Clinical, ultrasound and biochemical features of polycystic ovary syndrome in adolescents: implications for diagnosis

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1 Human Reproduction, Vol.26, No.6 pp , 2011 Advanced Access publication on April 8, 2011 doi: /humrep/der102 ORIGINAL ARTICLE Reproductive endocrinology Clinical, ultrasound and biochemical features of polycystic ovary syndrome in adolescents: implications for diagnosis M. Hickey 1, *, D.A. Doherty 2, H. Atkinson 2, D.M. Sloboda 3, S. Franks 4, R.J. Norman 5, and R. Hart 2 1 Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, VIC, Australia 2 School of Women s and Infants Health, and Women s and Infants Research Foundation, University of Western Australia, Perth, Australia 3 Liggins Institute, University of Auckland, and the National Research Centre for Growth and Development, Auckland, New Zealand 4 Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Hospital, London, UK 5 Robinson Institute, Research Centre for Reproductive Health, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia *Correspondence address. Tel: ; Fax: ; . martha.hickey@thewomens.org.au Submitted on October 28, 2010; resubmitted on February 25, 2011; accepted on March 8, 2011 background: Diagnosing polycystic ovary syndrome (PCOS) in adolescence is clinically challenging. The prevalence of clinical, ultrasound and biochemical features of PCOS in a community-based adolescent population using current diagnostic criteria has not previously been described. methods: This was a prospective cohort study with 244 unselected post-menarchal girls, mean age 15.2 years, of whom 91% were Caucasian. Subjects were recruited from a large population-based birth cohort (the Raine cohort). Clinical hyperandrogenism (HA) was quantified using Ferriman Gallwey scores. In the early follicular phase (Day 2 6), we measured circulating androgens and sex hormonebinding globulin by immunoassay, and ovarian morphology was assessed by transabdominal ultrasound examination. BMI and waist hip ratio were measured. results: Normal ranges for early follicular phase androgens in adolescence were derived for this population. The top 5 and 10% of circulating free testosterone levels were 45.6 and 34.5 pmol/l, respectively. Fifty-one percent of girls reported menstrual irregularity. Clinical HA was uncommon, being observed in only 3.5% of girls. Mean ovarian volume was greater than that reported by others in adult women and 35% of girls had polycystic ovary morphology on transabdominal ultrasound. Taking the upper 5% of free testosterone as HA, 42 girls (18.5%) would have met the Rotterdam criteria for PCOS, 11 girls (5%) the Androgen Excess Society criteria and 7 girls (3.1%) the National Institutes of Health criteria. conclusions: Menstrual irregularity is common in adolescence and does not relate to clinical or biochemical HA. Diagnostic criteria for PCOS which include ovarian volume and morphology may be of limited use in adolescence. Key words: polycystic ovary syndrome / hyperandrogenism / adolescence / diagnosis Introduction Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in adult women, affecting around 6 8% of women (Azziz et al., 2004; March, 2010). Whilst the symptoms of PCOS are commonly present in young women, the prevalence of features of PCOS in community-based adolescents has not been reported. This is of clinical significance because the diagnosis of PCOS presents a challenge in adolescence and early diagnosis may facilitate interventions to improve long-term health (Palmert et al., 2002; Ibanez et al., 2004). Since the diagnostic label of PCOS implies an increased risk for infertility, dysfunctional uterine bleeding, endometrial cancer, obesity, type II diabetes, dyslipidemia, hypertension and possibly cardiovascular disease, and may negatively affect access to healthcare coverage, diagnostic accuracy is clinically, socially and financially important. There is ongoing controversy regarding the most appropriate diagnostic criteria for PCOS in adults. Three sets of criteria have been & 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 1470 Hickey et al. proposed: the so-called National Institutes of Health (NIH) criteria [oligo-anovulation and biochemical or clinical hyperandrogenism (HA)] (Zawadzki and Dunaif, 1995) and the Rotterdam consensus criteria (two out of three of oligo-anovulation, clinical and/or biochemical HA or polycystic ovary appearance on ultrasound) (Group, 2004). In 2009, the Androgen Excess Society (AES) launched a new definition that considers PCOS primarily as a disorder of clinical and/or biochemical androgen excess, plus either chronic oligo-anovulation and/or polycystic ovaries (Azziz et al., 2009). All agree that other related endocrine disorders should be excluded. However, none of these diagnostic criteria can confidently be used in adolescence (Khan, 2007). Menstrual irregularity is common in the early years after menarche and oligo-anovulation may be normal (Apter and Vihko 1985). Clinical HA may be less common in younger women (Rosenfield et al., 2000). Biochemical HA is the most consistent biochemical abnormality in PCOS but there are no widely accepted normal values for adolescence (Azziz et al., 2009). Further, laboratory assays used to measure testosterone concentrations vary widely between laboratories. Compounding these limitations is the paucity of normative data for testosterone concentrations from populationbased adolescents (Khan, 2007). Ovarian appearance in the early post-menarchal years may differ from that seen in adult women. It is not well defined how a multifollicular appearance may differ from polycystic ovary (PCO) morphology (Holm et al., 1985). Further, assessment of ovarian morphology is limited to the transabdominal approach when girls are not sexually active. Hence, clinicians are increasingly faced with young women showing potential features of PCOS (Franks, 2008), with little guidance on how these features can be discriminated from the normal developmental process (Blank et al., 2008). No previous published studies have reported the prevalence of clinical, biochemical and ultrasound features of PCOS in a large, prospective, community-based, predominantly Caucasian adolescent population using current diagnostic criteria. The aim of this study was to evaluate the prevalence of clinical and biochemical features of PCOS in post-menarchal girls aged years. Further, we aimed to determine the normal range for circulating androgen levels in the early follicular phase. Materials and Methods Subjects Two hundred and forty-four unselected post-menarchal girls aged years from a large population-based birth cohort ( were recruited by advertising within the cohort. Girls in the cohort were included if they were at least 6 months post-menarchal were in good general health and were not taking any sex steroid hormones or other medication likely to interfere with pituitary, ovarian or endometrial function. Participants were part of a parallel study of prenatal factors impacting ovarian function (Hickey et al., 2009; Sloboda et al., 2009; Hart et al., 2010). Age at menarche was prospectively ascertained. This study was approved by the Raine Executive Committee and the ethics committees of King Edward Memorial Hospital and Princess Margaret Hospital. Subjects and their accompanying parent or guardian provided written consent. Examination and investigations were scheduled for Day 2 6 of the menstrual cycle by asking the girls to telephone the research nurse to book their visit on the first day of bleeding. This ensured that subjects with regular and irregular cycles were sampled during the early follicular phase. All visits were timed between 15:30 and 16:30 h to account for diurnal variations in androgen production (Davison and Bell, 2006) and fit in with school commitments. At this visit, age, personal and family medical history were recorded, and subject height, weight, waist circumference, hip circumference and blood pressure were measured. Subjects were asked to report their menstrual pattern using standardized definitions of regular and irregular menstruation (Treloar, 1981) and also completed a menstrual diary for the next 90 days. Clinical HA was evaluated by a trained nurse using the Ferriman Gallwey (F-G) scoring system (Ferriman and Galwey, 1961). Acne was measured using a validated scale and classed as mild, moderate or severe (Thiboutot et al., 2001). Assessment of ovarian morphology Ovarian volume and follicle count were evaluated using transabdominal ultrasound with a confirmed full bladder. All ovarian ultrasounds were performed during the early follicular phase (Days 2 6 of the menstrual cycle) and by one of two experienced gynecological ultrasonographers. At ultrasound ovarian and uterine volume and the number and diameter of ovarian follicles were recorded (Sloboda et al., 2009). All images were reviewed by one independent expert gynecological ultrasonographer. If a follicle.10 mm was seen, the ultrasound was repeated in the early follicular phase of the next cycle. PCO morphology was determined according to standardized international criteria for adult women as 1 or more ovaries with a volume.10 cm 3 or 12 or more follicles between 2 and 9 mm diameter (Balen et al., 2003). Biochemical measurements Plasma concentrations of sex hormone-binding globulin (SHBG) and androgen [total testosterone, androstenedione and dihydroepiandrosteronedione (DHEAS)] were measured during the early follicular phase in 226 girls. Total testosterone was measured using a commercially available double antibody radioimmunoassay previously validated and adapted to improve sensitivity by using larger sample volume or longer incubation (DSL-4100, Beckman, Australia) and was shown to have adequate sensitivity for this population (University of Adelaide; Wei et al., 2009). The lower limit of sensitivity was 347 pmol/l and the inter-assay and interpatient coefficients of variation were 6 and 15%, respectively, at the 1 nmol/l concentration. SHBG concentrations were determined using a non-competitive liquid-phase radioimmunometric assay (68562, Orion Diagnostica, Espoo, Finland). Concentrations of DHEAS (DSL-2700, Beckman, Australia) and androstenedione (DSL-4200, Beckman, Australia) were determined using commercial radioimmunoassay. Circulating free testosterone (cft) concentrations were calculated from the measured total testosterone and SHBG concentrations using the Vermeulen equation (Vermeulen et al., 1999) assuming a standard albumin concentration using the calculator made available by one of the authors ( Concentrations of prolactin and thyroid-stimulating hormone (TSH) were determined using commercially available enzyme-linked immunosorbent assays from DSL labs (Beckman, Australia). Diagnosis of PCOS Menstrual irregularity, clinical or biochemical HA and PCO morphology on ultrasound were recorded. Rotterdam criteria for PCOS were considered to be met if subjects had two out of three criteria; PCO morphology, clinical or biochemical HA; or oligo-anovulation (Group, 2004). NIH criteria for PCOS were met if subjects had menstrual cycles that were oligo-anovulatory (as defined below), plus either clinical or biochemical HA (as defined below) (Zawadzki and Dunaif, 1995). AES criteria were met if subjects displayed clinical and/or biochemical androgen excess,

3 Feature of polycystic ovary syndrome in adolescence 1471 plus either chronic oligo-anovulation and/or polycystic ovaries (Azziz et al., 2009). (1) PCO morphology: PCO morphology was defined as 1 ovary with a volume.10 cm 3 or 12 or more follicles between 2 and 9 mm diameter (Balen et al., 2003). (2) Hyperandrogenism: Clinical HA was defined by a F-G score of 8 (Ferriman and Galwey, 1961; van Hooff et al., 1999a,b; Azziz et al., 2004). Biochemical HA was defined as the top fifth percentile for calculated early follicular phase cft levels (Huang et al., 2010). (3) Oligo-anovulation: Ovulation was determined using menstrual cycle regularity, prospectively measured using a purpose-designed menstrual diary over 90 days. Irregular cycles were defined as those,21 or.35 days in duration or where the cycle length varied from month to month by.4 days (Treloar, 1981). Other causes of oligo-anovulation were excluded by measuring TSH and prolactin concentrations. Acne The presence and severity of acne was measured in 226 girls using standardized criteria (Thiboutot et al., 2001). Statistical methods Continuous data were summarized using mean and SD or median and interquartile range according to data normality. Categorical data were summarized using frequency distributions. Mann Whitney, t-tests and x 2 tests were used for univariate group comparisons, as appropriate. Bivariate correlation coefficients were used to examine relationships between androgens. Logistic regression analysis was used to examine the effects of BMI, categorized as overweight or obese, on the likelihood of presence of PCO morphology. Linear regression with log-transformed ovarian volume as the outcome was used to estimate the mean ovarian volume in our population. All hypothesis tests were two-sided and P-values of,0.05 were considered statistically significant. The Statistical Package for the Social Sciences statistical software (version 15.0, SPSS Inc., Chicago, IL, USA) was used for data analysis. Results Of 723 girls in the Raine cohort who were invited to participate in this study, a total of 244 consented. Twelve were excluded because of oral contraceptive pill (OCP) use and data from 232 girls were available for analysis (see Fig. 1). Of these 232 girls, 211 were Caucasian (90.9%), 16 were Asian (6.9%) 2 were Maori/Polynesian (0.8%, 1 each Maori and 1 Polynesian) and 1 was Anglo-Indian (0.4%). In addition, one girl was Chilean (0.4%) and one other was undisclosed (0.4%). Differences between recruited and non-recruited adolescents Subject characteristics are shown in Table I. Mean age at assessment was 15.2 (SD 0.48) years and mean age at menarche was 12.5 (SD 1.2) years, consistent with recent published studies in similar populations (Sloboda et al., 2007). All subjects were post-menarchal but recruited girls had a younger mean age at menarche (12.5 years, SD ¼ 1.2, range: years) compared with non-recruited girls from the entire Raine cohort (mean 12.9 years, SD ¼ 1.1, range: years) P, Mean time since menarche was 32 months (SD 15 months and range: 2 90 months). Two hundred and twenty-four girls (96%) were from singleton pregnancies. The great majority (206, 91.6%) were Caucasian. Mean birthweight was 3278 g (SD 601 g) corresponding to a mean z-score of 0.15 (SD 1.0) standardized for gestational age at delivery, nulliparity and gender. Mean birth length was cm (SD 2.83 cm) and mean head circumference was cm (SD 1.66 cm). Mean ponderal index at birth was 2.8 (SD 0.33) and ranged from 1.95 to No relationship between age at menarche and birthweight was seen in this study (r ¼ 0.039, P ¼ 0.56). There were no differences in age at menarche between those of average gestational age, small gestational age (SGA) or large gestational age at birth (P ¼ 0.95) or between SGA and all other birthweights (P ¼ 0.99). Menstrual irregularity Menstrual regularity was determined prospectively using menstrual diaries (69/130, 53%) or by self-report (Treloar, 1981) (51/102, 50%). Ninety-six girls (51.7%) reported menstrual irregularity at recruitment. Ten girls reported absent menses for.60 days (8%) including one girl with menses absent for.90 days. All thyroid function and prolactin levels were within the normal range. Clinical HA Hirsutism. The prevalence of hirsutism was very low, with 92% of F-G scores,5 (210/229). One hundred and thirty-four girls (57.8%) had an F-G score of 0, 76 girls (32.8%) had F-G scores of 1 4 and 19 girls (8.2%) had F-G score of 5. A significant correlation between free testosterone levels and higher FG score was observed (r ¼ 0.16, P ¼ 0.017). Acne. Mild acne was recorded in 109/226 (48%) and moderate to severe acne in 48 (21%). Approximately one-third of girls evaluated (30%) had no acne. There was no relationship between acne scores ( no acne, mild acne and moderate to severe acne ) and cft (P ¼ 0.31) or PCO morphology (P ¼ 0.52). Androgen levels in adolescence and biochemical HA Androgen levels were measured in the early follicular phase in 226 girls. Median values and ranges for total testosterone, cft, androstenedione, DHEAS and SHBG are shown in Table II. The top 5% of cft for this population was 45.6 pmol/l, the top 10% was 34.5 pmol/l and the top 15% was 31.2 pmol/l. PCO morphology on ultrasound Early follicular phase (Day 2 6) transabdominal pelvic ultrasound was performed on 230 girls from the cohort. In 225/230 (98%) cases the morphology of both ovaries was clearly identified. Only one ovary was adequately visualized in the remaining five cases. The median absolute difference in ovarian volume between right and left ovaries was 2.8 cm 3 (range: 0 18 cm 3 ). If a follicle of.10 cm 3 was seen the ultrasound was repeated. The median absolute difference between the number of follicles was two (interquartile range: 1 3 follicles) with a range of 0 10 follicles. There was no association between PCO morphology and birthweight (P ¼ 0.86). Eighty girls (35.4%) met diagnostic criteria for polycystic ovaries on ultrasound (Balen et al., 2003). Ovarian volume was not normally distributed in our adolescent population, and median volume was 6.7 cm 3 (interquartile range: , range: ). The estimated mean ovarian volume was 6.62 cm 3 [95% confidence interval (CI) cm 3 ]. Increased ovarian volume according to PCO criteria

4 1472 Hickey et al. Figure 1 Flow chart showing recruitment of post-menarchal adolescents from the Raine cohort. PCOS-R, PCOS diagnosed by Rotterdam Consensus criteria; PCOS-N, PCOS diagnosed by National Institutes of Health criteria; PCOS-AES, PCOS diagnosed by Androgen Excess Society criteria (Note: For exclusions, only the 12 girls on oral contraception were excluded. Those not consenting to blood tests/ultrasound were included but data are missing for these outcomes.) (.10 cm 3 ) was identified in 69 girls (30%) overall. The presence of 12 or more follicles between 2 and 9 mm diameter was seen in 22 girls (10%) overall. PCO morphology was largely attributable to increased ovarian volume rather than an increased number of follicles (P, 0.001). Sex hormone-binding globulin Circulating early follicular phase SHBG levels were measured in 226 girls. Controlling for subject age and time since menarche, level of SHBG was correlated with total testosterone (P ¼ 0.02), cft (P, 0.001) and with DHEAS (P ¼ 0.007). SHBG levels were not related to levels of androstenedione (P ¼ 0.68). 226 of the 232 girls included (97%). Six girls were excluded owing to insufficient data. The prevalence of each diagnostic criterion for PCOS is shown in Table II. Biochemical HA was diagnosed as those with the highest 5% (95th centile) of cft levels for this population. Using these criteria, 7 girls (3.1%) would have met the NIH diagnostic criteria for PCOS, 11 girls (5%) would meet the AES criteria and 42 girls (18.5%) would have met Rotterdam diagnostic criteria. These categories are not exclusive and some girls met.1 diagnostic criteria. If biochemical HA was defined as the highest 10% of cft levels ( 34.5 pmol/l), 14 girls (6.2%) would have met the NIH criteria for PCOS, 19 girls (8.2%) would meet AES criteria and 48 girls (21.1%) would have met the Rotterdam diagnostic criteria. Diagnosis of PCOS in adolescence using adult criteria The features of PCOS defined using adult criteria include oligo-anovulation, clinical or biochemical HA and PCO morphology on ultrasound. Data for each of these features were available for Body mass index Normal BMI was observed in 159 girls (71%). Forty-seven (21%) were overweight and 18 (8%) were obese. BMI was correlated to cft (r ¼ 0.53, P, 0.001) and to SHBG concentrations (r ¼ 20.58,

5 Feature of polycystic ovary syndrome in adolescence 1473 Table I Features of PCOS in adolescence according to three international adult diagnostic criteria (Hickey, 2009). All PCOS-R... PCOS-N... PCOS-AES... n No Yes P No Yes P No Yes P (n 5 179) (n 5 48) (n 5 216) (n 5 10) (n 5 216) (n 5 11)... Current age (years) 15.2 (0.48) 15.2 (0.43) 15.4 (0.62) (0.45) 15.7 (0.72) (0.43) 15.9 (0.89),0.001 Age at menarche 12.5 (1.2) 12.6 (1.2) 12.4 (1.1) (1.2) 11.9 (1.4) (1.2) 11.8 (1.3) (years) Months since 32.2 (15.0) 31.3 (15.0) 35.4 (15.0) (15.0) 46.1 (17.0) (14.4) 48.4 (17.8) menarche BMI (kg/m 2 ) 22.7 (3.8) 22.3 (3.0) 24.5 (5.7), (3.4) 29.4 (6.8), (3.4) 28.8 (6.7),0.001 BMI (z-score) 0.54 (0.8) 0.48 (0.8) 0.77 (0.9) (0.8) 1.45 (0.9) (0.8) 1.37 (0.9) BMI, n (%) Normal 163 (70.3) 134 (74.9) 26 (54.2), (72.7) 2 (20.0), (70.8) 3 (27.3),0.001 Overweight 48 (20.7) 37 (20.7) 10 (20.8) 44 (20.4) 3 (30.0) 44 (20.4) 3 (27.3) Obese 19 (8.2) 7 (3.9) 11 (22.9) 13 (6.0) 5 (50.0) 13 (6.0) 5 (45.5) Waist:hip ratio 0.85 (0.08) 0.86 (0.08) 0.85 (0.10) (0.08) 0.87 (0.08) (0.08) 0.87 (0.07) Acne score N (%) None 69 (29.7) 57 (31.8) 10 (20.8) (30.1) 2 (20.0) (29.6) 3 (27.3) Mild 109 (47.0) 83 (46.4) 23 (47.9) 103 (47.7) 4 (40.0) 101 (46.7) 4 (36.4) Moderate/severe 48 (20.7) 34 (19.0) 14 (29.2) 42 (19.4) 4 (40.0) 41 (19.0) 4 (36.4) Ferriman Gallwey (57.8) 105 (58.7) 25 (52.1) (58.3) 3 (30.0), (57.4) 3 (27.3), (32.8) 62 (34.6) 13 (27.1) 74 (34.3) 1 (10.0) 72 (33.3) 2 (18.2) 5 19 (8.2) 10 (5.6) 9 (18.8) 13 (6.0) 5 (60.0) 13 (6.0) 6 (54.5) 8 8 (3.4) 4 (2.2) 4 (8.3) (1.9) 4 (40.0), (1.9) 4 (36.4),0.001 PCO morphology 80 (34.5) 35 (19.6) 43 (89.6), (32.4) 10 (100.0), (30.6) 9 (81.1),0.001 PCOS-R, PCOS diagnosed by Rotterdam Consensus criteria; PCOS-N, PCOS diagnosed by National Institutes of Health criteria; PCOS-AES, PCOS diagnosed by Androgen Excess Society criteria. a Five, six, nine cases have missing diagnoses on Rotterdam, NIH and AES diagnostic criteria, respectively. Data shown as mean and SD, or n (%). P-values for t-tests or x 2 tests for continuous and categorical outcomes, respectively. P, 0.001). Higher concentrations of cft were observed in overweight and obese girls, with a linear relationship. Lower concentrations of SHBG were observed in overweight and obese girls, which decreased linearly. Rate of PCO morphology in girls with BMI within normal range was 32.1% (51 out of 159 girls). This rate was similar to PCO morphology observed in 36.2% girls who were overweight (17 out of 47 girls) [odds ratio (OR) ¼ 1.20, 95% CI , P ¼ 0.60], and lower than amongst 61.1% girls who were obese (11 out of 18 girls; OR ¼ 3.33, 95% CI , P ¼ 0.019). Discussion This is the largest study to date of menstrual irregularity, PCO morphology and clinical and biochemical HA in a large population-based sample of predominantly Caucasian adolescents. Our findings suggest that menstrual irregularity is very common and not consistently associated with other features of PCOS. Further, we found that adult criteria for PCO morphology may not be appropriate for young women as mean ovarian volume is larger in young women. These finding are largely consistent with those from other populationbased studies of PCO features in adolescence (van Hooff et al., 1999a,b, 2000a,b). Clinical HA is a diagnostic feature of PCOS in adult women. Hirsutism, scored by F-G, is the most commonly reported method of determining clinical HA, though it is influenced by other factors such as ethnicity (Yildiz et al., 2010). We measured clinical HA using validated scores of hirsutism and acne. Most girls had a F-G scoring of zero. The small number of girls with F-G scores 5 (around 8%) also had higher cft levels. Our findings are consistent with reports that hirsutism is uncommon in adolescents (van Hooff et al., 1999a,b) and is not related to features of PCOS in Caucausian adolescents (van Hooff et al., 1999a,b). This may be because hirsutism takes longer to develop in the presence of increased androgens (Lucky et al., 2001). In contrast, acne was common affecting around 70% of girls and did not associate with other features of PCOS. This is consistent with population-based studies of Dutch adolescents where acne was not related to menstrual pattern (van Hooff et al., 1999a,b). Population-based studies of menstrual cyclicity are important as the great majority of adolescents with menstrual irregularity or secondary amenorrhoea do not consult a doctor (van Hooff et al., 1999a,b). We defined menstrual irregularity as cycles,25 or.35 days in duration or where the cycle length varied from month to month by.4 days (Treloar, 1981; Michelmore et al., 1999). This definition has been widely used in young adult women but may have been too strict for

6 1474 Hickey et al. Table II Early follicular phase (Day 2 6) androgen levels and diagnostic categories for PCOS. ALL (n 5 214) a PCOS-R... PCOS-N... PCOS-AES... No (n 5 168) Yes (n 5 46) P No (n 5 204) Yes (n 5 10) P No (n 5 203) Yes (n 5 11) P... A4 (nmol/l) 2.9 [ ] 2.7 ( ) 3.5 ( ), ( ) 5.8 ( ), ( ) 6.8 ( ),0.001 DHEAS (mmol/l) 3.2 [ ] 3.2 ( ) 3.7 ( ) ( ) 4.9 ( ) ( ) 5.4 ( ) SHBG (nmol/l) 47.9 [ ] 50.0 ( ) 38.9 ( ) ( ) 22.9 ( ) ( ) 27.9 ( ) TT (pmol/l) 1215 [ ] 1169 ( ) 1529 ( ), ( ) 2311 ( ), ( ) 2575 ( ),0.001 cft (pmol/l) 17.2 [ ] 16.2 ( ) 28.5 ( ), ( ) 58.5 ( ), ( ) 59.8 ( ),0.001 A4, Androstenedione; DHEAS, Dihydroepiandrosteronedione; SHBG, Steroid hormone-binding globulin; TT, Total testosterone; cft, Circulating free testostetone. a Five, six, nine cases have missing diagnoses on Rotterdam, NIH and AES diagnostic criteria, respectively. Data shown median and range [min max], or median and interquartile range (25th 75th) percentiles. P-values for Mann Whitney tests. Metric conversion factors are as follows: A4 3.49, DHEAS , Testosterone an adolescent population where irregular cycles are more common. In an unselected population of 2248 Dutch adolescents (mean age 15 years, range: years) van Hooff et al. (1999a,b) selected oligomenorrhoea (defined as cycles days, around three menstrual periods per year) as their primary outcome measure because 90% of adult infertility patients with oligomenorrhoea have high concentrations of LH or androgens, compatible with PCOS, and up to 90% of this population have PCO on ultrasound (Franks, 1995). Irregular cycles (defined as those with an average cycle length of days and 2 cycles with a length of,22 or.41 days during the previous 12 months) were observed in 14% and oligomenorrhoea in 6% of girls. Free testosterone was not calculated in the van Hooff et al. (1999a,b) study, but the authors report that 57% of oligomenorrhoeic girls had LH or androgen concentrations.95th centile (van Hooff et al., 1999a,b, 2000a,b). This report on the prevalence of PCO morphology in a populationbased sample of adolescent girls is the largest to date using current adult diagnostic criteria for PCOS (Balen et al., 2003). We have applied the international consensus definition of PCO morphology to allow comparison between adolescent and adult women and because rates of PCO will differ widely according to diagnostic criteria used (Michelmore et al., 1999). Rates of PCO morphology in our population were high at 35%. However, a recent population report of 262 Caucasian women aged years with regular menstrual cycles reported that PCO morphology (by transabdominal ultrasound) using Rotterdam criteria was present in 32% of women and decreased with age. PCO was associated with higher anti-mullerian hormone levels but not with free androgen index or other hormonal or metabolic parameters (Johnstone et al., 2010). Similarly, in 154 younger Danish women aged years from a longitudinal cohort who underwent transvaginal ultrasound, the prevalence of PCO morphology was 68% overall and 80% in the 44 women scanned who were not using hormonal contraception (Kristensen et al., 2010). A smaller study of 33 ethnically heterogenous American girls with PCOS diagnosed using NIH criteria, 22 of whom were obese, found no differences in ovarian morphology or volume between PCOS subjects and 13 obese controls (Silfen et al., 2003). Our findings suggest that current definitions of PCO morphology may be common in normal women of all ages but particularly in younger women. In a UK community-based study of 230 young women aged years, 33% had PCO morphology defined as at least 10 follicles (2 8 mm in diameter) arranged peripherally or scattered throughout an echodense stroma (Michelmore et al., 1999); however, in this study 50% of participants were using some form of hormonal contraception which may affect ovarian morphology and volume. A strength of our study is that very few girls were using hormonal contraception and they were excluded from the analyses. Ovarian morphology was assessed using a transabdominal approach because most participants were not yet sexually active. Whilst the transvaginal route is known to be more accurate in defining ovarian morphology, many other published studies in adolescents and in adult women have successfully used a transabdominal approach (Michelmore et al., 1999; van Hooff et al., 2000a,b; Silfen et al.; 2003; Johnstone et al., 2010). Further, it would not have been practical or ethical to use a transvaginal approach in girls who are not yet sexually active and would very likely have further reduced the number of adolescents willing to participate in our study. The transabdominal

7 Feature of polycystic ovary syndrome in adolescence 1475 approach, using high resolution techniques and experienced operators, successfully demonstrated ovarian morphology in 97% of girls. Around one-third (35%) had PCO morphology, somewhat higher than the 20 30% reported in unselected healthy adult populations (Polson et al., 1988; Clayton et al., 1992; Farquhar et al., 1994; Koivunen et al., 1999; Azziz, 2006). In a subgroup of 58 adolescents from a larger community-based population (mean age 16.7 years) using transabdominal ultrasound, van Hooff et al. (2000a,b) compared ovarian morphology between girls with oligomenorrhoea (cycles of days), irregular cycles (average cycle length days and 2 cycles with a length of,22 or.41 days during the previous 12 months) and regular cycles. PCO morphology, defined as.10 peripheral cysts in both ovaries was related to menstrual patterns. They report PCO morphology in only 10% of those with regular cycles, 28% with irregular cycles and 45% with oligomenorrhoea (van Hooff et al., 2000a,b). We used diagnostic criteria for ovarian volume derived from adult normative values (Balen et al., 2003). A large community-based study of asymptomatic adult women (Taponen et al., 2004) showed mean ovarian volume of 5.05 cm 3 on the left and 5.23 cm 3 on the right. Mean ovarian volume (estimated using log-transformation) in our adolescent population was greater at 6.62 cm 3 (95% CI cm 3 ), with a median volume of 6.7 cm 3 (interquartile range: , range: ). Ovarian volume was enlarged (.10 cm 3 ) in one ovary in 19% of girls and in both ovaries in 10%. Our findings are similar to those reported in Dutch girls aged years, where mean ovarian volume in those with regular cycles was 8.1 cm 3 (+2.7), cm 3 in those with irregular cycles and mm 3 in those with oligomenorrhoea (van Hooff et al., 2000a,b). Recent crosssectional population-based studies in women aged years using transvaginal ultrasound suggest that ovarian volume decreases with reproductive age (Johnstone et al., 2010). There are limited longitudinal data to indicate how ovarian volume changes over time in individual women, and the contribution of PCO to this. However, our data suggest that adult criteria for PCO morphology based on ovarian volume may not be applicable in young women (Blank et al., 2008). In a population-based sample of 95 adults, the 95% centile of free testosterone was reported as.0.75 ng/dl (equivalent to 26 pmol/l; Huang et al., 2010). In our study, the 95th centile for free testosterone was pmol/l, considerably higher than that reported in these adult women. However, variations between assays make these figures difficult to compare. We measured androgen levels on only one occasion during the early follicular phase. Previous longitudinal studies have suggested that total testosterone levels are increased in adolescents with oligomenorrhoea but also increase following menarche for around 5 6 years (van Hooff et al., 2000a,b). Variations in androgen levels in our study could have reflected gynecological age (time since menarche) rather than differences in ovarian or adrenal androgen production related to feature of PCOS. Current published data for circulating androgen levels in PCOS uses radioimmunoassay (RIA), often without extraction and chromatography. Liquid chromatography-mass spectrometry (LC/MS) has been widely advocated for androgen measurement and is certainly more specific than un-extracted immunoassays but is not necessarily more precise or accurate when compared with extraction with or without chromatography. Recent direct comparisons between RIA and LC/MS have shown that measurement of testosterone by RIA is comparable to LC/MS assays. There is significant variability between LC/MS assays and poor precision with all assays at lowtestosterone levels. High-testosterone levels correlated with clinical HA (hirsutism) using both measurement techniques (Legro et al., 2010). The method used in our study allows comparison between androgen levels in our cohort and those reported in other studies of adolescents and adult women with features of PCOS. In the absence of LC MS, we feel our assay allows an adequate assessment of androgen status to assign subjects to normal or high androgen status. Only around one-third of girls from the Raine cohort participated in our study. This likely reflects the considerable demands of the study (timed blood samples and ultrasound) but is a higher participation rate than other large community-based studies (Clayton et al., 1992; Michelmore et al., 1999; van Hooff et al., 2000a,b) and likely reflects the difficulties inherent in surveying normal healthy volunteers. However, those recruited may not be representative of this unselected population. Access to a gynecologist may have led to a bias in recruitment towards those with menstrual complaints. Further, 64 girls (28%), were within 2 years of menarche and this may have increased the number of anovulatory cycles (Apter and Vihko, 1977). Persistent oligomenorrhoea.2 years post-menarche is a much stronger predictor of continued menstrual irregularity (Southam and Richart, 1966; van Hooff et al., 2004). In Dutch teenagers aged years, around 50% with oligomenorrhoea at age 15 years continued to report oligomenorrhoea at age 18 years. Adolescents with irregular menstrual cycles (35 41 days) were at increased risk of developing oligomenorrhoea compared with those with regular menstrual cycles. A raised BMI was predictive of persistent oligomenorrhoea at age 18 years but androgen concentrations or ovarian morphology were not predictive of later menstrual pattern (van Hooff et al., 2004). However, these observations may have been confounded by increasing OCP use with increasing age (61% at 3 years, most for around 1 year) and potentially greater OCP use in those with irregular cycles. Our cohort showed similar rates of obesity and overweight to that commonly reported in other developed countries. Our findings are consistent with other reports that features of PCOS are more common in overweight or obese girls (Blank et al., 2009). A trend towards increasing weight in adolescents means that more girls are likely to meet current adult diagnostic criteria for PCOS. In a large Finnish birth cohort those who were obese at age 14 years were more likely to be diagnosed with PCOS in their 30s (Laitinen et al., 2003). Our findings suggest that menstrual irregularity, ovarian morphology, acne or hirsutism are not useful diagnostic features of PCOS in adolescent girls. Many girls met adult criteria for PCO morphology, largely owing to a higher mean ovarian volume. Diagnostic features of PCOS used in adult women, including clinical HA, ovarian volume and morphology, may be of limited use in adolescence. Authors roles M.H. led the study design and data collection and drafted the manuscript. She is also the principal investigator leading funding for this study. D.A.D. analyzed the data and has contributed to data interpretation and manuscript writing. R.H. assisted with study design, data

8 1476 Hickey et al. acquisition, data interpretation and manuscript writing. H.A. and D.M.S. contributed to data acquisition and interpretation and D.M.S to drafting the manuscript. S.F. and R.N. contributed to study design data interpretation and preparation of manuscripts. Acknowledgements We are extremely grateful to all the families who took part in this study and the whole Raine Study team. We acknowledge both the Raine Medical Research Foundation at the University of Western Australia and the Telethon Institute of Child Health Research for financial support and general support over the years. We acknowledge LeeAnn Mahoney, Sarah Simpson and Helen Box for study recruitment, James Humphreys for database construction and maintenance and Prof Jan Dickinson for ultrasonography. Funding The collection of maternal data and samples was funded by the Women and Infants Research Foundation (WIRF) while the collection of adolescent data and samples was funded by NHMRC project grant number (CIA Hickey). M.H. is also funded by an NHMRC Clinical Career Development Award (number ). References Apter D, Vihko R. Serum pregnenolone, progesterone, 17-hydroxyprogesterone, testosterone and 5 alpha-dihydrotestosterone during female puberty. J Clin Endocrinol Metab 1977;45: Apter D, Vihko R. Premenarcheal endocrine changes in relation to age at menarche. Clin Endocrinol (Oxf) 1985;22: Azziz R. Controversy in clinical endocrinology: diagnosis of polycystic ovarian syndrome: the Rotterdam criteria are premature. J Clin Endocrinol Metab 2006;91: Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES, Yildiz BO. The prevalence and features of the polycystic ovary syndrome in an unselected population. 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