Serum insulin patterns and the relationship between insulin sensitivity and glycaemic profile in women with polycystic ovary syndrome

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1 DOI: /j x Fertility and reproductive medicine Serum insulin patterns and the relationship between insulin sensitivity and glycaemic profile in women with polycystic ovary syndrome HR Seneviratne, a D Lankeshwara, b S Wijeratne, a N Somasunderam, c D Athukorale a a Vindana Reproductive Health Centre, Colombo, Sri Lanka b De Soyza Maternity Hospital for Women, Colombo, Sri Lanka c National Hospital of Sri Lanka, Colombo, Sri Lanka Correspondence: Prof. HR Seneviratne, 32 First Chapel Lane, Colombo 06, Sri Lanka. vindana-ivf@sltnet.lk Accepted 24 June Published Online 23 September Objective To evaluate serum insulin levels and insulin sensitivity in women with polycystic ovary syndrome (PCOS) in relation to their glycaemic status. Design An observational study. Setting A tertiary-level reproductive health centre in Sri Lanka. Sample Infertile women diagnosed as having PCOS (n = 168) on the basis of the Rotterdam criteria were included in the study. Methods Glycaemic status and serum insulin values were assessed at fasting and at 2 hours after a 75-g oral glucose load and stratified as diabetes mellitus (DM) (10.12%), impaired glucose tolerance (IGT) (23.21%) and normoglycaemia (66.67%). The normoglycaemic group was restratified as groups A (10.7%), B (79.5%) and C (9.8%) on the basis of serum insulin levels, with group A having the lowest and group C the highest values. The Quantitative Insulin Sensitivity Check Index (QUICKI) scores of women with DM and IGT and those in groups A, B and C in the normoglycaemic category were compared. Main outcome measures Insulin sensitivity in these groups of women. Results Body mass index (BMI) exceeded 23 kg/m 2 in 77.38% of the women. In normoglycaemic women with PCOS, insulin sensitivity was highest in group A. In groups B and C, insulin sensitivities corresponded to those found for women with IGT and DM respectively. This pattern was also reflected in the BMI. Conclusions Normoglycaemic women with PCOS are heterogeneous regarding insulin sensitivity. The treatment offered to those with DM and IGT could be extended to subgroups B and C of normoglycaemic subjects. Normoglycaemic women with PCOS with high insulin sensitivity (group A) would not qualify for this treatment. Keywords Infertility, insulin resistance, insulin-sensitising drugs, polycystic ovary syndrome, Quantitative Insulin Sensitivity Index. Please cite this paper as: Seneviratne H, Lankeshwara D, Wijeratne S, Somasunderam N, Athukorale D. Serum insulin patterns and the relationship between insulin sensitivity and glycaemic profile in women with polycystic ovary syndrome. BJOG 2009;116: Introduction Polycystic ovary syndrome (PCOS) is an established cause of infertility. 1,2 Increased production of luteinising hormone (LH), hyperandrogenism and resistance to insulin are central issues in this condition. Diagnosis, however, remains problematic because of the inconsistency of individual clinical and laboratory features of PCOS. 2,3 Agreement on diagnostic criteria reached at a meeting held in Rotterdam in has helped to make diagnosis of PCOS more uniform worldwide. In addition to infertility, PCOS is associated with many long-term effects on health, such as type II diabetes mellitus (DM), dyslipidaemia, hypertension, cardiovascular disease, carcinoma of the endometrium and gestational diabetes. 5,6 In infertile women with PCOS, disturbed glycaemic control caused by resistance to insulin is closely linked to anovulation. 7,8 Insulin resistance to glycaemia which occurs at post-receptor sites in target cells results in hyperinsulinaemia. As ovarian tissue retains its sensitivity to insulin, it produces an exaggerated response in the presence of increased serum insulin levels. 9 This response includes increased androgen production. In an effort to counter, the effect of excess insulin in PCOS, insulin-sensitising agents such as metformin are commonly used. 10 It has been argued, however, that the practice of treating all subjects with PCOS with these medications is unjustified ª 2009 The Authors Journal compilation ª RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology

2 Rational basis for metformin use in PCOS Insulin-sensitising agents are commonly used to treat DM and impaired glucose tolerance (IGT) to enhance insulin sensitivity. Basal levels of insulin are usually taken to represent the degree of insulin resistance. 11 Normal fasting serum levels of insulin range from 5 to 20 lu/ml. Excess insulin alters follicular paracrine activity, oocyte maturation, ovulation and basal insulin, and post-meal or post-glucose-load insulin increases have a similar effect. Treatment to reduce the action of insulin on the ovaries can therefore be of benefit in managing infertility in women with PCOS. Basal serum insulin levels and those after an oral glucose load have been noted to be enhanced in women with PCOS. 7 This study aimed: 1. To assess the pattern of glycaemia and associated serum insulin levels at fasting and post-glucose load. 2. To evaluate insulin sensitivity status in normoglycaemic subjects identified using the oral glucose tolerance test by matching it against the Quantitative Insulin Sensitivity Check Index (QUICKI). Methods Data for women with PCOS attending a tertiary reproductive health centre in Sri Lanka from December 2002 to November 2005 were evaluated. Infertile women with PCOS strictly diagnosed using the Rotterdam criteria of were recruited. Hyperandrogenism was considered either on clinical or biochemical evidence. Biochemical evidence of hyperandrogenism was found only in 25% of the patients. However, all 168 fulfilled the criteria for inclusion in the study in view of the ultrasound criteria for identification of polycystic ovaries and the presence of oligo-amenorrhoea. Those women with causes other than PCOS leading to amenorrhoea, anovulation and hirsuitism were excluded. All women had a routine clinical and ultrasound evaluation. The body mass index (BMI) was determined by measuring height (m) and weight (kg) during the clinical examination by medical staff. Basic endocrine investigations included menstrual cycle day two serum follicular stimulating hormone (FSH), LH and prolactin. Levels of adrenal steroids, serum levels of testosterone and dehydroepiandrosterone sulphate (DHEAS) and 17-hydroxy progesterone levels were estimated. Women with alternative causes of hyperandrogenism and secondary causes of amenorrhoea were excluded. Cortisol levels were evaluated only if clinical features of Cushing syndrome were present. Glycaemic status was evaluated by performing the 75-g oral glucose tolerance test (OGTT) on all participants according to World Health Organisation (WHO) guidelines. 12 At the same time, serum insulin levels were assessed on fasting blood (basal insulin) and on blood samples obtained 2 hours after the glucose load (2-hour insulin). Gonadotrophic and glycaemic tests were performed on all women (n = 168). Because of the high cost of the investigations, assessment of androgenic status was restricted to participants with clinically identified features of hyperandrogenism. Venepunctures to obtain test samples were kept to a minimum. The glycaemic status was diagnosed for the first time in this population of women with PCOS. Based on the OGTT results (WHO criteria), the glycaemic status of each participant was defined as normal (normoglycaemia), IGT or DM. 13 Insulin status was categorised by stratifying the basal and 2-hour insulin values following the study by Dunaif et al., 7 where the response to an oral glucose load (40 g/m 2 ) was assessed. Accordingly, for the purpose of analysis of data in this study, fasting serum insulin levels were stratified into three categories: , and 20 lu/ ml. In a similar manner, 2-hour insulin levels were stratified into three categories: , and 100 lu/ ml. The group classed as normoglycaemic according to the OGTT was further divided into three strata based on fasting and 2-hour insulin levels (see Table 5). The participants with the lowest serum insulin levels (fasting insulin lu/ml and 2-hour insulin lu/ml) were placed in group A, while those with the highest levels (fasting insulin 20.0 lu/ml and 2-hour insulin 100 lu/ml) constituted group C. All other participants, with serum insulin values in the intermediate range, were placed in group B (see Table 5 below). The Quantitative Insulin Sensitivity Check Index (QUICKI) is a method of expressing the insulin sensitivity of an individual. It has been validated against other methods of expressing insulin sensitivity such as the euglycaemic insulin clamp method. 14 The euglycaemic insulin clamp method of determining insulin sensitivity is too unwieldly for use in routine clinical practice. The formula used to calculate QUICKI values was: QUICKI ¼ 1=½logðInsÞ þ logðgluþš where (Ins) and (Glu) refer to serum insulin and plasma glucose levels respectively. In the original study, 14 QUICKI values were calculated only for fasting plasma glucose and serum insulin values. In this study, the QUICKI formula was also used to calculate 2-hour values. The correlation coefficient for the relationship between fasting and 2-hour QUICKI values for the whole study population was determined. Serum glucose was assessed using the glucose oxidase (GOD PAP) method with the Roche/Hitachi 704 Clinical Chemistry Analyzer (Roche Diagnostics GmbH, Mannheim, Germany), while endocrine assessments were performed using the IMx system (Abbot Diagnostics, Abbott Park, IL, ª 2009 The Authors Journal compilation ª RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology 1723

3 Seneviratne et al. USA) and the Elecsys 2010 Immunochemistry Analyzer (Roche Diagnostics GmbH). In this observational study, the data were analysed using SPSS version 13 (SPSS, Chicago, IL, USA). The prevalences of the clinical and endocrine features expected for PCOS were derived as percentages. Serum insulin levels are expressed as the mean and standard deviation for the different groups. Percentages of patients assigned to each insulin level category are given. QUICKI values for the different categories of participants are expressed as the mean and standard error of the mean, and the statistical significance of differences in QUICKI values among categories was evaluated using analysis of variance (ANOVA). This study was confined to analysis of data obtained during routine clinical practice and investigation of infertile women with PCOS. All study participants had the ultrasound features of PCOS. Oligoamenorrhoea and clinical or biochemical features of hyperandrogenism were recorded when present. Not all the cases which were negative for the latter had been recorded. In this retrospective analysis, each participant had oligoamenorrhoea and/or features of hyperandrogenism recorded so that each one included was compatible with the Rotterdam criteria. Results The basic characteristics of all participants (n = 168) were within the accepted norms for ovulation potential [mean Table 1. The prevalence of PCOS-related clinical and endocrine characteristics of the study population (n = 168) Parameter Abnormal characteristic Result (%) Menstruation Amenorrhoea 80.5 (128 of 159) Clinical androgenic features Hirsuitism etc (94 of 146) Body mass index (BMI) >23 kg/m (130 of 168) Serum LH >6 miu/ml (86 of 168) Serum prolactin >23.0 ng/ml 5.5 (06 of 109) LH: FSH Ratio >2: (48 of 168) Serum testosterone >120 ng/dl (29 of 115) Serum 17-OH Progesterone >80 ng/dl (24 of 59) Serum DHEA SO 4 >430 mg/dl All normal Table 3. Mean age ± SD in years and body mass index (kg/m 2 )of normoglycaemic (groups A, B and C) IGT and DM Group No. subjects Age (years) Body mass index A ± ± 3.2 B ± ± 4.05 C ± ± 5.43 IGT ± ± 4.39 DM ± ± 3.77 Total ± ± 4.40 age 29.6 ± 4.0 years (range years); mean mensus day two FSH 4.62 ± 1.78 miu/ml (range miu/ml)]. The prevalences of PCOS-related clinical and endocrine features are presented in Table 1 as percentages of those who had the test performed. All participants included in the study had the ultrasound features of PCOS and oligomenorrhoea and/or features of hyperandrogenism as specified in the Rotterdam criteria for PCOS. The 75-g OGTT (Table 2) was normal in 66.7% (n = 112) of the subjects while in 23.2% (n = 39) IGT was detected. Only 10.1% (n = 17) had DM, and all these participants had their glycaemic state diagnosed for the first time. The mean BMI for the entire study population was kg/m 2 (range kg/m 2 ) (Table 2). A BMI of >23.0 kg/m 2 was found in 77.38% (n = 130) of the study participants. The BMI values of the different subgroups are presented in Table 3. The differences in BMI among the three subcategories of normoglycaemic subjects, groups A, B and C, were statistically significant (P < 0.05). The mean basal serum insulin level (n = 168) was ± 9.36 lu/ml while the mean 2-hour serum insulin level was ± lu/ml (Table 4). The correlation coefficient for the relationship between the serum fasting and 2-hour insulin levels was (P < 0.01). The basal serum insulin levels of the normoglycaemic, IGT and DM women were ± 7.8, ± 10.6 and ± lu/ml respectively (Table 4). The corresponding 2-hour serum insulin levels were ± 75.8, ± 97.7 and ± 96 lu/ml respectively (Table 4). The percentages of normoglycaemic patients in each Table 2. Body mass index (kg/m 2 ) distribution by glycaemic status (n = 168) Glycaemic status No. subjects (%) Mean ± SD (range) <23 (%) >23 (%) Normal 112 (66.7) ± 4.37 ( ) n = 25 (22.5) n = 87 (77.7) IGT 39 (23.2) ± 4.39 ( ) n = 12 (30.8) n = 27 (69.2) DM 17 (10.1) ± 3.77 ( ) n = 1 (5.9) n = 16 (94.1) Total 168 (100) ± 4.40 ( ) n = 38 (22.6) n = 130 (77.38) 1724 ª 2009 The Authors Journal compilation ª RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology

4 Rational basis for metformin use in PCOS Table 4. Distribution of glycaemic status versus fasting and 2 hour serum insulin Glyceamic status No. subjects (%) Fasting insulin Mean ± SD (range) 2 hour insulin Mean ± SD (range) Normal 112 (66.67) ± 7.8 ( ) ± 75.8 ( ) IGT 39 (23.21) ± 10.6 (3.9 51) ± 97.7 ( ) DM 17 (10.12) 21.7 ± ( ) ± 96.2 ( ) Total 168 (100) ± 9.36 (3.1 ± 56.2) ± (8.9 ± 405.9) Table 5. Comparison between the basal and 2 hour serum insulin (lu/ml) values in normal glycaemic participants (n = 112) 2 hour insulin levels <49 n = 19 (17.0%) n = 38 (33.9%) 100< n = 55 (49.1%) <9.9 n =35 (31.3%) 12 (10.7%) Group A 16 (14.3%) 7 (6.3%) Basal insulin levels n =65 (58.0%) 7 (6.3%) 21 (18.8%) 37 (33.0%) 20< n =12 (10.7%) 0 (0.0%) 1 (0.9%) 11 (9.8%) Group C normoglycaemic subcategory were as follows: group A, 10.7% (n = 12); group B, 79.5% (n = 89) and group C, 9.8% (n = 11) (Table 5). The fasting and 2-hour QUICKI values for the whole study group were ± and ± respectively. The correlation coefficient for the relationship between the fasting and 2-hour QUICKI values was (P < 0.01). The mean values and standard errors of the mean for normoglycaemic women (groups A, B and C) and those with IGT and DM are shown in Figures 1 and 2. Group A had the highest insulin sensitivity, as indicated by both fasting and 2-hour QUICKI values. Group B had a fasting QUICKI value that was similar to that of the IGT group. Group C had a fasting QUICKI value that showed no difference from that of the women with DM. At 2 hours, the QUICKI values of participants in group C, the IGT group and the DM group were similar, while those in group B had a moderate level of insulin sensitivity. Mean BMI was lowest in group A, higher in group B and highest in group C. Mean BMIs in the IGT and DM groups corresponded to those in groups B and C respectively (Table 3). Discussion and conclusions Insulin-sensitising agents reduce the need for higher insulin production and thereby reduce serum insulin levels. These medications are therefore needed when tissue sensitivity to insulin is low and thus serum insulin levels are elevated. The clinical relevance of this study relates to the more accurate selection of insulin-sensitising agents in PCOS. These medications could be avoided in normoglycaemic subjects with low insulin levels. This study provides an insight into insulin production in women with PCOS in relation to their glycaemic status. The treatment of PCOS is a complex therapeutic exercise as the pathophysiological events follow varying paths. Why the ovary remains sensitive to insulin when other sites have become non-responsive has been difficult to explain. 9 It is possible that insulin may act in the ovary via other receptors. Insulin receptors in fibroblasts have been shown to ± QUIKI value ± ± ± A(n = 12) B(n = 89) C(n = 11) IGT(n = 39) DM(n = 17) Group ± Figure 1. Fasting QUICKI value of Normoglycaemic groups A, B and C, IGT and DM (mean ± SEM). ª 2009 The Authors Journal compilation ª RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology 1725

5 Seneviratne et al. QUIKI value ± ± ± ± ± A(n = 12) B(n = 89) C(n = 11) IGT(n = 39) DM(n = 17) Group Figure 2. Two (2) hour QUICKI value of Normoglycaemic groups A, B and C, IGT and DM (mean + SEM). remain selectively sensitive. 9,15 Ovulation induction in women with PCOS has been facilitated by the use of insulin-sensitising agents. 10 The method of selection of patients to receive this therapy has been mostly through diagnosis of PCOS. Obesity is a key factor in the pathophysiological chain of events of PCOS 16 as well as in the aetiology of DM. Even women with minimal increase in BMI have been shown to have an increased risk of developing DM. 17 In the present study, weight reduction would be the primary mode of management in the women with a BMI of >23.0 kg/m 2. However, as 22.7% (n = 38) had a BMI of <23.0 kg/m 2 it did not prove to be a reliable parameter to commence insulin-sensitisation therapy. Further analysis of BMI demonstrated an interesting relationship between groups A, B and C of the normoglycaemic subjects and those with IGT and DM. Subjects with DM and those in group C had the highest BMI while those with IGT and those in group B had similar values. Group A had the lowest BMI values. The serum insulin levels were also related in a similar manner. In previous studies of women with PCOS, between 7.5% and 10% were reported to have type II DM and between 31% and 35% to have IGT at the time of PCOS diagnosis. 8,18 The 33.3% of participants in this study who had IGT or DM would benefit from dietary and/or medical therapeutic interventions. Such interventions might include treatment with metformin and other insulin-sensitising agents or insulin therapy. These therapeutic interventions would reduce the risk of the development of long-term cardiovascular disease and also improve the fertility potential of the patients. Hyperinsulinaemia associated with PCOS has been shown to correlate with increased cardiovascular risk independent of obesity. 19 Weight loss also benefits ovarian follicular structure and function. 20 These measures together with treatment with insulin-sensitising medications would therefore benefit those 33.3% of participants in this study who have been identified as having IGT or DM. The 12 participants who were unsuitable for metformin treatment were reviewed separately. None of them was dieting, but all had BMIs <27 kg/m 2. However, all these patients had fulfilled the criteria for the diagnosis of PCOS. Only two of these participants had biochemical evidence of hyperandrogenism. In the present study, the presence of polycystic ovaries in the normoglycaemic women indicated that pathophysiological mechanisms other than hyperglycaemia were responsible for PCOS in these women. The role of dietary interventions and insulin-sensitising therapy in the treatment of these normoglycaemic participants is uncertain. However, BMI was >23 kg/m 2 in 77.7% (n = 87) of the normoglycaemic participants and weight loss would be a serious consideration in their plan of management. The stratification of the normoglycaemic women into three groups (A, B and C) based on their fasting and 2- hour serum insulin levels facilitated the assessment of their insulin sensitivity status. The QUICKI calculation validated this categorisation. The QUICKI calculation has been validated against other tests for insulin sensitivity using only fasting values. 14 In the present study, this validation was extended to 2-hour serum insulin values in these subjects, as the correlation coefficient for the relationship between fasting and 2-hour QUICKI values was only moderate at (P < 0.01). Sensitivity to insulin, as indicated by fasting and 2-hour QUICKI values, was lower in women with IGT than in normoglycaemia women, and even lower in women with DM. A similar trend was seen in the normoglycaemic women when they were stratified into groups A, B and C. Groups B (79.5%; n = 89) and C (9.8%; n = 11) had fasting and 2-hour QUICKI values comparable with those of women with IGT and DM respectively. Therefore, although, on the basis of the OGTT, these two groups did not qualify to receive treatment with insulin-sensitising agents, including metformin, their use in groups B and C was justified ª 2009 The Authors Journal compilation ª RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology

6 Rational basis for metformin use in PCOS This study indicates that there is, perhaps unexpectedly, a subcategory of normoglycaemic women with PCOS (group A; 10.7%; n = 12) who show high levels of insulin sensitivity and low insulin levels. On the basis of the results of this study, there is no justification for treating these women with insulin-sensitising agents such as metformin as their blood-glucose levels are low and their sensitivity to insulin is high. An issue that requires serious consideration is the possible dose -related effect of serum insulin on ovarian follicular function. The mean fasting serum insulin value for all participants was ± 9.36 lu/ml, while the mean 2-hour serum insulin value was ± lu/ ml. The 2-hour serum insulin level exceeded lu/ ml in 54.17% of all participants. Similarly, 2-hour serum insulin levels exceeding lu/ml were noted in 58.82% of participants with DM, 66.67% of those with IGT and 49.1% of normoglycaemic women. Fasting insulin level alone appears to be inadequate for the assessment of normoglycaemic women as some women can have high 2-hour insulin values despite normal fasting values. It is improbable that ovarian follicular function would remain unaffected by high serum insulin levels 2 hour after a glucose load and possibly after a meal. This phenomenon demonstrated in the present study needs further evaluation. The relationship between PCOS and DM has been shown to be a dynamic one. 21 Insulin levels can be expected to rise as resistance develops, and the patient then moves from a normoglycaemic state towards IGT and eventually develops DM. Such a phenomenon was found in this study: serum insulin levels were lowest in normoglycaemic women, higher in those with IGT and highest in those with DM. The most severe insulin resistance occurs when DM develops and the pancreas fails to produce insulin, leading to a fall in serum insulin levels. Studies on the pathophysiology of PCOS have been extended to a search for a genetic basis for this syndrome, and review articles have attempted to correlate data from many studies with the different components of the syndrome. 22,23 Early follicular development in women with PCOS has been shown to be different from that in women with normal ovaries. 24 The density of the small pre-antral follicles has been demonstrated to increase progressively, and a reciprocal decline in mature follicles occurs as the ovarian structure changes from normal to polycystic with ovulation and then to polycystic with anovulation. It has been suggested that differential colonisation of the fetal ovary by the germ cells or a reduction in the loss of oocytes during late gestation, childhood and puberty may account for these findings. An increased serum insulin response to an oral glucose load and increased ovarian cytochrome P450c17a activity, leading to a chain of events stimulating ovarian androgen production, have been demonstrated in obese women with PCOS. 25 A reduction of the insulin response with metformin treatment was associated with blunting of P450c17a activity. Metformin may be beneficial in combination with medications used to enhance follicular development. 26 Therapeutic genetic applications are, however, yet to materialise. Treatment for PCOS is still therefore based on current knowledge of lifestyle changes and endocrine manipulation. The results indicate that traditional clinical features and other endocrine assessments help with the diagnosis of PCOS, but are poor indicators of the need for treatment with insulin-sensitising agents. This analysis has identified a subcategory of normoglycaemic women with PCOS who have high insulin levels (group C) and would therefore, in addition to those with DM and IGT, benefit from antiinsulin therapy. On the basis of the results of this study, treatment with insulin-sensitising agents is unjustified in normoglycaemic women with low insulin levels (group A). The use of anti-insulin therapy to treat anovulation in normoglycaemic women with intermediate serum insulin levels (group B) seems justified, although this needs further evaluation. To make these observations, therapeutically valid clinical trials using insulin-sensitisation medications should be performed in all these groups of infertile women with PCOS. Disclosure of interests The authors declare that they do not have any vested financial, pharmaceutical or other interest in relation to the participants in this study or to the materials used. Contribution to authorship The contributions of individual authors to this paper were as follows. Harshalal Rukka Seneviratne (principal author and for all correspondence): 1 Conception and design, acquisition of data, analysis and interpretation of data, development of the hypothesis and research plan, establishment of methodology and management of patients with PCOS. 2 Drafting of the manuscript and critical revision of the manuscript for intellectual content. Dinuka Lankeshwara (co-author): 1 Acquisition of data, analysis and interpretation of data, record keeping and compiling of data. 2 Assistance with writing and revising the manuscript. 3 Final approval of version to be published. Sumedha Wijeratne (co-author): 1 Acquisition of data, analysis and interpretation of data, and total responsibility for all laboratory procedures. 2 Assistance with writing and revising the manuscript. ª 2009 The Authors Journal compilation ª RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology 1727

7 Seneviratne et al. Noel Somasunderam (co-author): 1 Acquisition of data, analysis and interpretation of data, and joint management of the patients with PCOS. 2 Assistance with writing and revising the manuscript. Dinendra Athukorale (co-author): 1 Analysis and interpretation of data, record keeping and compiling of data, and assistance with maintaining the database. 2 Assistance with writing the manuscript, particularly with preparing the figures and tables. Details of ethics approval Ethical approval was obtained from the Ethics Committee of the Faculty of Medicine of the University of Colombo (ethical approval granted on 11 May 2006; Reference No: EC/06/043). Funding For this work, the results of patient investigations stored in the database of the institution were analysed. Maintenance of the database, analysis of data and computing were carried out by the co-authors and no significant expenses were incurred. No funding was obtained from any agency. Acknowledgements The assistance provided by Dr Udul Hewage, Endocrinologist, in the clinical management of participants and Professor Lalani Rajapakse, Professor in Community Medicine, with the statistical analysis is acknowledged with thanks. j References 1 Stein IF, Leventhal ML. Amenorrhea associated with bilateral polycystic ovaries. Am J Obstet Gynaecol 1935;29: Lewis V Polycystic ovary syndrome: a diagnostic challenge. In: Guzick DS editor. Polycystic Ovary Syndrome, Obstet Gynaecol Clin North Am 2001; 28: Goldzieher JW, Young RL. Selected aspects of polycystic ovarian disease. In: Reproductive Endocrinology, Endocrinol Metab Clin North Am 1992; 21: Rotterdam ESHRE/ASRM-sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod 2004;19: Solomon CG. The epidemiology of polycystic ovary syndrome. In: Dunaif A, editor. 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Increased risk of non-insulin dependant diabetes mellitus, arterial hypertension and coronary artery disease in perimenopausal women with a history of the polycystic ovary syndrome. Hum Reprod 2000;15: Strauss JF. Review: some new thoughts on the pathophysiology and genetics of polycystic ovary syndrome. Ann NY Acad Sci 2003;997: Diamanti-Kandarakis E, Piperi C. Review: genetics of polycystic ovary syndrome. Hum Reprod Update 2005;11: Webber LJ, Stubbs S, Stark J, Trew GH, Margara R, Hardy K, et al. Formation and early development of follicles in the polycystic ovary. The Lancet 2003;362: Nestler JE, Jakubowicz DJ. Decreases in ovarian cytochrome P450c17a serum free testosterone after reduction of insulin secretion in polycystic ovary syndrome. N Engl J Med 1996;335: Legro RS, Barnhart HX, Schlaff WD, Carr BR, Diamond MP, Carson SA, et al. Clomiphene, metformin, or both for infertility in polycystic ovary syndrome. N Engl J Med 2007;356: ª 2009 The Authors Journal compilation ª RCOG 2009 BJOG An International Journal of Obstetrics and Gynaecology