ENDOCRINE AND METABOLIC CHANGES IN WOMEN WITH POLYCYSTIC OVARIES AND POLYCYSTIC OVARY SYNDROME

RIITTAKOIVUNEN

Department of Obstetrics andGynaecology, University of Oulu

OULU 2001

RIITTA KOIVUNEN

ENDOCRINE AND METABOLIC CHANGES IN WOMEN WITH POLYCYSTIC OVARIES AND POLYCYSTIC OVARY SYNDROME

Academic Dissertation to be presented with the assent ofthe Faculty of Medicine, University of Oulu, for publicdiscussion in the Auditorium 4 of the University Hospitalof Oulu, on August 24th, 2001, at 12 noon.

OULUN YLIOPISTO, OULU 2001

Copyright © 2001University of Oulu, 2001

Manuscript received 20 June 2001Manuscript accepted 27 June 2001

Communicated byDocent Dan ApterDoctor Niklas Simberg

ISBN 951-42-6426-6 (URL: http://herkules.oulu.fi/isbn9514264266/)

ALSO AVAILABLE IN PRINTED FORMATISBN 951-42-6425-8ISSN 0355-3221 (URL: http://herkules.oulu.fi/issn03553221/)

OULU UNIVERSITY PRESSOULU 2001

Koivunen, Riitta, Endocrine and metabolic changes in women with polycystic ovariesand polycystic ovary syndrome Department of Obstetrics and Gynaecology, University of Oulu, P.O.Box 5000, FIN-90014University of Oulu, Finland 2001Oulu, Finland(Manuscript received 20 June 2001)

Abstract

The prevalence of the isolated ultrasonographic finding of polycystic ovaries (PCO) in the Finnishpopulation and among women with a history of gestational diabetes (GDM) and changes in thepresent carbohydrate metabolism were investigated in the present study. One aim of this study was toinvestigate the prevalence of the recently discovered variant type LH (v-LH) in PCOS and to comparepatient cohorts from Finland, the Netherlands, the United Kingdom and the United States of America.In addition, this study attempted to evaluate the nature of the ovarian streoidogenic response ofwomen with PCOS to exogenously administered human chorionic gonadotrophin (hCG), humanmenotrophin (hMG) and follicle stimulating hormone (FSH). The effect of metformin on ovariansteroidogenesis was also studied.

The prevalence of PCO was significantly higher in younger (≤ 35 years, 21.6%) than among olderwomen (in ≥ 36 years, 7.8%). The overall prevalence of PCO in Finnish women was 14.2%. Womenwith previous GDM revealed a high prevalence of PCO (39.4%). The carrier frequency of the v-LHballele in the entire study population was 18.5%. The frequency of the v-LH carrier was significantlylower in obese PCOS subjects in the Netherlands (2.0%) and Finland (4.5%). Women with previousGDM had impaired insulin sensitivity and β-cell function. They also had higher adrenal androgensecretion than the control women. Women with PCO and previous GDM had markedhyperinsulinemia which was not explained by obesity. Obese PCOS women achieved peak peripheralserum T concentrations at 48 hours after a hCG injection, preceded by peak levels of 17-OHP and E2at 24 hours. In contrast, all steroids measured in the control women reached their maximum serumconcentrations at 96 hours. HMG stimulated the production of ovarian androgens more efficientlythan a urinary FSH after pituitary suppression with a gonadotrophin releasing hormone agonist(GnRHa).

In conclusion, the prevalence of PCO is common in healthy Finnish women and even morecommon in women with a history of GDM. The ultrasonographic appearance of PCO may be apredictive factor with regards abnormal glucose tolerance during and after pregnancy and, thesewomen should therefore be advised as to possible consequences. The high overall frequency of thev-LH allele in women in general and its low frequency in obese PCOS patients suggests that v-LHplays a role in reproductive functions and may counteract the pathogenesis of PCOS in obeseindividuals. The differences observed in steroid responses to hCG between normal and PCOS womenmight be explained by higher theca cell activity or mass in polycystic ovaries. Women with PCOSdid not show a distinctly exaggerated steroidogenic response to hMG or FSH administrationcompared with control women. FSH administration also resulted in increased A and T production.

Keywords: steroidogenesis, PCO, PCOS, Variant-LH, gestational diabetes

To Sini

Acknowledgements

The research for this thesis was carried out at the Departments of Obstetrics and Gynae-cology, and Clinical Chemistry, Oulu University, during the years 1994-2001.

I owe my thanks to Professor Pentti Jouppila, M.D., Head of the Department of Obste-rics and Gynecology, and to Emeritus Professor Antti Kauppila, M.D., the former Head ofthe Department of Obstetrics and Gynaecology, for their support and encouragement andfor creating such good conditions to carry out research in the clinic.

My deepest feelings of gratitude are directed to both of my teachers and supervisors;Professor Juha Tapanainen, M.D. and Docent Hannu Martikainen, M.D., for unwaveringsupport and skilful guidance throughout the study. Professor Juha Tapanainen has provi-ded advice, critics and unfailing encouragement whenever needed. His clear and logicalthinking, positive attitude to life in general and his organizational abilities are attributeswell worth learning. Docent Hannu Martikainen guided my first steps in the world ofscience and tactfully kept me on the right road, when I got my “really good” ideas. Hiscreative way of thinking has impressed me during these years. Despite their multiple res-ponsibilities, both of my supervisors had always time to listen my problems and, due totheir wide knowledge in the field of endocrinology, they find solutions to them. Workingunder their supervision is a distinct privilege. I thank you both for suffering throughvarious versions of my manuscripts during these years.

I wish to express my sincere gratitude to Professor Aimo Ruokonen, M.D., Depart-ment of Clinical Chemistry, for providing the service of monitoring of blood samples andfor his help with the original manuscripts. I am grateful to Docent Timo Laatikainen,M.D., who took me under his wings at first and guided me into the field of scientificinvestigation.

I have been privileged to share desperate and joyful moments of this work with my fri-end Laure Morin-Papunen, M.D., Ph.D. Her optimistic and humanistic attitude to life hasbeen as an example to me. Her knowledge of PCOS, help with the recruitment of thepatients, redaction of the original manuscripts and constructive criticism has been andwill be in a great value to me.

I owe my sincere gratitude to my co-worker Ilkka Vauhkonen, M.D., Ph.D. His know-ledge of the confusing world of the insulin metabolism, his capability to “keep it simple”and his redaction of the original manusript have been invaluable to me. I warmly thankCândido Tomás, M.D., Ph.D., for patiently introducing me the world of computers andstatistics. I would also express my gratitude to my co-workers Professor Ilpo Huhta-niemi, M.D., Professor Richard Clayton, M.D., Professor Bart Fauser, M.D., Professor

Stephen Franks, M.D., Professor Ann Taylor, M.D., Docent Leena Anttila, M.D., DocentKim Pettersson, M.D., Davinia White, M.D., Madhurima Rajkhowa M.D., their co-opera-tion made the study II of this thesis possible. I wish to thank Jaana Juutinen, M.D., for herhelp and collaboration during this study.

I owe my respectful gratitude to the official reviewers of this thesis, Docent Dan Apter,M.D. and Doctor Niklas Simberg, M.D., Ph.D., for their critically constructive comments,which saved me from many errors and definitely helped to improve the final manuscript.

I am greatly indebted to Mirja Ahvensalmi, Anni Jurvakainen and the stuff of thedepartment 15 for taking such a good care of the patients during the clamp, to RitvaVasala, Pirjo Ylimartimo, Annu Asukas, Raija Tolkkinen and the rest of the stuff at theResearch Unit for being so flexible whenever I needed help. I warmly thank Anja Heikki-nen, of the Clinical Chemistry laboratory, for running majority of the hormone analysis. Iowe my special thanks to Maire Syväri and Anneli Tiittanen for their smiling secretarialhelp during these days when there is always someone knocking on their door.

I want to warmly thank Michael Spalding, M.D. and Nick Bolton, Ph.D., for carefullyand patiently revising the English language. I owe my sincere thanks to Sirkka Pramila,M.Sc., who passed away during this study, and to Risto Bloigu, M.Sc., for their assistancewith statistical analysis.

Above all, I am grateful to all those women who generously volunteered to these stu-dies, although some of the experiments were rather laborious for them.

The flexibility, loyality and joy of life of the “Ultrasound sisters and brothers” madethe years of this research and my specializing in gynecology to the one of the most enjo-yable time of my life. I want to thank Eila Suvanto-Luukkonen, M.D., Ph.D., for herloyalty and for checking up my “burn-out”-level regularly, Kaarin Mäkikallio-Anttila,M.D., for all those hours we have spent talking about the meaning of our lives, PäiviVuolo-Merilä, M.D., for taking care of my physical condition and mental regression whenneeded, Merja Kurkinen-Räty, M.D., Ph.D, for being herself and shearing the hours of thenight in the computer room, and Jari Johansson, M.D., for the help with computers. Thecompanionship of Anna Kivijärvi, Kirsti Paajanen, Antti Perheentupa, Anneli Pouta,Marja Simojoki, Heli Spalding, Marja Vääräsmäki, Merja Varho and Leena Väisälä havebrightened these years with this thesis.

The hilarious annual “Jyväskylä in my mind” meetings with Mervi, Teppo, Anne,Timo, Outi, Jaakko, Satu and Juha have been milestones during this research. I alwaysfinished something day before our get-together.

Finally, I wish to thank my dear parents Eila and Professor Kalevi Jokinen for theirpatient love and support. I wish I could offer at least half as good life to my child as theyhave given to me. I am grateful also to my excellent parents-in-law, Sirpa and Seppo Koi-vunen, for being always understanding, helpful and interested in what their daughter-in-law is doing, although they knew the time is taken away from their son and grandchild.

Without the love and an extraordinary patience of my husband Petri the combinationof our marriage and this thesis would never had been possible. Petri’s never-failing sup-port and his belief in my capabilities has given me the strength I have needed to continueand thanks to my sunshine and antidepressant, Sini, I finally forced myself to finish thisthesis.

This research has been supported by grants provided by the University of Oulu, TheFinnish Gynaecologic Association, the Sigrid Jusélius Foundation and the Academy ofFinland.

Oulu, August 2001 Riitta Koivunen

Abbreviations

A androstenedioneACTH adrenocorticotropic hormoneAUC area under curveBMI body mass indexCC clomiphene citrateCI confidence intervalCPA cyproterone acetateDHEA dehydroepiandrosteroneDHEAS dehydroepiandrosterone sulphateDHT 5α-dihydrotestosteroneDM diabetes mellitusDNA deoxyribonucleic acidE2 estradiolFAI free androgen indexFFA free fatty acidFOH functional ovarian hyperandrogenismFSH follicle stimulating hormoneGDM gestational diabetes mellitusGLUT glucose transporterGnRH gonadotrophin releasing hormoneGnRHa gonadotrophin releasing hormone agonistHDL high density lipoproteinHCG human chorionic gonadotrophinHMG human menopausal gonadotrophin17-OHP 17-hydroxyprogesteroneIFG impaired fasting glucoseIGF insulin-like growth factorIGFBP insulin-like growth factor binding proteinIGT impaired glucose toleranceIRS insulin receptor substrateIVF in vitro fertilization

IVGTT intravenous glucose tolerance testLDL low density lipoproteinLH luteinizing hormoneMab monoclonal antibodyMFO multifollicular ovariesNPY neuropeptide YOC oral contraceptiveOGTT oral glucose tolerance testPAI-1 plasminogen activator inhibitorPCO polycystic ovariesPCOS polycystic ovary syndromeP450scc cholesterol side chain cleavage enzymeP450c cytochrome P450RIA radioimmunoassayRNA ribonucleic acidRSA recurrent spontaneous abortionSD standard deviationSE standard errorSHBG sex hormone binding globulinT testosteroneTA total areaTrigly triglyceridesVLDL very low-density lipoproteinv-LH variant type of luteinizing hormoneWHR waist-hip ratio

List of original articles

The thesis is based on the following articles, which are referred to in the text by theirRoman numerals:I Koivunen R, Laatikainen T, Tomás C, Huhtaniemi I, Tapanainen J & Martikainen H

(1999) The prevalence of polycystic ovaries in healthy women. Acta Obstetricia etGynecologica Scandinavica 78: 137-141.

II Tapanainen J, Koivunen R, Fauser B, Taylor A, Clayton R, Rajkhowa M, White D,Franks S, Anttila L, Pettersson K & Huhtaniemi I (1999) A new contributing factor topolycystic ovary syndrome: the genetic variant of luteinizing hormone. Journal of Clin-ical Endocrinology & Metabolism 84: 1711-1715.

III Koivunen R, Juutinen J, Vauhkonen I, Ruokonen A & Tapanainen J (2001) Metabolicand steroidogenic alterations related to increased frequency of polycystic ovaries inwomen with history of gestational diabetes. Journal of Clinical Endocrinology & Me-tabolism 86: 2591-2599.

IV Koivunen R, Morin-Papunen L, Ruokonen A, Tapanainen J & Martikainen H. (2001)Ovarian steroidogenic response to human chorionic gonadotrophin in obese womenwith polycystic ovary syndrome: effect of metformin. Manuscript in press, Human Re-production.

V Koivunen R, Ruokonen A & Martikainen H. Steroidogenic response to gonadotrophinstimulation in normal and PCOS women undergoing IVF treatment. Manuscript sub-mitted.

Contents

Abstract Acknowledgements Abbreviations List of original articles 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 Review of the literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2.1 General background of polycystic ovary syndrome (PCOS) . . . . . . . . . . . . . . . 192.2 Epidemiology of PCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.3 Clinical features of PCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

2.3.1 Women with an isolated finding of polycystic ovaries . . . . . . . . . . . . . . . 212.3.2 Present definition of polycystic ovaries . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2.4 Biochemical and clinical features of PCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242.4.1 Gonadotrophins in PCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

2.4.1.1 Inappropriate gonadotrophin secretion . . . . . . . . . . . . . . . . . . . . . 242.4.1.2 Variant type of luteinizing hormone . . . . . . . . . . . . . . . . . . . . . . . 25

2.4.2 Steroidogenesis in PCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272.4.2.1 Androgen secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272.4.2.2 Ovarian steroidogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272.4.2.3 Adrenal steroidogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292.4.2.4 Peripheral steroid metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

2.4.3 Metabolic features in PCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312.4.3.1 Glucose tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312.4.3.2 Insulin resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322.4.3.3 Insulin secretion and clearance . . . . . . . . . . . . . . . . . . . . . . . . . . . 332.4.3.4 Molecular mechanisms of insulin resistance in PCOS . . . . . . . . . 342.4.3.5 Insulin action in polycystic ovary . . . . . . . . . . . . . . . . . . . . . . . . . 362.4.3.6 Causal association of androgens and insulin resistance . . . . . . . . . 372.4.3.7 Insulin resistance in women with PCO . . . . . . . . . . . . . . . . . . . . . 40

2.4.4 Insulin-like growth factors in PCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402.4.5 Leptin and PCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422.4.6 Lipids and PCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432.4.7 Genetics of PCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

2.5 Long-term sequelae and risks in PCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

2.5.1 Dyslipidemia and cardiovascular disease . . . . . . . . . . . . . . . . . . . . . . . . . . 452.5.2 Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452.5.3 Gestational diabetes and diabetes mellitus . . . . . . . . . . . . . . . . . . . . . . . . . 462.5.4 Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

2.6 Modern treatment of PCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472.6.1 Treatment of hyperandrogenism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472.6.2 Treatment of irregular cycles and anovulation . . . . . . . . . . . . . . . . . . . . . . 482.6.3 Insulin-lowering treatment in PCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

2.6.3.1 Weight loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502.6.3.2 Insulin lowering medication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

3 Purpose of the present study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544 Subjects and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

4.1 Subjects and study design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554.2 Clinical parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584.3 Vaginal ultrasonography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584.4 Fasting insulin to glucose ratio and the homeostasis

model assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594.5 Oral glucose tolerance test and early phase insulin and

C-peptide measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594.6 Euglycemic hyperinsulinemic clamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594.7 Calorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604.8 Human chorion gonadotrophin stimulation test . . . . . . . . . . . . . . . . . . . . . . . . . 604.9 Treatment protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614.10Laboratory methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614.11Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

5 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645.1 Clinical parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645.2 Prevalence of polycystic ovaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645.3 Variant type of luteinizing hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 655.4 Effects of previous gestational diabetes (GDM) . . . . . . . . . . . . . . . . . . . . . . . . . 66

5.4.1 Clinical characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665.4.1.1 Women with previous GDM versus control subjects . . . . . . . . . . 665.4.1.2 Control women: comparison between women with normal ovaries and

those with polycystic ovaries (PCO) . . . . . . . . . . . . . . . . . . . . . . . 665.4.1.3 Women with previous GDM: comparison between women with

normal ovaries and those with PCO . . . . . . . . . . . . . . . . . . . . . . . . 665.4.2 Oral glucose tolerance test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

5.4.2.1 Women with previous GDM versus controls . . . . . . . . . . . . . . . . . 695.4.2.2 Women with previous GDM: a comparison between women with

normal and polycystic ovaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705.4.3 Insulin secretion and insulin sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

5.4.3.1 Women with previous GDM versus control subjects . . . . . . . . . . 705.4.3.2 Women with previous GDM: comparison between women with

normal and polycystic ovaries . . . . . . . . . . . . . . . . . . . . . . . . . . . 725.5 Endocrine parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

5.5.1 Women with previous GDM versus controls . . . . . . . . . . . . . . . . . . . . . . . 72

5.5.1.1 Control women: a comparison between women withnormal and polycystic ovaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

5.5.1.2 Women with previous GDM: a comparison between women with normal and polycystic ovaries . . . . . . . . . . . . . . . . . . 72

5.6 Steroidogenesis in women with PCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735.6.1 Effects of human chorionic gonadotrophin . . . . . . . . . . . . . . . . . . . . . . . . 735.6.2 Effects of metformin on steroidogenesis and on serum insulin and leptin

concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735.6.3 Effects of human menopausal gonadotrophin and follicle stimulating

hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

6.1 Prevalence of an isolated finding of polycystic ovaries . . . . . . . . . . . . . . . . . . . 786.2 Variant type of luteinizing hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796.3 Insulin sensitivity, and metabolic and endocrine features in women with a history of

GDM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 806.3.1 Association between previous GDM and PCO . . . . . . . . . . . . . . . . . . . . . 81

6.4 The effect of stimulation with human chorion gonadotrophin, human menopausal gonadotrophin andfollicle stimulating hormone on steroidogenesisin women with PCOS and in control women . . . . . . . . . . . . . . . . . . . . . . . . . . . 836.4.1 The effect of short-term human chorion

gonadotrophin stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836.4.2 The effect of long-term human menopausal gonadotrophin

and follicle stimulating hormone stimulation . . . . . . . . . . . . . . . . . . . . . . . 846.4.3 The effect of metformin on steroidogenesis in women with PCOS . . . . . . 866.4.4 The effect of metformin on serum leptin concentrations . . . . . . . . . . . . . . 87

7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 888 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

1 Introduction

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among wom-en of fertile age. The prevalence of PCOS varies between 2.5 and 7.5% (Futterweit &Mechanick 1988, Knochenhauer et al. 1998, Taylor 2000a). Its clinical manifestationsinclude menstrual dysfunction and hyperandrogenic symptoms, and it can be associatedwith metabolic dysfunction in which hyperinsulinemia and peripheral insulin resistanceare central features. A significant proportion of women with PCOS have insulin resis-tance greater than that of their weight-matched controls. Furthermore PCOS is thought topresent an early manifestation of the metabolic syndrome (or syndrome X), which is acluster of abnormalities where the combination of insulin resistance and compensatoryhyperinsulinemia predisposes individuals to develop a high plasma triglyseride (Trigly)and a low high-density lipoprotein (HDL) cholesterol concentration, high blood pressureand coronary heart disease (Reaven 1994). Women with PCOS may represent one of thelargest unique groups of women at high risk for the development of early onset coronaryheart disease (Franks 1995, Talbott et al. 2000). It has been proposed that there is a mildform of PCOS which includes women who have mild hyperandrogenism and an isolatedultrasonic finding of polycystic ovaries but whose ovulatory function is maintained(PCO). These women may be susceptible to developing the syndrome as well. Thus, theymay also be subject to increased morbidity (Carmina & Lobo 1999).

The etiology of PCOS is still obscure. It has been well documented that inappropriategonadotrophin secretion, especially high luteinizing hormone (LH) secretion, is asso-ciated with the classic form of PCOS (MacArthur et al. 1958, Yen et al. 1970). The fre-quency of the recently discovered genetic variant form of LH (v-LH) is high in the Fin-nish population (Haavisto et al. 1995). In the United Kingdom, women heterozygous forthe v-LH allele have higher levels of serum testosterone (T), estradiol (E2) and sex-hor-mone-binding globulin (SHBG), which may indicate differences in ovarian LH actionbetween normal (or wild type, wt) LH and v-LH (Rajkhow et al. 1995).

Several studies have indicated that polycystic ovaries usually produce excess androgen(Kirschner et al. 1976, Chang et al. 1983, Rosenfield 1999). The mechanisms leading toincreased androgen production in PCOS are not completely understood. Chronic LH sti-mulation in PCOS induces sustained hypersecretion of androgens by theca compartment,probably augmented by insulin and insulin-like growth factors (IGFs) (Yen et al. 1970,

18

Poretsky et al. 1999). Most data suggests that the primary dysfunction may be at the ova-rian level (Rosenfield et al. 1990, Franks 1995) or all manifestations of the syndrome mayoccur secondary to hyperinsulinemia (Barbieri et al. 1986, Dunaif 1999).

PCOS women have been shown to have an exaggerated 17-hydroxyprogesterone (17-OHP) and androstenedione (A) response to gonadotrophin releasing hormone agonist(GnRHa) and human chorionic gonadotrophin (hCG). Based on the results of these stu-dies it has been suggested that women with PCOS have a primary dysregulation of ova-rian P450c17, leading to enhanced activities of both 17α-hydroxylase and 17,20-lyase inthe ovarian theca cells (Barnes et al. 1989b, Ibanez et al. 1996, Gilling-Smith et al. 1997,Levrant et al. 1997).

In the present study, we investigated the prevalence of PCO in the Finnish population.We also studied the frequency of v-LH in the Finnish population and compared it topatient cohorts from the Netherlands, the United Kingdom and the United States. Specialattention was paid to occurrence of v-LH in women with PCOS, since LH is considered toplay a central role in this syndrome. Due to the well-known association between insulinresistance and PCOS, the prevalence of PCO in women with a history of gestational dia-betes mellitus (GDM) and the present-day carbohydrate metabolism among these womenwere also examined. In addition, the ovarian steroidogenic response to exogenously admi-nistered gonadotrophins, i.e. hCG, human menopausal gonadotrophin (hMG) and folliclestimulating hormone (FSH) were examined.

2 Review of the literature

2.1 General background of polycystic ovary syndrome (PCOS)

As early as 1844, Chereau described sclerocystic changes in the human ovary (Chereau1844). Although occasional reports on this condition continued to appear over the years,more interest was aroused in 1935 when bilateral polycystic ovaries were related by Steinand Leventhal in a clinical syndrome consisting of “menstrual irregularity featuringamenorrhea, a history of infertility, masculine type hirsutism and, less consistently, obesi-ty (Stein & Leventhal 1935). The condition was for a long time called the Stein-Lev-enthal syndrome.

In 1958 McArthur and coworkers observed elevated LH levels in women with poly-cystic ovaries (MacArthur et al. 1958) and the introduction of radioimmunoassays (RIAs)in 1971 stimulated reliance on a biochemical diagnosis. Although it was suspected asearly as 1962 that there was a wide variety of clinical presentation of PCOS, the conceptof PCOS with normal LH concentrations was not conceived until 1976 (Rebar et al.1976). The next milestone was the discovery of the association of PCOS and insulin resis-tance by Kahn and coworkers (Kahn et al. 1976) and Burghen et al. (Burghen et al.1980). The ultrasonographic finding of polycystic ovaries was described for the first timein 1981 (Swanson et al. 1981). Adams and coworkers introduced a definition for the ult-rasonographic appearance of PCO in 1985 as one diagnostic criterion of PCOS (Adams etal. 1985). This has been widely used thereafter, especially in Europe.

2.2 Epidemiology of PCOS

Data on the prevalence of PCOS are variable due, in part, to the lack of well accepted cri-teria for diagnosis. If PCOS is defined histopathologically (i.e. by the presence of poly-cystic ovaries upon oophorectomy or wedge resection), between 1.4-3.5 of unselectedwomen (Vara & Niemineva 1951) and 0.6-4.3% of infertile women (Breteche 1952) suf-fer from this syndrome.

20

If PCOS is defined by the ultrasonographic appearance of PCO, the prevalence variesdepending on the study settings used. Polycystic ovaries are seen in 92% of women withidiopathic hirsutism, 87% of women with oligomenorrhea (Adams et al. 1986), 21-23%of randomly selected women (Clayton et al. 1992, Farquhar et al. 1994), 23 % of womenwho consider themselves normal and who report regular menstrual cycles (Polson et al.1988) and in 17% of women participating in routine PAP smear (Botsis et al. 1995). Up to25% of patients with this sonographic picture may be entirely asymptomatic (Swanson etal. 1981), however, nor do all patients with hyperandrogenism demonstrate PCO (Orsiniet al. 1985, el Tabbakh et al. 1986, Carmina & Lobo 1999).

When biochemical parameters have been used as diagnostic criteria, the prevalence ofPCOS varies from 2.5-7.5% (Futterweit & Mechanick 1988). It has recently been obser-ved in an unselected, minimally-biased population of consecutive women, that the ove-rall prevalence of PCOS appears to be approximately 4.6%, although it could be as low as3.5% and as high as 11.2% (Knochenhauer et al. 1998). It is accepted, however, thatPCOS is one of the most common reproductive endocrinological disorders in women.

2.3 Clinical features of PCOS

Polycystic ovary syndrome is a syndrome, not a disease, and reflects multiple potentialetiologies and variable clinical presentations. The heterogeneity of the disorder makes thepathogenesis as well as the definition of PCOS difficult. The National Institutes of Health- National Institute of Child Health and Human Development (NIH-NICHD) Conferenceon PCOS was held in April 1990. In that conference a clear-cut definition was notreached, but the majority of participants believed that PCOS should be defined by 1) ovu-latory dysfunction, 2) clinical evidence of hyperandrogenism and/or hyperandrogenemia,and 3) exclusion of related disorders, such as hyperprolactinemia, thyroid dysfunctionand nonclassical adrenal hyperplasia (Zawadzki & Dunaif 1992). A peripubertal onset ofthe symptoms has been used as a diagnostic citeria (Yen 1999). There is strong evidenceof a peripubertal onset of the PCOS (see 2.4.1.1).

Oligomenorrhea or dysfunctional bleeding are frequently early and dominant symp-toms of the anovulatory component of PCOS. The menstrual irregularity of the PCOS ischronic and can be manifested in several different ways. Probably the most common iserratic menstruation owing to anovulation. Some women with PCOS have prolongedamenorrhea associated with endometrial atrophy. Some women have regular cycles atfirst and experience menstrual irregularity in association with weight gain (Taylor 1998).The occurrence of oligomenorrhea may be explained by PCOS in approximately 85-90%of women, whereas 30-40% of amenorrheic patients have been reported to have the disor-der (Goldzieher & Green 1962).

Hyperandrogenism is the second defining characteristic of PCOS. Clinically, the mostcommon sign of hyperandrogenism in PCOS women is hirsutism. The range of the preva-lence of hirsutism in PCOS women varies between 17 and 83% (Goldzieher & Green1963, Guzick 1998). Hirsutism may develop peripubertally or during adolescence (Yen1980) or it may be absent until the third decade of life (McKenna et al. 1983). The Ferri-man and Gallwey scale is most commonly used for the assessment of hirsutism (Ferri-

21

man & Gallwey 1961). Another common sign of hyperandrogenism is acne. Overt signsof virilization, i.e. male pattern balding, alopecia, increased muscle mass, a deepeningvoice or clitoromegaly usually reflect the presence of an androgen-producing tumor orovarian hyperthecosis (Yen 1999).

Infertility was included in the original description of PCOS by Stein and Leventhal(Stein & Leventhal 1935). The prevalence of infertility, caused mainly by anovulation, inPCOS women varies between 35 and 94% (Goldzieher & Green 1963, Franks 1995,Guzick 1998). According to one retrospective study, however, women with PCOS are aslikely to have children as healthy women, although often after infertility treatment (Dahl-gren et al. 1992). Some studies have also described an increased miscarriage rate inPCOS, the mechanism of which is poorly understood. It has been suggested that high fol-licular phase concentrations of LH have a deleterious effect on rates of conception andmiscarriage (Homburg et al. 1988, Balen 1993).

Although there are no controlled systematic studies to determine the exact prevalenceof obesity, most investigators have found that 30-50% of PCOS women are obese (Franks1995). PCOS women tend to have an increased waist-hip ratio, (WHR) i.e. abdominal(visceral) obesity (Rebuffe-Scrive et al. 1989, Bringer et al. 1993).

According to a study of Gülekli et al. including subjects between 14-36 years old,PCOS is a disorder with perimenarchal onset and the clinical, endocrine and ultrasoundfeatures were not changed by the age of 36 years, although patients were prone to gainweight (Gülekli et al. 1993). However, it has also been shown that hyperandrogenismpartly resolves before menopause in women with PCOS and they tend to gain more regu-lar menstrual cycles with increasing age (40 years and more) (Elting et al. 2000, Winterset al. 2000). A decline in follicle cohort has been reported to occur while aging (Faddy etal. 1992). Several findings have suggested, however, that the common denominator inwomen with hyperandrogenic anovulation, whether or not they have typical PCOS, couldbe functional ovarian hyperandrogenism (FOH) (Barnes & Rosenfield 1989a, Ehrmann etal. 1995).

2.3.1 Women with an isolated finding of polycystic ovaries

The isolated finding of polycystic ovaries (PCO, also called polycystic appearance ova-ries, PAO), which meets the calssic ultrasonographic criteria, occurs in 10-33% of thenormal population (Table 1). Normal ovulatory women with PCO (referring only to theovarian morphology) are not considered to have PCOS. A subgroup of these women may,however, have subtle abnormalities resembling PCOS (Takahashi et al. 1992, Norman etal. 1995, Carmina et al. 1997). Women with PCO have exaggerated ovarian responses togonadotrophins and GnRHa (Suikkari et al. 1995, Chang et al. 2000) and disturbances ofglucose metabolism (see 2.4.3.8) as in women with PCOS (Lobo et al. 2000). Womenwith PCO may be susceptible to develop the syndrome and may also have increased mor-bidity associated with PCOS (see 2.5) (Carmina & Lobo 1999).

The presence of an isolated ultrasonographic finding of PCO may be associated withdifferent endocrine dysfunctions. Therefore, the ultrasonic diagnosis of PCO should be

22

supplemented with an endocrinologic biochemical evaluation to exclude other endocrinedysfunctions (Abdel Gadir et al. 1992).

Table 1. A summary of studies on the prevalence of ultrasonographic findings of polycysticovaries (PCO).

2.3.2 Present definition of polycystic ovaries

On histology, the major diagnostic macroscopic features of PCO are bilateral enlarge-ment, thickened ovarian capsule, multiple follicular cysts (usually ranging between 2-8mm in diameter) and an increased amount of stroma (Goldzieher & Green 1962) (Table2).

Table 2. Histologic features of polycystic ovary.

In clinical practice, ultrasonography has replaced the histologic evaluation of PCO andnumerous parameters have been used for definition (Table 3).

Study Number of subjects Age (yr, range) Patient selection PCO (%)

Polson et al. 1988 257 18-36 Volunteers 23

Clayton et al. 1992 190 19-36 Unselected,Random sample

22

Farquhar et al. 1994 183 18-45 Random sample 21

Botsis et al. 1995 1078 17-40 Unselected women participating to rou-tine PAP smear

17

Borgfeldt & Andolf1999

335 25-40 Random sample 10.2

Michelmore et al. 1999 230 18-25 Volunteers 33

Histologic features of polycystic ovary

Whole ovarian hypertrophy

Thickened capsule

Increased number of subcapsular follicle cysts

Scarcity of corporea lutea or albicantia

Hyperplasia and fibrosis of the ovarian stroma

Decreased thickness of the granulosa layer

Atretic pattern of the granulosa layer

Increased thickness of the theca interna

Premature luteinization of theca cells

23

Table 3. Ultrasonographic criteria used for the diagnosis of PCO.

Fig. 1. An ultrasonograph picture of a polycystic ovary.

The ultrasound definition of PCO which has enjoyed the most widespread credibility isthat of Adams and collagues: the presence of either multiple cysts (ten or more) from 2-8mm in diameter distributed evenly around the ovarian periphery with an increased amountof stroma, or (less commonly) multiple small cysts 2-4 mm in diameter distributedthroughout abundant stroma (Fig.1, Adams et al. 1985). The combination of multiple fol-licles and an increased amount of stroma contribute to the overall increase in the ovarian

External morphological signs Internal morphological signs

Increased ovarian area or volume Number of small, echoless regions < 10 mm in size per ovary

Increased roundness index(ovarian width / ovarian length ratio)

Peripheral position of microcysts

Increased echogenity of ovarian stroma

Decreased uterine width / ovarian length ratio (U / O) Increased surface of ovarian stroma on a cross-sec-tional cut, (computerized measure)

Modified from (Adams et al. 1985) (Robert et al. 1995) (Dewailly 1997)

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size, on the other hand, ovarian volume may be within the normal range in a significantproportion of women with all other morphological criteria for PCOS (Yeh et al. 1987,Pache et al. 1992).

It has been proposed that an increased ovarian stroma is the most valuable diagnosticcriteria for PCO to distinguish it from multifollicular ovaries (MFO) (Polson et al. 1988,Conway et al. 1989). MFO are usually detected in women with hypogonadotrophic ame-norrhoea, without increased stroma and filled by six or more follicles 4-10 mm in diame-ter. The observation of a stromal hypertrophy is considered visual and subjective and the-refore the reliability of the diagnosis of PCO is totally dependent on the experience of theultrasonographist. It has been reported that a visual analysis of the ovarian stroma by ult-rasound is more sensitive (not more specific), but also more subjective than a computeri-zed selective measurement (Robert et al. 1995). Increased total ovarian area (total area,TA > 5.5 cm2), which can be easily detected by carefully shaping a strict longitudinalovarian cut) has been shown to have the same diagnostic value as an increased stromalarea by computerized measurement (Robert et al. 1995). A recent study has indicated thatthe number of peripherally distributed follicles > 10 was considered the most sensitivefeature of PCO, while stromal brightness had the best specificity to detect PCO (Atiomoet al. 2000).

Ovarian stroma and volume determinations can be obtained more accurately throughthree-dimensional images than through traditional ultrasonography (Wu et al. 1998).

2.4 Biochemical and clinical features of PCOS

2.4.1 Gonadotrophins in PCOS

2.4.1.1 Inappropriate gonadotrophin secretion

An inappropriate gonadotrophin secretion is associated with the classic form of PCOS.Compared with the follicular phase of the normal menstrual cycle, women with PCOSexhibit a disproportionately high LH secretion with relatively constant low FSH secre-tion (MacArthur et al. 1958, Yen et al. 1970). Therefore, an elevated LH/FSH-ratio of 2-3:1 is commonly used to indicate abnormal gonadotrophin secretion. The prevalence ofincreased serum LH in PCOS ranges from 30% to 90 % (Conway et al. 1989, Franks1989). The reasons for these varied estimates probably include the heterogeneity ofPCOS, different blood sampling frequencies and the specificity of the gonadotrophinassays used. It has been shown, that the mean of two LH values in samples collected at30-min interval significantly reduces random sample variability (Apter et al. 1994b).

An increased LH-pulse frequency in PCOS women, independent of body mass index(BMI) or adiposity, is well established (Waldstreicher et al. 1988, Morales et al. 1996). Ithas been suggested that gonadotrophin defects, particularly an excess of serum LH, is apredominant finding in hyperandrogenic women, whether they be adolescents or olderperimenopausal women (Apter et al. 1994a, Taylor 2000b). The underlying cause of thispattern of gonadotrophin secretion is linked to an accelerated gonadotrophin releasing

25

hormone (GnRH) pulse generator activity and heightened pituitary response to GnRH.LH and FSH synthesis and secretion are highly dependent on the pattern of the GnRH sti-mulus, with rapid frequencies favoring LH and slower pulses FSH synthesis and secretion(Clarke et al. 1984, Waldstreicher et al. 1988). Obesity only seems to lower the LH-pulseamplitude and the peak increment of LH in response to GnRH stimulation, but the LH-pulse frequency in PCOS women is not influenced by BMI (Morales et al. 1996, Arroyoet al. 1997).

The mechanism(s) underlying the abnormal regulation of GnRH in PCOS women hasremained unclear. It has been postulated that altered inputs to the GnRH neuronal systemby insulin, IGFs and/or sex steroids during the critical development phase of adrenarche/puberty may induce a dysregulation of GnRH pulse generator activities, a propositionconsonant with observations made in peripubertal girls with PCOS (Apter et al. 1994a,Apter et al. 1995). Chronically elevated levels of estrone, a weak estrogen aromatizedperipherally from andostenedione (A) in women with PCOS, can augment pituitary sensi-tivity to GnRH both by a direct action on gonadotrophin synthesis and by enhancingGnRH-induced GnRH receptors (Lobo et al. 1981). Studies have attempted to defineabnormalities of hypothalamic neurotransmitter (i.e. dopamine, opioids, alpha-adrenergicsystem) function in the pathogenesis of PCOS. It has not yet been demonstrated, however,whether or not neurotransmitters play a significant role in the abnormal gonadotrophinsecretion in women with PCOS (Marshall & Eagleson 1999).

2.4.1.2 Variant type of luteinizing hormone

Luteinizing hormone is a member of the glycoprotein hormone family which includeshCG, FSH and thyreoid stimulating hormone. These hormones are all α/β heterodimerswith a common α subunit and a unique β subunit. The β subunit confers the hormonalspecificity. In 1992, an immunologically anomalous variant form of LH (v-LH) wasfound in a healthy Finnish woman who was fertile and had normal levels of all other hor-mones measured. A specific monoclonal antibody (Mab) to the α/β-dimer was unable todetect LH in her serum and urinary samples in a two-site immunometric assay. The geneencoding her LHβ subunit was sequenced and two base changes in the N-terminal regionwere identified. The first mutation in codon 8 (TGC → CGG) changes tryptophan to argi-nine, and the second in codon 15 (ATC → ACC) changes isoleucine to threonine (Petters-son et al. 1992). These mutations have been shown to have a world-wide distribution(Furui et al. 1994, Okuda et al. 1994, Nilsson et al. 1997) and, rather than representingneutral polymorphisms, they alter the biological function of LH.

Two immunofluorometric assays have been used to determine the LH phenotypes. Thereference method (assay 2) uses two LHβ-specific Mab that recognize normal (or wildtype, wt) LH and v-LH with similar stoichiometries (Nilsson et al. 1997). The other assay(assay 1) uses Mab which only recognize the intact LH α/β-dimer and the α-subunit, butnot v-LH (Pettersson et al. 1992). When a large material of serum samples from healthyFinnish volunteers was analyzed and the ratio of LH with assay 1 / assay 2 was measured,the results fell clearly into three categories (1) those between 1.0-2.0 (normal ratio indivi-duals), i.e. the subject has two normal LHβ alleles, (2) those at 0.5-0.75 (low ratio indivi-

26

duals), i.e. the subject is heterozygous for the mutant LHβ gene and (3) those with theratio near 0, i.e. the subject is homozygous for the mutant LHβ gene (Fig. 2).

Fig. 2. A schematic picture of normal and variant LH molecules. Because of a supposed addi-tional carbohydrate chain in the β-subunit of LH variant, the antibody against intact α/β-dimerdoes not find the epitope detectable in normal LH (modified from Haavisto 1996).

V-LH displays an elevated in vitro bioactivity compared to that of normal LH but itshalf-time in the circulation is significantly lower and thus v-LH could change the kineticsof its action in vivo. No apparent pathologies in functions of the pituitary-gonadal axis areassociated with subjects homozygous for v-LH gene (Haavisto et al. 1995). Nevertheless,evidence is emerging that the LH action in carriers of the v-LH allele differ from that inindividuals with normal LH. V-LH shows an association with elevated serum E2, T andSHBG in the follicular phase of the menstrual cycle, obese PCOS women (Rajkhowa etal. 1995), menstrual disorders (Furui et al. 1994, Suganuma et al. 1996), slow pace ofpuberty in boys (Raivio et al. 1996) and infertility (Takahashi et al. 1998).

Several mutations of the LHβ subunit gene associated with gonadal dysfunction and/orinfertility have been discovered during the 1990’s (Weiss et al. 1992, Roy et al. 1996,Liao et al. 1998, Ramanujam et al. 1999, Ramanujam et al. 2000). Findings in variousethnic groups do not always agree. The variant form of LH may thus be an example of theinfluence of genetic heterogeneity on reproductive functions (Themmen & Huhtaniemi2000).

0 2 4 6 8 100

2

4

6

8

10

Antibody against α/β-dimer

Antibody against β-subunit

Antibody against α-subunit

Carbohydrate chain

ββ αα

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2.4.2 Steroidogenesis in PCOS

2.4.2.1 Androgen secretion

Androgens and androgen precursors are secreted by both the ovaries and adrenal glandsin response to their respective trophic hormones, LH and adrenocorticotropic hormone(ACTH). The initial, and the rate-determining, step in the biosynthesis of all steroid hor-mones is the conversion of cholesterol to pregnelone carried out by the cholesterol sidechain cleavage enzyme (P450scc) (Figs 3 and 4) (Rosenfield 1999). P450c17, possessingboth 17α-hydroxylase and 17,20-lyase activities, is the rate-limiting step in the formationof steroid hormones in the adrenals and in the gonads. Both enzymatic activities of theprotein are encoded by a single gene, CYP17. The expression of this gene is absolutelydependent on the concentration of trophic hormones, LH in ovary (Magoffin 1989) andACTH in the adrenal cortex (John et al. 1986). Two enzymes that are not members of theP450 gene family are also important for adrenal and gonadal steroid synthesis: 3α-hydroxysteroid dehyrogenase type II (3β-HSD II), which is exclusively expressed inadrenal gland and gonads (Rheaume et al. 1991) and steroid acute regulatory protein, atransporter of cholesterol from the outer to inner mitochondrial membrane (Lin et al.1995).

2.4.2.2 Ovarian steroidogenesis

Normal ovarian function depends on the combined action of LH on the theca-interstitial-stromal cells and FSH on granulosa cells (Fig 3). According to the two-cell model of ova-rian function, steroidogenesis is organized thereby that the theca cell compartmentsecretes A in response to LH and A is then converted within granulosa cells to estrogenby aromatase under the influence of FSH. As dominant follicle emerges, both increasedamounts of A and E2 are secreted, but E2 comes to predominate. Androgen synthesismust be kept to the minimum necessary to optimize follicular development. Althoughandrogens are obligate substrates for E2 synthesis, an excess of androgens seem to inter-fere with the process of follicular maturation, preventing the selection of the dominantfollicle and committing the follicle to atresia (Hillier & Tetsuka 1997) as well as interfer-ing with the LH action on luteinized granulosa cells (Polan et al. 1986).

Several studies have indicated that polycystic ovaries usually produce excess androgen(Kirschner et al. 1976, Chang et al. 1983, Rosenfield 1999). Chronic LH stimulation inPCOS induces sustained hypersecretion of androgens by the theca compartment (Yen etal. 1970). Theca cells are shown to secrete abnormal amounts of steroids in culture, bothbefore and after LH stimulation (Gilling-Smith et al. 1994). Furthermore, women withPCOS have been shown to exhibit 17-OHP hyperresponsiveness to GnRHa and hCG(Barnes et al. 1989b, Rosenfield et al. 1994, Ibanez et al. 1996, Levrant et al. 1997).PCOS women showed 17-OHP hyperresponsiveness to hCG also after their baseline LHdecreased to a level similar to that in control women after 1 month of GnRH agonist treat-ment (Gilling-Smith et al. 1997). The pattern of steroid secretion in polycystic ovary isthought to suggest a generalized dysregulation of ovarian androgen secretion, which is

28

particularly prominent at the level of 17α-hydroxylase and 17,20-lyase activities. Inaccordance with results of all of these studies it has been proposed that there exists a pri-mary “dysregulation of ovarian P450c17” in the theca cell steroidogenesis in PCOS.However, with these experiments a persistent effect of an antecedent LH excess cannot beruled out. LH suppression by GnRHa is also known to be incomplete (Chang et al. 1983).Furthermore, the synergistic effect of insulin excess and increased IGFs on pre-existingLH-mediated theca cell hyperfunction could not be excluded (Barbieri et al. 1986, Ehr-mann et al. 1995).

Insulin also augments ovarian androgen production. It has been shown that insulin actsalone or synergistically with LH to increase androgen production in the ovary (Barbieri etal. 1986). Insulin may act via its own receptors widely distributed throughout all ovariancompartments (Poretsky et al. 1984, Poretsky et al. 1999), the IGF receptor or via ahybrid receptor containing a combination of α- and β-subunits of both receptors (Rosen-field 1999). Inhibin stimulates ovarian androgen production (Hsueh et al. 1987) andandrogens, in turn, stimulate ovarian inhibin production (Hillier et al. 1991).

The follicular cysts in the ovaries of PCOS women do not mature fully. Granulosacells in these arrested follicles are few in number and are virtually devoid of aromataseactivity (Yen 1999). At baseline, there is a tendency toward a mild E2 excess for the stageof follicular maturation; this may be partly the consequence of excess androgen substratefor E2 secretion. Granulosa cells, examined in vitro, are not apoptotic and express highlevels of FSH receptors. They are highly responsive to FSH in vitro (Almahbobi et al.1996). In vitro PCOS granulosa cells also respond normally or hyperrespond to IGF-1,which is known to stimulate granulosa cell proliferation and aromatase activity (Ericksonet al. 1990, Mason et al. 1993). However, aromatase activity in granulosa cells in thePCOS follicle is very low which results in a higher androgen to estrogen ratio and follicu-lar arrest. These findings have led to the hypothesis that locally active inhibitors of IGFsor FSH, most likely insulin-like growth factor binding proteins (IGFBPs), in small antralPCOS follicles block stimulation of aromatase (Cataldo 1997).

There is data showing that circulating concentrations of follistatin are higher, and ofactivin A are lower, in PCOS subjects compared to those with no evidence of thissyndrome (Norman et al. 2001). Activin A is produced by follicles in culture (Smitz et al.1998) and has been proposed to promote proliferation of granulosa cells, enhance FSHreceptor expression, decrease LH-induced androgen production, increase pituitary FSHsecretion and generally promote follicle growth (Li et al. 1995, Smitz et al. 1998). Follis-tatin is a binding protein for activin and inhibits most of these actions of activin (Hillier &Miro 1993, Hillier 1999). Overexpression of follistatin could be one mechanism wherebyfollicular growth is impeded. There are, however, previous studies that failed to show anydifference in follicular fluid concentrations of follistatin in unstimulated or stimulated,normal or polycystic ovaries (Erickson et al. 1995, Lambert-Messerlian et al. 1997).

Although the mechanism is unclear, the consequence of dysregulation of androgensynthesis is that the normal coordination of ovarian androgen secretion with granulosacell function is disturbed, with grave consequences for follicular maturation. There isgeneralized ovarian hyperresponsiveness to gonadotrophins. The thecal cell hyperres-ponse to LH enhanced by insulin accounts for androgen excess. Granulosa cells arehyperresponsive to FSH, but hyperestrogenism is prevented by a compensatory reductionin FSH levels (Rosenfield 1999).

29

Fig. 3. The major steroid biosynthetic pathways in the small antral follicle of the ovary, accord-ing to the two gonadotrophin, two-cell model of ovarian steroidogenesis. Luteinizing hormone(LH) stimulates androgen formation within theca cells by the steroidogenic pathway commonto the gonads and adrenal glands. Follicle-stimulating hormone (FSH) regulates estradiol syn-thesis from androgens in granulosa cells. The conversion of 17-ketosteroids to 17β-hydroxyster-oids by 17β-hydroxysteroid dehydrogenase (17βHSD) is essential for the formation of testoster-one, dihydrotestosterone (DHT) and estradiol. Androgen formation in response to LH appearsto be modulated by intraovarian feedback at the levels of 17-hydroxylase and 17,20-lyase, bothwhich are activities of cytochrome P450c17. The quantitative importance of androstenedioneformation from 17-hydroxyprogesterone (dashed arrow) in the intact follicle is unknown. An-drogens and estradiol inhibits and insulin, inhibin, and insulin-like growth factor-I (IGF-I)stimulate 17-hydroxylase and 17,20-lyase activities. (Modified from Rosenfield 1999). ATP =adenosine triphosphate; cAMP = cyclic adenosine monophosphate; StAR = steroidogenic acuteregulatory protein; 3β = ∆5-isomerase-3β- hydroxysteroid dehydrogenase; 5α-R = 5α-reduct-ase; P450arom = aromatase enzyme

2.4.2.3 Adrenal steroidogenesis

Adrenal steroidogenic abnormalities are a common finding in women with hyperandro-genism including those with PCOS/FOH (Ehrmann et al. 1995). Functional adrenalhyperandrogenism is shown to occur in approximately 55% of women with signs ofhyperandrogenism. Adrenal and ovarian androgenic abnormalities occurred concurrentlyin approximately one-third of hyperandrogenic women (Ehrmann et al. 1992).

The nature of adrenal androgenic abnormality in PCOS has remained unexplained forthe most part. A supranormal adrenocortical capacity for androgen synthesis has been

0 2 4 6 8 100

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(-)(+)

17-reductasecAMP

ATP

cAMP

ATP

5α-reductaseDHT

INSULIN

(-)

(+)(-)

IGF ?

17-βHSD

P450arom P450arom

17-βHSD

3β

3β

3β

17,20-lyase17,20-lyase

17α-hydroxylase17α-hydroxylase

side chain cleavageStAR

Granulosa Cell

FSH

INHIBIN

LH

Theca Cell

OESTRADIOLOESTRONE

TESTOSTERONE

ANDROSTENEDIONE

17-HYDROXY-PROGESTERONE

PROGESTERONE

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described, suggesting exaggerated adrenarche (Lachelin et al. 1979, Lucky et al. 1986).Several reports have shown that children with premature or exaggerated adrenarche are athigh risk for developing PCOS-like FOH at puberty (Ibanez et al. 1993, Oppenheimer etal. 1995). It has recently been reported that girls with prenatal growth reduction werefound to be prone to develop, in addition to hyperinsulinism, a variant of exaggeratedadrenarche (Ibanez et al. 1999). Serum levels of the adrenal sex steroid precursors dehyd-roepiandrosterone sulphate (DHEAS) and 11β-hydroxyandrosterone are elevated inadults, reflecting enhanced steroidogenesis by zona reticularis (Hoffman et al. 1984,Hudson et al. 1990). Analogous to the ovary, the mechanisms of adrenal hyperandrogene-mia have been suggested to be due to adrenal enzyme deficiencies and adrenal androgenhyperresponsiveness (Lucky et al. 1986, Ehrmann et al. 1995). There is clinical researchdata which suggests that overactivity of androgen formation by 17-hydroxylase and17,20-lyase, involving P450c17 occurs in the adrenal glands, analogous to that occurringin women with FOH (see 2.3.)(Ehrmann et al. 1995, Gonzalez et al. 1996). However,these activities may be regulated differently in the ovaries than in adrenal glands (Gonza-lez et al. 1996). Increased IGFs, insulin, or both may amplify ACTH-mediated P450c17expression and adrenal androgen synthesis (l'Allemand et al. 1996, Mesiano et al. 1997).It is also possible that other extra-adrenal factors such as β- endorphins, prolactin andgrowth hormone or intra-adrenal factors (blood flow, maturation of adrenal gland, cytoki-nes) could exert a differential control over cortisol and adrenal androgens (McKenna etal. 1997, Ehrhart-Bornstein et al. 1998).

Fig. 4. Major steroid pathways in the adrenal cortex. The square contains the core steroidogenicpathways also used by the gonads. In the adrenals, 17-hydroxyprogesterone (17-OHP) is situ-ated at a potential branch point at which cortisol and sex hormone synthesis may diverge de-pending on whether 17-OHP undergoes 21-hydroxylation (pathway to cortisol) or 17,20-lysis(pathway to 17-ketosteroids). Cytochrome 450 enzyme steps are side chain cleavage; 17α-hy-droxylase/17,20-lyase; 21-hydroxylase (21); 11β-hydroxylase/18-hydroxylase-dehydrogenase(11, 18). Non-P450 enzyme steps are steroidogenic acute regulatory protein (StAR); ∆5-isomer-ase-3β-hydroxysteroid dehydrogenase (3β), 17β-hydroxysteroid dehydrogenase (17β), sulfoki-nase (SK) and sulfolyase (SL). Dashed pathways are considered to be relatively minor. Conver-sion of dehydroepiandrosterone (DHEA) to DHEA sulphate and androstenedione to 11β-hy-droxyandrostenedione takes place in the zona reticularis. Deoxycorticosterone (DOC). (Modi-fied from Rosenfield 1999).

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ACTH is known to affect both cortisol and adrenal androgen production. Cortisolfeeds back to ensure its level is maintained within a physiological range. It has been sug-gested that an increase in the metabolic clearance of cortisol without any change in that ofbiologically active androgens, causing lower plasma cortisol levels, could stimulate theproduction of ACTH and induce excessive adrenal androgen production (Rodin et al.1994).

2.4.2.4 Peripheral steroid metabolism

Peripheral steroid metabolism is altered in PCOS. Hirsutism and acne are common symp-toms, reflecting hyperandrogenism which may or may not be progressive. Adipose tissuehas the capacity to form T and estrone from inactive precursors (Bleau et al. 1974). Evi-dence of marginally increased 5α-reductase activity, converting T to dihydrotestosterone(DHT) in the pilosebaceous unit, has been reported (Stewart et al. 1990, Rodin et al.1994). This activity is mediated by IGF-I. Increased bioavailability of IGF-I in hyperin-sulinemic women (such as PCOS women) may amplify the manifestation of hirsutism.

2.4.3 Metabolic features in PCOS

2.4.3.1 Glucose tolerance

In 1921 the French physicians Achard and Thiers provided the first description of therelationship between androgen excess in women and disturbances in carbohydrate metab-olism which was dubbed “diabete des femmes á barbe” (diabetes of the bearded lady)(Achard & Thiers 1921). In 1980 it was shown for the first time that obese women withPCOS had significantly increased glucose and plasma insulin levels during an oral glu-cose tolerance test (OGTT) compared with obese control women (Burghen et al. 1980). In1987 obese PCOS women were shown to have significantly increased glucose levels dur-ing an OGTT when compared with age- and weight matched ovulatory hyperandrogene-mic and control women. Twenty percent of these obese women with PCOS had impairedglucose tolerance (IGT) or type 2 diabetes mellitus (DM) by National Diabetes group cri-teria (Dunaif et al. 1987). It has recently been reported in the USA, that of 254 prospec-tively studied PCOS women, 40 % had glucose intolerance, 31% IGT and 7.5% type 2DM. These rates were significantly higher than those for an age-, weight, and ethnicity-comparable control population (Legro et al. 1999). Also in that study nonobese andyoung PCOS women had glucose intolerance, although obesity and age increased the riskfor abnormal glucose tolerance. According to the study, PCOS was a more important riskfactor for glucose intolerance than was ethnicity or race (Legro et al. 1999).

A familial association between PCOS and gestational diabetes (GDM) has been shown(Plehwe et al. 1985), and an increased prevalence of GDM, type 1 and type 2 DM hasbeen reported among PCOS women (Lanzone et al. 1995a, Lanzone et al. 1996, Conn et

32

al. 2000, Escobar-Morreal et al. 2000). However, there are controversial reports, as well(Wortsman et al. 1991, Vollenhoven et al. 2000).

2.4.3.2 Insulin resistance

Insulin, a polypeptide hormone secreted by the β-cells of the pancreas, plays a dominantrole in maintaining glucose homeostasis. Its classic target tissues include the liver, mus-cle and fat. Insulin stimulates peripheral glucose uptake in muscle and fat tissue andinduces protein synthesis, cell growth and differentiation. Insulin promotes glycogen stor-age and inhibits gluconeogenesis and glycogenolysis in the liver. It also inhibits lipolysis(Cheatham & Kahn 1995). The terms insulin sensitivity and insulin resistance generallyrefer to the actions of insulin on glucose homeostasis.

Before the identification of the association of insulin resistance with PCOS, severalclinical models of rare syndromes of extreme insulin resistance had been described. Inthese syndromes, manifestations of androgen excess, amenorrhea, bilateral PCO andacanthosis nigricans were observed. Type A syndrome of insulin resistance is due to apoint mutation in the DNA sequence coding for the α- and β-subunit of the insulin recep-tor (Kadowaki et al. 1988, Kahn & Goldstein 1989). Leprechaunism is a rare geneticsyndrome with a point mutation of the α-subunit (D'Ercole et al. 1979). Type B syndromeis due to the presence of autoantibodies against the insulin receptor associated withautoimmune diseases. These observations were followed by reports of insulin resistancein women with a more classical PCOS (Burghen et al. 1980, Pasquali et al. 1983). Inaddition to the reproductive consequences of the syndrome, PCOS is characterized by ametabolic disorder in which hyperinsulinemia and peripheral insulin resistance are centralfeatures (Holte 1996, Dunaif 1997). The characteristic disturbances of insulin secretionand action are much more prominent in PCOS women with amenorrhea or anovulatorymenses than in equally hyperandrogenic women with regular cycles (Dunaif et al. 1987,Robert et al. 1995).

Measurements of fasting insulin, fasting glucose/fasting insulin ratio (Legro et al.1998b), area under the curve (AUC) in OGTTs, rapid intravenous glucose tolerance test(IVGTT) (Bergman et al. 1987, Steil et al. 1993) and the euglycemic hyperinsulinemicclamp (DeFronzo et al. 1979) studies have demonstrated significant and substantial dec-reases in peripheral sensitivity to insulin in PCOS. This decrease (approximately 35%to40%) is of a similar magnitude to that seen in type 2 DM (Dunaif et al. 1989b). Obesity(fat mass per se), body fat location (upper versus lower body), and muscle mass all haveimportant independent effects on insulin sensitivity. Alterations in any of these parame-ters could potentially contribute to insulin resistance in PCOS (Dunaif 1997).

Thus, although it seems clear that PCOS and obesity have an additive, or synergistic,negative impact on insulin sensitivity, the important question – whether PCOS per se isassociated with aberrations in carbohydrate metabolism - is still under debate. Studies, inwhich body composition assessed by hydrostatic weighing, the most precise availablemethod, and in which WHR has been matched to that of control women, have shown thatlean women with PCOS also seem to be insulin resistant indepenedent of potentially con-founding parameters (Dunaif et al. 1989b). There are studies from Europe showing that

33

women with PCOS seem not to be insulin resistant in the absence of increased amount oftruncal-abdominal fat (Bringer et al. 1993, Ovesen et al. 1993, Morin-Papunen et al.2000) suggesting regional diffrences or heterogeneity and a complex etiology of thissyndrome. There is evidence that insulin sensitivity increases with a normalization oftruncal-abdominal fat in obese PCOS women (Holte et al. 1995). Thus insulin resistancein PCOS women may be a consequence of increased truncal-abdominal fat and highlevels of free-fatty acids (FFA). It has been shown that hyperandrogenicity in women isclearly associated with a preponderance of fat localized to truncal-abdominal sites (Evanset al. 1983). Since intra-abdominal fat has a high lipolytic activity (Arner 1995) and thereis evidence that enhanced FFA release from excess abdominal fat interferes with hepaticand peripheral insulin action on carbohydrate metabolism (Randle et al. 1963, Bevilacquaet al. 1987) such a pathogenetic mechanism for insulin resistance in PCOS would seemlikely. Recently, decreased serum FFA concentrations with concominant improvement ofoxidative utilization of glucose and reduction of hyperinsulinemia, was reported after 6months of treatment with metformin (Morin-Papunen et al. 2000).

2.4.3.3 Insulin secretion and clearance

Insulin is secreted in a normal β-cell in response to a glucose stimulus in a biphasicmode, with an early burst (early phase), followed by progressively increasing insulinsecretion (second phase) as long as the hyperglycemic stimulus is present (DeFronzo etal. 1979). In the presence of insulin resistance, pancreatic β-cell insulin secretion increas-es in a compensatory fashion and type 2 DM develops when the compensatory increase ininsulin levels is no longer sufficient to maintain euglycemia (Bergman 1989). Fastinghyperinsulinemia is present in obese PCOS women, and insulin responses during the mid-to-later phases of an oral glucose load are increased in both lean and obese PCOS wom-en. These patients generally exhibit a subnormal first-phase insulin secretion, either inabsolute terms or in relation to the degree of insulin resistance (Dunaif & Finegood1996a).

Several studies have suggested an indirect role for androgens through a modificationof β-cell sensitivity to glucose. Androgens may play a role in inducing an increased panc-reatic glucose sensitivity, resulting in periods of lower glucose levels, with possible impli-cations for an increased stimulus to carbohydrate intake and counter-regulatory hormonalresponses. This would favor the accumulation of abdominal fat and insulin resistance(Schwartz et al. 1987, Prelevic et al. 1992).

Hyperinsulinemia can result from a decrease in insulin clearance as well as from inc-reased insulin secretion. Hepatic insulin extraction in women with PCOS has been shownto be decreased (O'Meara et al. 1993, Morin-Papunen et al. 2000), normal (Peiris et al.1989), or heterogenic despite a similar degree of insulin sensitivity (Ciampelli et al.1997). High lipolytic activity in intra-abdominal fat and FFA released (see 2.4.3.2) mayinhibit hepatic insulin extraction, and thus contribute to peripheral hyperinsulinemia(Randle et al. 1963, Bevilacqua et al. 1987, Reaven 1988)

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2.4.3.4 Molecular mechanisms of insulin resistance in PCOS

Insulin action is initiated when insulin binds to its cell surface receptor (Fig 5). The insu-lin receptor belongs to a family of protein tyrosine kinase receptors which includes theIGF-I, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor,colony-stimulating growth factor-I as well as several cytokine receptors (Dunaif 1997,White 1998). The insulin receptor is a heterotetramer made up of two α,b dimers linkedby disulfide bonds. The α-subunit is extracellular and contains the ligand-bindingdomain, whereas the β-subunit spans the membrane and contains intrinsic proteintyrosine kinase activity. After insulin binds to and activates its receptor, the ligand-recep-tor complex is internalized through endocytosis. Insulin is then degraded, and most of thereceptors are returned to the cell surface. This action may be responsible for the insulinreceptor down-regulation seen in chronic hyperinsulinemia (Cheatham & Kahn 1995).

Fig. 5. Insulin receptor, its signaling pathways for glucose transport and hypothetical mecha-nisms of stimulation or inhibition of steroidogenesis. After insulin binds to the insulin receptorα-subunits; the β-subunit tyrosine kinase is activated; insulin receptor substrates (IRS-1 and –2) are phosphorylated; phosphatidylinositol-3-kinase (PI-3) is activated by IRS-2 (metabolicpathway); glucose transporters are translocated to the cell membrane, and glucose uptake isstimulated. The IRS-1 and Ras-mitogen-activated protein kinase (MAPK) appear to regulatecell growth and mitogenesis (mitogenic pathway). An alternative signaling system involvinggeneration of inositolglycan second messengers at the cell membrane independently of the β-subunit tyrosine kinase activation. This pathway may mediate insulin modulation of steroidog-enic enzymes (see text for more details and references). (Modified from Dunaif 1999, Poretskyet al. 1999, Yen 1999). ATP = adenosine triphosphate; ADP = adenosine diphosphate; scc = sidechain cleavage; c= cytochrome; arom = aromatase; -S-S-= disulphide bonds; Glucose-6-P =Glucose 6 phosphate

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Ligand binding induces autophosphorylation of the insulin receptor on specific tyro-sine residues and increases the intrinsic tyrosine kinase activity of the β-subunit. The acti-vated insulin receptor then tyrosine phosphorylates intracellular substrates to initiate sig-nal transduction. Over the last few years, several of these substrates have been characteri-zed, such as insulin receptor substrates 1 and 2 (IRS-1 and –2), which bind to signalingmolecules, such as the specific domain of the phosphatidylinositol 3-kinase (PI3 kinase),a necessary step in the initialization of glucose transport (Saltiel 1996a, White 1998).IRS-1 is required for insulin-mediated translocation of the intracellular pool of glucosetransporters (GLUT-4) to the cell surface and thereby increases glucose uptake in res-ponse to insulin (Rose et al. 1994). IRS-2 mediates metabolic pathways (Withers et al.1998).

An alternative signalling pathway has also been described, involving the generation ofinositolglycan second messengers at the cell membrane independently of the β-subunittyrosine kinase activation. This pathway may mediate stimulation of ovarian steroidoge-nesis, explaining why the stimulation of ovarian steroidogenesis by insulin is still opera-tive despite insulin resistance (Nestler et al. 1998a).

The mechanisms by which the insulin signal is terminated is not completely under-stood. Receptor-mediated endocytosis and recycling are known to occur and may beimportant for signal termination (Saltiel 1996a, White 1998).

Studies of insulin action in isolated adipocytes from women with PCOS have revealedmarked decreases in insulin sensitivity together with less striking, but significant decrea-ses in maximal rates of insulin-stimulated glucose transport (Ciaraldi et al. 1992). Thedecrease in maximal rates of adipocyte glucose uptake is secondary to a significant dec-rease in the abundance of GLUT-4 (Rosenbaum et al. 1993). Decreased adipocyte sensiti-vity to inhibition of lipolysis by insulin has been found in PCOS (Ek et al. 1997). Dec-reased insulin receptor autophosphorylation has been reported in approximately 50% ofPCOS fibroblasts. Decreased insulin-dependent receptor tyrosine phosphorylation andincreased constitutive receptor serine phosphorylation, which inhibits insulin receptor sig-naling, was found in PCOS fibroblasts (Dunaif et al. 1995b). Increased insulin-indepen-dent serine phosphorylation in PCOS fibroblasts seems to be a unique disorder of insulinaction as other insulin-resistant states, such as obesity, type 2 DM, type A syndrome andleprechaunism do not exhibit this abnormality (Dunaif et al. 1995b). A significant dec-rease in muscle IRS-1-associated PI3-kinase activation during insulin infusion has beenreported in PCOS women, consistent with a defect in the early steps of insulin receptorsignaling (Dunaif 1999). Follicles in polycystic ovaries were recently shown to have dec-reased staining for IRS-1 in granulosa cells and increased staining for IRS-2 in theca cellscompared with follicles from ovulatory ovaries (Wu et al. 2000).

Protein kinase A could be the factor, which induces serine phosphorylation in the insu-lin receptor, thereby causing insulin resistance, and also serine-phosphorylates P450c17α,causing hyperandrogenism. This could explain the association of PCOS and insulin resis-tance (Zhang et al. 1995, Dunaif 1997). It has been suggested that the factor responsiblefor the increased serine phosphorylation could be genetically programmed (Dunaif 1999).These findings are in accordance with recent twin and family studies where insulin resis-tance appeared to be a genetic defect in PCOS (Legro 1998a).

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2.4.3.5 Insulin action in polycystic ovary

The possible mechanisms causing postbinding defects in insulin action in women withPCOS are shown in Fig. 6. Insulin alone can act as a gonadotrophin in the normal ovarybut, under physiologic conditions, is likely to interact with gonadotrophins as well as withparacrine ovarian factors such as IGFs (Willis et al. 1996, Willis et al. 1998). Insulinseems to have little effect on FSH-induced steroidogenesis in cells from normal humanovaries. In contrast, insulin consistently and significantly enhanced the effect of LH onthe production of both E2 and progesterone in granulosa cells from normal ovaries (Will-is et al. 1996).

There is evidence suggesting that the ovary in women with PCOS remains sensitive tothe effects of insulin, despite peripheral resistance to the insulin action (Willis et al.1996). In an in vitro study insulin, when coincubated with LH, seemed to augment LH-induced E2 and P production in granulosa cells from normal ovaries and PCO in an addi-tive manner. Insulin and FSH acted synergistically on the production of progesterone bygranulosa cells from normal and PCO, but the response of E2 was dependent on the typeof ovary from which the granulosa cells were isolated. Insulin and FSH had a synergisticeffect on E2 production in granulosa cells from anovulatory PCO, which was not seen inthe majority of ovulatory patients (Willis et al. 1996). Insulin also seems to produce a gre-ater increase in androgen production by theca cells isolated from PCOS women than thatobserved in cells obtained from controls (Nestler et al. 1998a).

Although the insulin and type IGF-I receptors are structurally similar and insulin isshown to cross-react with type IGF-I receptor (Nissley & Lopaczynski 1991), it has beenconcluded that insulin action to the ovary is mediated by way of its own receptor, ratherthan by cross-reaction with IGF receptor, even when there is evidence of peripheral insu-lin resistance (Willis & Franks 1995, Nestler et al. 1998a). It is possible that insulin actionin the cells from PCOS ovaries may involve inositolglycans in postreceptor signaling andthis pathway could provide an alternate means of signal transduction in otherwise insulin-resistant tissues (Nestler et al. 1998a, Poretsky et al. 1999).

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Fig. 6. Insulin action in women with PCOS. Possible mechanisms causing postbinding defectsin insulin action in women with PCOS. Decreased insulin-dependent receptor tyrosine phos-phorylation (Tyr-P) and increased constitutive receptor serine phosphorylation (Ser-P), prob-ably secondary to a cell membrane associated factor, inhibits insulin receptor signaling. Inhibi-tion of receptor signaling leads to decreased amount of glucose transporters and decreased glu-cose uptake. Ser-P of insulin receptor substrate-1 (IRS-1) appears to be the mechanism for tu-mor necrosis factor α (TNF α) mediated insulin resistance. Protein kinase A could be the factorthat serine phosphorylates the insulin receptor. The membrane glycoprotein PC-1 also inhibitsinsulin receptor kinase activity, but it does not cause serine phosphorylation of the receptor. Analternative signaling pathway involving generation of inositolglycan second messengers at thecell membrane, independently of the β-subunit tyrosine kinase activation, possible mediatestimulation of ovarian steroidogenesis. (Modified from Dunaif 1999, see text for more detailsand references). Arom = aromatase; c = cytochrome; scc = side chain cleavage; -S-S- = disul-phide bonds

2.4.3.6 Causal association of androgens and insulin resistance

The syndromes of extreme insulin resistance are commonly associated with hyperandro-genism when they occur in premenopausal women (Dunaif 1997). It has been proposedthat hyperinsulinemia causes hyperandrogenism because insulin has a variety of directactions on steroidogenesis in humans as discussed above. It is suggested that if insulin isto produce ovarian hyperandrogenism, polycystic ovarian changes must be present whichpredispose the ovaries to secrete excess androgens. Insulin does not seem in vivo to haveany acute effects on ovarian function in normal women under physiologic circumstances(Dunaif & Graf 1989a). In a recently reported case report, surgical removal of insulino-ma from a 24 year-old woman resulted in resolution of the clinical and biochemical fea-tures of PCOS after 4 months, but minimal change was observed in the ovarian ultra-

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sound appearances (Murray et al. 2000). Menstrual disturbances, polycystic ovaries, andhyperandrogenism are shown to be encountered in women taking valproate for epilepsy(Isojärvi et al. 1993). Treatment with valproate, associated with weight gain and menstru-al disorders in 2 of the 3 women, has been shown to develope a disorder characterized byhyperandrogenism and polycystic ovaries. Replacing valproate with lamotrigine resultedin a decrease in serum T concentrations in all 3 women. The polycystic changes disap-peared from the ovaries in 2 of the women after valproate therapy was discontinued, andthe 2 women who had gained weight and developed amenorrhea while being treated withvalproate lost weight and resumed menstruating after the change in medication. It wassuggested that obesity and associated hyperinsulinemia could be implicated in the devel-opment of PCOS (Isojärvi et al. 2000).

Studies, in which insulin levels have been lowered with agents that either decreaseinsulin secretion (diazoxide, somatostatin) or improve insulin sensitivity (metformin,troglitazone), demonstrate decreased androgen levels as well in PCOS women (Nestler etal. 1989, Dunaif et al. 1996b, Morin-Papunen et al. 1998, Morin-Papunen et al. 2000).Abnormalities in apparent 17,20-lyase activity have improved after metformin treatmentin parallel with reduced circulating insulin levels, consistent with an insulin-mediated sti-mulation of this enzyme (Nestler & Jakubowicz 1996)

Inverse associations between concentrations of SHBG and insulin are almost consis-tently found (Dunaif et al. 1987, Holte et al. 1994). Insulin has a major role in regulatinghepatic production of SHBG and it increases the biological availability of potent steroids,mainly T, through the suppression of SHBG synthesis (Nestler 1993). Insulin seems toincrease adrenal sensitivity to ACTH in hyperandrogenic women (Moghetti et al. 1996a).

LH may act as an intermediary in insulin augmented hyperandrogenism. Insulin has adirect action on the pituitary enhancing GnRH-stimulated LH release (Adashi et al.1981), the consequent hyperandrogenism resulting from an increase in LH secretion. Asmentioned previously, insulin acts alone or synergistically with LH to increase androgenproduction in the ovary (Barbieri et al. 1986). It has also been shown in severely over-weight infertility patients that weight reduction with a very low calorie diet results in adecrease in LH concentrations, a reduction in the LH/FSH ratio, and FSH predominancefavoring folliculogenesis. The decrease in LH concentrations was inversely related to theseverity of insulin resistance (Butzow et al. 2000).

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Fig. 7. Possible mechanisms causing hyperandrogenism and hyperinsulinemia in women withPCOS. The increased luteinising hormone (LH) pulse frequency increases the androgen pro-duction from theca cells of the ovary. In the follicles increased levels of insulin-like growth fac-tor-binding protein-2 and -4 (IGFBP-2, -4) and the absence of IGFBP proteases may sequesterthe insulin-like growth factor-I (IGF-I) and thereby reduce its synergistic action with FSH lead-ing to arrest of follicle development. Adrenal androgen production is increased. Hyperandro-genism increases the proportion of insulin resistant muscle fibres in the muscle tissue and se-rum free fatty acid (FFA) levels. The increased FFAs in the serum compete with glucose for up-take and oxidation in the muscle cell, inducing further insulin resistance. In addition FFAs havebeen shown to decrease hepatic insulin extraction. Androgens may indirectly modify β-cell sen-sitivity to glucose. Hyperinsulinemia, either primary or secondary to insulin resistance, induceshyperandrogenism directly by increasing ovarian androgen secretion, probably via its own re-ceptor, and indirectly by decreasing the secretion of sex hormone-binding globulin (SHBG) andIGFBP-1 in the liver. Insulin downregulates the number of its own receptors on the membranesof the target cells. Obesity may contribute to androgen excess due to the capacity of the adiposetissue to form testosterone and oestrone from inactive precursors. Elevated level of estrogen canaugment pituitary sensitivity to gonadotrophin releasing hormone (GnRH). IR = insulin recep-tor

Androgens may produce a mild insulin resistance by increasing the number of lessinsulin-sensitive type IIb skeletal muscle fibers (Holmang et al. 1992) and by inhibitingmuscle glycogen synthase activity (Rincon et al. 1996). The mild insulin resistance due toandrogen administration is not at the same magnitude as that seen in PCOS (Polderman etal. 1994). There were no significant changes in peripheral or hepatic insulin action afterprolonged androgen suppression by the GnRHa (Dunaif et al. 1990). Modest improve-

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ments in insulin sensitivity have been reported with antiandrogen therapy in less insulinresistant, less obese or nonobese PCOS women (Elkind-Hirsch et al. 1993, Moghetti et al.1996b). It has been suggested that T may indirectly contribute to insulin resistancethrough facilitating FFA release from the abdominal fat (Rebuffe-Scrive et al. 1991).

2.4.3.7 Insulin resistance in women with PCO

As described above (chapter 2.3.1.) normal ovulatory women with the isolated finding ofPCO, can not be considered to have PCOS. However, a subgroup of these women mayhave subtle abnormalities resembling PCOS (Norman et al. 1995, Carmina et al. 1997). Ithas been concluded that insulin resistance is limited to women with polycystic ovarymorphology and chronic anovulation (Dunaif et al. 1987, Dunaif et al. 1989b, Robinsonet al. 1993). The finding that diet-induced weight loss accompanied by a reduction infasting and glucose-stimulated plasma insulin levels in obese PCOS women is associatedwith the resumption of ovulatory cycles lends further support to this hypothesis (Pasqualiet al. 1989, Kiddy et al. 1992). It has been shown, however, that women with PCO andPCOS have equivalent disturbances in glucose and insulin responses to OGTT as well asin lipid disturbances compared with control women (Norman et al. 1995, Holte et al.1998). Women with PCO seem to have lower serum IGFBP-1 concentrations than con-trol women, although not as low as the women with PCOS, which may be a result of amild insulin resistance (Carmina et al. 1997). In the studies on women with previousGDM, the prevalence of PCO were twice that of those reported for premenopausal wom-en overall (Anttila et al. 1998, Holte et al. 1998, Kousta et al. 2000). According to ahyperinsulinemic euglycemic clamp, the group of women with previous GDM and PCO,in which the typical clinical symptoms of PCOS were scarce, showed significantly lowerinsulin sensitivity than control women and women with previous GDM and normal ova-ries (Holte et al. 1998).

2.4.4 Insulin-like growth factors in PCOS

The IGF family includes IGF peptides, IGF receptors, a family of homologous IGF-bind-ing proteins (IGFBP-1, -2, -3, -4, -5 and -6) which bind IGFs with a high affinity, a groupof low-affinity IGFBP-related proteins (IGFBP-rP), and a family of IGFBP proteases.

Insulin-like growth factors (IGFs), previously named somatomedins, are single-chainpolypetides, which interact with specific cell membrane receptors to stimulate cellularmitosis and differentation in a variety of cell types. They can act via autocrine, paracrineand endocrine mechanisms (Sara & Hall 1990). The liver is the major source of circula-ting IGFs (Schwander et al. 1983). In the liver, growth hormone stimulates gene expres-sion and synthesis of the metabolic and mitogenic mediator IGF-I and - to a lesser extent -IGF-II (Yen 1999). Trophic hormones, such as ACTH, thyrotropin, LH and FSH, can sti-mulate the biosynthesis of IGF-I in their target organs. Circulating concentrations of IGF-II are two or three times higher than those for IGF-I.

41

Both IGF-I and IGF-II circulate in plasma tightly bound to specific binding proteins(BPs). Like IGFs, the IGFBPs are produced by a variety of cells and are suggested to actin an autocrine/paracrine mode as well as in an endocrine mode. The IGFBPs regulate thephysiological actions of the IGFs by inhibition or stimulation, depending on the type ofthe binding protein and the target cells (Drop 1991). Circulating concentrations ofIGFBPs are highest for IGFBP-3 and lowest for IGFBP-1. IGFBP-3 possesses the highestbinding affinity for IGFs and functions as a reservoir for long-term monitoring of IGFlevels. IGFBP-1 is a relatively low affinity binding protein and serves as an acute modula-tor of the bioactivity of IGFs (Lee et al. 1993). Insulin is a major regulator of hepaticIGFBP-1 production reducing hepatic production of IGFBP-1 and enhancing IGFBP-1translocation to the extravascular space (Lee et al. 1993). Ageing is associated withreductions in IGF-1, IGFBP-3 and IGFBP-5 and an increase in IGFBP-4 (Rosen et al.1998).

Most reports demonstrate that serum IGF-I levels in PCOS women are within the nor-mal range (Suikkari et al. 1989, Homburg et al. 1992). Levels of circulating IGFBP-1 aresignificantly lower in women with PCOS compared with controls, most likely owing tothe inhibitory actions of insulin on hepatic IGFBP-1 production (Suikkari et al. 1989, Tii-tinen et al. 1993, Morales et al. 1996). Decreased IGFBP serum concentrations in PCOSwomen may lead to increased IGF bioavailability in serum and thus the ovary (Giudice1999). In ovarian and adrenal venous effluents, levels of IGF-1, IGFBP-1 and –3 are simi-lar to the levels of circulation and IGF-II levels are elevated - although not significantly -in severly hyperandrogenic women (Martikainen et al. 1997).

IGFs are also synthesized in multiple extrahepatic tissues, including granulosa cells inovary where they are under the control of FSH and play a role in folliculogenesis (Giu-dice 1992). IGF-I and IGF-II are found in the follicular fluid and receptors for IGF-I and–II are expressed in the follicle cells (Adashi et al. 1985, el-Roeiy et al. 1994). IGF-II rat-her than IGF-I seems to be the predominant IGF in human granulosa cells. It has beenfound that, although IGF-II alone is generally less potent in stimulating steroidogenesisthan IGF-I, preincubation with insulin significantly enhanced the E2 response to IGF-II(Mason et al. 1994). IGFs stimulate granulosa proliferation and aromatase in vitro, pro-cesses that are characteristically absent in the PCOS follicle in vivo. In the PCOS follicle,FSH and IGF-1 are in the physiologic range (Eden et al. 1990). In the PCOS follicle aro-matase activity and E2 production by granulosa cells are very low resulting in a higherandrogen to estrogen ratio and follicular arrest. In vitro, however, PCOS granulosa cellsrespond normally or hyperrespond to IGF-1 and FSH (Erickson et al. 1979, Erickson etal. 1989). These findings have led to the hypothesis that locally active inhibitors of IGFsor FSH in small antral PCOS follicles block stimulation of aromatase. In this regard,IGFBPs are likely candidates (Cataldo 1997).

Five IGFBPs have been identified in human ovaries (Cataldo 1997) and IGFBPs 1, 2,3 and 4 have been found in follicular fluid (Holly et al. 1990, Cataldo & Giudice 1992).In the PCOS follicles, the IGFBP profiles are similar to those in androgen-dominant,small antral (atretic) follicles obtained from normally cycling women. In the androgen-dominant follicles, there are high levels of IGFBP-2 and –4 and low levels of IGFBP pro-teases compared with growing estrogen-dominant follicles (Cataldo & Giudice 1992, SanRoman & Magoffin 1993). It has been reported that IGFBP-4 localization in PCOS antralfollicles correlated with insulin sensitivity: insulin resistant women had greater IGFBP-4

42

staining in the theca than in granulosa, while the reverse was seen in non-insulin resistantsubjects (Peng et al. 1996). In women with PCOS, however, follicular fluid IGFBP-1levels have been shown to be 48% of those from cycling women, possibly reflecting aninhibitory effect of insulin on IGFBP-1 production (Holly et al. 1990). Based on availabledata in humans and other species, the IGF system (increased IGF-II, decreased IGFBP-2and increased IGFBP-4 protease) may be “turned on” at the time of dominant follicleselection, augmenting the effects of FSH on granulosa. High levels of IGFBPs and theabsence of IGFBP protease in PCOS follicles would sequester the IGF-1 and therebyreduce its synergistic action with FSH (Yen 1999).

Any full understanding of the role of the IGF system in ovarian processes must takeinto account the myriad of growth factors and cytokines that coexist along with the intra-ovarian IGF system.

2.4.5 Leptin and PCOS

Leptin, a 167 amino-acid protein transcribed from the ob gene, was discovered by Zhanget al. 1994. In obese, hyperphagic, homozygote ob/ob mice, two mutations of the ob genewas demonstrated to lead to a lack of leptin (Zhang et al. 1994). Leptin is produced main-ly in fat cells. The secretion of leptin has ultradian and circadian fluctuations (Sinha et al.1996, Licinio et al. 1997) and is enhanced by glucocorticoids and insulin (Boden et al.1997, Papaspyrou-Rao et al. 1997). Leptin plays an important role in the regulation ofbody-fat mass in animals and in humans (Halaas et al. 1995). Secreted leptin appears tobind to one or more proteins in the circulation (Sinha et al. 1996). Leptin achieves mostof its metabolic effects by interacting with specific receptors located in the central ner-vous system and in peripheral tissues (Tartaglia et al. 1995, Lee et al. 1996). The leptinreceptor is a class I cytokine receptor (Ihle 1996). Several lines of evidence suggest thatthe hypothalamus is a critical target for the satiety effects of leptin (Halaas et al. 1995,Erickson et al. 1996). The complete set of genes involved in mediating the downstreameffects of activation of the central leptin receptor and of transcription factors on energymetabolism is unknown. One candidate effector molecule is the hypothalamic neuropep-tide Y (NPY), a potent stimulator of food intake, of which synthesis is inhibited by leptin(Stephens et al. 1995, Erickson et al. 1996).

Insulin resistance is associated with elevated plasma leptin concentrations (Segal et al.1996, Vauhkonen et al. 1998b), and hyperleptinemia has been suggested to be part of theinsulin resistance syndrome (de Courten et al. 1997, Laivuori et al. 2000). Subcutaneousfat tissue has been shown to be a more important determinant of serum leptin levels thanintra-abdominal fat tissue (Vauhkonen et al. 1998b). The high levels of leptin in obesitymay reflect resistance of these subjects to leptin action (Bray & York 1997). Hepaticeffects of leptin has been suggested to contribute to insulin resistance (Cohen et al. 1996).Leptin could contribute to a hypersecretion of insulin by the pancreatic β-cell islets inobese, insulin resistant subjects (Larsson et al. 1996), but there is evidence that leptinmay also have favourable effects on insulin sensitivity (Shimabukuro et al. 1997).

Leptin administration to the leptin deficient ob/ob mice produces weight loss, as wellas restoring ovulation and fertility (Halaas et al. 1995, Chehab et al. 1996). A link bet-

43

ween serum leptin and LH concentrations during the menstrual cycle has been reported inhumans (Messinis et al. 1998, Teirmaa et al. 1998). A direct effect of leptin on ovariansteroidogenesis is possible, because messenger RNA for leptin receptors has been foundin both the theca- and granulosa cells of the ovaries (Karlsson et al. 1997) and leptin synt-hesis has also been demonstrated in ovarian granulosa cells (Cioffi et al. 1997). It hasbeen reported that leptin in vitro inhibits the synergistic action of IGF-I on steroidogene-sis in human granulosa and theca cells (Agarwal et al. 1999).

The first report concerning leptin and PCOS suggested that a substantial proportion ofwomen with PCOS have leptin levels higher than expected for their BMI (Brzechffa et al.1996). Later studies did not find significant differences in leptin levels between PCOSwomen and weight- (Mantzoros et al. 1997) or fat-mass matched controls (Laughlin et al.1997). However, a recent study has shown that although circulating leptin levels in PCOSwomen did not differ from those in age- and weight-matched controls, the plasma NPYconcentrations were significantly higher in non-obese and obese PCOS women than incontrol women. The NPY concentration in PCOS women remained unchanged, in spite ofan increased BMI. This may indicate a disturbed feedback system in the interaction bet-ween NPY and leptin in obese women with PCOS (Baranowska et al. 1999). In a cohortof women with PCOS serum leptin concentrations were found to be 20% lower than incontrols with similar BMI across a wide range of body weights. It is therefore possiblethat insulin-stimulated leptin secretion is limited by insulin resistance in adipocytes inPCOS women (Jacobs & Conway 1999). Furthermore, visceral fat has been shown to sec-rete less leptin than subcutaneous fat (Vauhkonen et al. 1998b, Van Harmelen et al. 1998),which may lead to decreased satiety signals in the hypothalamus, permitting further deve-lopment of obesity (Jacobs & Conway 1999).

A new signaling molecule, resistin, has recently been discovered which is inducedduring adipogenesis and secreted by adipocytes. Resistin levels are increased in diet-induced obesity as well as in genetic models of obesity and insulin resistance. In adipo-cytes, neutralization with resistin antiserum enhanced insulin-stimulated glucose uptake,and insulin action was blunted by recombinant resistin. The administration of resistin tomice impaired glucose tolerance (IGT) without reducing insulin levels, and decreasedsensitivity to the effects of insulin. Recent data suggest that resistin is a unique hormonewhose effects on glucose metabolism are antagonistic to those of insulin (Steppan et al.2001).

2.4.6 Lipids and PCOS

Women with PCOS have lower HDL and /or HDL2 levels, higher Trigly and low-densitylipoprotein (LDL) levels than age-, and weight-matched control women (Wild et al. 1985,Conway et al. 1992). To a large extent, lipid profiles in PCOS are found to be related tothe degree of insulin resistance /hyperinsulinemia, independent of androgen levels andBMI (Norman et al. 1995). Plasminogen activator inhibitor (PAI) levels are elevated(Dahlgren et al. 1994b, Sampson et al. 1996) and together with the alterations of lipidsmay be partly responsible for the increased incidence of hypertension, coronary heart dis-ease and thrombosis in PCOS women (Wild et al. 1985, Talbott et al. 2000).

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2.4.7 Genetics of PCOS

A genetic basis for the Stein-Leventhal syndrome was suggested by Cooper and col-leagues in 1968 (Cooper et al. 1968). They studied 18 families in which the syndromeappeared in a pattern consistent with a dominant mode of inheritance. Many later studieshave also suggested a dominant mode of inheritance (Ferriman & Purdie 1979, Hague etal. 1988, Lunde et al. 1989, Carey et al. 1993, Govind et al. 1999) and one study suggest-ed an X-linked mode (Givens 1988). There are obvious problems which make geneticstudies of PCOS difficult to perform. The heterogeneity and lack of universally accept-able clinical or biochemical diagnostic criteria have been discussed. PCOS is a disorderwhich primarly affects women of reproductive age and it is therefore difficult for segre-gation studies to span more than one generation. There is no commonly accepted malephenotype. Male pattern premature balding has been demonstrated in male relatives infamilial PCOS studies. Lastly, the high prevalence of PCO in the population means thatlarge pedigrees may include subjects with PCO arising from a different genotype thanthat of the proband (Franks et al. 1997).

Genetic analyse of candidate genes have been performed. Both linkage and associationstudies have suggested that PCOS can be explained by the interaction of a small numberof key genes with environmental, particularly nutritional, factors. The steroid synthesisgene CYP11a, coding for P450 cholesterol side chain cleavage and the insulin gene regu-latory region may be involved (Franks et al. 1997, Waterworth et al. 1997, Diamanti-Kan-darakis et al. 2000).

While we are unable to exclude an autosomal or X-linked dominant mode of inheri-tance, the heritability of PCOS is probably more complex, similar to that of type 2 DM orcardiovascular disease. However, a positive family history appears to be the most infor-mative risk factor for the development of PCOS (Azziz & Kashar-Miller 2000). Further-more, environmental factors alter the clinical and biochemical presentation in those withgenetic predisposition to PCOS. This is illustrated by the effect of obesity, or converselycalorie restriction, on insulin levels, insulin sensitivity and menstrual function (Dunaif etal. 1987, Holte et al. 1995).

Fig. 8. Natural history of women with PCOS.0

2

4

6

8

20

AGE (years)

6040

hypercholesterolemia

coronary heart diseasediabetes mellitushypertension

30

gestational diabetespregnancy induced hypertensioninfertility, miscarriage

hirsutism

10

menstrual irregularities, oligo-/amenorrheapronounced adrenarche ?

5

- INSULIN RESISTANCE -

- OBESITY -

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2.5 Long-term sequelae and risks in PCOS

2.5.1 Dyslipidemia and cardiovascular disease

Lipid abnormalities have been well described among women with PCOS (see chapter2.4.6). Central obesity, insulin resistance and hyperlipidemia constitute the basis for anincrease in cardiovascular risks (Goode et al. 1995). Elevated PAI-1 levels found inPCOS women together with altered lipids are considered to be an added independent car-diovascular risk factor by increasing the incidence of intravascular thrombosis (Sampsonet al. 1996).

A seven-fold increase in the risk for myocardial infarction in PCOS has been pre-dicted (Dahlgren et al. 1992). There are studies reporting a higher prevalence of athe-rosclerosis on cardiac catheterization among hirsute women and greater intima-mediathickness on carotis ultrasonography among PCOS women (Wild et al. 1990, Guzick etal. 1996, Talbott et al. 2000). However, long-term follow-up studies have not shown inc-reased cardiovascular mortality in PCOS women, although a history of non-fatal cerebro-vascular disease and cardiovascular risk factors including diabetes are more prevalentamong women with PCOS (Pierpoint et al. 1998, Cibula et al. 2000, Wild et al. 2000). Ina case-control study, women with PCOS have been shown to have poorer lipid profilesand otherwise increased risk factors for cardiovascular disease, probably from youngadulthood onward. As the women get older, the significant differences seemed to disap-pear, other than hyperinsulinemia (Talbott et al. 1998). It has been postulated that eitherthe action of unopposed estrogen secreted during anovulatory cycles or elevated levels ofvascular endothelial growth factor protects PCOS women from fatal circulatory disease(Pierpoint et al. 1998) (Wild et al. 2000). It also possible that - with advancing age - thedifference in cardiovascular risk factors between PCOS women and controls may dimi-nish, accounting for the failure to detect an excess risk of cardiovascular mortality amongwomen with PCOS (Talbott et al. 1998, Wild et al. 2000).

2.5.2 Hypertension

Several studies have reported an association between PCOS and hypertension (Dahlgrenet al. 1992, Wild et al. 2000). In part, this can be explained by the increased prevalence ofobesity in women with PCOS. In a smaller study involving 24 lean women with PCOSand 26 normally cycling women, no difference was found in ambulatory blood pressures(Sampson et al. 1996).

Paradisi and co-workers found that mean diastolic blood pressure throughout preg-nancy was significantly higher among women with PCOS as compared with controls(Paradisi et al. 1998). The incidence of pre-eclampsia has been reported to be higher inwomen with PCOS compared with the general pregnant population, although the overallrates of pregnancy-induced hypertension were comparable between the groups (Gjönna-ess 1989). In contrast, in a retrospective cohort study, rates of overall pregnancy- inducedhypertension, not of pre-eclampsia alone, were significantly higher among PCOS womenthan in controls (Urman et al. 1997). It has been shown that pre-eclampsia is a state of

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increased insulin resistance persisting for at least 3 months after pregnancy (Kaaja et al.1999). Women with prior pre-eclamptic pregnancy seem to be characterized by hyperin-sulinemia and mild hyperandrogenism for up to 17 years after delivery (Laivuori et al.1996, Laivuori et al. 1998). It was recently concluded in a retrospective study, however,that PCOS does not have an important effect on the rate of premature delivery and pre-eclampsia (Mikola et al. 2001).

2.5.3 Gestational diabetes and diabetes mellitus

Gestational diabetes mellitus, defined as glucose intolerance with an onset or first recog-nition during pregnancy (O'Sullivan & Mahan 1965, Buchanan & Kjos 1994) occurs in0.2-8% of pregnancies (Hadden 1985). The pathogenetic mechanisms underlying GDMinvolve an imbalance between the capacity of the pancreatic β-cells and the increaseddemands for insulin due to decreased insulin sensitivity during pregnancy (Buchanan etal. 1990, Damm et al. 1992). Some studies have suggested that insulin resistance is theprimary cause in the deterioration of glucose metabolism in GDM (Byrne et al. 1995,Ryan et al. 1995), while others have emphasized the role of defective insulin secretion(Kuhl 1977, Byrne et al. 1995, Damm et al. 1995). Therefore, it may be that GDM isactually a heterogeneous disorder, which may at least partly explain the contradictoryresults regarding the primary defect in GDM.

Women with GDM have a significantly increased risk of developing type 2 DM laterin life (Stowers et al. 1985, Buschard et al. 1987, Damm et al. 1992). Furthermore, GDMis thought to present an early manifestation of the metabolic syndrome (or syndrome X),which is a cluster of abnormalities in which a combination of insulin resistance and com-pensatory hyperinsulinemia predisposes individuals to develop a high plasma Trigly and alow HDL cholesterol concentration, high blood pressure and coronary heart disease (Rea-ven 1994).

Women with PCOS seem to share some of the same metabolic abnormalities aswomen with GDM. An increased frequency of PCO, hirsutism and irregular cyclesamong women with a history of GDM, as compared with women with uncomplicatedpregnancy, has been reported (Anttila et al. 1998, Holte et al. 1998, Kousta et al. 2000).Some prospective and retrospective data support an increased risk for GDM in PCOS(Gjönnaess 1989, Paradisi et al. 1998, Mikola et al. 2001), especially if pregravid or earlypregnancy hyperinsulinemia exists (Lanzone et al. 1995a, Paradisi et al. 1998). There is,however, retrospective data which demonstrates that women with PCOS are not signifi-cantly more likely than other patients to have gestational diabetes (Wortsman et al. 1991,Vollenhoven et al. 2000).

Because of insulin resistance and central obesity, all women with PCOS are consideredto be at increased risk for IGT and overt type 2 DM. Dunaif suggested that up to 20% ofobese women with PCOS have IGT or DM by their third decade (Dunaif 1993). Accor-dingly, women with PCOS have significantly increased prevalence rates of IGT and, oftenundiagnosed, DM than do their age-, weight- and ethnicity-comparable reproductivelynormal women, even at a young age (Legro et al. 1999, Wild et al. 2000). It has recentlybeen shown that women with type 2 DM have a higher prevalence of PCO than that

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reported in the general population (82%)(Conn et al. 2000). Women with type 1 DM havebeen shown to have a high prevalence of hyperandrogenic disorders (38.8%), includingPCOS and hirutism (Escobar-Morreale et al. 2000). The number of deaths in which diabe-tes was considered to be a contributing cause has been demonstrated to be significantlyhigher among women with PCOS than that expected based on national data (OR, 3.6;95% CI 1.5 to 8.4) (Pierpoint et al. 1998).

2.5.4 Cancer

Obesity is a well documented risk factor for endometrial cancer (Folsom et al. 1989).Type 2 DM is associated with a twofold increase in the risk for endometrial cancer, whichis, at least in part, independent of concomitant obesity (O'Mara et al. 1985). One possiblemechanism by which PCOS increases the risk for endometrial cancer is the associatedhigh and unopposed levels of endogenous estrogen, which has been directly linked toendometrial cancer risk (Hammond et al. 1979). Epidemiologic studies have shown anincreased risk for endometrial cancer in women with chronic anovulation (Coulam et al.1983). In a case series of 97 young women with endometrial hyperplasia, 25% had PCOS(Chamlian & Taylor 1970). In a study involving 175 women with endometrial cancer and1746 population-based controls, several features suggestive of PCOS were more com-mon among the cases (Dahlgren et al. 1991).

It has been hypothesized that the androgenic milieu in the ovary of PCOS subjectsmight also predispose them to certain types of ovarian cancer. In the Cancer and SteroidHormone Study, women with epithelial ovarian cancer were significantly more likely toreport a prior diagnosis of PCOS than controls (Schildkraut et al. 1996). However, thereare several studies which could not show increased ovarian cancer risk in PCOS women(Coulam et al. 1983, Pierpoint et al. 1998). However, the possibly increased cancer riskemphasizes the usefulness of contraceptive pills in the treatment of PCOS (see 2.6.1), as areduction of the incidence of endometrial and possibly of ovarian cancer (Hulka et al.1982, Godsland et al. 1992).

2.6 Modern treatment of PCOS

Aside from establishing fertility, inducing ovulatory cycles and improving hirsutism, oneimportant aim in the treatment of PCOS women is to reduce the incidence of the long-term consequences of metabolic sequelae.

2.6.1 Treatment of hyperandrogenism

The symptoms of hyperandrogenism frequently bring the patient to medical attention.Drug treatment is directed at suppressing ovarian or adrenal sources of androgens orblocking androgen action in the skin.

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Oral contraceptives (OC) have been shown to be moderatly effective in reducing circu-lating androgen levels by up to 50% and in alleviating hirsutism (Givens et al. 1974).They suppress gonadotrophin secretion (Givens et al. 1974), reduce ovarian androgensecretion (Wild et al. 1982), increase SHBG synthesis thereby decreasing androgen actionand inhibit DHT binding to androgen receptors (Eil & Edelson 1984). The administrationof OCs alone is often effective in the treatment of acne. The combination of OCs withantiandrogen seems to be necessary in moderately or severe cystic acne and hirsutism(Rittmaster 1995).

Cyproterone acetate (CPA) is a potent progestin, a moderatly potent antiandrogen anda weak glucocorticoid. It acts as a competitive inhibitor of androgens by binding to theirreceptors, reduces 5α-reductase in the skin, and lowers ovarian androgen secretion byinhibiting gonadotrophin release (Neumann et al. 1970). It can be orally administered at adaily dose of 25-50 mg from days 1 to 10 of the cycle and is available as an OC (2 mgCPA in combination with 35 µg of ethinylestradiol) (Rittmaster 1995). An improvementof hirsutism is usually seen after 3 to 6 months of treatment (Kuttenn et al. 1980). Sup-pression of ovarian function by the use of GnRHa has also been succesful in the improve-ment of hirsutism (Adashi 1990, Rittmaster & Thompson 1990, Tiitinen et al. 1994).

Spironolactone binds competitively to the androgen receptor with 67% of the affinityof DHT (Eil & Edelson 1984). It is also a weak progestin and weak inhibitor of T biosynt-hesis. Spironolactone is usually administered orally at 25 to 100 mg twice daily (Crosby& Rittmaster 1991) and can be used together with OCs. Spironolactone alone is as effec-tive in the treatment of hirsutism as OC with CPA (Spritzer et al. 2000).

Flutamide is a nonsteroidal antiandrogen widely used in the treatment of prostate can-cer. It has no progestational, estrogenic, glucocorticoid or antigonadotropic activities. It isadministered orally in the treatment of hirsutism at a dose of 125-250 mg twice daily withor without OC. Its efficacy is comparable with spironolactone and CPA (Erenus et al.1994, Grigoriou et al. 1996, Moghetti et al. 2000).

Finasteride, a 5α-reductase inhibitor, inhibits the conversion of T to DHT, the activeandrogen in the hair follicle (Rittmaster 1997). It is administered 5 mg daily in the treat-ment of hirsutism and seems to be as effective as flutamide (Erenus et al. 1997, Falsetti etal. 1997, Sahin et al. 1998). Combined with OC including CPA, finasteride significantlydecreased the hirsutism score after 3 months of treatment, while OC alone induced thiseffect after 6 months of treatment (Tartagni et al. 2000).

The antifungal agent ketoconazole seems to be an effective antiandrogen in the treat-ment of hirsutism in PCOS women using a dose of 400 mg daily (Gokmen et al. 1996). Itis a known inhibitor of steroidogenic enzymes of the P450 family (Feldman 1986).

All the medications above may have teratogenic effects. It is therefore suggested thatthese medications should be used with a proper contraception, usually OC, in women whoare sexually active (Taylor 1998).

2.6.2 Treatment of irregular cycles and anovulation

When infertility is not an issue, the treatment of irregular menstruation is best achievedusing a low-dose OC or by the cyclic administration of oral progestins. Progestin therapy

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improves irregular menstrual bleeding and reduces the risk of endometrial cancer (Dahl-gren & Janson 1994a). The most conservative approach with progestins is to induce blee-ding monthly (Taylor 1998). A low dose OC normalizes menstrual cycles as well as pre-vents endometrial hyperplasia (Hulka et al. 1982). It also suppresses hirsutism and provi-des contraception in addition to its other known benefits. There is conflicting data as towhether or not the treatment with OCs increases insulin resistance. Some data exists sug-gesting a worsening in carbohydrate tolerance (Morin-Papunen et al. 2000) and enhancedinsulin resistance in women who receive OC (Godsland et al. 1992, Korytkowski et al.1995), although OC seemed not to be associated with an adverse effect on the lipid profileof PCOS women.

The first-line treatment for the induction of ovulation in PCOS women with infertilityis the antiestrogen clomiphene citrate (CC). The ovulation rate is high (75-80%) with acumulative conception rate approaching normal (Franks et al. 1985, Hull 1992). There issome evidence that combining dexamethasone to CC to suppress adrenal androgen secre-tion increased ovulation and pregnancy rates in PCOS women with elevated DHEAS orresistance to CC (Lobo et al. 1982, Daly et al. 1984, Trott et al. 1996). CC has beenshown to reduce plasma IGF-1 concentrations and increase serum SHBG and plasmaIGFBP-1 concentrations (Bützow T et al. 1995, De Leo et al. 2000).

Approximately 20-25% of women with PCOS are, however, resistant to clomiphene.For these women, the induction of ovulation is performed with the exogenous administra-tion of gonadotrophin preparations, either with or without GnRHa to inhibit endogenousovarian function, with a 50% cumulative pregnancy rate (Wang & Gemzell 1980). As therisk of ovarian hyperstimulation in response to exogenous gonadotrophins is greater inwomen with PCOS, these women are good candidates for in vitro fertilization (IVF)techniques (Taylor 1998). In PCOS associated with hypersecretion of LH, purified FSHpreparations have theoretical advantages over the use of hMG preparations containingboth FSH and LH. It remains uncertain whether or not this claimed advantage extendsinto clinical practice. Clinical results (including pregnancy rate) in IVF cycles have beensimilar in PCOS women using purified or recombinant FSH compared with PCOSwomen using hMG preparations (Homburg et al. 1990, Sagle et al. 1991, Fulghesu et al.1992, Teissier et al. 1999).

Surgical treatment by bilateral wedge resection, although relatively succesful in resto-ring ovulation, has fallen from grace due to its propensity for adhesion formation (But-tram & Vaquero 1975, Adashi et al. 1981). Laparoscopic ovarian diathermy was introdu-ced by Gjönnaes (Gjönnaess 1984). In his small study and in larger studies since (Aak-vaag & Gjönnaess 1985, Kovacs et al. 1991, Naether et al. 1993a, Felemban et al. 2000)ovulation rates of 70-90% and cumulative pregnancy rates of 40-70% have been achie-ved. Postoperative laparoscopy has revealed the presence of mild intraperitoneal adhe-sions in about 20% of these cases (Dabirashrafi et al. 1991, Naether & Fischer 1993b). Acase of unilateral ovarian atrophy has been reported after the procedure (Dabirashrafi1989). The benefit of ovarian diathermy is limited to approximately 6 months, but it is apromising mode of therapy for clomiphen resistant women with PCOS and large ovaries.It was recently shown that laparoscopic ovarian drilling with an insulated needle cauteryis an effective treatment in clompihen resistant women with PCOS. This treatment wasassociated with a minimal amount of adhesion formation (Felemban et al. 2000).

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2.6.3 Insulin-lowering treatment in PCOS

2.6.3.1 Weight loss

In numerous studies of women with PCOS, caloric restriction (even without weight loss)or weight-reducing diets have resulted in a normalization of insulin sensitivity. Theimprovement in endocrine-metabolic parameters occurs after 4 to 12 weeks of dietaryrestriction (Kiddy et al. 1992), an increase in SHBG is accompanied by a fall in free T,insulin and IGF-I levels. Serum concentrations of IGFBP-1 rise significantly (Hamilton-Fairley et al. 1993). Consequent to these changes, weight loss is attended by a reductionof hyperandrogenism and a restoration of ovulation (Bates & Whitworth 1982) (Harlasset al. 1984, Jakubowicz & Nestler 1997). Hirsutism also appears to improve significantlyin most of the patients losing weight (Pasquali et al. 1989).

Weight loss may also decrease the LH pulse amplitude (Harlass et al. 1984). It hasbeen demonstrated in severly overweight infertility patients that weight reduction with avery low calorie diet results in a decrease in LH concentrations, a reduction in the LH/FSH ratio, and FSH predominance favoring folliculogenesis. The decrease in LH con-centrations was inversely related to the severity of insulin resistance (Butzow et al. 2000).

Moderate exercise, a process of increased fuel expenditure, has been shown to induce arise in serum IGFBP-1 with a decrease in serum IGF-I concentrations (Suikkari et al.1989). Thus, when combined with dietary restriction, exercise serves as an importantadjunct therapy for PCOS (Yen 1999).

2.6.3.2 Insulin lowering medication

Agents which lower circulating insulin without affecting insulin sensitivity

The first anti-diabetic drugs studied in PCOS women were diazoxide and the somatosta-tin analogue, octreotide, which can both directly inhibit pancreatic insulin secretion. Theyalso reduce serum androgen and LH concentrations. Octreotide has been shown to restoreovulation (Nestler et al. 1989, Prelevic et al. 1995). The long-term use of these drugsmay, however, worsen glucose tolerance and further increase the risk of developing dia-betes (Lillioja et al. 1993, Dunaif 1995a).

The demonstration of β-endorphins in the human pancreas (Ipp et al. 1978) and theevidence that they may stimulate insulin and glucagon release in humans (Giugliano et al.1987) suggest that opioids may play a role in glycoregulation. The opioid antagonist nalt-rexone has been demonstrated to decrease insulin response during OGTT and may do solargely by increasing the rate of insulin clearance (Lanzone et al. 1991, Lanzone et al.1995b, Fulghesu et al. 1998). Opioid antagonists do not seem to affect glucose utilizationduring a clamp study (Fulghesu et al. 1998). Although these antagonists have not beenassociated with a lowering of LH or androgens (Fulghesu et al. 1993, Cagnacci et al.1994), they appear to reduce the LH response to the GnRH test in hyperinsulinemic patie-nts (Lanzone et al. 1995b). Improvements in both spontaneous ovulation and responsive-

51

ness to CC have also been noted in association with the decline in circulating insulin(Roozenburg et al. 1997).

Metformin

The biguanide molecule was first synthesized in 1879. Metformin and phenphormin, thetwo main biguanides, were introduced in the 1920s (Bailey & Nattrass 1988). Metformin(dimethylbiguanide) is approved for the treatment of type 2 DM and is in use worldwide.Although it is not technically considered an “insulin sensitizer”, meformin has severalmechanisms of action which tend to result in improvements in hyperinsulinemia. It hasbeen suggested that metformin should be regarded as an “antihyperglycemic” agent(Bailey & Nattrass 1988). The effect is seen in the liver where it suppresses hepatic glu-cose output, possibly due to an inhibition of gluconeogenesis (Stumvoll et al. 1995) sec-ondarily to the decrease in serum FFA levels (Perriello et al. 1994, Wiesenthal et al.1999). It has also been suggested that metformin decreases FFA uptake and oxidation inthe liver and muscle and improves insulin-mediated glucose utilization in these tissuesvia a substrate-competition effect (Randle et al. 1963, Abbasi et al. 1997, Abbasi et al.1998). There are studies, however, in which no effect of metformin on hepatic glucoseproduction was observed (Riccio et al. 1991, Abbasi et al. 1997). In vivo, metforminappears to delay glucose absorption and increase glucose utilization by the intestine(Bailey & Turner 1996). Metformin may also improve peripheral insulin resistance(Inzucchi et al. 1998).

In vitro and in vivo evidence exists according to which metformin improves insulinaction and direct effects on glucose utilization in lymphocytes or adipocytes (Nosadini etal. 1987, Purrello et al. 1987, Matthaei et al. 1991). Metformin is shown to increase insu-lin-mediated glucose uptake in peripheral tissues with predominantly enhanced nonoxida-tive (Riccio et al. 1991, Widen et al. 1992, Bell & Hadden 1997) or oxidative glucosemetabolism (Perriello et al. 1994). Others have failed to confirm these results (Wu et al.1990). It is possible that meformin improves insulin-receptor binding in vitro by increa-sing the number of low-affinity insulin binding sites in lymphocytes, erythrocytes andhuman breast cells (Holle et al. 1981, Bailey & Nattrass 1988). Metformin could alsopotentiate insulin action by postreceptor effects, as demonstrated by an increase in bothglycogen synthesis and glucose oxidation in the soleus muscle of mice (Bailey et al.1986). A possible postreceptor mechanism has not been defined. Metformin may inhibitlipolysis in adipose tissue and decrease both FFA concentrations and lipid oxidation (Ric-cio et al. 1991, Perriello et al. 1994, Abbasi et al. 1998). Metformin significantly reducescirculating lipid levels because of a reduced hepatic synthesis of very low-density lipo-protein (VLDL) and Trigly (Fedele et al. 1976). Decreases in plasma Trigly, total andLDL cholesterol concentrations and increases in HDL and the HDL/LDL cholesterolratio, independently of improved glycemic control, have been observed (DeFronzo &Goodman 1995).

Velasquez published the first study of metformin (1.5 g daily) in women with PCOS(Velazquez et al. 1994), demonstrating an improvement in T and free T levels in obesepatients. Subsequent studies have demonstrated that metformin (1.0 to 2.25 g daily) canincrease SHBG levels and reduce free T levels (Nestler & Jakubowicz 1996), improveovulation rates (Velazquez et al. 1997, Morin-Papunen et al. 1998, Moghetti et al. 2000)

52

and improve response to CC (Nestler et al. 1998b) or exogenous gonadotropins (De Leoet al. 1999). It has recently been reported that metformin (1275 mg daily for 6 months)reduced hyperinsulinemia, hirsutism and hyperandrogenism, and restored eumenorrheafor the time of the treatment in nonobese adolescent girls with a history of precociouspubarche who had a tendency to hyperinsulinism (Ibanez et al. 2000). It has been shownthat insulin reduction with metformin increases follicular and luteal phase serum glycode-lin and IGFBP-1 concentrations and enhances luteal phase uterine vascularity and bloodflow in PCOS. It was concluded that these changes may reflect an improved endometrialmilieu for the establishment and maintenance of pregnancy (Jakubowicz et al. 2001). Notall studies, however, have demonstrated a benefit of this medication (Crave et al. 1995,Ehrmann et al. 1997a).

The androgen lowering effects of metformin are still unclear. In general, studies inwhich metformin therapy results in a reduction in serum insulin concentrations are thesame studies, which demonstrate a significant decrease in serum T concentrations (Tay-lor 2000a). Androgen responses to ACTH have been demonstrated to decrease after 4weeks of metformin (1-1.5 g daily) treatment (la Marca et al. 1999) or not change after 12weeks of metformin treatment (Unluhizarci et al. 1999a). Studies evaluating the ovarianandrogen response to leuprolide acetate, buserelin or to hCG have shown either decreased(Nestler & Jakubowicz 1996, la Marca et al. 2000) or unchanged ovarian androgen res-ponse after 4 to 12 weeks of metformin (1-1.5 g daily) treatment (Unluhizarci et al.1999b). In a recent randomized, double-blind and placebo-controlled 6-month trial inPCOS women with abdominal obesity, it was shown that long-term treatment with met-formin (1.7 g daily) added to a hypocaloric diet induced (in comparison with placebo) agreater reduction of body weight and abdominal fat and a more consistent decrease ofserum insulin, T, and leptin concentrations. These changes were associated with a moresignificant improvement of hirsutism and menstrual irregularities. In that study, metfor-min significantly decreased insulin and the C-peptide response to oral glucose administra-tion, which indicates a contemporary improvement of both insulin resistance and β-cellfunction (Pasquali et al. 2000). It is difficult to explain why metformin succesfully lowersinsulin and androgen levels in some studies but not in others. Variations in body weight,dosing, entry criteria or genetic background may be the explanation (Taylor 2000a).

Thiazolidinediones

The thiazolidinediones are true “insulin sensitiziers” which improve peripheral glucoseuptake, while also having some effect on hepatic glucose production (Inzucchi et al.1998). To date, five studies exist documenting the efficacy of troglitazone, the first-gener-ation thiazolidinedione in obese women with PCOS (Dunaif et al. 1996b, Ehrmann et al.1997b, Hasegawa et al. 1999, Mitwally et al. 1999, Azziz et al. 2001). Troglitazoneimproves the action of insulin in the liver, skeletal muscle, and adipose tissue directly. Itacts primarily as a ligand for the nuclear peroxisome proliferator activated receptor,which, when activated, enhances the transcription of factors promoting glucose disposal,primarily in the muscle (Saltiel & Olefsky 1996b).

All of the studies examining the effect of troglitazone (150-600 mg for 12-14 weeks)on PCOS women have demonstrated a reduction in fasting insulin levels or the insulinarea under the curve in OGTT, as well as a reduction in free T levels without a change in

53

body weight (Dunaif et al. 1996b, Ehrmann et al. 1997b, Hasegawa et al. 1999 Azziz etal. 2001). Troglitazone suppresses ovarian androgen response to leuprolide acetate andreduces the functional activity of PAI-1 in blood (Ehrmann et al. 1997b). Although trogli-tazone seems effective in these studies of women with PCOS, reports of fatal hepatotoxi-city have significantly limited its usefulness. A large multicenter study of troglitazoneshowed improvement of ovulation and hirsutism in women with PCOS (Azziz et al.2001). According to its package insert, troglitazone reduces serum levels of ethinyl estra-diol and norethindrone by approximately 30% each. Therefore, women with PCOS usingOCs might experience contraceptive failure or breakthrough bleeding (Taylor 2000a).There are two new thiazolidinediones approved for use for type 2 DM in USA, rosiglita-zone and pioglitazone, which are theoretically attractive alternatives to troglitazone due totheir apparently reduced risk of hepatotoxicity. However, no published reports are avai-lable for these drugs in the treatment of PCOS (Taylor 2000a).

D-chiro-inositol

The medication most recently studied in women with PCOS is d-chiro-inositol, a natural-ly-occurring substance which seems to improve insulin action by increasing insulin sig-nal transduction at the postreceptor level. When insulin binds to its receptor, inositolphosphoglycan mediators are generated by hydrolysis of glycosylphosphatidylinositollipids located at the cell membrane. An inositol phosphoglycan molecule containing d-chiro-inositol and galactosamine is known to play a role in activating key enzymes con-trolling the oxidative and nonoxidative metabolism of glucose (Ortmeyer et al. 1993). In20 obese women with PCOS, d-chiro-inositol (1200 mg) improved serum insulin andserum androgen concentrations, decreased WHR and improved ovulation (Nestler et al.1999).

3 Purpose of the present study

Polycystic ovary syndrome (PCOS) reflects multiple potential etiologies and variableclinical presentations. Possibly the mildest form of this condition is the isolated ultra-sonographic finding of polycystic ovaries (PCO) with no other clinical symptoms ofPCOS. One purpose of this study was to investigate the prevalence of PCO in the Finnishpopulation. Due to the known association between insulin resistance and PCOS, the prev-alence of PCO among women with a history of altered glucose metabolism, i.e. gestation-al diabetes (GDM) was also examined. The frequency of the recently discovered v-LH ishigh in the Finnish population, for this reason and due to the well known genetic isola-tion in Finland, it was interesting to study the occurrence of v-LH in PCOS, a conditionin which LH is known to play a central role. A excessive production of androgens is char-acteristic to PCOS. This study attempted to evaluate the nature of the ovarian stre-oidogenic response of women with PCOS to different gonadotrophins. Specifically, thedetailed aims of this study were:1. To determine the prevalence of PCO in the Finnish population among women with no

other clinical symptoms of PCOS (Study I).2. To study the prevalence of v-LH in PCOS and to compare patient cohorts from

Finland, the Netherlands, the United Kingdoms and the United States (Study II).3. To evaluate the prevalence of PCO, insulin sensitivity and secretion in women with

previous GDM (Study III).4. To evaluate the role of exogenously administered LH and/or FSH in ovarian steroid

hormone secretion in women with PCOS. Special attention was paid to the androgensecretion pattern after hCG, hMG and FSH stimulation (Studies IV and V).

4 Subjects and methods

4.1 Subjects and study design

The study population in the study I consisted of 189 healthy volunteers invited through anadvertisement in the University Hospital of Oulu. They had not been treated for menstru-al disturbances, infertility or hirsutism. Only women with no gynecological problemswere recruited. In study II, a total of 1466 serum samples from Finland (n=425), theNetherlands (n=347), the United Kingdom (n=404) and the United States (n=290 cauca-sians from Boston, MA) were analysed. 363 women fullfilled the criteria for PCOS, 79women had PCO with no other symptoms of PCOS and 944 healthy women with regularcycles served as controls.

The subjects for study III were recruited using a questionnaire sent to 180 women deli-vered at Oulu University Hospital between the years 1990 and 1993 and who had hadGDM for the first time during the pregnancy. 180 age (+ 2 years), parity (nulliparous, 1-3or > 3 deliveries) and delivery date-matched women with previous uncomplicated preg-nancy without GDM served as controls. Finally, 48 controls and 31 women with GDMfullfilled the inclusion criteria and were able to attend the study. The euglycemic hyperin-sulinemic clamp was performed in 15 women with previous GDM and in 23 controlwomen. Ovarian ultrasonography had not been performed to the subjects before this studyand - based on the questionnaire - none of the subjects had been diagnosed with PCOSpreviously.

All of the PCOS subjects in studies IV and V were recruited from the ReproductiveEndocrinology Unit of the University Hospital of Oulu. The controls for study IV werehealthy volunteers contacted through an advertisement in the Oulu University Hospital.Controls in study V participated in the IVF-program in the Reproductive EndocrinologyUnit of the University Hospital of Oulu. All controls were healthy and had a regularmenstrual cycle.

A total of 399 women with PCOS, 91 women with isolated finding of polycysticappearing ovaries and 1262 healthy control women were examined during the years 1994to 1998. A summary of the subjects, methods and main results presented in the originalcommunications is shown in Table 4.

56

The diagnostic criteria for PCOS was polycystic ovaries by transvaginal ultrasound(Adamas et al. 1985), oligoamenorrhea (intermenstrual interval > 36), and hirsutism (Fer-riman-Gallwey score > 7) and/or raised serum T (> 2.7 nmol/l).

GDM had been verified by OGTT (75g) during the second trimester based on bloodglucose values: 0 min > 5.5 or at 60 min > 11 or at 120 min > 9.0 mmol/l.

Subjects using medication (including OCs), a hormonal intrauterine device and preg-nant or lactating subjects were excluded from the studies. Diabetic subjects were includedin the clinical data of the subjects in the study III but were excluded from the hormonaland metabolic data. Diabetic subjects were excluded from the other studies. Late onsetadrenal hyperplasia patients were excluded from studies II, IV and V on the basis of anormal basal serum 17-OHP level (17-OHP < 9.0 nmol/l), and patients with disorders ofthe pituitary and thyroid glands on the basis of normal serum levels of prolactin and thy-roid-stimulating hormone.

The subjects gave their informed written consent and the study protocols were appro-ved by the ethical committee of the Medical Faculty of the University of Oulu.

Table 4. Subjects, methods and m

ain results of the studies. Data is show

n as mean +

SD.

StudyStudy I

Study IIStudy III

Study IVStudy V

Study typecross-sectional

cross-sectional m

ulticenter studycase-control study

case-control study case-control study

No of subjects

normal

ovaries: 162PC

O: 27

Controls: 944

PCO

S: 363C

ontrols: 48G

DM

: 31C

ontrols: 27PC

OS: 12

Controls: 81

PCO

S: 23

Age (yr)

normal

ovaries: 36.4 ± 6.5PC

O: 33.6 ± 5.1

Controls: 32.3 ± 7.7

PCO

S: 27.6 ± 5.1 **C

ontrols: 37.1 ± 5.3G

DM

: 34.9 ± 3.6 *C

ontrols: 31.5 ± 6.0PC

OS: 29.2 ± 4.8 *

Controls: 32.4 ± 4.6

PCO

S: 30.2 ± 4.3

BM

I (kg/m

2)norm

alovaries: 23.4 ± 3.2PC

O: 22.2 ± 2.3

Controls B

MI < 27: 22.1 ± 2.1

PCO

S BM

I < 27: 22.0 ± 2.3 C

ontrols BM

I > 27: 32.5 ± 5.2PC

OS B

MI > 27: 33.4 ± 5.9

Controls: 24.4 ± 4.6

Controls + PC

O: 24.6 ± 3.3

GD

M: 25.9 ± 4.6

GD

M + PC

O: 27.5 ± 4.9

Controls: 23 ± 3.0

PCO

S: 33.1 ± 5.3 **C

ontrols: 23.1 ± 3.3PC

OS: 27.3 ± 5.1 *

Treatment

nonenone

noneM

etformin 1.5g/day for 2

months in PC

OS group

HC

G (5000 IU

) stimulation

test

GnR

H agonist +

HM

G or FSH

stimulation

Methods

Ultrasonography

Horm

onal assaysH

ormonal assays

Ultrasonography

Horm

onal assaysO

GTTEuglycem

ic hyperinsulinemic

clamp

Horm

onal assaysH

ormonal assays

Main results

The overall preva-lence of PC

O in the

study group was

14.2% and

21.5% in w

omen

aged < 35 and 7.9%

in wom

en aged > 36.

The overall carrier frequency of v-LH

allele was 18.5%

; it w

as highest in Finland (28.9%)

and lowest in the N

etherlands (11.2%

). The frequency was

low in obese PC

OS w

omen (2-

5%) in Finland, the N

etherlands and the U

SA.

Wom

en with previous G

DM

had a higher frequency of PC

O (39.4%

), abnorm

al OGTT and higher serum

levels of adrenal androgens com

-pared w

ith controls. They were

more insulin resistant w

ith a lower

acute insulin response to glucose than control w

omen

PCO

S wom

en had a male-

type steroidogenic response pattern to a single injection of hC

G. Two m

onths treat-m

ent with m

etformin did not

modify this response pattern.

FSH w

ith neglible LH activ-

ity stimulated ovarian andro-

gen production. PCO

S w

omen did not show

a dis-tinctly exaggerated steroid response to gonadotrophins and no sign of steroidogenic defects w

as observed.*p<0.05,**p<0.01 com

pared with controls.

57

58

4.2 Clinical parameters

All subjects were examined between cycle days 1 and 7 after spontaneous menstruationor after progestin-induced (dydrogesterone, 10 mg/day for 10 days) bleeding or at anyconvenient time if dydrogesterone failed to cause bleeding (two PCOS women in studyIV). The aim of using progestin in amenorrhoic subjects was to avoid examinations(ultrasonography and hormone assays) during the spontaneous luteal phase. Dydrogester-one was used as it has a negligible effect on insulin sensitivity (Crook et al. 1997). Theexaminations were performed at least 7 days after the last progestin pill to assure a mini-mal progestin effect. A regular menstrual cycle was defined as a cycle with an intermen-strual interval of 21-35 days and the variation of cycle length from one period to anotherwas < 4 days in study I and < 7 days in studies III, IV and V. A cycle was considered oli-gomenorrhoic if the intermenstrual interval was > 36. The subject was considered amen-orrhoic if the intermenstrual interval was > 6 months. BMI was calculated as the ratio ofweight (kg)/height (m)2. Obesity was defined by a cut-off of 27 kg/m2, above which insu-lin sensitivity decreases significantly in humans (Campbell & Gerich 1990). Thus, obesewomen were defined by a BMI > 27 kg/m2 and non-obese women by a BMI < 27 kg/m2.To calculate the WHR, waist and hip circumferences were measured to the nearest centi-metre with a soft tape at the narrowest part of the torso and at the widest part of the glu-teal region (Ohlson et al. 1985). Hirsutism was graded using the Ferriman & Gallweyscoring system (Ferriman & Gallwey 1961). A woman was considered hirsute if the scorewas > 7 counted from hormone sensitive areas (i.e. face, lower abdomen, anterior thighs,chest, breasts and pubic area).

4.3 Vaginal ultrasonography

Transvaginal ultrasonography of the ovaries was carried out to measure ovarian volumesand the number of follicles (Toshiba SSA-270A, Toshiba Co., Tokyo, equipped with a 6MHz curvilinear transvaginal probe, PVF-651VT, with a scanning angle of 120° in stud-ies I and III, and in the Finnish subjects in study II, and a General Electric RT- x 200,Milwaukee, Wisconsin, with a 6.5 MHz probe in studies IV and V). Polycystic ovarieswere defined as 10 or more follicles (8 or more in study II) 2-8 mm in diameter in oneplane of each ovary in association with increased and/or hyperechogenic ovarian stroma,evaluated visually (Adams et al. 1985). Volume determinations in studies I, III, IV and V,and in Finnish subjects in study II were carried out using the formula for the volume of anellipsoid: 0.523 x length x width x thickness (Robert et al. 1995). The TA of the ovarieswas assessed by carefully shaping a strict longitudinal cut in studies I, III and IV (Robertet al. 1995). The volume and the TA of the ovaries in women with ovarian cysts > 30 mmin diameter were not included.

59

4.4 Fasting insulin to glucose ratio and the homeostasis model assessment

In study IV, the fasting glucose to insulin ratio was calculated to assess insulin resistance.We used a cut-off value < 0.250 mmol/mU (= < 4.5 mg/µU), which has been demonstrat-ed to provide the best combination of sensitivity and specificity, as well as the best posi-tive and negative predictive values as a screening test for predicting insulin resistance inPCOS (Legro et al. 1998b). Another formula was used to describe insulin resistance, i.e.the homeostasis model assessment (HOMA): insulin resistance = (fasting glucose x fast-ing insulin) / 22.5 (Matthews et al. 1985).

4.5 Oral glucose tolerance test and early phase insulin and C-peptide measurements

The OGTT (a load of 75 g glucose in 300 ml water) was performed in study III after anovernight fast of 10-12 hours. Venous blood samples for blood glucose, serum insulin andserum C-peptide assays were drawn at 0, 15, 30, 60 and 120 min. Glucose tolerance wasdefined according to the new American Diabetes Association criteria (1997), as in thefollowing. Diabetes mellitus: blood glucose 0 min > 6.1 and/or at 120 min > 10 mmol/l;impaired glucose tolerance (IGT): 0 min < 6.1 and at 120 min 6.7-10.0 mmol/l; impairedfasting glycemia (IFG): 0 min > 5.6 and < 6.1, at 120 min < 6.7 mmol/l; normal glucosetolerance: 0 min < 5.6 and at 120 min < 6.7 mmol/l (Anonymous 1997).

Early phase insulin secretion (the insulinogenic index) was calculated as the ratio ofthe increment of serum insulin 30 min after an oral glucose load, to blood glucose con-centration 30 min after the glucose load [(30 min insulin - fasting insulin) / 30 min glu-cose] (Wareham et al. 1995). The insulinogenic index has previously been shown tostrongly correlate with the first phase insulin response following IVGTT (r=0.88) (Kosakaet al. 1996). Early phase C-peptide secretion was similarly calculated [(30 min C-peptide- fasting C-peptide ) / 30 min glucose]. The formula [(insulin 30 min – insulin 0 min) /(glucose 30 min- glucose 0 min)] for insulinogenic index is used more commonly (Kado-waki et al. 1984), but because the denominator becomes negative in some subjects whenearly phase insulin secretion and early phase C-peptide secretion was calculated, thisalternative formula was used.

The incremental insulin (AUCins) and glucose (AUGgluc) areas under the curve werecalculated using a trapezoidal model. The fasting serum C-peptide/fasting serum insulinratio was calculated as an index of hepatic insulin extraction in the fasting state (Shusteret al. 1988).

4.6 Euglycemic hyperinsulinemic clamp

In study III, insulin sensitivity was assessed using the euglycemic hyperinsulinemicclamp technique, which is considered as the gold standard for measuring insulin sensitivi-

60

ty in vivo (DeFronzo et al. 1979). A priming dose of insulin infusion (Actrapid 100 IU/ml; Novo Nordisk, Gentofte, Denmark) was administered during the initial 10 minutes toquickly raise plasma insulin to the desired level, where it was maintained via a continu-ous insulin infusion at a rate of 80 mU/m2 body surface area per minute. Blood glucosewas clamped at 5 mmol/l for the next 180 minutes by adjusting the rate of 20% glucoseinfusion according to blood glucose measurements performed every 5 minutes using aphotometric assay (HemoCue AB, Ängelholm, Sweden). The M-value (amount of glu-cose infused, i.e. whole body glucose disposal, µmol/kg/min) was calculated as the meanvalue for each 20 minute interval during the last 60 minutes of the clamp. The insulinsensitivity index (M/I) was calculated by dividing the M-value by the mean steady stateinsulin concentration during the last 60 minutes of the clamp (glucose µmol/l x 100 / kgof body weight / min / insulin mU/l). The coefficient of variation for blood glucose was <4% during the last 60 minutes of the clamp. It has been shown previously that endoge-nous glucose production is negligible in non-diabetic hyperandrogenic subjects at thisinsulin infusion rate, and the amount of glucose infused may be considered equivalent towhole body glucose uptake, i.e. whole body glucose disposal (M-value) (Moghetti et al.1996a). Blood samples for assay of serum lactate, insulin and free fatty acids (FFA) weredrawn at 0, 120, 140, 160 and 180 minutes.

4.7 Calorimetry

Indirect calorimetry was performed with a computerised flow-through canopy gas analy-ser system (DELTATRAC®, TM Datex, Helsinki, Finland) in connection with the eugly-cemic clamp as previously described (Laakso et al. 1988). This device has a precision of2.5% for O2 consumption and 1.0% for CO2 production. On the day of the experiment,gas exchange (O2 consumption and CO2 production) was measured after a 12 hour fastbefore the clamp and during the last 30 minutes of the clamp. The values obtained duringthe first 10 minutes of both time periods were discarded, and the mean values for theremaining 20 minutes were used for calculation. Protein, glucose and lipid oxidation werecalculated according to Ferrannini (Ferrannini 1988). Protein oxidation was calculated onthe basis of the urinary non-protein nitrogen excretion rate (Ferrannini 1988). The frac-tion of carbohydrate non-oxidation during the euglycemic clamp was estimated by sub-tracting the carbohydrate oxidation rate (determined by indirect calorimetry) from theglucose infusion rate (determined by the euglycemic clamp). Both values were adjustedfor the prevailing insulin levels during the clamp by dividing the values by the meansteady state insulin concentration during the last 60 minutes of the clamp (glucose oxida-tion index and non-oxidation index, respectively).

4.8 Human chorion gonadotrophin stimulation test

In study IV, 5000 IU hCG (Pregnyl, Organon, Oss, Netherlands) was injected intramuscu-larly on day 1-3 of a spontaneous cycle or after progestin-induced menstrual bleeding.Blood samples for 17-OHP, A, T and E2 assays were collected at 0, 24, 48 and 96 hours.

61

4.9 Treatment protocols

In study IV, women with PCOS received 500 mg metformin (metformin hydrochloride,Diformin®, Leiras, Finland) 3 times a day for 2 months. The subjects were evaluatedbefore and after the treatment.

In study V, 90 women (71 control and 19 PCOS) were given nafarelin intranasally(Synarela®, Searle, Sweden) 400 µg twice a day for pituitary suppression while 14women (10 control and 4 PCOS) received buserelin acetate nasal spray (Suprecur®,Hoechst, Frankfurt am Main, FRG) 300 µg four times per day from cycle day 23 until thepituitary-ovarian axis suppression was achieved. Suppression was considered complete iftransvaginal ultrasound revealed a thin endometrium (< 4 mm), the diameter of the fol-licles were < 10 mm and the serum E2 level was < 0.1 nmol/l. The dosage of both GnR-Has was then diminished to a half and continued until hCG administration.

When suppression was achieved, the patients began daily intramuscular injections ofhMG (Humegon®, Organon, Oss, Netherlands or Pergonal, Serono, Aubonne, Switzer-land) containing 75 IU of both LH and FSH activity per ampoule or urinary FSH (Folle-gon®, Organon, Oss, Netherlands) containing 75 IU per ampoule of FSH activity and <0.1 IU of LH activity (Harlin, Khan et al. 1986). An initial dose of two ampoules per daywas used, and the dose was increased depending on the ovarian response verified bytransvaginal ultrasound. The maximum daily dose was four ampoules. When the diameterof two or more follicles exceeded 18 mm, 5000 IU or 10 000 IU hCG (Pregnyl®, Orga-non, Oss, Netherlands) was given. Moreover, recombinant FSH (recFSH) was given to 8control women after pituitary suppression with buserelin.

Blood samples for 17-OHP, dehydroepiandrosterone (DHEA), A, T and E2 assayswere taken at 7.30-9.00 a.m. before beginning the gonadotropin treatment and on thesame day as the ultrasonographic examination before hCG injection.

4.10 Laboratory methods

Serum samples were frozen at -20° C until analysed. Blood samples were analyzedimmediately after sampling. Details of the assays used are given in Table 5. All assayswere performed according to the instructions of the reagent manufacturers.

62

Table 5. Characteristics of the assays used.

Serum LDL cholesterol was calculated by the Friedewald formula if the serum Triglylevel was < 4 mmol/l; if the Trigly level was > 4 mmol/l, LDL cholesterol was determinedby precipitating LD-lipoproteins and measuring cholesterol in the liquid phase and subt-racting it from the total cholesterol (reference range > 3.5 mmol/l). The free androgenindex (FAI) was calculated according to the equation (Tx100)/SHBG (reference range forpremenopausal women 1.4-7.3).

Two immunofluorometric assays have been used to determine the LH phenotypes. Thereference method (assay 2) used two LHβ-specific Mab which recognize normal (=wildtype, wt) LH and v-LH with similar stoichiometries. The other assay (assay 1) uses Mab,which only recognizes the intact LH α/β-dimer and the α-subunit, but not v-LH (Petters-son et al. 1992). When a large material of serum samples from healthy Finnish volunteerswas analyzed and the ratio of LH with assay 1 / assay 2 was measured, the results fellclearly into three categories: (1) those between 1.0-2.0 (normal ratio individuals), i.e. thesubject has two normal LHβ alleles, (2) those at 0.5-0.75 (low ratio individuals), i.e. the

Analyse Method Sensitivity Coefficient of intra-assayvariation (%)

Coefficient of interassay variation (%)

Reference rangein follicular phase

S-T FIA*RIA**LIA***

0.2 nmol/l0.35 nmol/l

4.54.0

6.45.6

0.3-3.1 nmol/l0.4-2.7 nmol/l

S-E2 RIA 20 pmol/l 5.7 6.4 0.04-0.26 nmol/l

S-Cortisol RIA 5.0 nmol/l 4.0 4.3 0.15-0.65 nmol/lS-DHEA RIA 0.1 nmol/l 6.5 7.9 1.7-36 nmol/lS-DHEAS RIA 0.03 µmol/l 5.3 7.0 1.0-14 µmol/lS-A RIA 0.07 nmol/l 5.0 8.6 0.7-16 nmol/lS-17-OHP RIA 0.2 nmol/l 5.0 5.4 0.3-3.6 nmol/lS-C-peptide RIA 0.07 nmol/l 5.3 7.2 0.2-1.0 nmol/l S-FSH FIA 0.05 IU/l 3.8 4.3 2-10 IU/lS-LH FIA 0.05 IU/l 4.9 6.5 2-10 IU/LS-SHBG FIA 0.5 nmol/l 1.3 5.1 20-140 nmol/lS-Leptin RIA 0.5 µg/l 5.0 6.0S-IGFBP-1 EIA 0.4 µg/l 3.4 7.4 0.8-13 µg/lS-Insulin RIA 2 mU/l 5.3 7.6 5-20 mU/lB-Lactate EA 2.2 3.8 0.33-1.33 mmol/lS-FFA EA 3.8 5.5 0.08-0.70 mmol/lB-Glucose EA 1.5 2.3 3.3-5.6 mmol/lS-Chol EA 0.7 2.3 3.0-7.0 mmol/lS-HDL EA 0.5 3.6 1.0-2.5 mmol/lS-Trigly EA 0.9 2.1 0.4-1.7 mmol/l* used in study I, ** used in study III and *** in studies IV and V. B-: blood, EA: enzymatic assay, EIA:enzyme immunoassay, FIA: fluoroimmunoassay, LIA: luminescence immunoassay, RIA: radioimmunoassay, S-: serum

63

subject is heterozygous for the mutant LHβ gene and (3) those with the ration near 0, i.e.the subject is homozygous for the mutant LHβ gene (Figure 1) (Haavisto et al. 1995)

4.11 Statistical analysis

All statistical analyses in studies III and IV were performed using SPSS version 8.0 forWindows 98 (Statistical Package for Social Science, Inc., Chicago, IL). The statisticalanalyses in studies I, II and V were performed using a StatView IITM for Macintosh soft-ware package (Abacus Concepts, Inc., Berkeley, CA, USA).

Student's two-tailed t-test was used for a comparison between groups of normallydistributed variables, with or without log transformation. The Mann-Whitney U-test wasused for variables with a persisting skewed distribution after log transformation.

Student's paired t-test and Wilcoxon's paired non-parametric rank sum test were usedfor comparison within groups when appropriate. Wilcoxon's paired test was used forvariables with a persisting skewed distribution after log transformation.

In study III a linear regression method was used to identify the influence of age, BMIand WHR on the variables in the control and PCOS groups. If the level of significancewas < 0.05, covariance analysis was carried out to evaluate the impact of these variableson the results.

Statistical analysis of frequency differences between the groups was evaluated usingthe χ2 test.

The confidence intervals (CI) for the differences of the frequencies of v-LH among thecountries were calculated using the Goodman statistic (Miller 1985).

The chosen level of significance was p < 0.05 in all studies.

5 Results

5.1 Clinical parameters

The differences in age and BMI between controls and women with PCOS are presented inTable 4. (chapter 4.1). In studies II, III, IV and V, women with PCOS were younger andhad higher BMI than the controls. Women with PCO in study I were younger, althoughnot significantly, but the BMI was similar between the groups. In study IV, metformin didnot change BMI (35 ± 4.7 vs. 34.0 ± 4.9 kg/m2, p = 0.4) or WHR (0.84 ± 0.09 vs. 0.86 ±0.07, p = 0.4) after 2 months of treatment. Women with PCO/PCOS in studies I, III andIV had significantly higher ovarian volumes and ovarian TAs than control women.

In study I, women with PCO had minor irregularities in menstrual cycles significantlymore often than women with normal ovaries (44 % vs. 19%, p = 0.03). Women with PCOalso had difficulties to conceive more often than did women with normal ovaries (25.9%vs. 9.3%, p = 0.01). All of these women conceived spontaneously within 3 years, howe-ver, without infertility treatments. In study III, control women with PCO also had irregu-lar periods more often (42.9% vs. 22.5%, p= 0.03).

5.2 Prevalence of polycystic ovaries

Table 6 shows the clinical parameters of subjects in studies I and III. The prevalence ofPCO among healthy women not treated for menstrual disturbances, infertility or hirsut-ism, was 14.2% (27/189). The prevalence of PCO was 21.6 % (19/88) in women aged <35 years and 7.9% (8/101) in women aged > 36 years (p=0.04). In women with a previousGDM, the prevalence of PCO was 39.4% (13/33) and 16.7% (8/48, p= 0.03) in controlwomen.

65

Table 6. Clinical parameters, serum LH/FSH-ratio and testosterone concentrations inwomen with normal and polycystic ovaries from studies I and III. Values are presented asmean ± SD or as percentages. Women fulfilling the criteria for PCOS were excluded (n=2)from the analysis in study III.

5.3 Variant type of luteinizing hormone

The frequencies of v-LH in different countries are presented in Table 7. The PCOS wom-en had significantly higher serum levels of LH (7.5 IU/L; 95% CI 7.0-8.0), than did theircontrols (4.4 IU/L: 95% CI 4.2-4.6, p < 0.001).

Table 7. The carrier frequency of normal (wild type, wt) and variant (v) LH alleles indifferent countries, as determined by the ratio of two LH immunoassays (assay1/assay2,see chapter 4.10). Results are shown as percentages.

The v-LH carrier frequency was similar in obese and nonobese controls in each countrystudied, but was significantly lower in obese PCOS subjects in the Netherlands and Fin-land, where the proportions of v-LH carriers in obese PCOS subjects were only 2.0% and4.5%, respectively (Fig 9). In contrast to these countries, no such difference, or even atendency, was observed in the United Kingdom, while such a tendency was apparent inthe United States (Fig 9).

Variable Study I Study III

normal ovariesn=162

PCOn=27

p-value normal ovariesn= 60

PCOn=21

p-value

BMI (kg/m2) 23.4 ± 3.2 22.2 ± 2.3 0.5 24.9 ± 5.0 26.8 ± 4.5 0.01

Irregular cycles (%) 19 48 0.002 18.3 42.8 0.009

Hirsutism (%) 0.6 3.7 0.2 1.6 10.4 0.3

Ovarian volume (sum, cm3) 12.8 ± 5.1 17.5± 11.2 0.04 9.3 ± 6.8 14.8 ± 6.1 0.02

Total area (sum, cm2) 9.9. ± 4.3 12.4 ± 4.0 0.008 7.8 ± 2.2 10.9 ± 2.0 0.01

LH/FSH 0.8 ± 0.5 1.1 ± 0.5 0.04 0.7 ± 0.3 0.9 ± 0.3 0.1

T (nmol/l) 1.7 ± 0.7 2.1 ± 0.6 0.03 1.3 ± 0.5 1.5 ± 0.6 0.3

Ratio of assay 1 / assay 2 Finlandn=425

Netherlandsn=347

United Kingdomsn=404

USAn=290

1.0-2.0 (= homozygotes for wt-LH, %) 71.1 88.8 83.2 85.9

0.5-0.75 (= heterozygotes for v-LH, %) 26.1 10.95 16.3 10.7

0 (= homozygotes for v-LH, %) 2.8 0.3 0.5 3.5

Heterozygotes + homozygotes for v-LH (%) 28.9 11.2 16.8 14.1

Allele frequency (%) 15.9 5.8 8.7 8.8

66

5.4 Effects of previous gestational diabetes (GDM)

5.4.1. Clinical characteristics

5.4.1.1 Women with previous GDM versus control subjects

Women with previous GDM were younger (34.9 ± 3.6 vs. 37.1 ± 5.3, p = 0.01) and had ahigher WHR (0.83 ± 0.05 vs. 0.79 ± 0.05, p = 0.002) than control women. BMI did notdiffer significantly between study and control group (GDM 26.5 ± 5.3 vs. controls 24.6 ±4.6 kg/m2, p = 0.1). PCO (39.4 % vs. 16.7 %, p = 0.03) and a family history of type 2 DM(87.8 % vs. 20.8%, p = 0.001) were more frequent in the GDM group than in the controlgroup.

5.4.1.2 Control women: comparison between women with normal ovaries and those with polycystic ovaries (PCO)

Clinical and biochemical characteristics are shown in Table 8. The mean age, BMI andWHR were similar between these two groups. Women with PCO had irregular periodsmore often (42.9% vs. 22.5%, p= 0.03) and a greater total ovarian volume (11.0, 95% CI8.7-13.3 vs. 9.7, 7.0-12.3 cm3, p=0.04) than women with normal ovaries.

5.4.1.3 Women with previous GDM: comparison between women with normal ovaries and those with PCO

Clinical and biochemical characteristics are shown in Table 8. Women with PCO wereyounger, had a higher total ovarian volume (12.4, 95% CI 9.6-15.9 vs. 8.4, 4.8-9.2 cm3,p< 0.004) and more irregular cycles (46.2% vs. 10 %, p<0.001). Only two women in thePCO group fulfilled the criteria of PCOS (i.e. oligomenorrhea, hirsutism and/or hyperan-drogenism, and PCO).

67

Fig. 9. The frequency (percentage) of v-LH (homozygotes and heterozygotes) in different coun-tries among controls and PCOS women with a BMI of 27 or less or with a BMI more than 27kg/m2. The number of subjects is indicated inside the bars. The frequency between the groupswas compared using the χ2 test: a, P < 0.05; b, P < 0.01; c, P < 0.03; after continuity correctionP = 0.07 compared to controls BMI > 27 kg/m2.

0

5

10

15

20

25

30

35

40

2233PCOS > 27control > 27PCOS < 27control < 27

39266a

FINLAND

%

0

5

10

15

20

25

30

35

40

PCOS > 27control > 27PCOS < 27control < 27

725750176

UNITED KINGDOM

%

--

0

5

10

15

20

25

30

35

40

PCOS > 27control > 27PCOS < 27control < 27

b495868146

NETHERLANDS

%

heterozygote homozygote

0

5

10

15

20

25

30

35

40

PCOS > 27control > 27PCOS < 27control < 27

c352728181

USA%

Table 8. Clinical (m

ean ± SD) and m

etabolic (95% C

I in parentheses) characteristics in control and GD

M w

omen w

ith or without

polycystic ovaries (PCO

).

Control

GD

MVariable

PCO

- (n=40)PC

O + (n=8)

p-valuePC

O - (n=20)

PCO

+ (n=13)p-value

Age (yr)

37.1 ± 3.236.7 ± 3.3

0.636.1 ± 2.8

33.0 ± 3.30.02

BM

I (kg/m2)

24.4 ± 4.624.6 ± 3.3

0.825.9 ± 4.6

27.5 ± 4.90.1

WH

R0.80 ± 0.05

0.79 ± 0.040.7

0.83 ± 0.050.82 ± 0.04

0.7

Fasting glucose (mm

ol/l)4.9 (4.8-5.1)

4.5 (4.1-4.9)0.7

5.3 (5.0-5.6)5.2 (4.8-5.6)

0.4

Fasting insulin (pmol/L)

57.4 (48.8-66.0)53.3 (40.1-66.4)

0.468.9 (55.0-82.7)

90.0 (57.8-122.4)0.3

Early phase insulin secretion (pm

ol/mm

ol)36.8 (26.7-46.9)

42.3 (16.8-67.8)0.6

21.5 (13.1-29.8)41.3 (20.5-62.1)

0.06

Fasting C-peptide (pm

ol/L)371.8 (307.8-435.7)

371.4 (205.0-537.9)0.9

394.4 (279.4-509.5)327.3 (157.1-497.5)

0.4Early phase C

-peptide secretion (pm

ol/mm

ol)161.3 (133.5-189.2)

171.6 (91.5-251.6)0.8

84.4 (63.0-105.7)117.0 (61.0-173.0)

0.4

Hepatic insulin extraction

6.6 (5.7-7.5)6.9 (4.5-9.2)

0.85.6 (4.4-6.8)

4.0 (2.2-5.9)0.07

Insulin sensitivity index (µm

ol x kgBW

-1 x min -1 /pm

ol /L)0.072 (0.061-0.083)

0.077 (0.057-0.097)0.7

0.055 (0.034-0.075)0.063 (0.033-0.094)

0.5

68

69

5.4.2. Oral glucose tolerance test

5.4.2.1 Women with previous GDM versus controls

Table 9 presents the results of OGTTs. An abnormal glucose tolerance was more frequentin the women with previous GDM (57.6%) than in the control group (12.5%, p <0.001).The GDM group had higher fasting glucose (5.3, CI 95% 5.0-5.5 vs. 4.9, 4.7-5.0 mmol/l,p = 0.008), fasting insulin (76.7, 62.6-90.7 vs. 56.8, 49.3-64.2 pmol/l, p = 0.003) and low-er hepatic insulin extraction (5.0, 4.0-6.0 vs. 6.7, 5.8-7.4 µmol x kgBW-1 x min-1 / pmol /L, p = 0.03) than did the control group. The difference in fasting insulin concentrationdisappeared after correction for WHR (p = 0.1).

Table 9. Results of OGTTs in women with a history of GDM treated with diet or withinsulin, and controls.

The oral glucose response, expressed as an incremental glucose area under the curve(AUCgluc) was significantly higher in the GDM group than in the control group (829, CI95% 776-891 vs. 673, 631-708 mmol/L x hour; p < 0.001, Fig 10, Study III). The diffe-rence remained statistically significant after correction for WHR. The incremental insulinarea under the curve (AUCins) was higher, but not significantly, in the GDM group than inthe control group (579, CI 95% 428-729 vs. 433, 339-527 pmol/L x hour, p = 0.1)

Fig. 10. Blood glucose (mean ± SE) and serum insulin concentrations during OGTT in controlwomen (black squares) and in women with previous GDM (black circles). * p < 0.05, ** p < 0.01,*** p < 0.001 compared with controls.

Result of OGTT GDM (diet)n = 18

GDM (insulin)n = 15

Controlsn = 48

Normal n (%) 12 (66.7) 2 (13.3)* 42 (87.5)IFG n (%) 3 (16.7) 3 (20.0) 0IGT n (%) 1 (5.6) 9 (60.0) 5 (10.4)DM n (%) 2 (11.1) 1 (6.7) 1 (2.1)* p < 0.002 compared with women treated by diet, and controls(IFG= impaired fasting glucose, IGT= impaired glucose tolerance)

0 30 60 120

5

6

7

8

9

10 ******

AUC p < 0.0001**

BLO

OD

GLU

CO

SE (m

mol

/L)

minutes0 30 60 120

50

100

150

200

250

300

350

400

450***

** controls GDM

SER

UM

INSU

LIN

(pm

ol/L

)

minutes

70

5.4.2.2 Women with previous GDM: a comparison between women with normal and polycystic ovaries

The frequency of abnormal OGTT results was similar among women with PCO and thosewith normal ovaries (53.8% vs. 60%, respectively). Fig. 11 shows the blood glucose andserum insulin responses during OGTTs in women with previous GDM with or withoutPCO. Women with previous GDM and PCO had significantly higher AUCins than wom-en with previous GDM and normal ovaries (776.9, CI 95% 446.7-1107.1 vs. 463.8,320.1-607.9 pmol/L/h, p=0.03).

Fig. 11. Blood glucose (mean ± SE) and serum insulin concentrations during OGTT in womenwith previous GDM and normal ovaries (black squares) and those with previous GDM andPCO (black circles). * p < 0.05 compared with controls.

5.4.3. Insulin secretion and insulin sensitivity

5.4.3.1 Women with previous GDM versus control subjects

Early phase insulin secretion did not differ between the study groups, but early phase C-peptide secretion was significantly lower in the GDM group than in the control group(96.7, CI 95% 73.1-120.4 vs. 162.8, 137.6-188.2 pmol/mmol glucose, p < 0.002, Fig 12).Serum C-peptide concentrations at 30 min in the OGTT were significantly lower in wom-en with previous GDM than in control women (1130, 911-1350 vs. 1424, 1229-1618pmol/L, p= 0.05), even after correction for BMI and WHR.

The insulin sensitivity index (M/I) tended to be lower in the GDM group than in thecontrol group (0.059, CI 95% 0.043-0.074 vs. 0.074, 0.065-0.083 µmol x kgBW-1 x min-1/pmol/L, p=0.09, Fig 12). This difference persisted after correction for BMI but disap-peared after correction for WHR. The lower M/I in the GDM group was due to lower glu-cose nonoxidation index (0.040, 0.026-0.051 vs. 0.054, 0.049-0.061 µmol x kgBW-1 xmin-1/pmol/L, p=0.05). Blood lactate concentrations did not differ between the groups in

0 30 60 120

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

BLO

OD

GL

UC

OSE

(mm

ol/L

)

minutes0 30 60 120

50100150200250300350400450500550600650700

*

AUC, p = 0.03

normal PCO

SER

UM

INSU

LIN

(pm

ol/L

)

minutes

71

the fasting state or during the euglycemic clamp. Women participating in the euglycemicclamp study did not differ from those women who did not participate with respect toBMI, WHR, OGTT, AUCgluk and AUCins.

Fig. 12. Early phase insulin and C-peptide secretion (mean ± SE) during the OGTT in controlwomen (white bars) and in women with previous GDM (gray bars). The third panel shows theinsulin sensitivity index (M/I, glucose oxidation and nonoxidation) assessed by hyperinsuline-mic euglycemic clamp. * p < 0.05 (glucose nonoxidation only), ** p < 0.01 compared with con-trols.

0,0

0,1

0,2

0,3

0,4

GDMcontrolsInsulin sensitivity index

*

M /

I

Glucose nonoxidation Glucose oxidation

0

10

20

30

40

50

GDMcontrolsEarly phase insulin secretion

pmol

/mm

ol g

luco

se

0

50

100

150

GDMcontrols

pmol

/mm

ol g

luco

se **

Early phase C-peptide secretion

72

5.4.3.2 Women with previous GDM: comparison between women with normal and polycystic ovaries

Women with PCO tended to have higher early phase insulin secretion in the OGTT thanthe control women, but early phase C-peptide secretion did not differ between thesegroups (Table 8). Hepatic insulin extraction was slightly lower in the PCO women com-pared with that in women with normal ovaries (p=0.07, Table 8.). The insulin sensitivity index was comparable between these groups. Women with normalovaries tended to have a lower glucose oxidation index (0.018, CI 95% 0.008-0.027 vs.0.027, 0.01-0.043 µmol x kgBW-1 x min-1 / pmol / L, p=0.1), however, as well as a low-er glucose nonoxidation index (0.037, 0.023-0.05 vs. 0.044, 0.01-0.077 µmol x kgBW-1 xmin-1 / pmol / L, p=0.2) during the clamp than did those women with PCO.

5.5 Endocrine parameters

5.5.1. Women with previous GDM versus controls

The GDM group had significantly higher serum cortisol (0.55, CI 95% 0.51-0.59 vs.0.47, 0.43-0.51 µmol/l, p = 0.006), DHEA (34.7, 28.0-41.4 vs. 19.9, 15.3-24.7 nmol/l, p =0.001), DHEAS (6.2, 5.2-7.4 vs. 4.6, 3.9-5.3 nmol/l, p= 0.01) and A (9.7, 8.7-10.9 vs. 7.3,6.3-8.2 nmol/l, p = 0.001) concentrations than the control group. The GDM group alsohad significantly lower S-HDL concentrations than the control women (1.3, 1.2-1.4 vs.1.5, 1.4-1.6 mmol/l, p = 0.009).

5.5.1.1 Control women: a comparison between women withnormal and polycystic ovaries

Serum concentrations of T (PCO 1.5, 0.9-2.2 vs. normal ovaries 1.3, 1.1-1.5 nmol/l,p=0.2), DHEAS and A, and the LH/FSH ratio and FAI (2.5, 1.3-3.7 vs. 2.8, 1.9-3.6,p=0.6) did not differ between the groups.

5.5.1.2 Women with previous GDM: a comparison between women with normal and polycystic ovaries

Serum concentrations of leptin (PCO 19.8, 12.9- 30.9 ng/mL vs. normal ovaries13.3,10.2- 17.4 ng/mL, p=0.09), T (1.4, 1.0-1.8 vs. 1.39, 1.1-1.6 nmol/l, p=0.9) andSHBG (42.7,30.9- 58.9 nmol/L vs. 53.1,42.7- 66.1 nmol/L, p=0.2), and FAI (3.9, 1.7-6.1vs. 2.9, 1.9-3.9, p=0.3) did not differ significantly between women with PCO and normalovaries.

73

5.6 Steroidogenesis in women with PCOS

5.6.1. Effects of human chorionic gonadotrophin

To study the ovarian steroidogenic response pattern, a hCG (5000 IU) injection was giv-en to PCOS and control women. 24 hours after the injection a peak in the serum 17-OHPand E2 concentrations was observed in PCOS women, while the maximum concentrationsof all steroids measured in the control women were reached at 96 hours. Serum basal and48 hour A were significantly higher in the PCOS women than in controls. Serum T con-centrations were constantly significantly higher in PCOS women than in controls andreached a maximum level at 48 hours (Fig 13).

5.6.2. Effects of metformin on steroidogenesis and on serum insulin and leptin concentrations

Serum basal T (from 2.1, CI 95% 1.7-2.7 to 1.8, 0.8-2.3 nmol/l, p = 0.05), insulin (from28.6, 20.5-31.4 to 21.5, 14.3-24.8 mU/l, p= 0.04) and leptin (from 35.9, 26.1-45.7 to 28.1,17.5-39.6 ng/mL, p = 0.03) concentrations decreased significantly after 2 months of treat-ment with metformin (500 mg x 3 daily). Serum 48-h and 96-h T and 48-h A alsodecreased significantly after this treatment (Fig. 13).

Area under A curve (AUCA, from 894.4, CI 95% 636.8-1007.8 to 738.4, 504.1-899.5nmol/l x h, p = 0.05) and AUCT (from 152.5, 115.7-172.7 to 114.1, 77.4-140.0 nmol/l xh, p = 0.04) decreased significantly after 2 months of treatment with metformin (Fig. 13).The fasting glucose to insulin ratio was 0.19, CI 95% 0.13-0.25 mmol/mU before themetformin treatment, but this ratio increased to the cut-off value 0.250, 0.15-0.33 mmol/mU during treatment (p = 0.2). HOMA also decreased significantly during the treatment,indicating improved insulin sensitivity (6.5, CI 95% 5.4-7.5 vs. 4.7, 3.7-5.7 mmol x mU/l2, p = 0.01).

74

Fig. 13. Serum 17-OHP, androstenedione, testosterone and estradiol responses (mean ± SE) toa single injection of hCG (5000 IU) in control women (black squares, n = 27) and in PCOS wom-en before (black circles, n = 12) and after (black triangles) 2 months of metformin treatment.AUCA and AUCT were calculated in PCOS women before and after metformin treatment. * p< 0.05 compared with PCOS women after metformin treatment, ** p < 0.01 compared withPCOS women before metformin treatment.

5.6.3. Effects of human menopausal gonadotrophin and follicle stimulating hormone

After pituitary-ovarian axis suppression with GnRHa, women with PCOS and their con-trols received daily injections of FSH alone or FSH together with LH (hMG) to elucidateovarian steroidogenic responses to these gonadotrophins. After nine days of treatmentwith hMG and FSH the serum concentrations of 17-OHP, A, T and E2 increased signifi-cantly in both control women and women with PCOS compared with basal values. Thebaseline values of serum 17-OHP, DHEA, A, T and E2 did not differ after pituitary-ovari-an axis suppression with the GnRHa between the groups. Serum DHEA levels did notincrease during gonadotrophin treatment in either of the groups (Table 10).

The serum concentrations of 17-OHP, A, T and E2 increased more during hMG in boththe control and PCOS women than during FSH stimulation. The response to FSH wasabout 50-60% of that to hMG (Table 10, Fig 14).

0 1 2 4

4

6

8

10

12 a)

**

**

17-O

HP

(nm

ol/l)

days

controls PCOS before PCOS after metformin

0 1 2 48

10

12

14

16

18 b)

**

**

*

AUC, p = 0.05

andr

oste

nedi

one

(nm

ol/l)

days

0 1 2 4

1,2

1,6

2,0

2,4

2,8c)

AUC, p < 0.05

**

*

******

**

test

oste

rone

(nm

ol/l)

days0 1 2 4

0,100,150,200,250,300,350,400,450,50 d)

**

**

estr

adio

l (nm

ol/l)

days

75

Fig. 14. The change (post-treatment minus basal concentrations) in serum 17α-hydoxyproges-terone (17-OHP), androstenedione, testosterone and estradiol during either human menopau-sal gonadotrophin (hMG, white bars) or purified urinary follicle stimulating hormone (FSH,gray bars) stimulation after pituitary suppression with GnRHa in the control and in the PCOSwomen. * p < 0.05 compared with controls stimulated with hMG.

0

2

4

6

8

*

PCOSCONTROLS

∆ an

dros

tene

dion

e (n

mol

/L)

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

*

PCOSCONTROLS

∆ te

stos

tero

ne (n

mol

/L)

0

1

2

3

4

PCOSCONTROLS

∆ 17

-OH

P (n

mol

/L)

HMG FSH

0

1

2

3

4

5

PCOSCONTROLS

∆ es

trad

iol (

nmol

/l)

Table 10. Horm

onal parameters (m

ean ± SD) am

ong control and PCO

S wom

en in the basal state (after GnRH

a suppression) and afterhum

an menopausal gonadotrophin (hM

G) and follicle stim

ulating hormone (FSH

) stimulations.

VariableH

MG

basalH

MG

post-treatment

FSHbasal

FSH

post-treatment

controls (n=46)

PCO

S(n=14)

controls(n=46)

PCO

S(n=14)

controls(n=35)

PCO

S(n=9)

controls(n=35)

PCO

S(n=9)

17-OH

P (nmol/L)

2.5 ± 1.33.0 ± 1.1

5.8 ± 3.4 *6.8 ± 3.7 *

2.2 ± 1.12.3 ± 1.0

5.1 ± 3.0 *3.7 ± 2.3 *

A (nm

ol/L)7.0 ±3.0

8.6 ± 3.014.4 ± 6.0 *

15.6 ± 7.7 *5.9 ± 2.6

10.9 ± 3.8 9.8 ± 4.0 *

15.0 ± 5.6 *

DH

EA (nm

ol/L)18.4 ± 9.2

22.7 ± 10.620.1 ± 10.1

23.4 ± 13.818.3 ± 8.7

30.8 ± 17.1 19.5 ± 8.4

30.0 ± 13.2

T (nmol/L)

1.7 ± 0.71.6 ± 0.5

2.6 ± 1.0 *3.4 ± 2.2 *

1.6 ± 0.61.9 ± 0.8

2.2 ± 0.7 * 2.6 ± 1.4 *

E2 (nm

ol/L)0.06 ± 0.01

0.06 ±0.014.3 ± 3.0 **

4.7 ± 2.3 **0.06 ± 0.02

0.06 ± 0.02 3.1 ± 2.6 **

2.9 ± 1.8 **

* p< 0.05 compared to the basal level in the corresponding group, ** p < 0.01 com

pared to the basal level in the corresponding group

76

77

There were no differences in the ∆17-OHP/∆A ratio or in ∆E2/∆A ratio between the twogroups or between the two gonadotrophins (Fig 15).

Fig. 15. ∆ 17-hydroxyprogesterone / ∆ androstenedione and ∆ estradiol / ∆ testosterone ratios(mean ± SE) in control women and women with PCOS using human menopausal gonadotropin(hMG) or follicle stimulating hormone (FSH) for stimulation after GnRHa suppression.

The clinical results of the IVF cycles are given in Table 11. There were no differencesbetween control women and women with PCOS in the number of hMG and FSH ampou-les used for the stimulation.

Table 11. Clinical characteristics and outcome of IVF cycles (mean ± SD) in controlwomen and women with PCOS stimulated with human menopausal gonadotrophin (hMG)or follicle stimulating hormone (FSH) after pituitary suppression with GnRHa.

Variable HMGControln=45

PCOSn=14

p-valueFSHControln=35

PCOSn=9

p-value

Age (yr) 31.7 ± 4.3 30.8 ± 5.1 NS 33.3 ± 4.8 29.3 ± 2.4 0.02

BMI (kg/m2) 23.3 ± 3.2 26.6 ± 5.4 0.03 23.3 ± 3.5 28.4 ± 5.4 0.01

No of ampoules (á 75 IU) 22.5 ± 7.5 22.4 ± 5.3 NS 23.1 ± 5.8 24.6 ± 4.7 NS

No of oocytes (n) 9.2 ± 4.7 10.8 ± 4.7 NS 9.6 ± 4.2 11.3 ± 8.0 NS

No of fertilized oocytes (n) 4.4 ± 4.0 6.1 ± 3.9 NS 6.1 ± 3.6 5.3 ± 5.0 NS

No of cleaved oocytes (n) 3.7 ± 3.8 5.7 ± 4.0 0.04 5.0 ± 3.3 3.4 ± 2.5 NS

Clinical pregnancies/ET (%) 23.7 38.5 NS 29.0 28.6 NS

NS = nonsignificant, ET = embryo transfer

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

PCOSCONTROLS

∆17-

OH

P / ∆

A

0,0

0,5

1,0

1,5

PCOSCONTROLS

∆E2

/ ∆T

HMG FSH

6 Discussion

6.1 Prevalence of an isolated finding of polycystic ovaries

The present results demonstrate that the prevalence of an isolated finding of polycysticovaries (PCO) is dependent on age in healthy women, the prevalence being significantlyhigher in the younger age group (< 35 years) than among older women (> 36 years). Theoverall prevalence (14.2%) of PCO in this study group was slightly lower than thatreported (16-23%) in previous studies (Polson et al. 1988, Abdel Gadir et al. 1992, Clay-ton et al. 1992, Farquhar et al. 1994, Botsis et al. 1995). The prevalence of PCO in 48control women with uncomplicated pregnancies, who were parity and delivery date-matched with women with previous GDM, was also 16.7 %. The selection criteria for thestudy subjects have varied in different studies, which makes a comparison between thestudies difficult. All of the subjects in our study were healthy and had none of the typicalsymptoms of PCOS. This may explain the lower prevalence of PCO compared with thatin an unselected population (Clayton et al. 1992, Farquhar et al. 1994). In a study by Pol-son et al. (1988), the selection criteria were similar to ours and the prevalence of PCOwas 23%.

The prognostic significance of PCO among asymptomatic women is unclear. The hor-monal parameters and clinical findings mimic those associated with PCOS. Women withPCO in the present study had a higher LH/FSH ratio, serum T concentrations, incidenceof minor cycle irregularities and significantly more problems in conceiving than womenwith normal ovaries. Women with PCO seem to have exaggerated ovarian responses togonadotrophins (Suikkari et al. 1995), to GnRHa (Chang et al. 2000) and to have compa-rable serum IGFBP-1 levels with women with PCOS rather than with their weight mat-ched controls, reflecting a similarity between PCO and PCOS (Carmina et al. 1997). Fut-hermore, 33% of women with PCO had an elevation of at least one serum androgen value(Carmina et al. 1997). These results suggest that women with an isolated ultrasonic fin-ding of PCO have subtle endocrine disturbances similar to PCOS and may be susceptibleto develop the syndrome and thus, these women may also have an increased morbiditysuch as that associated with PCOS (Carmina & Lobo 1999). In the present study, womenwith previous GDM revealed a high prevalence of PCO (39.4%), i.e. twofold higher thanthat reported in healthy, premenopausal women. This confirms observations in three

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recent studies on women with a history of GDM (Anttila et al. 1998, Holte et al. 1998,Kousta et al. 2000). In the present study, only two women with PCO fulfilled the criteriafor PCOS. The clinical symptoms were also scarce among women with PCO in previousstudies (Anttila et al. 1998, Holte et al. 1998, Kousta et al. 2000).

PCOS has a peripubertal onset of symptoms. It has been proposed that various“insults” need to come into play after puberty for women with PCO to develop PCOS(Lobo 1995). Usually more than one factor may be involved, and the list of these insultsis long (insulin resistance, obesity, stress and dopaminergic dysregulation)(Carmina &Lobo 1999). On the other hand, obviously not all the women with PCO will developPCOS. According to our results, the prevalence of PCO lowers with increasing age,which may be due in part to the physiological decline of the follicular cohort (Faddy et al.1992). It has furthermore been reported that hyperandrogenism may partly resolve beforemenopause in women with PCOS and they gain more regular menstrual cycles with inc-reasing age (Elting et al. 2000, Winters et al. 2000). In conclusion, an isolated finding ofPCO is common among Finnish women. Hormonal parameters and clinical findings inwomen with PCO mimicked those associated with PCOS. It may be possible, however,that the ultrasonic appearance of PCO disappear with increasing age, probably in womenwho will not gain weight or will not became predisposed to the other risk-factors relatedto PCOS. Our study was a cross-sectional study, however, and longitudinal studies areneeded to clarify what will happen over the long term to women with PCO.

6.2 Variant type of luteinizing hormone

The present study on control women and women with PCOS confirms our previous find-ings on a common occurrence of v-LH and its considerable variation among differentpopulations and even within Caucasian populations. With regard PCOS, the most strik-ing finding was the low v-LH frequency of 2-4.5% in obese patients from the Netherlandsand Finland, with a similar tendency in the United States. It has previously been reportedfrom the United Kingdom that obese women with PCOS exhibit a higher frequency of v-LH than obese controls (Rajkhowa et al. 1995), which was also the case with subjectsfrom the United Kingdom in our study (Study II). The discrepancy between the countriesis difficult to explain, but given the multifactorial pathogenesis of PCOS, it is possiblethat another genetic factor, enriched in the United Kingdom population, alters the patho-genesis of PCOS.

It is unclear whether the high activity of v-LH at the receptor site but shorter half-timein the circulation results in a net increase or decrease in the overall bioactivity of itsaction in vivo. It has been proposed that the biological properties of v-LH may inducesubtle changes in LH action, either predisposing the affected individuals to or protectingthem from the disease conditions related to LH action (Haavisto et al. 1995). In the pre-sent study, the low frequency of v-LH in obese PCOS women in 3 of the populations sug-gests that obese women with v-LH may be somehow protected from developing sympto-matic PCOS, while those with normal LH are more likely to develop the disease.

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Obesity per se was not related to the variant heterozygosity, as the carrier frequencywas the same in lean and obese controls. It has been reported, however, that the variantLH is more common in obese (BMI > 25 kg/m2) women with recurrent spontaneousabortion (RSA) and regular menstruation (60%) than in lean women with RSA and regu-lar menstruation (30%) (Tulppala et al. 1998). In contrast to this, it has also been sugge-sted that women with a normal or below-normal BMI, who possess v-LH, may have sub-fertility (Cramer et al. 2000). Although several studies show an association between diffe-rent types of v-LH and gonadal dysfunction and/or infertility, larger numbers of observa-tions from various ethnic groups are needed to resolve the role of v-LH in pathologies ofreproductive functions (Themmen & Huhtaniemi 2000).

In conclusion, the high frequency of v-LH in various populations must be kept inmind, as many widely used immunoassay reagents do not detect this LH form. One diag-nostic criterion for PCOS, the elevated LH/FSH ratio, may remain undetected if such aLH assay is used, as has been recently emphasized (Kurioka et al. 1999).

6.3 Insulin sensitivity, and metabolic and endocrine features in women with a history of GDM

The present study confirms that women with previous GDM have impaired insulin sensi-tivity (Byrne et al. 1995, Ryan et al. 1995), and, in addition, that this is mainly due to adecreased glucose nonoxidation, reflecting a defect in glycogen storage. A similar phe-nomenon has been shown in first-degree relatives of type 2 diabetic patients (Eriksson etal. 1989, Schalin-Jantti et al. 1994, Vauhkonen et al. 1998a). Note, however, that womenwith previous GDM were more obese, especially abdominally, than the control women,and further, a correction for WHR (but not for BMI) abolished the difference in insulinsensitivity between these groups. This observation strongly suggests that the impairedinsulin sensitivity in women with previous GDM is due - at least in part - to abdominalobesity. It has been shown that especially abdominal fat tissue has high lipolytic activitywhich may lead to an increased flux of FFAs from the adipose tissue, with subsequentcompetition between FFAs and glucose for oxidation in the muscle cells (Randle et al.1963). This, in turn, may lead to impaired insulin sensitivity.

Apart from an impaired insulin sensitivity, women with a previous GDM had defectiveearly phase insulin secretion, reflected in their serum C-peptide response and in a delayedserum insulin response to an oral glucose load. These observations reflect impaired β-cellfunction in these women. They also had a low fasting serum C-peptide/insulin ratio,which indicates impaired hepatic insulin extraction. It is well known that hepatic insulinextraction is lowered in many conditions such as obesity (Escobar et al. 1999), and it isthus possible that women with previous GDM had a low hepatic insulin extraction, due -at least in part - to obesity.

Women with previous GDM had an abnormal OGTT result significantly more often(57.6%) than the controls (12.5%). This supports the concept that women with GDM areat risk of developing DM later in life (Dornhorst et al. 1990, O'Sullivan 1991, Damm etal. 1992). Women who had insulin treatment during pregnancy had an abnormal OGTTmore often than diet-treated and control women, and insulin treatment during pregnancy

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is strongly associated with subsequent diabetes (Farrell et al. 1986). The high prevalenceof an abnormal OGTT in diet-treated women (33.3%) in our study, which was similar tothe incidence reported in earlier studies (29.7-34.3%) (Damm et al. 1992, Damm et al.1995), underlines the importance of a regular postpartum assessment of glucose tolerancein women with diet-treated GDM.

Women in the GDM group had significantly higher serum adrenal steroid concentra-tions (cortisol, DHEA, DHEAS and A) than did control women, which may have beendue to insulin resistance, hyperinsulinemia and abdominal fat accumulation (i.e. increasedWHR) in this group. Multiple endocrine perturbations have been associated with abdomi-nal fat accumulation, including elevated serum cortisol and androgen levels in women,and - in particular - hyperinsulinemia secondary to pronounced insulin resistance (Kisse-bah & Peiris 1989, Bjorntorp 1991, Bjorntorp 1996). It is possible that androgens areoverproduced by the adrenals as a component of the hypersensitive hypothalamo-pitui-tary-adrenal (HPA) axis (Cameron et al. 1984, Roy et al. 1990, Ljung et al. 1996). Asregards the role of insulin in adrenal steroid secretion, obese hyperinsulinemic womenwith PCOS have been found to display an excessive secretion of adrenal androgens (Mar-tikainen et al. 1996). Furthermore, a positive relationship has been shown between seruminsulin and DHEAS levels in healthy obese women (Leenen et al. 1994). There are stu-dies, however, in which an improvement of insulin resistance and hyperinsulinemia achie-ved by way of pharmacologic therapy has not altered circulating DHEA or DHEASlevels in women (Beer et al. 1994). The possible stimulatory effect of insulin on adrenalsteroidogenesis may be mediated via insulin receptors and/or the insulin growth factor(IGF) system. In our study, the lower serum IGFBP-1 concentrations in the GDM groupreflects higher free IGF-1 concentrations, which may contribute to adrenal steroid secre-tion. It has been shown that IGF-1 enhances the steroidogenic response to ACTH and theexpression and activity of cytochrome P450C17 in adult human adrenal cortical cells(l'Allemand et al. 1996, Mesiano et al. 1997).

In conclusion, our data demonstrate that women with previous GDM often have abnor-mal OGTT, they are insulin resistant, mainly as a result of lowered glucose nonoxidationin the peripheral tissues, and, furthermore, they display inappropriately low insulin res-ponses to glucose, which is due to impaired ß-cell function. Women with previous GDMalso have higher adrenal androgen secretion than do control women. The clinical impor-tance of this observation, and whether it is due to abdominal obesity, insulin resistanceand/or hyperinsulinemia, remains to be clarified.

6.3.1. Association between previous GDM and PCO

Apart from having metabolic defects typical of type 2 DM, women with previous GDMalso revealed a high prevalence of PCO, which was two-fold higher than that reported inhealthy premenopausal women (Polson et al. 1988, Clayton et al. 1992). This confirmsthe observations of three recent studies on women with GDM (Anttila et al. 1998, Holteet al. 1998, Kousta et al. 2000)

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Interestingly, although women with PCO and previous GDM showed insulin sensiti-vity and blood glucose responses in the OGTT comparable with those of the women withnormal ovaries and previous GDM, they had a more marked hyperinsulinemia, which wasnot explained by obesity. This may indicate that these women have a lowered metabolicclearance rate of insulin. Notably, they also had a low serum C-peptide/insulin ratio in thefasting state, implying that the lowered metabolic clearance rate of insulin was due to itsimpaired hepatic extraction. The pathogenic mechanism behind this novel finding, howe-ver, remains largely unclear. It has recently been shown that an elevation of serum FFAconcentrations may impair hepatic insulin extraction (Wiesenthal et al. 1999, Morin-Papunen et al. 2000), but the serum FFA levels in the present study were comparableamong GDM women with and without PCO (data not shown). On the other hand, thewomen with PCO tended to have a higher lipid oxidation rate in the fasting state than thewomen with normal ovaries (data not shown), implying that hyperinsulinemia in womenwith PCO and previous GDM may indeed be associated with altered adipose tissue meta-bolism.

In accordance with a study previously conducted in Sweden (Holte et al. 1998), thefirst phase insulin secretion was lower in women with previous GDM and normal ovariessuggesting that β-cell dysfunction may be a more dominant component in the womenwith normal ovaries. In contrast to the Swedish study, however, women with previousGDM and PCO in our study were not more insulin resistant than women with previousGDM and normal ovaries.

A study from United Kingdom did not demonstrate any differences in the metabolicparameters between their post-GDM PCO group and post-GDM normal ovaries group(Kousta et al. 2000). The discrepancies between these studies may be due to regional dif-ferences in the study populations and to the complex etiology of both PCO and GDM.Relatively low sample sizes in all of the studies in question may also have influence onthe results.

The clinical symptoms typical of PCOS were scarce in the women with PCO. This isin accordance with previous studies (Holte et al. 1998, Kousta et al. 2000). However,these women with previous GDM and PCO had subtle clinical, endocrine and metabolicfeatures characteristic of PCOS. Women with full-blown PCOS have been shown to havea higher risk of developing impaired glucose metabolism either before conception orduring pregnancy (Levran et al. 1990, Lanzone et al. 1995a, Lanzone et al. 1996). Ourfindings suggest that the ultrasonographic appearance of PCO may be a predictive factoras regards abnormal glucose tolerance during and after pregnancy and thus, women withPCO should be checked regularly. Our findings also ascertain that women with PCO havemetabolic abnormalities resembling PCOS and thus, they may be at risk to an increasedmorbidity associated with PCOS.

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6.4 The effect of stimulation with human chorion gonadotrophin, human menopausal gonadotrophin and

follicle stimulating hormone on steroidogenesisin women with PCOS and in control women

6.4.1. The effect of short-term human choriongonadotrophin stimulation

Obese women with PCOS displayed a distinctly different steroidogenic response patternto a single dose of hCG compared with the control women followed up for 4 days. Peakperipheral serum A and T concentrations were achieved at 48 hours after an injection ofhCG in the PCOS women, with peak levels of 17-OHP and E2 at 24 hours. In contrast, allsteroids measured in the control women reached their maximum serum concentrations at96 hours.

The rapid 17-OHP and E2 responses to hCG (with similar biological properties as LH)in PCOS women are in accordance with the results of previous studies in which short-term responses (up to 24–48 h) to hCG (Ibanez et al. 1996, Gilling-Smith et al. 1997,Levrant et al. 1997) or to GnRHa (Barnes et al. 1989b, Rosenfield et al. 1994, Ibanez etal. 1996) have been studied. These findings have been interpreted to imply that "dysregu-lation" of the enzyme P450c17α, leading to enhanced activities of both 17α-hydroxylaseand 17,20-lyase, plays a central role in the pathogenesis of the ovarian hyperandrogenismassociated with PCOS. The male-type steroidogenic response pattern to hCG seems to berelated to PCOS and not to obesity, since a similar response to hCG was found in both thelean and the obese control women.

The steroid responses to hCG in this study were followed longer than in previous stu-dies, and it was observed that the levels of 17-OHP and E2 began to decline at 48 hours,in contrast to the control women, who demonstrated peak 17-OHP and E2 values at 96hours after hCG. The steroid response patterns observed in obese PCOS women wereidentical to those found in normal men (Smals et al. 1979, Martikainen et al. 1980). It hasbeen suggested that E2 inhibits 17-lyase activity, leading to an accumulation of 17-OHP,possibly preventing its further conversion to T in the human testis (Forest et al. 1979,Martikainen et al. 1980, Tapanainen et al. 1983). It is tempting to speculate that a similarregulatory mechanism may be operative in the polycystic ovary, with increased theca cellactivity and mass. Since a physiological decline of follicular cohort has been shown tooccur while ageing (Faddy et al. 1992), it would be interesting to see if the responses tohCG are different in younger women than in older women. Furthermore, the time courseof 17-OHP and E2 responses indicates that E2, in contrast to a previous suggestion (Iba-nez et al. 1996), may be an important regulator of 17,20-lyase activity in the humanovary. These statements are still speculative, however, and further studies are required tosupport them.

The observed rapid and significant increase in E2 after hCG in obese PCOS women isnot in good agreement with the two-cell model of ovarian steroidogenesis in which LHstimulates theca cell androgen production and FSH mainly stimulates granulosa cellestrogen production. There is a considerable amount of data, however, indicating that

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human theca cells are capable of forming E2 throughout the life span of the antral follicle(McNatty et al. 1979, Gilling-Smith et al. 1994). Thus, the rapid release of E2 from poly-cystic ovaries after hCG-stimulation observed in this study may reflect the existence of areleasable pool of steroids in the theca cells. Alternatively, hCG may directly stimulateovarian aromatase activity (Tapanainen et al. 1991), or androgens might exert a rapidparacrine effect on granulosa cells by up-regulating their aromatase activity (Haning,Hackett et al. 1993). Furthermore, the response of E2 to hCG may also be due to a directactivation of granulosa cells, since granulosa cells from small individual folliclesobtained from anovulatory polycystic ovaries have been shown to be prematurely respon-sive to LH (Willis et al. 1998).

In conclusion, the present study indicates that obese PCOS women have a male-typesteroidogenic response pattern to a single dose of hCG, which may be explained by highertheca cell activity or mass in polycystic ovaries.

6.4.2. The effect of long-term human menopausal gonadotrophin and follicle stimulating hormone stimulation

It is obvious that higher amounts of gonadotrophins in the hMG preparation (FSH 75IUand LH 75IU) than in the FSH preparation (FSH 75IU and LH < 0.1 IU) have an influ-ence on the stimulation results. It is noteable that FSH administration alone also led to adistinctly increased production of 17-OHP, A and T in both endocrinologically normaland PCOS women. According to the two-cell theory of ovarian steroidogenesis, LH stim-ulates the theca cell production of A and T and FSH stimulates granulosa cell function.As there was no significant supply of LH in the FSH preparation used for stimulation inthis study, it was likely that FSH alone is also capable of stimulating ovarian theca cellsteroid synthesis under these experimental conditions. This was confirmed by stimula-tions with recombinant FSH preparation. Our results are in accordance with the study byTanbo et al. (Tanbo et al. 1990), in which the experimental conditions were similar toours.

The mechanism(s) by which FSH stimulates theca cell function is unclear. It has beenpostulated that when granulosa cells are exposed to FSH they can generate estrogens onlywhen supplied with androgens as the aromatase substrate (Erickson & Ryan 1976). Ingonadotrophin-deficient woman it was shown that FSH alone, without any LH, is not ableto stimulate follicular steroid synthesis (Schoot et al. 1992). In the presence of profoundgonadotrophin deficiency, pharmacological doses of highly purified FSH with minute LHcontamination have been reported to be capable of stimulating ovarian follicular matura-tion (Couzinet et al. 1988). FSH is known to potentiate LH action by inducing LH recep-tors (Knecht et al. 1986). Thus, minute amounts of endogenously secreted or exogenouslyadministered LH, because of its presence in the highly purified FSH preparation, couldresult in a significant but subnormal production of ovarian androgens (Couzinet et al.1988, Teissier et al. 1999). LH suppression by GnRHa is known to be incomplete (Changet al. 1983, Matikainen et al. 1992, Bützow et al. 1999) and endogenously secreted LHtherefore probably had an influence on ovarian androgen production in our study.

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Estradiol has been shown to augment A production and the stimulatory effect of FSHon steroid production in theca cells may thus be mediated by E2 produced in granulosacells (Gilling-Smith et al. 1997). FSH also seems to be capable of influencing ovarianandrogen synthesis via a paracrine mechanism involving an enhanced expression of the-cal/interstitial P450c17α. It is unclear which locally produced factor(s) mediates thisaction of FSH. Likely candidates are regulatory proteins produced by granulosa cells res-ponding to a direct stimulation by FSH (i.e. inhibin, IGF-1) (Adashi et al. 1985, Hsueh etal. 1987, Smyth et al. 1993). It has recently been suggested that theca cells secrete fac-tor(s) inhibiting the differentation of immature, while promoting that of matured granu-losa cells; those results also suggested that granulosa cells secrete factor(s) promotingboth the differentation and growth of theca cells throughout the follicular maturation pro-cess (Yada et al. 1999).

In this study with daily injections of gonadotrophins after pituitary suppression,women with PCOS did not show a distinctly exaggerated steroidogenic response togonadotrophin stimulation compared to endocrinologically normal women. We wereunable to detect any difference in the serum A and 17-OHP concentrations or in the ?17-OHP/?A ratio between the two patient groups (control vs. PCOS) studied, indicatingunchanged 17-20-lyase activity in PCOS. This result is in contrast with those of short-term GnRHa or hCG stimulation studies where PCOS women showed an exaggerated 17-OHP response compared with control women (Barnes et al. 1989b, Rosenfield et al.1994, Ibanez et al. 1996, Gilling-Smith et al. 1997).

The discrepancies between this and previous studies is most likely due to experimen-tal conditions. GnRHa used for suppression prior to gonadotropin stimulation maymodify ovarian responsivness to gonadotrophins, for example, by inhibiting LH receptorformation (Hsueh & Erickson 1979, Rabin & McNeil 1980, Amsterdam et al. 1981).However, PCOS women have been shown to display 17-OHP hyperresponsiveness to asingle dose of hCG (10 000 IU) also after 1 month of GnRH agonist treatment (Gilling-Smith et al. 1997). The lesser increase in 17-OHP and A in the present study may be dueto the lower dosage of gonadotrophin (150-300 IU/daily) given. On the other hand, consi-dering the heterogeneity of PCOS, the sample size in this study may be too small to give asignificant result. One explanation for our results may be ovarian desensitization togonadotrophins or the down-regulation of LH receptors after repeatedly injected exoge-nous gonadotrophins. It has been shown that stimulation of male rat testicular Leydigcells with exogenous gonadotrophins (LH or hCG) is followed by a loss of LH receptorsand a decreased maximum T response (Hsueh et al. 1976, Hsueh et al. 1977, Tsuruhara etal. 1977). It is tempting to suggest, however, that the rapid 17-OHP response to hCG sti-mulation in previous studies (Barnes et al. 1989b, Rosenfield et al. 1994, Ibanez et al.1996, Gilling-Smith et al. 1997) may possibly only reflect the releasable pool of steroidsfrom the thecal cells of polycystic ovaries and not an increased P450c17α-activity in thesingle thecal cell.

A low estrogen/androgen ratio has a negative influence on follicle developement inPCOS women (Cataldo 1997). The ∆E2/∆T ratio was slightly - but not - significantlyhigher in both controls and in PCOS women in hMG stimulated cycles than in FSH sti-mulated cycles, suggesting that hMG does not have unfavourable effects on aromataseactivity as compared to FSH. The higher increase of serum A concentrations in hMG sti-mulated cycles may, however, have some unfavourable effects on endometrial cell

86

growth, as has recently been reported (Tuckerman et al. 2000). Nevertheless, the clinicalresults (including pregnancy rate) were similar in all treatment groups, which is in accor-dance with previous studies (Homburg et al. 1990, McFaul et al. 1990, Tanbo et al.1990).

In conclusion, the present findings indicate that both hMG and FSH stimulate ovarianandrogen production after pituitary suppression in normal and PCOS women. The PCOSwomen did not show a distinctly exaggerated steroid response to gonadotrophins and nosign of steroidogenic defects was observed under these experimental conditions.

6.4.3. The effect of metformin on steroidogenesis in women with PCOS

The improvement of hyperandrogenism observed during metformin treatment has beenconsidered to be the result of a decrease in serum insulin concentrations (Velazquez et al.1994, Nestler & Jakubowicz 1996, Morin-Papunen et al. 1998, Pasquali et al. 2000),although not all studies support this concept (Acbay & Gundogdu 1996, Ehrmann et al.1997a). In the present study, we observed a slight, but non-significant decrease in basalserum androgen concentrations during metformin treatment. In addition, we observed sig-nificant decreases in AUCT and AUCA after hCG during metformin treatment. Duringthat treatment fasting serum insulin concentration decreased and insulin sensitivityimproved in obese and insulin-resistant PCOS women, suggesting that the slight allevia-tion of hyperandrogenism brought about by metformin may be mediated by a decreasedinsulin action. No change in BMI or in WHR was observed during the study.

Given that significant decreases in GnRHa and hCG-stimulated 17-OHP responseshave been observed during metformin treatment, it has been suggested that an improve-ment of hyperinsulinemia by metformin treatment could reduce ovarian cytochromeP450c17α activity (Nestler & Jakubowicz 1996, Nestler & Jakubowicz 1997, la Marca etal. 2000). In contrast to these studies, however, the response of 17-OHP to hCG was notsignificantly decreased by metformin in the present study, although insulin levels dec-reased. The results indicate that a 2-month treatment period with metformin does notmodify the response pattern of 17-OHP. These data suggest that - if the increased res-ponse of 17-OHP to hCG in women with PCOS is partly due to hyperactivity ofP450c17α (Rosenfield et al. 1994, Levrant et al. 1997) - short-term metformin treatmentmay not improve hyperandrogenism by affecting this step of androgen biosynthesis. Insu-lin is known to augment the expression of P450c17α (Ehrmann et al. 1995), possessingboth 17α-hydroxylase and 17,20-lyase activities in the ovary, and thus, decreased seruminsulin concentrations after metformin treatment in our study may be the result of an imp-rovement of hyperandrogenism.

The present results, as well as those of our previous studies (Morin-Papunen et al.1998, Morin-Papunen et al. 2000), showed that serum SHBG concentrations and the FAIdid not change significantly during the 2 months of metformin treatment. This is in agree-ment with a recent study of long-term metformin treatment (Pasquali et al. 2000) and incontrast to some previous studies where increased serum SHBG concentrations duringmetformin treatment have been observed (la Marca et al. 1999, Unluhizarci et al.1999a,b, la Marca et al. 2000). One explanation for this discrepancy may be that abdomi-

87

nal obesity is associated with reduced SHBG concentrations (Pasquali et al. 1990), andsince no weight loss was observed after the treatment in our study, SHBG did not change.Moreover, the women with PCOS in our study were probably still relatively hyperinsuli-nemic after their treatment and no change in SHBG was therefore noticed.

The present results suggest that the slight alleviation of hyperandrogenism broughtabout by metformin therapy may be explained by decreased ovarian steroidogenesis, pro-bably due to a reduced serum insulin concentration.

6.4.4. The effect of metformin on serum leptin concentrations

During metformin treatment a significant decrease of serum insulin and leptin concentra-tions at two months of treatment was observed in parallel with T changes. This is inaccordance with previous studies (Morin-Papunen et al. 1998, Morin-Papunen et al.2000, Pasquali et al. 2000). Messenger RNA for leptin receptors has been found in bothovarian granulosa and theca cells (Agarwal et al. 1999), suggesting a possible direct roleof leptin in ovarian function. This may explain the parallelism observed between changesin leptin and T concentrations.

Insulin stimulates leptin production in vitro (Kennedy et al. 1997) and in vivo (Kola-cynski et al. 1996). The improvement of hyperinsulinemia in our study may also dec-rease serum leptin concentrations. The relationship between hyperinsulinemia, insulinresistance and serum leptin concentrations is not clear, however, and as the role of leptinin PCOS is not understood, more studies are needed to clarify whether the improvementof hyperinsulinemia can decrease serum leptin concentrations.

As metformin may inhibit lipolysis in adipose tissue and thus play some role in themetabolism of fat cells (Riccio et al. 1991), a direct effect of metformin on the secretionof leptin in fat tissue cannot be excluded.

7. Conclusions

1. The present results demonstrate that the prevalence of PCO is dependent on ageamong healthy women, the prevalence being significantly higher in the younger agegroup (< 35 years) than it was among older women (> 36 years). This may be due to aphysiological decline of the follicular cohort, leading to a normalized ovarianultrasonographic appearance with advanging age. The overall prevalence of PCO inFinnish women with no other overt symptoms of PCOS was 14.2%. This is slightlylower than that reported in previous studies (16-23%). The hormonal parameters andclinical findings in these women with an isolated finding of an ultrasonographicappearance of PCO mimic those associated with PCOS. Women with PCO havehigher a LH/FSH ratio, serum T concentrations, incidence of minor cycleirregularities and problems in conceiving significantly more often than women withnormal ovaries.

2. The carrier frequency of the v-LHβ allele in the entire study population was 18.5%.The v-LH carrier frequency was similar in obese and nonobese controls in eachcountry studied, but was significantly lower in obese PCOS subjects in theNetherlands and Finland, where the proportions of v-LH carriers in obese PCOSsubjects were only 2.0% and 4.5%, respectively. In contrast to these countries, nosuch difference was observed in the United Kingdom. Both the present results andthose from previous studies on other pathological gonadal conditions indicate that theprevalence of v-LH varies among different ethnic groups. The high overall frequencyof the v-LH allele in women in general and its low frequency in obese PCOS patientsalso suggests that v-LH may somehow protect obese individuals from developingPCOS. It must be kept in mind, however, that many widely used immunoassayreagents do not detect v-LH and thus, an elevated LH/FSH ratio may not bediscovered among PCOS women.

3. Women with previous GDM displayed a high prevalence of PCO (39.4%), being two-fold higher than that reported in healthy premenopausal women. Women with PCOand previous GDM showed insulin sensitivity and blood glucose responses in theOGTT comparable with those of women with normal ovaries and previous GDM.They had a more marked hyperinsulinemia, however, which could not be explained

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by obesity. This may indicate that these women have a lowered metabolic clearancerate of insulin. They also had subtle clinical, endocrine and metabolic featurescharacteristic of PCOS, although clinical symptoms typical of PCOS were scarce.These findings suggest that the ultrasonographic presence of PCO may be a predictivefactor as regards abnormal glucose tolerance during and after pregnancy and thesewomen should therefore be advised as to the possible consequences. As women withPCO have metabolic abnormalities resembling PCOS, they may be at risk forincreased morbidity associated with PCOS, i.e. DM, hypertension, coronary arterydisease etc. with ageing. Our data also demonstrate that women with previous GDMoften have abnormal OGTT results in general, they are insulin resistant, mainly as aresult of lowered glucose nonoxidation in the peripheral tissues, and, furthermore,they show inappropriately low insulin responses to glucose, due to impaired ß-cellfunction. They also demonstrate a higher adrenal androgen secretion than the controlwomen. Health education and the follow-up of these women is of great importance.

4. Obese women with PCOS displayed a distinctly different steroidogenic responsepattern to a single dose of hCG compared with the control women followed for 4days. Peak peripheral serum A and T concentrations were achieved in the PCOSwomen at 48 hours after hCG injection, preceded by peak levels of 17-OHP and E2 at24 hours. In contrast, all steroids measured in the control women reached theirmaximum serum concentrations at 96 hours. It remained unclear, however, if thisexaggerated 17-OHP and E2 response to hCG in PCOS women is due to adysregulation of certain enzyme(s), or to higher theca cell activity or mass inpolycystic ovaries. The slight alleviation of hyperandrogenism brought about bymetformin therapy may be explained by decreased ovarian steroidogenesis due toreduced serum insulin concentrations.

In addition to hMG containing equal amounts of both FSH and LH, FSH alonealso stimulated the production of ovarian androgens after suppression of the pituitary-ovarian axis with GnRHa. Under this experimental condition, women with PCOS didnot display any distinctly exaggerated steroidogenic response to gonadotrophinstimulation compared to endocrinologically normal women.

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