ORIGINAL ARTICLE Reproductive endocrinology. Submitted on February 15, 2013; resubmitted on May 15, 2013; accepted on May 20, 2013

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1 Human Reproduction, Vol.28, No.9 pp , 2013 Advanced Access publication on June 11, 2013 doi: /humrep/det255 ORIGINAL ARTICLE Reproductive endocrinology Assessment of glucose metabolism in polycystic ovary syndrome: HbA1c or fasting glucose compared with the oral glucose tolerance test as a screening method E. Lerchbaum 1, *, V. Schwetz 1, A. Giuliani 2, and B. Obermayer-Pietsch 1 1 Division of Endocrinology and Metabolism, Department of Internal Medicine, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria 2 Department of Gynaecology and Obstetrics, Medical University of Graz, 8036 Graz, Austria *Correspondence address. Tel: ; Fax: ; elisabeth.lerchbaum@medunigraz.at Submitted on February 15, 2013; resubmitted on May 15, 2013; accepted on May 20, 2013 studyquestion: Are HbA1c and fasting glucose (FG) useful in predicting the presence of prediabetes and type 2 diabetes (T2DM) in a large cohort of women with polycystic ovary syndrome (PCOS)? summaryanswer: HbA1c and FGarenot suitable as screening tools for prediabetes in a large cohort of PCOS women but do showa good level of agreement with T2DM. what is known already: Women with PCOS have an increased risk of prediabetes and T2DM. As performing an oral glucose tolerance test (OGTT) is time consuming, HbA1c and FG have been suggested as screening tools for prediabetes and T2DM. study design, size, duration: This was a cross-sectional study of 671 women with PCOS conducted from 2006 to participants/materials, setting, methods: The study was carried out at the endocrinological outpatient department of the Medical University of Graz, Austria. We performed 75 g 2-h OGTTs and measured HbA1c in 671 women with PCOS aged years with a median BMI of 24.2 ( ) kg/m 2. PCOS was defined according to the Rotterdam criteria. Prediabetes (FG mg/dl and/or 2-h glucose mg/dl and/or HbA1c %) and T2DM (FG 126 mg/dl and/or 2-h glucose 200 mg/dl and/or HbA1c 6.5%) were diagnosed according to the American Diabetes Association (ADA) criteria. Levels of agreement between different definitions were analyzed using k-index. main results and the role of chance: According to the ADA criteria, we found prediabetes and T2DM in 12.8% (n ¼ 76) and 1.5% (n ¼ 9) of PCOS women, respectively. When using elevated HbA1c ( %) for defining prediabetes, 19 (3.2%) of all PCOS women had prediabetes with a k-index of When using elevated FG ( mg/dl) for defining prediabetes, 31 (5.2%) of all the PCOS women were diagnosed with prediabetes with a k-index of Further, elevated HbA1c ( 6.5% defining T2DM) was found in six (0.9%) PCOS women (k-index 0.80), and elevated FG ( 126 mg/dl diagnosing T2DM) was found in seven PCOS women (1%; k-index 0.82). limitations, reasons for caution: Our results are limited to an Austrian cohort of PCOS women diagnosed by Rotterdam criteria with a median BMI in the normal weight range. wider implications of the findings: Our results are in line with results from previous smaller PCOS cohorts. Our findings do not support the recommendation that FG or HbA1c can be used for the screening of prediabetes in women with PCOS. For such women, OGTT should be performed for screening of prediabetes. Whether this finding is generalizable to other cohorts remains to be determined in further studies. study funding/competing interest(s): The authors declare no study funding and no competing interests. trial registration number: Not applicable. Key words: polycystic ovary syndrome / oral glucose tolerance test / prediabetes / type 2 diabetes / HbA1c & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oup.com

2 2538 Lerchbaum et al. Introduction Polycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders with a prevalence of 20% when the Rotterdam criteria (Yildiz et al., 2012) are used. PCOS is characterized by increased ovarian and adrenal androgen secretion, hyperandrogenic symptoms, menstrual irregularity and polycystic ovaries (Ehrmann, 2005). Although the pathophysiology of PCOS is not fully understood, insulin resistance and obesity, central obesity in particular, seem to play a key role in the development of PCOS (Gambineri et al., 2002; Ehrmann, 2005). The tendency to accumulate body fat in intra-abdominal fat stores, which is frequently observed in PCOS, is linked to an increased risk of type 2 diabetes (T2DM) and cardiovascular disease (Williams, 2004). Because insulin resistance and central obesity are associated with an increased risk of prediabetes and T2DM (Wehr et al., 2011; Lerchbaum et al., 2013), screening of all PCOS women using a 2-h oral glucose tolerance test (OGTT) was recommended by the Androgen Excess Society (AES) in 2007 (Salley et al., 2007). A more recent consensus statement by the AES (2010) recommends the performance of a 2-h OGTT only in obese (BMI. 30 kg/m 2 ) or in lean/overweight PCOS women with at least one additional risk factor such as a personal history of gestational diabetes (GDM), a family history of T2DM or advanced age (.40 years; Wild et al., 2010). This approach might seem reasonable due to the fact that an OGTT is time-consuming and difficult to perform in all affected women considering the high prevalence of PCOS. Recently, the American Diabetes Association (ADA) suggested that hemoglobin A1c (HbA1C) and fasting glucose (FG) are as appropriate as a 75 g 2-h OGTT to screen for T2DM or prediabetes (American Diabetes Association, 2013). However, FG has been classified as an insufficient screening tool in a recent systematic review on previous small studies in PCOS women (Tomlinson et al., 2010). HbA1c might be considered a screening tool due to its advantages over FG and OGTT such as greater convenience (fasting is not required) and less day-to-day variability during periods of stress or illness. Recently, Celik et al. (2013) investigated a cohort including 252 Turkish women with PCOS and reported that HbA1c is not a reliable screening tool in PCOS women for detecting prediabetes. To our knowledge, to date there is no study of the utility of HbA1c or FG versus OGTT in detecting prediabetes and T2DM in central European cohorts. Thus, we aimed to examine the utility of HbA1c and FG in estimating the risk of prediabetes and T2DM in a large cohort including 671 PCOS women from Austria diagnosed with the Rotterdam criteria. Further, we evaluated the prevalence of prediabetes and T2DM in PCOS women according to BMI and the prevalence of additional risk factors as suggested by the AES in 2010 (Wild et al., 2010). Materials and Methods Subjects The study cohort consisted of 671 women with PCOS, aged years who were routinely referred to our outpatient clinic for PCOS evaluation from 2006 to We included consecutive patients who fulfilled all of the inclusion criteria and none of the study exclusion criteria. Written informed consent was obtained from each woman before study entry. The women with PCOS consulted our outpatient clinic for PCOS-related symptoms such as hirsutism, acne, obesity, infertility or menstrual irregularities. PCOS was diagnosed using the Rotterdam criteria (The Rotterdam ESHRE/ASRM-Sponsored consensus workshop group, 2004) in order to obtain a large and broad cohort of PCOS women. Two out of the following three characteristics are required to confirm the diagnosis: clinical and/or biochemical signs of hyperandrogenism, oligo- and/or anovulation, polycystic ovaries (by ultrasound). Hyperandrogenism was defined by the clinical presence of hirsutism (modified Ferriman Gallwey score 6), acne or alopecia and/or elevated androgen levels (normal range of total testosterone:,0.77 ng/ml, free testosterone:,0.013 ng/ml and DHEAS:,2.75 mg/ ml). Oligo- and/or anovulation were defined by the presence of oligomenorrhea or amenorrhea. Polycystic ovarian morphology was examined by ultrasound. Polycystic ovaries were defined as the presence of 12 or more follicles in each ovary measuring 2 9 mm in diameter and/or increased ovarian volume (.10 ml; calculated using the formula 0.5 length width thickness; The Rotterdam ESHRE/ASRM-Sponsored consensus workshop group, 2004). Hyperprolactinemia, Cushing s syndrome, congenital adrenal hyperplasia and androgen-secreting tumors were excluded by specific laboratory analyses [prolactin, cortisol, adrenocorticotropic hormone (ACTH), 17alpha-OH progesterone]. PCOS women did not take any medication known to affect endocrine parameters, carbohydrate metabolism or serum lipid profile for at least 3 months before entering the study. Ethical approval The study protocol was approved by the ethics committee of the Medical University of Graz and written informed consent was obtained from each patient. Procedures Standard anthropometric data [height, weight, waist circumference (WC), hip circumference (HC), blood pressure] were obtained from each subject. WC was measured in a standing position midway between the lower costal margin and the iliac crest. HC was measured in a standing position at the maximum circumference over the buttocks. The BMI was calculated as the weight in kilograms divided by the square of the height in meters. Hirsutism was quantified with the modified Ferriman Gallwey score. Moreover, basal blood samples for assays of hormones [total testosterone, sex hormone-binding globulin (SHBG), androstenedione, DHEAS, ft3, ft4, TSH, 17alpha-OH progesterone, prolactin, cortisol, ACTH], glucose, HbA1c and serum lipids were collected between 8.00 and 9.00 a.m. after an overnight fast. Assays were performed in the same laboratory as where they came in and laboratory kits and the assays did not change between 2006 and Total testosterone and SHBG were measured on a daily basis (assays were run concurrently on the same day) and stored at 48C until analysis. Free testosterone values were calculated from total testosterone, SHBG and albumin according to Vermeulen (1999). The free androgen index (FAI) was calculated as total testosterone (nmol/l)/shbg (nmol/l) * 100. All the participants underwent a fasting 75 g OGTT. Blood samples were drawn after 30, 60 and 120 min for glucose determination. Data on HbA1c were available for 612 PCOS women. Normal weight was defined as BMI, 25 kg/m 2 and overweight/obesity as BMI 25 kg/m 2. Prediabetes and T2DM were defined according to the ADA (American Diabetes Association, 2013) as follows: Prediabetes: FG mg/dl and/or 2-h glucose during 75 g OGTT between 140 mg/ dl-199 mg/dl and/or HbA1c %; T2DM: FG 126 mg/dl and/or 2 h glucose during 75 g OGTT 200 mg/dl and/or HbA1c 6.5%. Further, PCOS women were stratified according to the prevalence of risk factors suggested by the AES 2010 (Wild et al., 2010) into two groups: no risk factors or at least one risk factor (BMI. 30 kg/m 2 and/or personal history of GDM and/or family history of T2DM and/or advanced age.40 years).

3 HbA1c, FG and OGTT as screening methods in PCOS 2539 Biochemical analyses Total testosterone and prolactin, TSH, ft3, ft4 and cortisol (Siemens, Erlangen, Germany) were measured by luminescence immunoassay. Intra- and inter-assay coefficients of variation (CVs) for total testosterone measurements were 6.2 and 4.7%, respectively. SHBG was measured by luminescence immunoassay (Roche, Basel, Switzerland) with an intra- and inter-assay CV of 1.3 and 2.1%, respectively. Albumin was measured by photometric assay (Roche, Vienna, Austria). DHEAS (LDN Labor Diagnostika Nord GmbH, Nordhorn, Germany), androstenedione and 17a-OH progesterone (DiaMetra, BioVendor, Brno, Czech Republic) were measured by ELISA with intra- and inter-assay CVs of,10%. Hypotheses The hypothesis of our study is that HbA1c and FG are as suitable as a 75 g OGTT in estimating the risk of prediabetes and T2DM in PCOS women. Further, we evaluate the prevalence of prediabetes and T2DM in PCOS women according to BMI and the prevalence of additional risk factors as suggested by the AES in 2010 (Wild et al., 2010). Statistical analyses Data are presented as median with interquartile range unless otherwise stated. The distribution of data was analyzed by descriptive statistics and the Kolmogorov Smirnov test. All parameters were found to be nonnormally distributed. The Kruskal Wallis test, Mann Whitney U-test and x 2 test were used for comparisons between groups. To evaluate whether HbA1c and FG are as suitable as OGTT in detecting prediabetes and T2DM, we used k-statistics. Levels of agreement between the different definitions of prediabetes and T2DM were analyzed using sensitivity, specificity and the k-index, which is considered excellent for values.0.80, good for values , moderate for values between 0.41 and 0.60, fair for values between 0.21 and 0.40 and weak for levels All statistical procedures were performed with SPSS version 20 (SPSS Inc., Chicago, IL, USA). A P-value of,0.05 was considered statistically significant. Results The baseline characteristics of PCOS women according to their glucose tolerance are presented in Table I. As expected, women with prediabetes or T2DM had a higher prevalence of (central) obesity, higher blood pressure and an adverse lipid profile compared with PCOS women with normal glucose tolerance. Further, women with T2DM had significantly higher FAI and free testosterone levels and lower SHBG levels than women with normal glucose tolerance or prediabetes. Testosterone levels were significantly higher in women with T2DM than in women with normal glucose tolerance, and SHBG levels were significantly lower in women with prediabetes or T2DM than in women with normal glucose tolerance. Overweight/obesity was found in 55.6% of PCOS women. Data on the prevalence of prediabetes and T2DM according to the ADA criteria (American Diabetes Association, 2013) and elevated HbA1c and FG levels stratified by BMI status as well as hyperandrogenism are shown in Table II. We present data on sensitivity, specificity and level of agreement of prediabetes and T2DM diagnosed by the ADA criteria (elevated FG and/or 2-h glucose and/or HbA1c) and HbA1c elevation alone in Table III. In PCOS women with prevalent T2DM, we found a good level of agreement, whereas the level of agreement of elevated HbA1c in women with prediabetes was poor. We reanalyzed our data comparing elevated HbA1c with elevated FG and/or 2 h glucose for definition of prediabetes and T2DM and found similar results (data not shown). Table IV shows sensitivity, specificity and levels of agreement of elevated FG alone with prediabetes and T2DM. When comparing elevated FG alone with the ADA definition of prediabetes and T2DM, we found again good levels of agreement when diagnosing T2DM and very poor k-indices when analyzing prediabetes. When prediabetes and T2DM were defined by elevated FG and/or 2 h glucose and compared with FG elevation alone, we found a good level of agreement with prediabetes and an excellent k-index for T2DM (data not shown). We found at least one AES risk factor (Wild et al., 2010) in 51.7% of all PCOS women. In women with prediabetes (according to the ADA criteria), 69.6% had at least one risk factor and 30.4% had no risk factor (P ¼ 0.001; k ¼ 0.10).InwomenwithT2DM(accordingtotheADA criteria), 75% had at least one risk factor and 25% had no risk factor (P ¼ 0.183, k ¼ 0.01). Discussion We present evidence that HbA1c and FG are not suitable as screening tools for prediabetes in a large cohort of PCOS women. In contrast, when evaluating T2DM, HbA1c and FG were both suitable screening tools showing a good level of agreement. Further, the performance of OGTT only in PCOS women with obesity or at least one risk factor as suggested by the AES (Wild et al., 2010) would also underestimate the prevalence of prediabetes and T2DM. We want to stress that it is very important to detect a disturbed glucose metabolism at an early stage in these young women in order to start lifestyle intervention and/or metformin treatment as soon as possible in order to prevent the progression to T2DM. HbA1c is a commonly used marker of chronic glycemia, and it reflects the average blood glucose levels over a 2 3-month period. HbA1c has been suggested as a screening tool by the ADA (American Diabetes Association, 2013) due to its advantages over FG and OGTT such as greater convenience (fasting is not required) and less day-to-day variability during periods of stress or illness. Our findings suggest, however, that HbA1c is an inappropriate screening tool in women with PCOS, in particular regarding the detection of prediabetes. Our findings are in line with a previous smaller study of 252 PCOS women from Turkey showing that HbA1c is an insufficient screening tool for women with PCOS (Celik et al., 2013). Our data further suggest that FG is an insufficient screening tool for prediabetes but shows good agreement with T2DM. Previously, it has been shown that FG rather than OGTT underestimates the prevalence of T2DM in PCOS by.50% (Legro et al., 1999). In that study, 3.2% of women were diagnosed with T2DM compared with 7.5% using the OGTT criteria. We have, however, to acknowledge that in our Austrian cohort, T2DM prevalence was relatively low, which might, at least in part, explain those differences. As the majority of PCOS women who are affected by a disturbed glucose metabolism present with prediabetes and not with T2DM, our data suggest that FG is an insufficient screening test, which is in linewith the conclusion of a recent systematic review conducted in 2010 (Tomlinson et al., 2010). One possible explanation for the failure of FG as a screening tool might be the fact that insulin secretory defects have been suggested to be present in PCOS (Holte et al., 1994), and this may explain why

4 Table I Baseline characteristics of PCOS women according to their glucose status according to the ADA definition (normal versus prediabetes versus T2DM). All women (n 5 603) Normal glucose tolerance Prediabetes (n 5 76) T2DM (n 5 9) P-value (Kruskal Wallis (n 5 518) test) Median Interquartile range Median Interquartile range Median Interquartile range Median Interquartile range... Age (years) BMI (kg/m 2 ) * **, ***,0.001 WC (cm) * **, ***,0.001 HC (cm) * **, *** Waist-to-hip ratio * **, ***,0.001 Systolic blood pressure (mmhg) * **, *** Diastolic blood pressure (mmhg) * **,0.001 FG (mg/dl) * **, ***, h glucose (mg/dl) * **, ***,0.001 HbA1c (%) * **, ***,0.001 Total cholesterol (mg/dl) **, *** LDL cholesterol (mg/dl) **, *** Total cholesterol/hdl ratio * **, ***,0.001 HDL (mg/dl) * **,0.001 Triglycerides (mg/dl) * **, ***,0.001 Triglycerides/HDL ratio * **, ***,0.001 Total testosterone (ng/ml) ** SHBG (nmol/l) * **, ***,0.001 Free testosterone (ng/ml) **, *** FAI **, *** 0.06 Biochemical hyperandrogenism a (%) *** Clinical hyperandrogenism (%) *** Menstrual irregularities (%) **, *** Polycystic ovaries (%) Obesity (BMI.30 kg/m 2 ) (%) * 77.8**, *** Family history of T2DM (%) * 44.4 Personal history of GDM (%) * 0 Age.40 years (%) Differences between groups were calculated by the Kruskal Wallis test, Mann Whitney U test or x 2 -test. ADA, American DiabetesAssociation; PCOS, polycysticovarysyndrome; T2DM, type2diabetes; LDL, low-densitylipoproteincholesterol; HDL, high-densitylipoproteincholesterol; SHBG, sexhormone-binding globulin; FAI, freeandrogenindex; GDM, gestational diabetes; FG, fasting glucose; WC, waist circumference; HC, hip circumference. *P, 0.05 for prediabetes versus normal glucose tolerance. ** P, 0.05 for T2DM versus normal glucose tolerance. *** P, 0.05 for prediabetes versus T2DM. a Elevated total testosterone and/or free testosterone and/or DHEAS (dehydroepiandrosterone) Lerchbaum et al. Downloaded from at Pennsylvania State University on February 20, 2016

5 HbA1c, FG and OGTT as screening methods in PCOS 2541 Table II Prevalence of prediabetes and T2DM in PCOS women stratified by BMI and hyperandrogenism. Overweight/ obese 1 hyperandrogenism Clinical hyperandrogenism Biochemical hyperandrogenism Overweight/obese (BMI 25 kg/m 2 ) All women Normal weight (BMI < 25 kg/m 2 )... Prediabetes 76 (12.8%) 24 (7.2%) 52 (19.4%) 32 (12.9%) 43 (12.6%) 35 (20.3%) Elevated HbA1c ( %) 19 (3.2%) 1 (0.3%) 18 (6.7%) 13 (5.2%) 9 (2.6%) 11 (6.4%) Elevated FG ( mg/dl) 31 (5.2%) 12 (3.6%) 19 (7.1%) 15 (6.0%) 23 (6.6%) 17 (9.7%) T2DM 9 (1.5%) 0 9 (3.2%) 6 (2.3%) 5 (1.4%) 6 (4.5%) Elevated HbA1c ( 6.5%) 6 (0.9%) 0 6 (2.1%) 4 (1.5%) 4 (1.1%) 4 (2.9%) Elevated FG ( 126 mg/dl) 7 (1%) 0 7 (2.5%) 4 (1.4%) 5 (1.3%) 4 (2.7%) Prediabetes and T2DM were defined according to the ADA criteria (American Diabetes Association, 2013). Biochemical hyperandrogenism was defined as elevated total testosterone and/or free testosterone and/or DHEAS (dehydroepiandrosterone). T2DM, type 2 diabetes; PCOS, polycystic ovary syndrome; FG, fasting glucose. postprandial glucose rather than FG is more indicative of prediabetes risk. This notion is supported by the fact that several studies report normal FG levels despite elevated postprandial glucose levels in PCOS women (reviewed by Tomlinson et al. (2010)). Further, it has been suggested that HbA1c and glucose levels reflect different aspects of glucose metabolism (Cowie et al., 2010) and the respective contribution of preprandial and postprandial glucose alterations to the HbA1c levels is controversial. Indeed, HbA1c has been shown to underestimate T2DM risk not only in women with PCOS (Celik et al., 2013) but also in non-pcos populations such as NHANES (Cowie et al., 2010). Further, HbA1c has been shown to have low sensitivity when screening women after delivery with previous GDM (Picón et al., 2012). The ADA recommendation suggesting that a 75 g 2-h OGTT, FG or HbA1c can be used for the screening for prediabetes and T2DM (American Diabetes Association, 2013) is in contrast to the AES guidelines from 2007 (Salley et al., 2007). In those PCOS-specific guidelines, performing an OGTT is recommended in all PCOS women, which is highly supported by our data. T2DM is a major risk factor for cardiovascular disease in women (Huxley et al., 2006). As women with PCOS have an increased prevalence of cardiovascular risk factors and probably a consequent high risk of cardiovascular disease (Wild et al., 2010), early detection and treatment of prediabetes is of high importance. Moreover, it has been shown that a high percentage of PCOS women with prediabetes and without adequate treatment will develop T2DM in the following few years (Tomlinson et al., 2010). In women with PCOS, the annual progression rate to T2DM has been found to be 2 3% (Legro et al., 1999; Norman et al., 2001). In 2010, a consensus statement by the AES recommended that a 2-h OGTT should be performed in PCOS women with a BMI of.30 kg/m 2, or alternatively in lean or overweight PCOS women with advanced age (.40 year), a personal history of GDM or a family history of T2DM (Wild et al., 2010). We found a significant difference in the prevalence of obesity, a personal history of GDM and in family history of T2DM between women with normal glucose tolerance and prediabetes or T2DM and no significant difference regarding age. Nevertheless, in our study, 30.4% of PCOS women with prediabetes and 25.0% of PCOS women with T2DM had none of the suggested risk factors. Thus, screening only women with at least one risk factor as suggested by the AES 2010 (Wild et al., 2010) would also lead to an underestimation of the prevalence of prediabetes and T2DM. Interestingly, we found significantly higher FAI and free testosterone levels in PCOS women with T2DM compared with PCOS women with prediabetes or normal glucose tolerance. Nevertheless, our results suggest that overweight/obesity seems to be a more important risk factor of prediabetes and T2DM than hyperandrogenism. This notion is supported by the fact that the prevalence of prediabetes and T2DM was similar in women with and without hyperandrogenism. Our data are of high clinical importance as strategies such as lifestyle intervention and metformin use exist to prevent T2DM (Knowler et al., 2002; Tomlinson et al., 2010). The ADA recommends a weight loss of 7% and increasing physical activity to at least 150 min/week of moderate activity (American Diabetes Association, 2013). Lifestyle changes and treatment with metformin both reduced the incidence of T2DM in persons at high risk by 58 and 31%, respectively (Knowler et al., 2002). Although long-term studies of the reduction of T2DM incidence in PCOS by weight reduction are missing, previous short-term

6 2542 Lerchbaum et al. Table III Sensitivity, specificity and level of agreement for prediabetes (elevated FG and/or elevated 2-h glucose and/or elevated HbA1c) and T2DM (elevated FG and/or elevated 2 h glucose and/or elevated HbA1c) with HbA1c elevation alone in PCOS women. Prediabetes... Yes No Sensitivity (%) Specificity (%) k-index P-value... All PCOS HbA1c % Yes ,0.001 No BMI, 25 kg/m 2 HbA1c % Yes ,0.001 No BMI 25 kg/m 2 HbA1c % Yes ,0.001 No T2DM All PCOS women HbA1c 6.5% Yes ,0.001 No Data available for 612 PCOS women. Levels of agreement were analyzed using sensitivity, specificity and the k-index. P-values are given for k-statistics. FG, fasting glucose; T2DM, type 2 diabetes; PCOS, polycystic ovary syndrome. studies suggest that hypocaloric diets and exercise improve parameters of metabolic syndrome (Thomson et al., 2008). Obesity, and central obesity in particular, suggesting intra-abdominal fat accumulation is associated with an increased risk of prediabetes and T2DM (Wehr et al., 2011; Lerchbaum et al., 2013). These findings are supported by our data showing a higher prevalence of prediabetes in overweight/obese women compared with normal weight women and that none of the normal weight had T2DM. Of note, we found prediabetes in 7% of normal weight PCOS women. According to the ADA guidelines, in asymptomatic adults, diabetes screening should be performed only in PCOS women with BMI 25 kg/m 2. As we found prediabetes in 7% of normal weight PCOS women, in those women, their prediabetes would remain undiagnosed. A previous study among specialists in obstetrics and gynecology and in reproductive endocrinology and infertility suggests that 57 71% of PCOS women are screened for T2DM; OGTT was the most common test used for screening (68%), while 8 22% used FG and 19 20% used HbA1c (Abdel-Rahman et al., 2012). Screening frequency in other specialities might be even lower. Considering the high prevalence of prediabetes and T2DM in women with PCOS and the poor performance of FG and HbA1c as well as the risk factors suggested by the AES in 2010 (Wild et al., 2010), the AES 2007 recommendation (Salley et al., 2007) to perform an OGTT in all PCOS women might be considered the best approach. So far, there is no consensus regarding rescreening of PCOS women for glucose disturbances. Some authors (Tomlinson et al., 2010) suggested performing annual OGTTs in women with PCOS and at least one additional risk factor, which might be a reasonable approach. In contrast, the AES suggests that PCOS women with normal glucose tolerance should be rescreened every 2 years or sooner if additional risk factors are identified and those with prediabetes should be screened annually for developing T2DM (Salley et al., 2007; Wild et al., 2010). Our study has limitations that should be noted. First, our findings are restricted to a cohort of young and lean PCOS women and might not be generalizable to other cohorts including older or more obese women. As PCOS was diagnosed according to the Rotterdam criteria, our findings might differ from other PCOS cohorts diagnosed by different criteria (AES or NIH). Of note, the efficacy of screening tools might be different in PCOS cohorts diagnosed by AES or NIH criteria. Further, HbA1c levels were not available for all women. As we have no follow-up data on PCOS women, we cannot answer the question how many PCOS women with currently normal glucose tolerance will develop prediabetes or T2DM in the following years and which risk factors will predict this conversion. The strength of our study is the large sample size and the performance of OGTT in all women. In conclusion, we present evidence from a large cohort of PCOS women that HbA1c and FG are insufficient screening tools for prediabetes. Further, performing OGTT only in PCOS women with risk factors as suggested by the AES in 2010 was not sufficient in our large PCOS cohort. Our data strongly suggest that OGTTs should be

7 HbA1c, FG and OGTT as screening methods in PCOS 2543 Table IV Sensitivity, specificity and level of agreement for prediabetes (elevated FG and/or elevated 2-h glucose and/or elevated HbA1c) and T2DM (elevated FG and/or elevated 2 h glucose and/or elevated HbA1c) with FG elevation alone in PCOS women. Prediabetes... Yes No Sensitivity (%) Specificity (%) k-index P-value... All PCOS women FG mg/dl Yes ,0.001 No BMI,25 kg/m 2 FG mg/dl Yes ,0.001 No BMI 25 kg/m 2 FG mg/dl Yes ,0.001 No T2DM All PCOS women FG 126 mg/dl Yes ,0.001 No Data available for 612 PCOS women. Levels of agreement were analyzed using sensitivity, specificity and the k-index. P-values are given for k-statistics. FG, fasting glucose; T2DM, type 2 diabetes; PCOS, polycystic ovary syndrome. performed in all women with PCOS, which is in line with the 2007 AES recommendation. Acknowledgements The authors have nothing to acknowledge. Authors roles E.L. contributed to study conception and design, acquisition, analysis and interpretation of data and drafting of the article. V.S. contributed to acquisition of data and revising the article critically for important intellectual content. A.G. contributed to study conception, acquisition of data and revising the article critically for important intellectual content. B.O.-P. contributed to study conception and design and revising the article for important intellectual content. All the authors approved the final version of the manuscript. Funding The authors declare no funding. Conflict of interest The authors declare no conflict of interest. References Abdel-Rahman MY, Jackson L, Rodewald KJ, Abdellah MA, Ismail SA, Hurd WW. Polycystic ovary syndrome and diabetes screening: a survey of gynecologists and reproductive endocrinologists. Eur J Obstet Gynecol Reprod Biol 2012;162: American Diabetes Association. Standards of medical care in diabetes Diabetes Care 2013;36:S11 S66. Celik C, Abali R, Bastu E, Tasdemir N, Tasdemir UG, Gul A. Assessment of impaired glucose tolerance prevalence with hemoglobin A1c and oral glucose tolerance test in 252 Turkish women with polycystic ovary syndrome: a prospective, controlled study. Hum Reprod 2013; 28: CowieCC, Rust K, Byrd-Holt DD, GreggEW, Ford ES, Geiss LS, Bainbridge KE, Fradkin JE. Prevalence of diabetes andhigh risk for diabetesusinga1c criteria in the U.S. population in Diabetes Care 2010;33: Ehrmann DA. Polycystic ovary syndrome. N Engl J Med 2005; 352: Gambineri A, Pelusi C, Vicennati V, Pagotto U, Pasquali R. Obesity and the polycystic ovary syndrome. Int J Obes Relat Metab Disord 2002;26: Holte J, BerghT, Berne C, BerglundL, Lithell H. Enhanced early insulin response to glucose in relation to insulin resistance in women with polycystic ovary syndrome and normal glucose tolerance. J Clin Endocrinol Metab 1994; 78: Huxley R, Barzi F, Woodward M. Excess risk of fatal coronary heart disease associated with diabetes in men and women: meta-analysis of 37 prospective cohort studies. Br Med J 2006;332:73 78.

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