Endocrine and metabolic changes in women with polycystic ovaries and with polycystic ovary syndrome

Riitta Koivunen

Department of Obstetrics and Gynaecology and Department of Clinical Chemistry, University of Oulu, P.O.Box, FIN-90014 University of Oulu, Finland

Abstract

The prevalence of the isolated ultrasonographic finding of polycystic ovaries (PCO) in the Finnish population and among women with a history of gestational diabetes (GDM) and changes in the present carbohydrate metabolism were investigated in the present study. One aim of this study was to investigate the prevalence of the recently discovered variant type LH (v-LH) in PCOS and to compare patient 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 of women with PCOS to exogenously administered human chorionic gonadotrophin (hCG), human menotrophin (hMG) and follicle stimulating hormone (FSH). The effect of metformin on ovarian steroidogenesis was also studied.

The prevalence of PCO was significantly higher in younger (= 35 years, 21.6%) than among older women (in = 36 years, 7.8%). The overall prevalence of PCO in Finnish women was 14.2%. Women with previous GDM revealed a high prevalence of PCO (39.4%). The carrier frequency of the v-LHb allele in the entire study population was 18.5%. The frequency of the v-LH carrier was significantly lower in obese PCOS subjects in the Netherlands (2.0%) and Finland (4.5%). Women with previous GDM had impaired insulin sensitivity and β -cell function. They also had higher adrenal androgen secretion than the control women. Women with PCO and previous GDM had marked hyperinsulinemia which was not explained by obesity. Obese PCOS women achieved peak peripheral serum T concentrations at 48 hours after a hCG injection, preceded by peak levels of 17-OHP and E2 at 24 hours. In contrast, all steroids measured in the control women reached their maximum serum concentrations at 96 hours. HMG stimulated the production of ovarian androgens more efficiently than 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 more common in women with a history of GDM. The ultrasonographic appearance of PCO may be a predictive factor with regards abnormal glucose tolerance during and after pregnancy and, these women should therefore be advised as to possible consequences. The high overall frequency of the v-LH allele in women in general and its low frequency in obese PCOS patients suggests that v-LH plays a role in reproductive functions and may counteract the pathogenesis of PCOS in obese individuals. The differences observed in steroid responses to hCG between normal and PCOS women might be explained by higher theca cell activity or mass in polycystic ovaries. Women with PCOS did not show a distinctly exaggerated steroidogenic response to hMG or FSH administration compared with control women. FSH administration also resulted in increased A and T production.

Dedication

To Sini

Table of Contents
Acknowledgements
Abbreviations
List of original articles
1. Introduction
2. Review of the literature
2.1. General background of polycystic ovary syndrome (PCOS)
2.2. Epidemiology of PCOS
2.3. Clinical features of PCOS
2.3.1. Women with an isolated finding of polycystic ovaries
2.3.2. Present definition of polycystic ovaries
2.4. Biochemical and clinical features of PCOS
2.4.1. Gonadotrophins in PCOS
2.4.2. Steroidogenesis in PCOS
2.4.3. Metabolic features in PCOS
2.4.4. Insulin-like growth factors in PCOS
2.4.5. Leptin and PCOS
2.4.6. Lipids and PCOS
2.4.7. Genetics of PCOS
2.5. Long-term sequelae and risks in PCOS
2.5.1. Dyslipidemia and cardiovascular disease
2.5.2. Hypertension
2.5.3. Gestational diabetes and diabetes mellitus
2.5.4. Cancer
2.6. Modern treatment of PCOS
2.6.1. Treatment of hyperandrogenism
2.6.2. Treatment of irregular cycles and anovulation
2.6.3. Insulin-lowering treatment in PCOS
3. Purpose of the present study
4. Subjects and methods
4.1. Subjects and study design
4.2. Clinical parameters
4.3. Vaginal ultrasonography
4.4. Fasting insulin to glucose ratio and the homeostasis model assessment
4.5. Oral glucose tolerance test and early phase insulin and C-peptide measurements
4.6. Euglycemic hyperinsulinemic clamp
4.7. Calorimetry
4.8. Human chorion gonadotrophin stimulation test
4.9. Treatment protocols
4.10. Laboratory methods
4.11. Statistical analysis
5. Results
5.1. Clinical parameters
5.2. Prevalence of polycystic ovaries
5.3. Variant type of luteinizing hormone
5.4. Effects of previous gestational diabetes (GDM)
5.4.1. Clinical characteristics
5.4.2. Oral glucose tolerance test
5.4.3. Insulin secretion and insulin sensitivity
5.5. Endocrine parameters
5.5.1. Women with previous GDM versus controls
5.6. Steroidogenesis in women with PCOS
5.6.1. Effects of human chorionic gonadotrophin
5.6.2. Effects of metformin on steroidogenesis and on serum insulin and leptin concentrations
5.6.3. Effects of human menopausal gonadotrophin and follicle stimulating hormone
6. Discussion
6.1. Prevalence of an isolated finding of polycystic ovaries
6.2. Variant type of luteinizing hormone
6.3. Insulin sensitivity, and metabolic and endocrine features in women with a history of GDM
6.3.1. Association between previous GDM and PCO
6.4. The effect of stimulation with human chorion gonadotrophin, human menopausal gonadotrophin and follicle stimulating hormone on steroidogenesis in women with PCOS and in control women
6.4.1. The effect of short-term human chorion gonadotrophin stimulation
6.4.2. The effect of long-term human menopausal gonadotrophin and follicle stimulating hormone stimulation
6.4.3. The effect of metformin on steroidogenesis in women with PCOS
6.4.4. The effect of metformin on serum leptin concentrations
7. Conclusions
References
List of Tables
1. A summary of studies on the prevalence of ultrasonographic findings of polycystic ovaries (PCO).
2. Histologic features of polycystic ovary.
3. Ultrasonographic criteria used for the diagnosis of PCO.
4. Subjects, methods and main results of the studies. Data is shown as mean SD.
5. Characteristics of the assays used.
6. Clinical parameters, serum LH/FSH-ratio and testosterone concentrations in women with normal and polycystic ovaries from studies I and III. Values are presented as mean SD or as percentages. Women fulfilling the criteria for PCOS were excluded (n=2) from the analysis in study III.
7. The carrier frequency of normal (wild type, wt) and variant (v) LH alleles in different countries, as determined by the ratio of two LH immunoassays (assay1/assay2, see chapter 4.10). Results are shown as percentages.
8. Clinical (mean SD) and metabolic (95% CI in parentheses) characteristics in control and GDM women with or without polycystic ovaries (PCO).
9. Results of OGTTs in women with a history of GDM treated with diet or with insulin, and controls.
10. Hormonal parameters (mean SD) among control and PCOS women in the basal state (after GnRHa suppression) and after human menopausal gonadotrophin (hMG) and follicle stimulating hormone (FSH) stimulations.
11. Clinical characteristics and outcome of IVF cycles (mean SD) in control women and women with PCOS stimulated with human menopausal gonadotrophin (hMG) or follicle stimulating hormone (FSH) after pituitary suppression with GnRHa.
List of Figures
1. An ultrasonograph picture of a polycystic ovary.
2. A schematic picture of normal and variant LH molecules. Because of a supposed additional carbohydrate chain in the β -subunit of LH variant, the antibody against intact α/β -dimer does not find the epitope detectable in normal LH (modified from Haavisto 1996).
3. The major steroid biosynthetic pathways in the small antral follicle of the ovary, according to the two gonadotrophin, two-cell model of ovarian steroidogenesis. Luteinizing hormone (LH) stimulates androgen formation within theca cells by the steroidogenic pathway common to the gonads and adrenal glands. Follicle-stimulating hormone (FSH) regulates estradiol synthesis from androgens in granulosa cells. The conversion of 17-ketosteroids to 17β -hydroxysteroids by 17β -hydroxysteroid dehydrogenase (17β HSD) is essential for the formation of testosterone, dihydrotestosterone (DHT) and estradiol. Androgen formation in response to LH appears to be modulated by intraovarian feedback at the levels of 17-hydroxylase and 17,20-lyase, both which are activities of cytochrome P450c17. The quantitative importance of androstenedione formation from 17-hydroxyprogesterone (dashed arrow) in the intact follicle is unknown. Androgens 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 acute regulatory protein; 3β = Δ5-isomerase-3β - hydroxysteroid dehydrogenase; 5α-R = 5α-reductase; P450arom = aromatase enzyme
4. Major steroid pathways in the adrenal cortex. The square contains the core steroidogenic pathways also used by the gonads. In the adrenals, 17-hydroxyprogesterone (17-OHP) is situated at a potential branch point at which cortisol and sex hormone synthesis may diverge depending 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-hydroxylase/17,20-lyase; 21-hydroxylase (21); 11β -hydroxylase/18-hydroxylase-dehydrogenase (11, 18). Non-P450 enzyme steps are steroidogenic acute regulatory protein (StAR); Δ5-isomerase-3β -hydroxysteroid dehydrogenase (3β ), 17β -hydroxysteroid dehydrogenase (17β ), sulfokinase (SK) and sulfolyase (SL). Dashed pathways are considered to be relatively minor. Conversion of dehydroepiandrosterone (DHEA) to DHEA sulphate and androstenedione to 11β -hydroxyandrostenedione takes place in the zona reticularis. Deoxycorticosterone (DOC). (Modified from Rosenfield 1999).
5. Insulin receptor, its signaling pathways for glucose transport and hypothetical mechanisms of stimulation or inhibition of steroidogenesis. After insulin binds to the insulin receptor α-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 (metabolic pathway); glucose transporters are translocated to the cell membrane, and glucose uptake is stimulated. The IRS-1 and Ras-mitogen-activated protein kinase (MAPK) appear to regulate cell growth and mitogenesis (mitogenic pathway). An alternative signaling system involving generation of inositolglycan second messengers at the cell membrane independently of the β -subunit tyrosine kinase activation. This pathway may mediate insulin modulation of steroidogenic enzymes (see text for more details and references). (Modified from Dunaif 1999, Poretsky et al. 1999, Yen 1999). ATP = adenosine triphosphate; ADP = adenosine diphosphate; scc = side chain cleavage; c= cytochrome; arom = aromatase; -S-S-= disulphide bonds; Glucose-6-P = Glucose 6 phosphate
6. Insulin action in women with PCOS. Possible mechanisms causing postbinding defects in insulin action in women with PCOS. Decreased insulin-dependent receptor tyrosine phosphorylation (Tyr-P) and increased constitutive receptor serine phosphorylation (Ser-P), probably secondary to a cell membrane associated factor, inhibits insulin receptor signaling. Inhibition of receptor signaling leads to decreased amount of glucose transporters and decreased glucose uptake. Ser-P of insulin receptor substrate-1 (IRS-1) appears to be the mechanism for tumor necrosis factor α (TNF α) mediated insulin resistance. Protein kinase A could be the factor that serine phosphorylates the insulin receptor. The membrane glycoprotein PC-1 also inhibits insulin receptor kinase activity, but it does not cause serine phosphorylation of the receptor. An alternative signaling pathway involving generation of inositolglycan second messengers at the cell membrane, independently of the β -subunit tyrosine kinase activation, possible mediate stimulation of ovarian steroidogenesis. (Modified from Dunaif 1999, see text for more details and references). Arom = aromatase; c = cytochrome; scc = side chain cleavage; -S-S- = disulphide bonds
7. Possible mechanisms causing hyperandrogenism and hyperinsulinemia in women with PCOS. The increased luteinising hormone (LH) pulse frequency increases the androgen production from theca cells of the ovary. In the follicles increased levels of insulin-like growth factor-binding protein-2 and -4 (IGFBP-2, -4) and the absence of IGFBP proteases may sequester the insulin-like growth factor-I (IGF-I) and thereby reduce its synergistic action with FSH leading to arrest of follicle development. Adrenal androgen production is increased. Hyperandrogenism increases the proportion of insulin resistant muscle fibres in the muscle tissue and serum free fatty acid (FFA) levels. The increased FFAs in the serum compete with glucose for uptake and oxidation in the muscle cell, inducing further insulin resistance. In addition FFAs have been shown to decrease hepatic insulin extraction. Androgens may indirectly modify β -cell sensitivity to glucose. Hyperinsulinemia, either primary or secondary to insulin resistance, induces hyperandrogenism directly by increasing ovarian androgen secretion, probably via its own receptor, and indirectly by decreasing the secretion of sex hormone-binding globulin (SHBG) and IGFBP-1 in the liver. Insulin downregulates the number of its own receptors on the membranes of the target cells. Obesity may contribute to androgen excess due to the capacity of the adipose tissue to form testosterone and oestrone from inactive precursors. Elevated level of estrogen can augment pituitary sensitivity to gonadotrophin releasing hormone (GnRH). IR = insulin receptor
8. Natural history of women with PCOS.
9. The frequency (percentage) of v-LH (homozygotes and heterozygotes) in different countries among controls and PCOS women with a BMI of 27 or less or with a BMI more than 27 kg/m2. The number of subjects is indicated inside the bars. The frequency between the groups was compared using the χ2 test: a, P < 0.05; b, P < 0.01; c, P < 0.03; after continuity correction P = 0.07 compared to controls BMI > 27 kg/m2.
10. Blood glucose (mean SE) and serum insulin concentrations during OGTT in control women (black squares) and in women with previous GDM (black circles). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with controls.
11. Blood glucose (mean SE) and serum insulin concentrations during OGTT in women with previous GDM and normal ovaries (black squares) and those with previous GDM and PCO (black circles). * p < 0.05 compared with controls.
12. Early phase insulin and C-peptide secretion (mean SE) during the OGTT in control women (white bars) and in women with previous GDM (gray bars). The third panel shows the insulin sensitivity index (M/I, glucose oxidation and nonoxidation) assessed by hyperinsulinemic euglycemic clamp. * p < 0.05 (glucose nonoxidation only), ** p < 0.01 compared with controls.
13. Serum 17-OHP, androstenedione, testosterone and estradiol responses (mean SE) to a single injection of hCG (5000 IU) in control women (black squares, n = 27) and in PCOS women 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 with PCOS women before metformin treatment.
14. The change (post-treatment minus basal concentrations) in serum 17α-hydoxyprogesterone (17-OHP), androstenedione, testosterone and estradiol during either human menopausal 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 PCOS women. * p < 0.05 compared with controls stimulated with hMG.
15. Δ 17-hydroxyprogesterone / Δ androstenedione and Δ estradiol / Δ 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.
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