asadi N, Ghaffarpasand F & Kazerooni Y
(2010) Effects of metformin plus simvastatin on polycystic ovary syndrome: a
prospective, randomized, double-blind, placebo-controlled study. Fertil Steril 94(6):
2208–2213.
Kelly CC, Lyall H, Petrie JR, Gould GW, Connell JM & Sattar N (2001) Low grade
chronic inflammation in women with polycystic ovarian syndrome. J Clin Endocrinol
Metab 86(6): 2453–2455.
Kelly CJ, Speirs A, Gould GW, Petrie JR, Lyall H & Connell JM (2002) Altered vascular
function in young women with polycystic ovary syndrome. J Clin Endocrinol Metab
87(2): 742–746.
Kero J, Poutanen M, Zhang FP, Rahman N, McNicol AM, Nilson JH, Keri RA &
Huhtaniemi IT (2000) Elevated luteinizing hormone induces expression of its receptor
and promotes steroidogenesis in the adrenal cortex. J Clin Invest 105(5): 633–641.
Kiriwat O & Petyim S (2010) The effects of transdermal contraception on lipid profiles,
carbohydrate metabolism and coagulogram in Thai women. Gynecol Endocrinol 26(5):
361–365.
Kjerulff LE, Sanchez-Ramos L & Duffy D (2011) Pregnancy outcomes in women with
polycystic ovary syndrome: a metaanalysis. Am J Obstet Gynecol 204(6): 558.e1–
558.e6.
Kjøtrød SB, Carlsen SM, Rasmussen PE, Holst-Larsen T, Mellembakken J, ThurinKjellberg A, Haapaniemikouru K, Morin-Papunen L, Humaidan P, Sunde A & von
During V (2011) Use of metformin before and during assisted reproductive
technology in non-obese young infertile women with polycystic ovary syndrome: a
prospective, randomized, double-blind, multi-centre study. Hum Reprod 26(8): 2045–
2053.
Knochenhauer ES, Key TJ, Kahsar-Miller M, Waggoner W, Boots LR & Azziz R (1998)
Prevalence of the polycystic ovary syndrome in unselected black and white women of
the southeastern United States: a prospective study. J Clin Endocrinol Metab 83(9):
3078–3082.
Koivunen R, Laatikainen T, Tomas C, Huhtaniemi I, Tapanainen J & Martikainen H (1999)
The prevalence of polycystic ovaries in healthy women. Acta Obstet Gynecol Scand
78(2): 137–141.
98
Koivunen RM, Juutinen J, Vauhkonen I, Morin-Papunen LC, Ruokonen A & Tapanainen
JS (2001) Metabolic and steroidogenic alterations related to increased frequency of
polycystic ovaries in women with a history of gestational diabetes. J Clin Endocrinol
Metab 86(6): 2591–2599.
Kojima T, Lindheim SR, Duffy DM, Vijod MA, Stanczyk FZ & Lobo RA (1993) Insulin
sensitivity is decreased in normal women by doses of ethinyl estradiol used in oral
contraceptives. Am J Obstet Gynecol 169(6): 1540–1544.
Krentz AJ, von Muhlen D & Barrett-Connor E (2007) Searching for polycystic ovary
syndrome in postmenopausal women: evidence of a dose-effect association with
prevalent cardiovascular disease. Menopause 14(2): 284–292.
Krintus M, Sypniewska G & Kuligowska-Prusinska M (2010) Effect of second and third
generation oral contraceptives on C-reactive protein, lipids and apolipoproteins in
young, non-obese, non-smoking apparently healthy women. Clin Biochem 43(6):
626–628.
Kumar A, Woods KS, Bartolucci AA & Azziz R (2005) Prevalence of adrenal androgen
excess in patients with the polycystic ovary syndrome (PCOS). Clin Endocrinol (Oxf)
62(6): 644–649.
Kumarapeli V, Seneviratne Rde A, Wijeyaratne CN, Yapa RM & Dodampahala SH (2008)
A simple screening approach for assessing community prevalence and phenotype of
polycystic ovary syndrome in a semi-urban population in Sri Lanka. Am J Epidemiol
168(3): 321–328.
Kwintkiewicz J, Foyouzi N, Piotrowski P, Rzepczynska I & Duleba AJ (2006) Mevastatin
inhibits proliferation of rat ovarian theca-interstitial cells by blocking the mitogenactivated protein kinase pathway. Fertil Steril 86(4 Suppl): 1053–1058.
Lakhani K, Seifalian AM & Hardiman P (2002) Impaired carotid viscoelastic properties in
women with polycystic ovaries. Circulation 106(1): 81–85.
Lanzone A, Petraglia F, Fulghesu AM, Ciampelli M, Caruso A & Mancuso S (1995)
Corticotropin-releasing
hormone
induces
an
exaggerated
response
of
adrenocorticotropic hormone and cortisol in polycystic ovary syndrome. Fertil Steril
63(6): 1195–1199.
Laurent S, Boutouyrie P, Asmar R, Gautier I, Laloux B, Guize L, Ducimetiere P &
Benetos A (2001) Aortic stiffness is an independent predictor of all-cause and
cardiovascular mortality in hypertensive patients. Hypertension 37(5): 1236–1241.
Lauritsen MP, Bentzen JG, Pinborg A, Loft A, Forman JL, Thuesen LL, Cohen A,
Hougaard DM & Nyboe Andersen A (2014) The prevalence of polycystic ovary
syndrome in a normal population according to the Rotterdam criteria versus revised
criteria including anti-Mullerian hormone. Hum Reprod 29(4): 791–801.
Legro RS, Arslanian SA, Ehrmann DA, Hoeger KM, Murad MH, Pasquali R, Welt CK &
Endocrine Society (2013) Diagnosis and treatment of polycystic ovary syndrome: an
Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 98(12): 4565–
4592.
99
Legro RS, Finegood D & Dunaif A (1998) A fasting glucose to insulin ratio is a useful
measure of insulin sensitivity in women with polycystic ovary syndrome. J Clin
Endocrinol Metab 83(8): 2694–2698.
Legro RS, Kunselman AR, Dodson WC & Dunaif A (1999) Prevalence and predictors of
risk for type 2 diabetes mellitus and impaired glucose tolerance in polycystic ovary
syndrome: a prospective, controlled study in 254 affected women. J Clin Endocrinol
Metab 84(1): 165–169.
Legro RS, Kunselman AR & Dunaif A (2001) Prevalence and predictors of dyslipidemia
in women with polycystic ovary syndrome. Am J Med 111(8): 607–613.
Legro RS, Myers ER, Barnhart HX, Carson SA, Diamond MP, Carr BR, Schlaff WD,
Coutifaris C, McGovern PG, Cataldo NA, Steinkampf MP, Nestler JE, Gosman G,
Guidice LC, Leppert PC & Reproductive Medicine Network (2006) The Pregnancy in
Polycystic Ovary Syndrome study: baseline characteristics of the randomized cohort
including racial effects. Fertil Steril 86(4): 914–933.
Lillioja S, Mott DM, Spraul M, Ferraro R, Foley JE, Ravussin E, Knowler WC, Bennett
PH & Bogardus C (1993) Insulin resistance and insulin secretory dysfunction as
precursors of non-insulin-dependent diabetes mellitus. Prospective studies of Pima
Indians. N Engl J Med 329(27): 1988–1992.
Lim SS, Norman RJ, Davies MJ & Moran LJ (2013) The effect of obesity on polycystic
ovary syndrome: a systematic review and meta-analysis. Obes Rev 14(2): 95–109.
Liou TH, Yang JH, Hsieh CH, Lee CY, Hsu CS & Hsu MI (2009) Clinical and
biochemical presentations of polycystic ovary syndrome among obese and nonobese
women. Fertil Steril 92(6): 1960–1965.
Lo JC, Feigenbaum SL, Escobar GJ, Yang J, Crites YM & Ferrara A (2006a) Increased
prevalence of gestational diabetes mellitus among women with diagnosed polycystic
ovary syndrome: a population-based study. Diabetes Care 29(8): 1915–1917.
Lo JC, Feigenbaum SL, Yang J, Pressman AR, Selby JV & Go AS (2006b) Epidemiology
and adverse cardiovascular risk profile of diagnosed polycystic ovary syndrome. J
Clin Endocrinol Metab 91(4): 1357–1363.
Lönn M, Mehlig K, Bengtsson C & Lissner L (2010) Adipocyte size predicts incidence of
type 2 diabetes in women. FASEB J 24(1): 326–331.
Lopez LM, Grimes DA & Schulz KF (2014) Steroidal contraceptives: effect on
carbohydrate metabolism in women without diabetes mellitus. Cochrane Database
Syst Rev 4: CD006133.
Luque-Ramírez M, Alvarez-Blasco F, Mendieta-Azcona C, Botella-Carretero JI &
Escobar-Morreale HF (2007) Obesity is the major determinant of the abnormalities in
blood pressure found in young women with the polycystic ovary syndrome. J Clin
Endocrinol Metab 92(6): 2141–2148.
Maas KH, Chuan SS, Cook-Andersen H, Su HI, Duleba A & Chang RJ (2015)
Relationship between 17-hydroxyprogesterone responses to human chorionic
gonadotropin and markers of ovarian follicle morphology in women with polycystic
ovary syndrome. J Clin Endocrinol Metab 100(1): 293–300.
100
Maciel GA, Baracat EC, Benda JA, Markham SM, Hensinger K, Chang RJ & Erickson GF
(2004) Stockpiling of transitional and classic primary follicles in ovaries of women
with polycystic ovary syndrome. J Clin Endocrinol Metab 89(11): 5321–5327.
Macut D, Damjanovic S, Panidis D, Spanos N, Glisic B, Petakov M, Rousso D, Kourtis A,
Bjekic J & Milic N (2006) Oxidised low-density lipoprotein concentration – early
marker of an altered lipid metabolism in young women with PCOS. Eur J Endocrinol
155(1): 131–136.
Malin SK, Kirwan JP, Sia CL & Gonzalez F (2015) Pancreatic beta-cell dysfunction in
polycystic ovary syndrome: Role of hyperglycemia-induced nuclear factor-kappaB
activation and systemic inflammation. Am J Physiol Endocrinol Metab :
ajpendo.00510.2014.
Mannerås-Holm L, Leonhardt H, Kullberg J, Jennische E, Odén A, Holm G, Hellstrom M,
Lönn L, Olivecrona G, Stener-Victorin E & Lönn M (2011) Adipose tissue has
aberrant morphology and function in PCOS: enlarged adipocytes and low serum
adiponectin, but not circulating sex steroids, are strongly associated with insulin
resistance. J Clin Endocrinol Metab 96(2): E304–11.
Mantovani A, Garlanda C, Doni A & Bottazzi B (2008) Pentraxins in innate immunity:
from C-reactive protein to the long pentraxin PTX3. J Clin Immunol 28(1): 1–13.
March WA, Moore VM, Willson KJ, Phillips DI, Norman RJ & Davies MJ (2010) The
prevalence of polycystic ovary syndrome in a community sample assessed under
contrasting diagnostic criteria. Hum Reprod 25(2): 544–551.
Markopoulos MC, Rizos D, Valsamakis G, Deligeoroglou E, Grigoriou O, Chrousos GP,
Creatsas G & Mastorakos G (2011) Hyperandrogenism in women with polycystic
ovary syndrome persists after menopause. J Clin Endocrinol Metab 96(3): 623–631.
Markopoulos MC, Valsamakis G, Kouskouni E, Boutsiadis A, Papassotiriou I, Creatsas G
& Mastorakos G (2012) Study of carbohydrate metabolism indices and adipocytokine
profile and their relationship with androgens in polycystic ovary syndrome after
menopause. Eur J Endocrinol 168(1): 83–90.
Martikainen H, Salmela P, Nuojua-Huttunen S, Perala J, Leinonen S, Knip M & Ruokonen
A (1996) Adrenal steroidogenesis is related to insulin in hyperandrogenic women.
Fertil Steril 66(4): 564–570.
Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF & Turner RC (1985)
Homeostasis model assessment: insulin resistance and beta-cell function from fasting
plasma glucose and insulin concentrations in man. Diabetologia 28(7): 412–419.
McCartney CR, Bellows AB, Gingrich MB, Hu Y, Evans WS, Marshall JC & Veldhuis JD
(2004) Exaggerated 17-hydroxyprogesterone response to intravenous infusions of
recombinant human LH in women with polycystic ovary syndrome. Am J Physiol
Endocrinol Metab 286(6): E902–8.
McCredie RJ, McCrohon JA, Turner L, Griffiths KA, Handelsman DJ & Celermajer DS
(1998) Vascular reactivity is impaired in genetic females taking high-dose androgens.
J Am Coll Cardiol 32(5): 1331–1335.
Messinis IE, Messini CI & Dafopoulos K (2014) Novel aspects of the endocrinology of the
menstrual cycle. Reprod Biomed Online 28(6): 714–722.
101
Miller WL & Auchus RJ (2011) The molecular biology, biochemistry, and physiology of
human steroidogenesis and its disorders. Endocr Rev 32(1): 81–151.
Moghetti P, Tosi F, Castello R, Magnani CM, Negri C, Brun E, Furlani L, Caputo M &
Muggeo M (1996) The insulin resistance in women with hyperandrogenism is
partially reversed by antiandrogen treatment: evidence that androgens impair insulin
action in women. J Clin Endocrinol Metab 81(3): 952–960.
Moller DE & Flier JS (1991) Insulin resistance – mechanisms, syndromes, and
implications. N Engl J Med 325(13): 938–948.
Morales AJ, Laughlin GA, Butzow T, Maheshwari H, Baumann G & Yen SS (1996)
Insulin, somatotropic, and luteinizing hormone axes in lean and obese women with
polycystic ovary syndrome: common and distinct features. J Clin Endocrinol Metab
81(8): 2854–2864.
Moran C, Knochenhauer E, Boots LR & Azziz R (1999) Adrenal androgen excess in
hyperandrogenism: relation to age and body mass. Fertil Steril 71(4): 671–674.
Moran C, Reyna R, Boots LS & Azziz R (2004) Adrenocortical hyperresponsiveness to
corticotropin in polycystic ovary syndrome patients with adrenal androgen excess.
Fertil Steril 81(1): 126–131.
Moran LJ, Misso ML, Wild RA & Norman RJ (2010) Impaired glucose tolerance, type 2
diabetes and metabolic syndrome in polycystic ovary syndrome: a systematic review
and meta-analysis. Hum Reprod Update 16(4): 347–363.
Moran LJ, Pasquali R, Teede HJ, Hoeger KM & Norman RJ (2009) Treatment of obesity
in polycystic ovary syndrome: a position statement of the Androgen Excess and
Polycystic Ovary Syndrome Society. Fertil Steril 92(6): 1966–1982.
Morin-Papunen L, Martikainen H, McCarthy MI, Franks S, Sovio U, Hartikainen AL,
Ruokonen A, Leinonen M, Laitinen J, Jarvelin MR & Pouta A (2008) Comparison of
metabolic and inflammatory outcomes in women who used oral contraceptives and the
levonorgestrel-releasing intrauterine device in a general population. Am J Obstet
Gynecol 199(5): 529.e1–529.e10.
Morin-Papunen L, Rantala AS, Unkila-Kallio L, Tiitinen A, Hippelainen M, Perheentupa
A, Tinkanen H, Bloigu R, Puukka K, Ruokonen A & Tapanainen JS (2012)
Metformin improves pregnancy and live-birth rates in women with polycystic ovary
syndrome (PCOS): a multicenter, double-blind, placebo-controlled randomized trial. J
Clin Endocrinol Metab 97(5): 1492–1500.
Morin-Papunen L, Rautio K, Ruokonen A, Hedberg P, Puukka M & Tapanainen JS (2003)
Metformin reduces serum C-reactive protein levels in women with polycystic ovary
syndrome. J Clin Endocrinol Metab 88(10): 4649–4654.
Morin-Papunen LC, Vauhkonen I, Koivunen RM, Ruokonen A & Tapanainen JS (2000)
Insulin sensitivity, insulin secretion, and metabolic and hormonal parameters in
healthy women and women with polycystic ovarian syndrome. Hum Reprod 15(6):
1266–1274.
Muniyappa R, Lee S, Chen H & Quon MJ (2008) Current approaches for assessing insulin
sensitivity and resistance in vivo: advantages, limitations, and appropriate usage. Am
J Physiol Endocrinol Metab 294(1): E15–26.
102
Mutharasan P, Galdones E, Penalver Bernabe B, Garcia OA, Jafari N, Shea LD, Woodruff
TK, Legro RS, Dunaif A & Urbanek M (2013) Evidence for chromosome 2p16.3
polycystic ovary syndrome susceptibility locus in affected women of European
ancestry. J Clin Endocrinol Metab 98(1): E185–90.
Nahum R, Thong KJ & Hillier SG (1995) Metabolic regulation of androgen production by
human thecal cells in vitro. Hum Reprod 10(1): 75–81.
National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation,
and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) (2002)
Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on
Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult
Treatment Panel III) final report. Circulation 106(25): 3143–3421.
Nelson VL, Legro RS, Strauss JF,3rd & McAllister JM (1999) Augmented androgen
production is a stable steroidogenic phenotype of propagated theca cells from
polycystic ovaries. Mol Endocrinol 13(6): 946–957.
Nelson VL, Qin KN, Rosenfield RL, Wood JR, Penning TM, Legro RS, Strauss JF,3rd &
McAllister JM (2001) The biochemical basis for increased testosterone production in
theca cells propagated from patients with polycystic ovary syndrome. J Clin
Endocrinol Metab 86(12): 5925–5933.
Nelson-Degrave VL, Wickenheisser JK, Hendricks KL, Asano T, Fujishiro M, Legro RS,
Kimball SR, Strauss JF,3rd & McAllister JM (2005) Alterations in mitogen-activated
protein kinase kinase and extracellular regulated kinase signaling in theca cells
contribute to excessive androgen production in polycystic ovary syndrome. Mol
Endocrinol 19(2): 379–390.
Nestler JE & Jakubowicz DJ (1996) Decreases in ovarian cytochrome P450c17 alpha
activity and serum free testosterone after reduction of insulin secretion in polycystic
ovary syndrome. N Engl J Med 335(9): 617–623.
Nestler JE, Jakubowicz DJ, de Vargas AF, Brik C, Quintero N & Medina F (1998) Insulin
stimulates testosterone biosynthesis by human thecal cells from women with
polycystic ovary syndrome by activating its own receptor and using inositolglycan
mediators as the signal transduction system. J Clin Endocrinol Metab 83(6): 2001–
2005.
Norman RJ, Dewailly D, Legro RS & Hickey TE (2007) Polycystic ovary syndrome.
Lancet 370(9588): 685–697.
Norman RJ, Masters L, Milner CR, Wang JX & Davies MJ (2001) Relative risk of
conversion from normoglycaemia to impaired glucose tolerance or non-insulin
dependent diabetes mellitus in polycystic ovarian syndrome. Hum Reprod 16(9):
1995–1998.
Nowis D, Malenda A, Furs K, Oleszczak B, Sadowski R, Chlebowska J, Firczuk M,
Bujnicki JM, Staruch AD, Zagozdzon R, Glodkowska-Mrowka E, Szablewski L &
Golab J (2014) Statins impair glucose uptake in human cells. BMJ Open Diabetes Res
Care 2(1): e000017.
103
O'Connor A, Phelan N, Tun TK, Boran G, Gibney J & Roche HM (2010) High-molecularweight adiponectin is selectively reduced in women with polycystic ovary syndrome
independent of body mass index and severity of insulin resistance. J Clin Endocrinol
Metab 95(3): 1378–1385.
Odlind V, Milsom I, Persson I & Victor A (2002) Can changes in sex hormone binding
globulin predict the risk of venous thromboembolism with combined oral
contraceptive pills? Acta Obstet Gynecol Scand 81(6): 482–490.
O'Meara NM, Blackman JD, Ehrmann DA, Barnes RB, Jaspan JB, Rosenfield RL &
Polonsky KS (1993) Defects in beta-cell function in functional ovarian
hyperandrogenism. J Clin Endocrinol Metab 76(5): 1241–1247.
Orentreich N, Brind JL, Rizer RL & Vogelman JH (1984) Age changes and sex differences
in serum dehydroepiandrosterone sulfate concentrations throughout adulthood. J Clin
Endocrinol Metab 59(3): 551–555.
Orio F,Jr, Palomba S, Cascella T, De Simone B, Di Biase S, Russo T, Labella D, Zullo F,
Lombardi G & Colao A (2004) Early impairment of endothelial structure and function
in young normal-weight women with polycystic ovary syndrome. J Clin Endocrinol
Metab 89(9): 4588–4593.
Ortega I, Cress AB, Wong DH, Villanueva JA, Sokalska A, Moeller BC, Stanley SD &
Duleba AJ (2012) Simvastatin reduces steroidogenesis by inhibiting Cyp17a1 gene
expression in rat ovarian theca-interstitial cells. Biol Reprod 86(1): 1–9.
Ortega I, Villanueva JA, Wong DH, Cress AB, Sokalska A, Stanley SD & Duleba AJ
(2014) Resveratrol potentiates effects of simvastatin on inhibition of rat ovarian thecainterstitial cells steroidogenesis. J Ovarian Res 7: 21-2215-7-21.
Pabon JE, Li X, Lei ZM, Sanfilippo JS, Yussman MA & Rao CV (1996) Novel presence
of luteinizing hormone/chorionic gonadotropin receptors in human adrenal glands. J
Clin Endocrinol Metab 81(6): 2397–2400.
Palan PR, Strube F, Letko J, Sadikovic A & Mikhail MS (2010) Effects of oral, vaginal,
and transdermal hormonal contraception on serum levels of coenzyme q(10), vitamin
e, and total antioxidant activity. Obstet Gynecol Int 2010: 925635. Epub 2010 Aug 9.
Palomba S, Falbo A, Zullo F & Orio F,Jr (2009) Evidence-based and potential benefits of
metformin in the polycystic ovary syndrome: a comprehensive review. Endocr Rev
30(1): 1–50.
Paradisi G, Steinberg HO, Hempfling A, Cronin J, Hook G, Shepard MK & Baron AD
(2001) Polycystic ovary syndrome is associated with endothelial dysfunction.
Circulation 103(10): 1410–1415.
Pasanen S, Ylikomi T, Syvala H & Tuohimaa P (1997) Distribution of progesterone
receptor in chicken: novel target organs for progesterone and estrogen action. Mol
Cell Endocrinol 135(1): 79–91.
Pasceri V, Willerson JT & Yeh ET (2000) Direct proinflammatory effect of C-reactive
protein on human endothelial cells. Circulation 102(18): 2165–2168.
104
Patel SM, Ratcliffe SJ, Reilly MP, Weinstein R, Bhasin S, Blackman MR, Cauley JA,
Sutton-Tyrrell K, Robbins J, Fried LP & Cappola AR (2009) Higher serum
testosterone concentration in older women is associated with insulin resistance,
metabolic syndrome, and cardiovascular disease. J Clin Endocrinol Metab 94(12):
4776–4784.
Payne AH & Hales DB (2004) Overview of steroidogenic enzymes in the pathway from
cholesterol to active steroid hormones. Endocr Rev 25(6): 947–970.
Pessin JE & Saltiel AR (2000) Signaling pathways in insulin action: molecular targets of
insulin resistance. J Clin Invest 106(2): 165–169.
Phelan N, O'Connor A, Kyaw-Tun T, Correia N, Boran G, Roche HM & Gibney J (2010)
Lipoprotein subclass patterns in women with polycystic ovary syndrome (PCOS)
compared with equally insulin-resistant women without PCOS. J Clin Endocrinol
Metab 95(8): 3933–3939.
Pierpoint T, McKeigue PM, Isaacs AJ, Wild SH & Jacobs HS (1998) Mortality of women
with polycystic ovary syndrome at long-term follow-up. J Clin Epidemiol 51(7): 581–
586.
Pigny P, Merlen E, Robert Y, Cortet-Rudelli C, Decanter C, Jonard S & Dewailly D (2003)
Elevated serum level of anti-mullerian hormone in patients with polycystic ovary
syndrome: relationship to the ovarian follicle excess and to the follicular arrest. J Clin
Endocrinol Metab 88(12): 5957–5962.
Piltonen T, Koivunen R, Morin-Papunen L, Ruokonen A, Huhtaniemi IT & Tapanainen JS
(2002) Ovarian and adrenal steroid production: regulatory role of LH/HCG. Hum
Reprod 17(3): 620–624.
Piltonen T, Koivunen R, Perheentupa A, Morin-Papunen L, Ruokonen A & Tapanainen JS
(2004) Ovarian age-related responsiveness to human chorionic gonadotropin in
women with polycystic ovary syndrome. J Clin Endocrinol Metab 89(8): 3769–3775.
Piltonen T, Koivunen R, Ruokonen A & Tapanainen JS (2003) Ovarian age-related
responsiveness to human chorionic gonadotropin. J Clin Endocrinol Metab 88(7):
3327–3332.
Piltonen T, Morin-Papunen L, Koivunen R, Perheentupa A, Ruokonen A & Tapanainen JS
(2005) Serum anti-Mullerian hormone levels remain high until late reproductive age
and decrease during metformin therapy in women with polycystic ovary syndrome.
Hum Reprod 20(7): 1820–1826.
Pirwany IR, Fleming R, Greer IA, Packard CJ & Sattar N (2001) Lipids and lipoprotein
subfractions in women with PCOS: relationship to metabolic and endocrine
parameters. Clin Endocrinol (Oxf) 54(4): 447–453.
Polderman KH, Gooren LJ, Asscheman H, Bakker A & Heine RJ (1994) Induction of
insulin resistance by androgens and estrogens. J Clin Endocrinol Metab 79(1): 265–
271.
Polson DW, Adams J, Wadsworth J & Franks S (1988) Polycystic ovaries – a common
finding in normal women. Lancet 1(8590): 870–872.
Pradhan AD, Manson JE, Rifai N, Buring JE & Ridker PM (2001) C-reactive protein,
interleukin 6, and risk of developing type 2 diabetes mellitus. JAMA 286(3): 327–334.
105
Raja-Khan N, Kunselman AR, Hogeman CS, Stetter CM, Demers LM & Legro RS (2011)
Effects of atorvastatin on vascular function, inflammation, and androgens in women
with polycystic ovary syndrome: a double-blind, randomized, placebo-controlled trial.
Fertil Steril 95(5): 1849–1852.
Rajala U, Laakso M, Paivansalo M, Pelkonen O, Suramo I & Keinanen-Kiukaanniemi S
(2002) Low insulin sensitivity measured by both quantitative insulin sensitivity check
index and homeostasis model assessment method as a risk factor of increased intimamedia thickness of the carotid artery. J Clin Endocrinol Metab 87(11): 5092–5097.
Randeva HS, Tan BK, Weickert MO, Lois K, Nestler JE, Sattar N & Lehnert H (2012)
Cardiometabolic aspects of the polycystic ovary syndrome. Endocr Rev 33(5): 812–
841.
Rautio K, Tapanainen JS, Ruokonen A & Morin-Papunen LC (2005) Effects of metformin
and ethinyl estradiol-cyproterone acetate on lipid levels in obese and non-obese
women with polycystic ovary syndrome. Eur J Endocrinol 152(2): 269–275.
Rautio K, Tapanainen JS, Ruokonen A & Morin-Papunen LC (2006) Endocrine and
metabolic effects of rosiglitazone in overweight women with PCOS: a randomized
placebo-controlled study. Hum Reprod 21(6): 1400–1407.
Raval AD, Hunter T, Stuckey B & Hart RJ (2011) Statins for women with polycystic ovary
syndrome not actively trying to conceive. Cochrane Database Syst Rev (10)(10):
CD008565.
Ridker PM, Buring JE, Shih J, Matias M & Hennekens CH (1998) Prospective study of Creactive protein and the risk of future cardiovascular events among apparently healthy
women. Circulation 98(8): 731–733.
Ridker PM, Danielson E, Fonseca FA, Genest J, Gotto AM,Jr, Kastelein JJ, Koenig W,
Libby P, Lorenzatti AJ, MacFadyen JG, Nordestgaard BG, Shepherd J, Willerson JT,
Glynn RJ & JUPITER Study Group (2008) Rosuvastatin to prevent vascular events in
men and women with elevated C-reactive protein. N Engl J Med 359(21): 2195–2207.
Ridker PM, Hennekens CH, Buring JE & Rifai N (2000) C-reactive protein and other
markers of inflammation in the prediction of cardiovascular disease in women. N Engl
J Med 342(12): 836–843.
Ridker PM, Rifai N, Rose L, Buring JE & Cook NR (2002) Comparison of C-reactive
protein and low-density lipoprotein cholesterol levels in the prediction of first
cardiovascular events. N Engl J Med 347(20): 1557–1565.
Rifai N, Buring JE, Lee IM, Manson JE & Ridker PM (2002) Is C-reactive protein specific
for vascular disease in women? Ann Intern Med 136(7): 529–533.
Rimm EB, Manson JE, Stampfer MJ, Colditz GA, Willett WC, Rosner B, Hennekens CH
& Speizer FE (1992) Oral contraceptive use and the risk of type 2 (non-insulindependent) diabetes mellitus in a large prospective study of women. Diabetologia
35(10): 967–972.
Rizzo M & Berneis K (2006) Low-density lipoprotein size and cardiovascular risk
assessment. QJM 99(1): 1–14.
106
Rocha MP, Marcondes JA, Barcellos CR, Hayashida SA, Curi DD, da Fonseca AM,
Bagnoli VR & Baracat EC (2011) Dyslipidemia in women with polycystic ovary
syndrome: incidence, pattern and predictors. Gynecol Endocrinol 27(10): 814–819.
Roger VL, Go AS, Lloyd-Jones DM, Benjamin EJ, Berry JD, Borden WB, Bravata DM,
Dai S, Ford ES, Fox CS, Fullerton HJ, Gillespie C, Hailpern SM, Heit JA, Howard VJ,
Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Makuc DM,
Marcus GM, Marelli A, Matchar DB, Moy CS, Mozaffarian D, Mussolino ME,
Nichol G, Paynter NP, Soliman EZ, Sorlie PD, Sotoodehnia N, Turan TN, Virani SS,
Wong ND, Woo D, Turner MB & American Heart Association Statistics Committee
and Stroke Statistics Subcommittee (2012) Heart disease and stroke statistics – 2012
update: a report from the American Heart Association. Circulation 125(1): e2–e220.
Ross R (1999) Atherosclerosis – an inflammatory disease. N Engl J Med 340(2): 115–126.
Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group (2004) Revised
2003 consensus on diagnostic criteria and long-term health risks related to polycystic
ovary syndrome (PCOS). Hum Reprod 19(1): 41–47.
Salans LB, Knittle JL & Hirsch J (1968) The role of adipose cell size and adipose tissue
insulin sensitivity in the carbohydrate intolerance of human obesity. J Clin Invest
47(1): 153–165.
Salley KE, Wickham EP, Cheang KI, Essah PA, Karjane NW & Nestler JE (2007) Glucose
intolerance in polycystic ovary syndrome – a position statement of the Androgen
Excess Society. J Clin Endocrinol Metab 92(12): 4546–4556.
Sathyapalan T, Kilpatrick ES, Coady AM & Atkin SL (2009) The effect of atorvastatin in
patients with polycystic ovary syndrome: a randomized double-blind placebocontrolled study. J Clin Endocrinol Metab 94(1): 103–108.
Sathyapalan T, Smith KA, Coady AM, Kilpatrick ES & Atkin SL (2012) Atorvastatin
therapy decreases androstenedione and dehydroepiandrosterone sulphate
concentrations in patients with polycystic ovary syndrome: randomized controlled
study. Ann Clin Biochem 49(Pt 1): 80–85.
Sattar N, Preiss D, Murray HM, Welsh P, Buckley BM, de Craen AJ, Seshasai SR,
McMurray JJ, Freeman DJ, Jukema JW, Macfarlane PW, Packard CJ, Stott DJ,
Westendorp RG, Shepherd J, Davis BR, Pressel SL, Marchioli R, Marfisi RM,
Maggioni AP, Tavazzi L, Tognoni G, Kjekshus J, Pedersen TR, Cook TJ, Gotto AM,
Clearfield MB, Downs JR, Nakamura H, Ohashi Y, Mizuno K, Ray KK & Ford I
(2010) Statins and risk of incident diabetes: a collaborative meta-analysis of
randomised statin trials. Lancet 375(9716): 735–742.
Schachter M (2005) Chemical, pharmacokinetic and pharmacodynamic properties of
statins: an update. Fundam Clin Pharmacol 19(1): 117–125.
Schmidt J, Brannstrom M, Landin-Wilhelmsen K & Dahlgren E (2011) Reproductive
hormone levels and anthropometry in postmenopausal women with polycystic ovary
syndrome (PCOS): a 21-year follow-up study of women diagnosed with PCOS around
50 years ago and their age-matched controls. J Clin Endocrinol Metab 96(7): 2178–
2185.
107
Shaw LJ, Bairey Merz CN, Azziz R, Stanczyk FZ, Sopko G, Braunstein GD, Kelsey SF,
Kip KE, Cooper-Dehoff RM, Johnson BD, Vaccarino V, Reis SE, Bittner V, Hodgson
TK, Rogers W & Pepine CJ (2008) Postmenopausal women with a history of irregular
menses and elevated androgen measurements at high risk for worsening
cardiovascular event-free survival: results from the National Institutes of Health –
National Heart, Lung, and Blood Institute sponsored Women's Ischemia Syndrome
Evaluation. J Clin Endocrinol Metab 93(4): 1276–1284.
Sirmans SM, Parish RC, Blake S & Wang X (2014) Epidemiology and comorbidities of
polycystic ovary syndrome in an indigent population. J Investig Med 62(6): 868–874.
Sirmans SM & Pate KA (2013) Epidemiology, diagnosis, and management of polycystic
ovary syndrome. Clin Epidemiol 6: 1–13.
Sitruk-Ware R & Nath A (2011) Metabolic effects of contraceptive steroids. Rev Endocr
Metab Disord 12(2): 63–75.
Sitruk-Ware R & Nath A (2013) Characteristics and metabolic effects of estrogen and
progestins contained in oral contraceptive pills. Best Pract Res Clin Endocrinol Metab
27(1): 13–24.
Smith U (1994) Carbohydrates, fat, and insulin action. Am J Clin Nutr 59(3 Suppl): 686S–
689S.
Sokalska A, Piotrowski PC, Rzepczynska IJ, Cress A & Duleba AJ (2010) Statins inhibit
growth of human theca-interstitial cells in PCOS and non-PCOS tissues independently
of cholesterol availability. J Clin Endocrinol Metab 95(12): 5390–5394.
Sokalska A, Stanley SD, Villanueva JA, Ortega I & Duleba AJ (2014) Comparison of
effects of different statins on growth and steroidogenesis of rat ovarian thecainterstitial cells. Biol Reprod 90(2): 44.
Solomon CG, Hu FB, Dunaif A, Rich-Edwards J, Willett WC, Hunter DJ, Colditz GA,
Speizer FE & Manson JE (2001) Long or highly irregular menstrual cycles as a
marker for risk of type 2 diabetes mellitus. JAMA 286(19): 2421–2426.
Stein I & Leventhal M (1935) Amenorrhoea associated with bilateral polycystic ovaries.
29: 181–185.
Stepto NK, Cassar S, Joham AE, Hutchison SK, Harrison CL, Goldstein RF & Teede HJ
(2013) Women with polycystic ovary syndrome have intrinsic insulin resistance on
euglycaemic-hyperinsulaemic clamp. Hum Reprod 28(3): 777–784.
Stewart PM, Penn R, Holder R, Parton A, Ratcliffe JG & London DR (1993) The
hypothalamo-pituitary-adrenal axis across the normal menstrual cycle and in
polycystic ovary syndrome. Clin Endocrinol (Oxf) 38(4): 387–391.
Svendsen PF, Nilas L, Madsbad S & Holst JJ (2009) Incretin hormone secretion in women
with polycystic ovary syndrome: roles of obesity, insulin sensitivity, and treatment
with metformin. Metabolism 58(5): 586–593.
Sverrisdottir YB, Mogren T, Kataoka J, Janson PO & Stener-Victorin E (2008) Is
polycystic ovary syndrome associated with high sympathetic nerve activity and size at
birth? Am J Physiol Endocrinol Metab 294(3): E576–81.
108
Talbott EO, Guzick DS, Sutton-Tyrrell K, McHugh-Pemu KP, Zborowski JV, Remsberg
KE & Kuller LH (2000) Evidence for association between polycystic ovary syndrome
and premature carotid atherosclerosis in middle-aged women. Arterioscler Thromb
Vasc Biol 20(11): 2414–2421.
Talbott EO, Zborowski JV, Rager JR, Boudreaux MY, Edmundowicz DA & Guzick DS
(2004) Evidence for an association between metabolic cardiovascular syndrome and
coronary and aortic calcification among women with polycystic ovary syndrome. J
Clin Endocrinol Metab 89(11): 5454–5461.
Tang T, Lord JM, Norman RJ, Yasmin E & Balen AH (2012) Insulin-sensitising drugs
(metformin, rosiglitazone, pioglitazone, D-chiro-inositol) for women with polycystic
ovary syndrome, oligo amenorrhoea and subfertility. Cochrane Database Syst Rev 5:
CD003053.
Taponen S, Ahonkallio S, Martikainen H, Koivunen R, Ruokonen A, Sovio U, Hartikainen
AL, Pouta A, Laitinen J, King V, Franks S, McCarthy MI & Jarvelin MR (2004a)
Prevalence of polycystic ovaries in women with self-reported symptoms of
oligomenorrhoea and/or hirsutism: Northern Finland Birth Cohort 1966 Study. Hum
Reprod 19(5): 1083–1088.
Taponen S, Martikainen H, Jarvelin MR, Sovio U, Laitinen J, Pouta A, Hartikainen AL,
McCarthy MI, Franks S, Paldanius M, Ruokonen A & Northern Finland Birth Cohort
1966 Study (2004b) Metabolic cardiovascular disease risk factors in women with selfreported symptoms of oligomenorrhea and/or hirsutism: Northern Finland Birth
Cohort 1966 Study. J Clin Endocrinol Metab 89(5): 2114–2118.
Taylor F, Huffman MD, Macedo AF, Moore TH, Burke M, Davey Smith G, Ward K &
Ebrahim S (2013) Statins for the primary prevention of cardiovascular disease.
Cochrane Database Syst Rev 1: CD004816.
Tehrani FR, Simbar M, Tohidi M, Hosseinpanah F & Azizi F (2011) The prevalence of
polycystic ovary syndrome in a community sample of Iranian population: Iranian
PCOS prevalence study. Reprod Biol Endocrinol 9: 39-7827-9-39.
Tenenbaum A, Klempfner R & Fisman EZ (2014) Hypertriglyceridemia: a too long
unfairly neglected major cardiovascular risk factor. Cardiovasc Diabetol 13(1): 159.
Thierry van Dessel HJ, Lee PD, Faessen G, Fauser BC & Giudice LC (1999) Elevated
serum levels of free insulin-like growth factor I in polycystic ovary syndrome. J Clin
Endocrinol Metab 84(9): 3030–3035.
Tock L, Carneiro G, Pereira AZ, Tufik S & Zanella MT (2014) Adrenocortical production
is associated with higher levels of luteinizing hormone in nonobese women with
polycystic ovary syndrome. Int J Endocrinol 2014: 620605.
Torchen LC, Fogel NR, Brickman WJ, Paparodis R & Dunaif A (2014) Persistent
Apparent Pancreatic beta-Cell Defects in Premenarchal PCOS Relatives. J Clin
Endocrinol Metab 99(10): 3855–3862.
Tracy RP, Psaty BM, Macy E, Bovill EG, Cushman M, Cornell ES & Kuller LH (1997)
Lifetime smoking exposure affects the association of C-reactive protein with
cardiovascular disease risk factors and subclinical disease in healthy elderly subjects.
Arterioscler Thromb Vasc Biol 17(10): 2167–2176.
109
Tsilchorozidou T, Honour JW & Conway GS (2003) Altered cortisol metabolism in
polycystic ovary syndrome: insulin enhances 5alpha-reduction but not the elevated
adrenal steroid production rates. J Clin Endocrinol Metab 88(12): 5907–5913.
Tso LO, Costello MF, Albuquerque LE, Andriolo RB & Macedo CR (2014) Metformin
treatment before and during IVF or ICSI in women with polycystic ovary syndrome.
Cochrane Database Syst Rev 11: CD006105.
Tuppurainen M, Klimscheffskij R, Venhola M & Dieben TO (2004) The combined
contraceptive vaginal ring (NuvaRing) and lipid metabolism: a comparative study.
Contraception 69(5): 389–394.
van den Heuvel MW, van Bragt AJ, Alnabawy AK & Kaptein MC (2005) Comparison of
ethinylestradiol pharmacokinetics in three hormonal contraceptive formulations: the
vaginal ring, the transdermal patch and an oral contraceptive. Contraception 72(3):
168–174.
van den Oord SC, Sijbrands EJ, ten Kate GL, van Klaveren D, van Domburg RT, van der
Steen AF & Schinkel AF (2013) Carotid intima-media thickness for cardiovascular
risk assessment: systematic review and meta-analysis. Atherosclerosis 228(1): 1–11.
van der Wal AC, Becker AE, van der Loos CM & Das PK (1994) Site of intimal rupture or
erosion of thrombosed coronary atherosclerotic plaques is characterized by an
inflammatory process irrespective of the dominant plaque morphology. Circulation
89(1): 36–44.
Vaskivuo TE, Anttonen M, Herva R, Billig H, Dorland M, te Velde ER, Stenback F,
Heikinheimo M & Tapanainen JS (2001) Survival of human ovarian follicles from
fetal to adult life: apoptosis, apoptosis-related proteins, and transcription factor
GATA-4. J Clin Endocrinol Metab 86(7): 3421–3429.
Velazquez EM, Mendoza S, Hamer T, Sosa F & Glueck CJ (1994) Metformin therapy in
polycystic ovary syndrome reduces hyperinsulinemia, insulin resistance,
hyperandrogenemia, and systolic blood pressure, while facilitating normal menses and
pregnancy. Metabolism 43(5): 647–654.
Velling Magnussen L, Mumm H, Andersen M & Glintborg D (2011) Hemoglobin A1c as a
tool for the diagnosis of type 2 diabetes in 208 premenopausal women with polycystic
ovary syndrome. Fertil Steril 96(5): 1275–1280.
Verma S, Wang CH, Li SH, Dumont AS, Fedak PW, Badiwala MV, Dhillon B, Weisel RD,
Li RK, Mickle DA & Stewart DJ (2002) A self-fulfilling prophecy: C-reactive protein
attenuates nitric oxide production and inhibits angiogenesis. Circulation 106(8): 913–
919.
Visser M, Bouter LM, McQuillan GM, Wener MH & Harris TB (1999) Elevated Creactive protein levels in overweight and obese adults. JAMA 282(22): 2131–2135.
Vrbikova J, Hill M, Bendlova B, Grimmichova T, Dvorakova K, Vondra K & Pacini G
(2008) Incretin levels in polycystic ovary syndrome. Eur J Endocrinol 159(2): 121–
127.
Webber LJ, Stubbs S, Stark J, Trew GH, Margara R, Hardy K & Franks S (2003)
Formation and early development of follicles in the polycystic ovary. Lancet
362(9389): 1017–1021.
110
White T, Jain JK & Stanczyk FZ (2005) Effect of oral versus transdermal steroidal
contraceptives on androgenic markers. Am J Obstet Gynecol 192(6): 2055–2059.
White T, Ozel B, Jain JK & Stanczyk FZ (2006) Effects of transdermal and oral
contraceptives on estrogen-sensitive hepatic proteins. Contraception 74(4): 293–296.
Wickenheisser JK, Nelson-Degrave VL & McAllister JM (2005) Dysregulation of
cytochrome P450 17alpha-hydroxylase messenger ribonucleic acid stability in theca
cells isolated from women with polycystic ovary syndrome. J Clin Endocrinol Metab
90(3): 1720–1727.
Wickenheisser JK, Nelson-DeGrave VL, Quinn PG & McAllister JM (2004) Increased
cytochrome P450 17alpha-hydroxylase promoter function in theca cells isolated from
patients with polycystic ovary syndrome involves nuclear factor-1. Mol Endocrinol
18(3): 588–605.
Wickenheisser JK, Quinn PG, Nelson VL, Legro RS, Strauss JF,3rd & McAllister JM
(2000) Differential activity of the cytochrome P450 17alpha-hydroxylase and
steroidogenic acute regulatory protein gene promoters in normal and polycystic ovary
syndrome theca cells. J Clin Endocrinol Metab 85(6): 2304–2311.
Wiegratz I, Kutschera E, Lee JH, Moore C, Mellinger U, Winkler UH & Kuhl H (2003)
Effect of four different oral contraceptives on various sex hormones and serumbinding globulins. Contraception 67(1): 25–32.
Wild RA, Carmina E, Diamanti-Kandarakis E, Dokras A, Escobar-Morreale HF,
Futterweit W, Lobo R, Norman RJ, Talbott E & Dumesic DA (2010) Assessment of
cardiovascular risk and prevention of cardiovascular disease in women with the
polycystic ovary syndrome: a consensus statement by the Androgen Excess and
Polycystic Ovary Syndrome (AE-PCOS) Society. J Clin Endocrinol Metab 95(5):
2038–2049.
Wild RA, Grubb B, Hartz A, Van Nort JJ, Bachman W & Bartholomew M (1990) Clinical
signs of androgen excess as risk factors for coronary artery disease. Fertil Steril 54(2):
255–259.
Wild RA, Rizzo M, Clifton S & Carmina E (2011) Lipid levels in polycystic ovary
syndrome: systematic review and meta-analysis. Fertil Steril 95(3): 1073-9.e1-11.
Wild S, Pierpoint T, McKeigue P & Jacobs H (2000) Cardiovascular disease in women
with polycystic ovary syndrome at long-term follow-up: a retrospective cohort study.
Clin Endocrinol (Oxf) 52(5): 595–600.
Winters SJ, Talbott E, Guzick DS, Zborowski J & McHugh KP (2000) Serum testosterone
levels decrease in middle age in women with the polycystic ovary syndrome. Fertil
Steril 73(4): 724–729.
Wood JR & Strauss JF,3rd (2002) Multiple signal transduction pathways regulate ovarian
steroidogenesis. Rev Endocr Metab Disord 3(1): 33–46.
World Health Organization (2006) Definition and diagnosis of diabetes mellitus and
intermediate hyperglycaemia.
World HO (2005) WHO STEPS Surveillance Manual: The WHO STEPSwise approach to
chronic disease risk factor surveillance. Geneva, World Health Organizaqtion.
111
Wu S, Divall S, Nwaopara A, Radovick S, Wondisford F, Ko C & Wolfe A (2014)
Obesity-induced infertility and hyperandrogenism are corrected by deletion of the
insulin receptor in the ovarian theca cell. Diabetes 63(4): 1270–1282.
Xu XF, De Pergola G & Bjorntorp P (1991) Testosterone increases lipolysis and the
number of beta-adrenoceptors in male rat adipocytes. Endocrinology 128(1): 379–382.
Yada T, Nakata M, Shiraishi T & Kakei M (1999) Inhibition by simvastatin, but not
pravastatin, of glucose-induced cytosolic Ca2+ signalling and insulin secretion due to
blockade of L-type Ca2+ channels in rat islet beta-cells. Br J Pharmacol 126(5):
1205–1213.
Yalamanchi SK, Sam S, Cardenas MO, Holaday LW, Urbanek M & Dunaif A (2012)
Association of fibrillin-3 and transcription factor-7-like 2 gene variants with
metabolic phenotypes in PCOS. Obesity (Silver Spring) 20(6): 1273–1278.
Yarali H, Yildirir A, Aybar F, Kabakci G, Bukulmez O, Akgul E & Oto A (2001) Diastolic
dysfunction and increased serum homocysteine concentrations may contribute to
increased cardiovascular risk in patients with polycystic ovary syndrome. Fertil Steril
76(3): 511–516.
Yildirim B, Sabir N & Kaleli B (2003) Relation of intra-abdominal fat distribution to
metabolic disorders in nonobese patients with polycystic ovary syndrome. Fertil Steril
79(6): 1358–1364.
Yildiz BO (2015) Approach to the patient: contraception in women with polycystic ovary
syndrome. J Clin Endocrinol Metab 100(3): 794–802.
Yildiz BO, Bozdag G, Yapici Z, Esinler I & Yarali H (2012) Prevalence, phenotype and
cardiometabolic risk of polycystic ovary syndrome under different diagnostic criteria.
Hum Reprod 27(10): 3067–3073.
Yildiz BO, Knochenhauer ES & Azziz R (2008) Impact of obesity on the risk for
polycystic ovary syndrome. J Clin Endocrinol Metab 93(1): 162–168.
Yildiz BO, Woods KS, Stanczyk F, Bartolucci A & Azziz R (2004) Stability of
adrenocortical steroidogenesis over time in healthy women and women with
polycystic ovary syndrome. J Clin Endocrinol Metab 89(11): 5558–5562.
Yildiz BO, Yarali H, Oguz H & Bayraktar M (2003) Glucose intolerance, insulin
resistance, and hyperandrogenemia in first degree relatives of women with polycystic
ovary syndrome. J Clin Endocrinol Metab 88(5): 2031–2036.
Zawadzki JK & Dunaif A (1992) Diagnostic criteria for polycystic ovary syndrome. In:
Dunaif A, Givens J, Haseltine F & Merriam GR (eds) Polycystic Ovary Syndrome.
Boston, Blackwell Scientific Publications: 377–384.
Zwaka TP, Hombach V & Torzewski J (2001) C-reactive protein-mediated low density
lipoprotein uptake by macrophages: implications for atherosclerosis. Circulation
103(9): 1194–1197.
112
Original publications
I
Puurunen J, Piltonen T, Jaakkola P, Ruokonen A, Morin-Papunen L & Tapanainen JS
(2009) Adrenal androgen production capacity remains high up to menopause in
women with polycystic ovary syndrome. J Clin Endocrinol Metab 94(6):1973–8.
II Puurunen J, Piltonen T, Morin-Papunen L, Perheentupa A, Järvelä I, Ruokonen A &
Tapanainen JS (2011) Unfavorable hormonal, metabolic, and inflammatory alterations
persist after menopause in women with PCOS. J Clin Endocrinol Metab 96(6):1827–
34.
III Puurunen J, Piltonen T, Puukka K, Ruokonen A, Savolainen MJ, Bloigu R, MorinPapunen L & Tapanainen JS (2013) Statin therapy worsens insulin sensitivity in
women with polycystic ovary syndrome (PCOS): a prospective, randomized, doubleblind, placebo-controlled study. J Clin Endocrinol Metab 98(12):4798–807.
IV Piltonen T*, Puurunen J*, Hedberg P, Ruokonen A, Mutt SJ, Herzig KH, Nissinen A,
Morin-Papunen L & Tapanainen JS (2012) Oral, transdermal and vaginal combined
contraceptives induce an increase in markers of chronic inflammation and impair
insulin sensitivity in young healthy normal-weight women: a randomized study. Hum
Reprod 27(10):3046–56.
*equal contribution
Reprinted with permission from The Endocrine Society (I–III) and Oxford
University Press (IV).
Original publications are not included in the electronic version of the dissertation.
113
114
ACTA UNIVERSITATIS OULUENSIS
SERIES D MEDICA
1283. Orajärvi, Marko (2015) Effect of estrogen and dietary loading on rat condylar
cartilage
1284. Bujtár, Péter (2015) Biomechanical investigation of the mandible, a related donor
site and reconstructions for optimal load-bearing
1285. Ylimäki, Eeva-Leena (2015) Ohjausintervention vaikuttavuus elintapoihin ja
elintapamuutokseen sitoutumiseen
1286. Rautiainen, Jari (2015) Novel magnetic resonance imaging techniques for articular
cartilage and subchondral bone : studies on MRI Relaxometry and short echo
time imaging
1287. Juola, Pauliina (2015) Outcomes and their predictors in schizophrenia in the
Northern Finland Birth Cohort 1966
1288. Karsikas, Sara (2015) Hypoxia-inducible factor prolyl 4-hydroxylase-2 in cardiac
and skeletal muscle ischemia and metabolism
1289. Takatalo, Jani (2015) Degenerative findings on MRI of the lumbar spine :
prevalence, environmental determinants and association with low back symptoms
1290. Pesonen, Hanna-Mari (2015) Managing life with a memory disorder : the mutual
processes of those with memory disorders and their family caregivers following a
diagnosis
1291. Pruikkonen, Hannele (2015) Viral infection induced respiratory distress in
childhood
1292. Vähänikkilä, Hannu (2015) Statistical methods in dental research, with special
reference to time-to-event methods
1293. Sipola-Leppänen, Marika (2015) Preterm birth and cardiometabolic risk factors in
adolescence and early adulthood
1294. Koskenkorva, Timo (2015) Outcome after tonsillectomy in adult patients with
recurrent pharyngitis
1295. Pietilä, Riikka (2015) Angiopoietin 1 and 2-regulated Tie2 receptor translocation
in endothelial cells and investigation of Angiopoietin-2 splice variant 443
1296. Timlin, Ulla (2015) Adolescent's adherence to treatment in psychiatric care
1297. Aatsinki, Sanna-Mari (2015) Regulation of hepatic glucose homeostasis and
Cytochrome P450 enzymes by energy-sensing coactivator PGC-1?
1298. Rissanen, Ina (2015) Nervous system medications and suicidal ideation and
behaviour : the Northern Finland Birth Cohort 1966
Book orders:
Granum: Virtual book store
http://granum.uta.fi/granum/
D 1299
OULU 2015
UNIVERSITY OF OUL U P.O. Box 8000 FI-90014 UNIVERSITY OF OULU FINLA ND
U N I V E R S I TAT I S
O U L U E N S I S
ACTA
A C TA
D 1299
ACTA
U N I V E R S I T AT I S O U L U E N S I S
Johanna Puurunen
University Lecturer Santeri Palviainen
Postdoctoral research fellow Sanna Taskila
Professor Olli Vuolteenaho
University Lecturer Veli-Matti Ulvinen
Johanna Puurunen
Professor Esa Hohtola
ANDROGEN SECRETION
AND CARDIOVASCULAR RISK
FACTORS IN WOMEN WITH
AND WITHOUT PCOS
STUDIES ON AGE-RELATED CHANGES AND
MEDICAL INTERVENTION
Director Sinikka Eskelinen
Professor Jari Juga
University Lecturer Anu Soikkeli
Professor Olli Vuolteenaho
Publications Editor Kirsti Nurkkala
ISBN 978-952-62-0808-4 (Paperback)
ISBN 978-952-62-0809-1 (PDF)
ISSN 0355-3221 (Print)
ISSN 1796-2234 (Online)
UNIVERSITY OF OULU GRADUATE SCHOOL;
UNIVERSITY OF OULU,
FACULTY OF MEDICINE;
MEDICAL RESEARCH CENTER OULU;
OULU UNIVERSITY HOSPITAL;
NATIONAL GRADUATE SCHOOL OF CLINICAL INVESTIGATION
D
MEDICA