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The Diagnostic Value of Anti-Müllerian Hormone in Early Post Menarche Adolescent Girls with Polycystic Ovarian Syndrome
Accepted Manuscript The Diagnostic Value of Anti-Mullerian Hormone in Early Post Menarche Adolescent Girls with Polycystic Ovarian Syndrome Pınar Kocaay, MD, Zeynep Siklar, MD, Sema Buyukfirat, Merih Berberoglu, MD PII:
S1083-3188(18)30159-1
DOI:
10.1016/j.jpag.2018.02.126
Reference:
PEDADO 2209
To appear in:
Journal of Pediatric and Adolescent Gynecology
Received Date: 16 November 2017 Revised Date:
18 January 2018
Accepted Date: 10 February 2018
Please cite this article as: Kocaay P, Siklar Z, Buyukfirat S, Berberoglu M, The Diagnostic Value of AntiMullerian Hormone in Early Post Menarche Adolescent Girls with Polycystic Ovarian Syndrome, Journal of Pediatric and Adolescent Gynecology (2018), doi: 10.1016/j.jpag.2018.02.126. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title of article: The Diagnostic Value of Anti-Mullerian Hormone in Early Post Menarche Adolescent Girls with Polycystic Ovarian Syndrome
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Authors:
Pınar Kocaay, MDa; Zeynep Siklar, MDa; Sema Buyukfiratb, Merih Berberoglu, MDa
Department of Pediatric Endocrinology, Ankara University School of Medicine, Ankara, Turkey
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a
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Turkey
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Department of Endocrinology Laboratories, Ankara University School of Medicine, Ankara,
Financial Support and Disclaimers: Financial support was provided by Ankara University
Pınar Kocaay, MD
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Corresponding author:
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BAPRO, grant number: 13H3330007
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Hoşdere caddesi, Aziziye mahallesi, Refik Belendir sokak, 69/11 Cankaya/Ankara, Turkey Phone: +90 505 720 2569
Email address: [email protected]
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Abstract: Study Objective: Polycystic ovarian syndrome (PCOS) is a common endocrine disorder
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characterized by hyperandrogenism and chronic anovulation, which affects 5-10% of reproductive age females. Diagnosis of adult patients with PCOS is made easily with clinical and laboratory methods and the antimullerian hormone (AMH) level are accepted as a good indicator. However, there is still no complete consensus on the diagnosis of PCOS in
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adolescents.
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Design and Settings: Prospective cohort study, December 2013 to November 2014 Participants: The study was conducted on adolescent girls with oligomenorrhea, with at least two years since menarche. The study group consisted of adolescent girls with complete PCOS and incomplete PCOS. A control group was formed of healthy adolescent girls. Complete PCOS
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was diagnosed according to the Rotterdam criteria, as the presence of all the following characteristics; oligomenorrhea, hyperandrogenism, and polycystic ovarian morphology (PCOM) on ultrasound image. Incomplete PCOS was accepted as “oligomenorrhea and PCOM”, or
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“oligomenorrhea and hyperandrogenism”.
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Interventions and Main Outcome Measures: All patients underwent a physical examination and the anthropometric assessments, insulin resistance, and acanthosis nigricans were recorded. It was also noted whether or not the patient had an acne score The Ferriman-Gallwey (FG) score was applied to evaluate hirsutism. Result: The results of this study showed no statistically significant difference was found between the PCOS and incomplete PCOS groups and the control group in respect of AMH levels.
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Conclusion: The use of adult specific diagnostic methods in adolescence may result in an incomplete diagnosis and inadequate treatment plan. Although the serum AMH level clearly
still controversial and further studies are needed.
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facilitates the diagnosis of PCOS, the use of the AMH level in adolescence in PCOS diagnosis is
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Key words: Polycystic ovarian syndrome, Antimullerian hormone, adolescence, diagnosis
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Introduction Polycystic ovarian syndrome (PCOS) is a common endocrinological disorder which affects 5-
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10% of reproductive age females, and is characterized by hyperandrogenism and chronic anovulation. It can cause infertility problems and may also be seen with some metabolic conditions such as obesity, type 2 diabetes mellitus (DM), hypertension, dyslipidemia, and cardiovascular disorders.1 Stein and Leventhal first described this syndrome in 1935 in women
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changed many times since then (Table 1).2-5
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with amenorrhea, hirsutism and bilateral polycystic ovary, and the diagnostic criteria have
The diagnostic criteria for adults have generated several phenotypes of PCOS. Phenotype 1 is classical PCOS as “Hyperandrogenism+ Oligomenorrhea+ Polycystic ovary morphology”. Phenotype 2 is classified as Hyperandrogenism+ Oligomenorrhea, Phenotype 3 (ovulatory
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PCOS) as Hyperandrogenism+ Polycystic ovary morphology, and Phenotype 4 (nonhyperandrogenic PCOS) as Oligomenorrhea+ Polycystic ovary morphology.6,7 In adolescents, these broad definitions may be problematic6,7 because these PCOS criteria may also be seen
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physiologically in adolescence, so PCOS diagnosis in this age group may present some difficulties. Exaggerated pubertal symptoms in this period can correspond to PCOS diagnosis.
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The common features of adult hyperandrogenism are less reliable in adolescent girls than women. In addition, menstrual irregularity, hirsutism and acne can be temporary without progression. This situation causes difficulties in diagnosis and does not present an accurate prevalence. Even with regular cycles after the first two years following menarche, there can be asymptomatic anovulation in half of adolescents. Multifollicular ovaries may be present in adolescents which might not be distinguishable from PCO morphology. It may also not be
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possible to perform transvaginal ultrasonography in many adolescents and thus screening is challenging especially in obese adolescents.7,8
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Therefore, in the 2012 consensus meeting in Amsterdam, it was stated that it was necessary for all three of the Rotterdam criteria to be met for a diagnosis of PCOS to be made in adolescents.5 However, there may be patients who present with menarche more than two years previously and
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trying to fulfill the three criteria in these cases can cause diagnostic errors. Furthermore, there may be adolescent cases who do not meet all the criteria, but complain of hyperandrogenism and
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menstrual irregularity. In some cases, the period of hyperandrogenism may be short and therefore does not meet the Ferriman–Gallwey (FG) score of 8.
With an accurate diagnosis of PCOS, the existing risk can be reduced and treatment can be given to prevent future infertility, metabolic syndrome, type 2 DM and cardiovascular disorders.9
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However, over-diagnosis may lead to unnecessary treatment and psychological stress in adolescents. Anti Mullerian Hormone (AMH) is a supportive marker in the diagnosis of PCOS. AMH is a protein of dimeric glycoprotein structure, which is secreted from ovarian small antral
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and preantral cells of granulosa cells from the 36th week of gestation11 The secretion is independent of gonadotropins and the menstrual cycle. It is related with antral follicle number
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and is therefore a good marker of ovarian reserve and function.10 Although there have been many studies of AMH levels in adults, there are conflicting data about the adolescent period. In addition, adolescent girls who do not meet all the diagnostic criteria for PCOS but are evaluated as at risk could carry metabolic risk factors and therefore require early diagnosis. In this study, the AMH levels in confirmed PCOS cases which met the 3 Rotterdam diagnostic criteria and cases with PCOS which met 2 of the 3 diagnostic criteria and had oligomenorrhea,
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were compared with the AMH levels of a control group. From these comparisons, it was evaluated if AMH levels could be a used as a marker in adolescent cases of PCOS.
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Materials and Methods This study was conducted on adolescent females who presented at Ankara University School of Medicine, Department of Child Health and Diseases, Division of Pediatric Endocrinology,
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adolescent clinic between December 2013 and November 2014.
All subjects were informed of the study protocol and written informed consent was obtained
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from their parents. All authors complied with the World Medical Association Declaration of Helsinki regarding the ethical conduct of research involving human subjects. In all cases, menarche age was recorded, and cases were included if menarche was at least 2 years previously. Family history of PCOS, hyperandrogenism, obesity, hypertension,
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dyslipidemia, and type 2 DM was also questioned. Patients with non-classical congenital adrenal hyperplasia were excluded from the study.
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The study group comprised adolescent females aged 14.3-17.2 years, with PCOS of either a complete or incomplete phenotype. A control group was formed of healthy adolescents aged
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13.5-17.6 years who agreed to participate in the study. The diagnosis of PCOS was made according to the revised Rotterdam criteria.5 Patients with all 3 criteria were evaluated as complete PCOS (Group 1) and patients with 2 of the PCOS criteria as incomplete PCOS (Group 2). Within Group 2 subgroups were formed as those with oligomenorrhea and PCOM categorized as Group 2a, and those with oligomenorrhea and clinical or laboratory-evaluated hyperandrogenism as Group 2b,
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Group 1: Oligomenorrhea + PCO morphology + hyperandrogenism Group 2a: Oligomenorrhea + PCO morphology
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Group 2b: Oligomenorrhea + hyperandrogenism The physical examination and anthropometric assessments of all cases were made by one of the researchers (PK). Findings of acne and acanthosis nigricans were recorded. Hyperandrogenism
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was clinically evaluated using the FG score. According to the FG method hair distribution in a total of 9 areas (upper lip, chin, chest, upper and lower parts of the back, lower and upper
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abdomen, upper part of arms and legs) is scored from 0 to 4 points. A score of >8 is considered as hirsutism.13 Oligomenorrhea was accepted as <8 cycles in 1 year)14,15 and amenorrhea as the absence of cycles for >3 months. BMI was calculated with the formula of body weight (kg) / height (m2).16 The weight to height percentage was calculated using the formula: the patient's
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measured BMI (kg /m2) / required BMI of the patient x 100. A value within the range of 90-110 was considered normal, 110-120 as overweight, >120 as obese, and >140 as morbidly obese.17 The blood pressure measurements were evaluated using the Tumer standards and appropriate age
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and gender norms. The average systolic or diastolic BP indicated HT if it was ≥95th percentile
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for adjusted age, gender and height percentile for healthy children.18 Blood samples were taken between 08:00-10:00 after 10-12 hours of fasting during the early follicular phase (days 3-5 of the menarcheal period) for examination of fasting blood glucose, lipid profile, prolactin, LH, FSH, E2, testosterone, 17-OH progesterone, and DHEAS. Biochemical assessments were made in the biochemistry and endocrinology laboratories of Ankara University School of Medicine. Total testosterone over 55 ng/mL, and free testosterone over 3 ng/mL was evaluated as hyperandrogenism.13,19
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Serum AMH was measured using a double antibody enzyme linked immunosorbent assay kit. Following collection and processing, serum samples for AMH were stored at -20° C until hormone analyses were performed. All serum AMH was performed in the same laboratory using
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the same assay. The results were expressed as ng/mL. The lower limit of detectability for this assay is 0.08 ng/mL (range 0.08 ng/mL to 20ng/mL). The intra and interassay coefficients of variation were 10% and 12% respectively. The sensitivity of this assay was defined as the lowest
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protein concentration that could be differentiated from zero. It was determined by subtracting two standard deviations to the standard replicates and calculating the corresponding
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concentration.
Ultrasonography was performed in a single center by a single expert. Ovarian and uterine size and polycystic ovarian morphology (PCOM) were evaluated. PCOM was accepted as the following conditions: 2-9 mm in diameter, with 12 or more follicles and/or increased ovarian
Statistical Method
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volume (>10 mL).20 Ovarian volume was calculated as 0.52 x length x width x thickness.
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Data analysis was applied using SPSS Windows 15 software. Descriptive statistics were stated as
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mean ±standard deviation (SD) and percentage (%). The Mann-Whitney U test was used to assess the significance of differences between means in two groups. When there were more than two groups, the significance of difference between the means was analyzed with ANOVA variance analysis while the significance of medians was analyzed with the Kruskal Wallis test. Nominal variables were analyzed with Pearson Chi-square or Fisher Exact tests.
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The correlation between continuous variables was analyzed with the Spearman correlation test for variables not showing normal distribution while the Pearson correlation test was used for
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normally distributed variables. A value of p<0.05 was accepted as statistically significant. Results:
The study was conducted in the adolescent clinic of Pediatric Endocrinology between December
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2013 and November 2014. A total of 53 cases were diagnosed with complete or incomplete PCOS and were compared with 55 healthy, age-matched adolescent girls as a control group.
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Within the study groups, Group 1 comprised 29 cases (complete PCOS); Group 2a, 16 cases, and Group 2b, 8 cases (incomplete PCOS). All the patients in the study groups had complaints of irregular menstruation. The second most frequent complaint was hirsutism (n: 27/53, 50.9%). Acne was reported in 2 cases (3.7%), and hair loss in 1 case (1.8%). The mean post-menarche
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period was 3.1±1.08 years for all cases. No statistically significant difference was determined between the study and control groups in respect of the post-menarche period (p: 0.301). Family history revealed that 28% (15/53) of first degree relatives of the patients had obesity,
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24% (13/53) had PCOS, 5.6% (3/53) had Type 2 DM and 12% (7/53) had dyslipidemia,
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hypertension or cardiovascular disease. The mean age on admission was 15.54±1.32 years in the study group, 15.55±1.33 years in complete PCOS group, 15.79±0.97 years in incomplete PCOS group, and 15.4±1.45 years in the control group. No significant difference was determined between the groups in respect of mean age (p: 0.485). The mean height SD of the whole study group was 0.25±0.83 SD. There was no significant difference in height SDS between the groups (p: 0.393) (Table 2). The frequency of obesity and overweight was determined as 51% (27/53) and 11.3% (6/53) respectively in the
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PCOS groups. In the control group, 43.6% (24/55) of the cases were of normal weight, 25.4% (14/55) were overweight, and 30.9% (17/55) were obese. There was no significant difference
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between RBMI between the groups (p: 0.557). Physical examination revealed that 22 (41%) cases in the study group had acanthosis nigricans. Serum DHEAS and free testosterone levels within the PCOS groups were similar. The total testosterone level of Group 1 was higher than that of Group 2a (p: 0.03), and Group 2b (p: 0.001)
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(Table 4). Serum 17-OH progesterone level was higher in Group 1 compared to Group 2a, with
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no significant difference between the other groups. FSH, LH, estradiol, fasting blood glucose, fasting insulin, total testosterone, HDL, LDL, and VLDL levels were similar in all the groups. No correlation was determined between AMH level and other laboratory parameters or between AMH level and RBMI (Table 3). There was no significant difference between the control group
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and all the PCOS groups in respect of AHM levels and no significant difference in AMH levels between the PCOS groups (Table 4). Discussion:
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In adults, the diagnosis of PCOS can be made easily with clinical and laboratory parameters and
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AMH levels are accepted as a good indicator. However, in the adolescent age group, there are still controversies about PCOS diagnosis. Using adult diagnostic procedures for adolescents may result in under-diagnosis, fail to make a diagnosis or over-diagnosis. During the first years following menarche in adolescent girls, anovulatory cycles are a physiological occurrence and polycystic ovary morphology may be seen on ultrasound and considered as signs of PCOS. Another error at this time is seeing acne as a sign of PCOS rather than a physiological finding. Over-diagnosis of PCOS can have negative effects both physiologically and psychologically.
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The aim of this study was to determine the AMH levels in patients with complete PCOS where all 3 diagnostic criteria are met and incomplete PCOS with 2 of the 3 criteria and to compare these with the levels in a control group. The results showed no statistically significant differences
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between either PCOS groups and the control group or between the different PCOS groups in respect of serum AMH levels.
Serum AMH levels can be affected by some parameters. The relationship between serum AMH
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levels and androgen levels is a matter of controversy. While some studies have shown a positive
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relationship between serum AMH levels and androgen levels in cases with PCOS, others have not.21-23 Serum androgen levels are reported to be more related with AMH levels and not with intraovarian androgen levels because of the inhibition of aromatase by AMH.24 In the current study no relationship was found between these parameters. This can probably be attributed to milder hyperandrogenism in the current study cases or that the hyperandrogenism period was
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still short and the FG score might not have reached 8. The laboratory values for hyperandrogenism have been derived from studies based on adults. There are no reliable assays with well-defined normal ranges for the documentation of hyperandrogenism in adolescence and
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accurate determinations of testosterone levels are often problematic.7
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As a significant number of PCOS cases are obese, it may not always be possible to obtain efficient results from suprapubic ultrasound. Transvaginal ultrasound cannot be applied to virgin adolescents. Furthermore, there is no exact value for the volume of normal ovaries during adolescence. In adults, either 12 or more follicles measuring 2-9 mm in diameter, and/or increased ovarian volume (greater than 10 mL) are considered as PCOM.8 To date, some studies have accepted different volumes between 7.8-9.6 mL as maximum volume in the adolescent
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period.6,25 Therefore, during adolescence, the diagnostic increase of ovarian volume for PCOS could be >10 mL.6
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In many studies a positive correlation has been found between serum AMH levels and ovarian volume.21,26 The reason behind this is the increased ovarian volume which is one of the criteria of PCOS. In the current study, ovarian volume of ≥10 mL was accepted as PCO. Thus controversial evaluations were excluded. PCO morphology in the current study was evaluated by follicle
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number and ovary volume but no relationship was found with either. This could be because
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many patients in this study were obese and could not be evaluated clearly with ultrasound or because of an insufficient number of cases.
Obesity, which is frequently associated with PCOS, is another factor that may have an effect on serum AMH levels27. There may be a negative correlation between AMH levels and obesity, but
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in the current study, no such relationship was found. Furthermore, there was no difference between obese cases with PCOS and obese cases without PCOS in respect of serum AMH levels. This situation might be attributable to the equal number of obese and overweight cases in both
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the study and control groups.
A positive correlation has been previously determined between cases with PCOS and serum
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AMH levels28, although this difference has not been observed in some studies. It has been considered that the reason for different results could originate from differences in age and ethnicity of the cases in the studies. The current study cases were in the early menarcheal period which could explain why there were no differences between the groups and the control patients. The differences may appear over a longer time.
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The cutoff value for AMH in the diagnosis of PCOS is controversial, as different kits have been used for AMH measurements. Many studies have been conducted to make PCOS diagnosis with precise AMH levels, and many authors think AMH levels are useful for the diagnosis of
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PCOS.11,21,28,29 Cutoff values for AMH ranging from 2.8 ng/mL-10.7 ng/mL have been suggested in those studies.11,30,31 In a recent meta-analysis, the cutoff value for AMH levels was accepted as 4.7 ng/mL.32 In some studies, it has been shown that there can be an overlap of AMH levels in
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PCOS cases and control group patients and has therefore been claimed that it cannot be used as an independent marker. In a study of 207 PCOS cases, it was shown that the AMH value had low
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sensitivity, specificity and a positive predictive value according to both NIH and the Rotterdam criteria.33 Explanations for these low values included selection bias, and increased prevalence of menstrual irregularities in adolescents.
In a study by Pigny et al, the cutoff value was accepted as 8.4 ng/mL with 92% specificity and
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67% sensitivity.34,35 In another study, the cutoff value was accepted as 8 ng/mL with 70% specificity and 61.7% sensitivity.29 In the current study, serum AMH was measured using a double antibody enzyme linked immunosorbent assay kit. However, no cut off value was
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determined as there was no significant difference between the groups in respect of AMH levels. Previously reported differences may have arisen from ethnic differences between study
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groups.11,29-31
There may be a correlation between serum AMH level and age, as is thought that AMH level is the marker of ovarian age36,37 and serum AMH levels are known to decrease with age. However, this decrease is slower in cases with PCOS compared to cases without PCOS.29 In the current study; the mean age of patients was 15.54 years, so no aging effect on serum AMH levels was expected.
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The reason for the low specificity and sensitivity of the results of AMH levels for PCOS diagnosis is the menstrual irregularity that can be frequently seen in adolescents. During the early reproductive years, menstrual irregularity may be a normal reflection of anovulatory
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cycles.38 Over-diagnosis of PCOS has been attributed to inadequate identification of adolescent polycystic ovarian morphology on ultrasound images even though it is clearly identifiable for adults. A large multi-follicular ovarian structure in adolescence has been indicated as normal in
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many studies.39
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To date, AMH levels have been reported to not change with the menstrual cycle.31,40 However, it has been recently reported that estradiol expresses AMH secretion within the in vitro environment.41 It can be thought that AMH levels may be low in the middle of the cycle. Low and similar AMH levels within the current study groups may have been due to the collection of blood specimens in the middle of the cycles and the number of cases may not have been
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sufficient to show a difference.
In conclusion, the serum AMH levels were found to be similar in the PCOS groups and the
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control group. During adolescence, especially at an early post-menarcheal age, the use of AMH levels as a diagnostic tool for PCOS is still controversial and more studies on this topic are
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needed.
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35. Al-Qahtani A, Muttukrishna S, Appasamy M, et al: Development of a sensitive enzyme immunoassay for anti-mullarian hormone and evaluation of pottential clinical applications in males and females. Clin Endocrinol (Oxf) 2005; 63:267. 36. Visser JA, De Jong FH, Laven JS, et al: Anti-Müllerian hormone: a new marker for ovary function. Reproduction 2006; 131:1. 37. de Vet A, Laven JS, de Jong FH, et al: Antimüllerian hormone serum levels: a putative marker for ovarian aging. Fertil Steril 2002; 77:357. 38. Adams Hillard PJ: Adolescent menstrual health. Pediatr Endocrinol Rev 2006; 3:138. 39. Sampalo P, Livieri C, Montanari L, et al: Precocious signs of polycystic ovaries in obese girls. Ultrasound Obstet Gynecol 1994; 4:310. 40. Hagen CP, Aksglaede L, Sorensen K, et al: Serum levels of anti-Mullerian hormone as a marker of ovarian function in 926 healty females from birth to adulthood and in 172 Turner syndrome patients. J Clin Endocrinol Metab 2010; 95:5003. 41. Grynber M, Pierre A, Rey R, et al: Differential regulation of ovarian anti müllerian hormone (AMH) by estradiol through α and β estrogen receptors. J Clin Endocrinol Metab 2012; 97:1649.
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Contributions: Pınar Kocaay made substantial contributions to the conception, design, analysis and
and gave final approval of the version to be published.
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interpretation of data, drafted the article, revised it critically for important intellectual content
Zeynep Siklar made substantial contributions to the conception, design, analysis and
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interpretation of data, drafted the article and gave final approval of the version to be published. Sema Buyukfirat made substantial contributions to the conception, design, analysis and
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interpretation of data.
Merih Berberoglu made substantial contributions to the conception, design, analysis and
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interpretation of data, drafted the article and gave final approval of the version to be published.
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Table Legends Table 1. Diagnostic criteria for PCOS
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Table 2. Clinical characteristics of the cases Table 3. Correlations between serum AMH levels and other laboratory parameters
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Table 4. Laboratory findings of the cases
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Table 1. Diagnostic criteria for PCOS PCOS DEFINITION
HYPERANDROGENISM
+
ROTTERDAM (2003)
+
First ESHRE/ASRM PCOS consensus workshops
-
+
+
2 of 3 criteria
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AE-PCOS SOCIETY (2009)
POLYCYSTIC OVARY MORPHOLOGY
+
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NIH CRITERIA (1990)
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Clinical or Biochemical
OLIGOMENORRHEA
+
+
+
AMSTERDAM (2012) Third ESHRE/ASRM PCOS consensus workshops
+
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1 of 2 criteria +
+
All criteria for adolescent
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*Adult Phenotypes: Phenotype 1: “Hyperandrogenism+ Oligomenorrhea+ Polycystic ovary morphology”, Phenotype 2: “Hyperandrogenism+ Oligomenorrhea”, Phenotype 3: “Hyperandrogenism+ Polycystic ovary morphology”, Phenotype 4: “Oligomenorrhea+ Polycystic ovary morphology”
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Incomplete
Control
29
24
55
Age (year)
15.55±1.3
15.79±0.97
15.4±1.45
0.485
Height SDS
0.33±0,95
0.38±0.80
0.14±0.76
0.393
RBMI (%)
121±21.2
122.6±32.6
115±23.5
0.394
3.2±1.1
3.08±1.07
2.8±1.07
0.301
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Post-Menarcheal Age (%)
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Number of patient (n)
p
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Complete
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p value
Age
0.168
0.083
RBMI
-0,108
0.442
LH (mIU/mL)
-0.104
0.457
FSH (mIU/mL)
0.008
0.957
Estradiol (pg/mL)
-0,067
0.635
Total testesterone (ng/mL)
0.144
0.487
Freetestesterone (ng/mL)
0.122
0.745
DHEAS (mcg/dL)
0.07
0.657
0.104
0.432
0.144
0.302
-0.136
0.331
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HDL-kolesterol (mg/dL)
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17 OH progesteron (ng/dL)
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Correlation coefficient
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Variable
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Table 3. The Correlations between serum AMH levels and other laboratory parameters
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Triglyceride (mg/dL)
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Table 4. Laboratory findings of cases
Group 2b
AMH (ng/dL)
7.05±4.72
8.4±4.7
5.3±5.9
LH (mIU/mL)
9.8±7.8
10.9±9.5
12.9±7.4
FSH (mIU/mL)
5.2±1.96
4.9±1.6
4.93±2.1
17 OHP (ng/mL)
1.47±0.77 0.87±0.37
Control
p
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Group 2a
7.0±3.84
0.33
-
0.48
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Group 1
0.82
1.03±0.34
-
0.01
1.65±0.6
1.76±0.77
-
0.18
Total Testosterone (ng/mL) 69.1±25.1 46.3±18.5
46.7±21.2
-
0.04
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Free Testosterone (ng/mL) 2.6±1.57
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S1083-3188(18)30159-1
DOI:
10.1016/j.jpag.2018.02.126
Reference:
PEDADO 2209
To appear in:
Journal of Pediatric and Adolescent Gynecology
Received Date: 16 November 2017 Revised Date:
18 January 2018
Accepted Date: 10 February 2018
Please cite this article as: Kocaay P, Siklar Z, Buyukfirat S, Berberoglu M, The Diagnostic Value of AntiMullerian Hormone in Early Post Menarche Adolescent Girls with Polycystic Ovarian Syndrome, Journal of Pediatric and Adolescent Gynecology (2018), doi: 10.1016/j.jpag.2018.02.126. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title of article: The Diagnostic Value of Anti-Mullerian Hormone in Early Post Menarche Adolescent Girls with Polycystic Ovarian Syndrome
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Authors:
Pınar Kocaay, MDa; Zeynep Siklar, MDa; Sema Buyukfiratb, Merih Berberoglu, MDa
Department of Pediatric Endocrinology, Ankara University School of Medicine, Ankara, Turkey
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a
b
Turkey
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Department of Endocrinology Laboratories, Ankara University School of Medicine, Ankara,
Financial Support and Disclaimers: Financial support was provided by Ankara University
Pınar Kocaay, MD
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Corresponding author:
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BAPRO, grant number: 13H3330007
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Hoşdere caddesi, Aziziye mahallesi, Refik Belendir sokak, 69/11 Cankaya/Ankara, Turkey Phone: +90 505 720 2569
Email address: [email protected]
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Abstract: Study Objective: Polycystic ovarian syndrome (PCOS) is a common endocrine disorder
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characterized by hyperandrogenism and chronic anovulation, which affects 5-10% of reproductive age females. Diagnosis of adult patients with PCOS is made easily with clinical and laboratory methods and the antimullerian hormone (AMH) level are accepted as a good indicator. However, there is still no complete consensus on the diagnosis of PCOS in
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adolescents.
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Design and Settings: Prospective cohort study, December 2013 to November 2014 Participants: The study was conducted on adolescent girls with oligomenorrhea, with at least two years since menarche. The study group consisted of adolescent girls with complete PCOS and incomplete PCOS. A control group was formed of healthy adolescent girls. Complete PCOS
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was diagnosed according to the Rotterdam criteria, as the presence of all the following characteristics; oligomenorrhea, hyperandrogenism, and polycystic ovarian morphology (PCOM) on ultrasound image. Incomplete PCOS was accepted as “oligomenorrhea and PCOM”, or
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“oligomenorrhea and hyperandrogenism”.
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Interventions and Main Outcome Measures: All patients underwent a physical examination and the anthropometric assessments, insulin resistance, and acanthosis nigricans were recorded. It was also noted whether or not the patient had an acne score The Ferriman-Gallwey (FG) score was applied to evaluate hirsutism. Result: The results of this study showed no statistically significant difference was found between the PCOS and incomplete PCOS groups and the control group in respect of AMH levels.
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Conclusion: The use of adult specific diagnostic methods in adolescence may result in an incomplete diagnosis and inadequate treatment plan. Although the serum AMH level clearly
still controversial and further studies are needed.
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facilitates the diagnosis of PCOS, the use of the AMH level in adolescence in PCOS diagnosis is
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Key words: Polycystic ovarian syndrome, Antimullerian hormone, adolescence, diagnosis
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Introduction Polycystic ovarian syndrome (PCOS) is a common endocrinological disorder which affects 5-
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10% of reproductive age females, and is characterized by hyperandrogenism and chronic anovulation. It can cause infertility problems and may also be seen with some metabolic conditions such as obesity, type 2 diabetes mellitus (DM), hypertension, dyslipidemia, and cardiovascular disorders.1 Stein and Leventhal first described this syndrome in 1935 in women
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changed many times since then (Table 1).2-5
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with amenorrhea, hirsutism and bilateral polycystic ovary, and the diagnostic criteria have
The diagnostic criteria for adults have generated several phenotypes of PCOS. Phenotype 1 is classical PCOS as “Hyperandrogenism+ Oligomenorrhea+ Polycystic ovary morphology”. Phenotype 2 is classified as Hyperandrogenism+ Oligomenorrhea, Phenotype 3 (ovulatory
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PCOS) as Hyperandrogenism+ Polycystic ovary morphology, and Phenotype 4 (nonhyperandrogenic PCOS) as Oligomenorrhea+ Polycystic ovary morphology.6,7 In adolescents, these broad definitions may be problematic6,7 because these PCOS criteria may also be seen
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physiologically in adolescence, so PCOS diagnosis in this age group may present some difficulties. Exaggerated pubertal symptoms in this period can correspond to PCOS diagnosis.
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The common features of adult hyperandrogenism are less reliable in adolescent girls than women. In addition, menstrual irregularity, hirsutism and acne can be temporary without progression. This situation causes difficulties in diagnosis and does not present an accurate prevalence. Even with regular cycles after the first two years following menarche, there can be asymptomatic anovulation in half of adolescents. Multifollicular ovaries may be present in adolescents which might not be distinguishable from PCO morphology. It may also not be
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possible to perform transvaginal ultrasonography in many adolescents and thus screening is challenging especially in obese adolescents.7,8
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Therefore, in the 2012 consensus meeting in Amsterdam, it was stated that it was necessary for all three of the Rotterdam criteria to be met for a diagnosis of PCOS to be made in adolescents.5 However, there may be patients who present with menarche more than two years previously and
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trying to fulfill the three criteria in these cases can cause diagnostic errors. Furthermore, there may be adolescent cases who do not meet all the criteria, but complain of hyperandrogenism and
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menstrual irregularity. In some cases, the period of hyperandrogenism may be short and therefore does not meet the Ferriman–Gallwey (FG) score of 8.
With an accurate diagnosis of PCOS, the existing risk can be reduced and treatment can be given to prevent future infertility, metabolic syndrome, type 2 DM and cardiovascular disorders.9
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However, over-diagnosis may lead to unnecessary treatment and psychological stress in adolescents. Anti Mullerian Hormone (AMH) is a supportive marker in the diagnosis of PCOS. AMH is a protein of dimeric glycoprotein structure, which is secreted from ovarian small antral
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and preantral cells of granulosa cells from the 36th week of gestation11 The secretion is independent of gonadotropins and the menstrual cycle. It is related with antral follicle number
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and is therefore a good marker of ovarian reserve and function.10 Although there have been many studies of AMH levels in adults, there are conflicting data about the adolescent period. In addition, adolescent girls who do not meet all the diagnostic criteria for PCOS but are evaluated as at risk could carry metabolic risk factors and therefore require early diagnosis. In this study, the AMH levels in confirmed PCOS cases which met the 3 Rotterdam diagnostic criteria and cases with PCOS which met 2 of the 3 diagnostic criteria and had oligomenorrhea,
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were compared with the AMH levels of a control group. From these comparisons, it was evaluated if AMH levels could be a used as a marker in adolescent cases of PCOS.
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Materials and Methods This study was conducted on adolescent females who presented at Ankara University School of Medicine, Department of Child Health and Diseases, Division of Pediatric Endocrinology,
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adolescent clinic between December 2013 and November 2014.
All subjects were informed of the study protocol and written informed consent was obtained
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from their parents. All authors complied with the World Medical Association Declaration of Helsinki regarding the ethical conduct of research involving human subjects. In all cases, menarche age was recorded, and cases were included if menarche was at least 2 years previously. Family history of PCOS, hyperandrogenism, obesity, hypertension,
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dyslipidemia, and type 2 DM was also questioned. Patients with non-classical congenital adrenal hyperplasia were excluded from the study.
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The study group comprised adolescent females aged 14.3-17.2 years, with PCOS of either a complete or incomplete phenotype. A control group was formed of healthy adolescents aged
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13.5-17.6 years who agreed to participate in the study. The diagnosis of PCOS was made according to the revised Rotterdam criteria.5 Patients with all 3 criteria were evaluated as complete PCOS (Group 1) and patients with 2 of the PCOS criteria as incomplete PCOS (Group 2). Within Group 2 subgroups were formed as those with oligomenorrhea and PCOM categorized as Group 2a, and those with oligomenorrhea and clinical or laboratory-evaluated hyperandrogenism as Group 2b,
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Group 1: Oligomenorrhea + PCO morphology + hyperandrogenism Group 2a: Oligomenorrhea + PCO morphology
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Group 2b: Oligomenorrhea + hyperandrogenism The physical examination and anthropometric assessments of all cases were made by one of the researchers (PK). Findings of acne and acanthosis nigricans were recorded. Hyperandrogenism
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was clinically evaluated using the FG score. According to the FG method hair distribution in a total of 9 areas (upper lip, chin, chest, upper and lower parts of the back, lower and upper
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abdomen, upper part of arms and legs) is scored from 0 to 4 points. A score of >8 is considered as hirsutism.13 Oligomenorrhea was accepted as <8 cycles in 1 year)14,15 and amenorrhea as the absence of cycles for >3 months. BMI was calculated with the formula of body weight (kg) / height (m2).16 The weight to height percentage was calculated using the formula: the patient's
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measured BMI (kg /m2) / required BMI of the patient x 100. A value within the range of 90-110 was considered normal, 110-120 as overweight, >120 as obese, and >140 as morbidly obese.17 The blood pressure measurements were evaluated using the Tumer standards and appropriate age
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and gender norms. The average systolic or diastolic BP indicated HT if it was ≥95th percentile
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for adjusted age, gender and height percentile for healthy children.18 Blood samples were taken between 08:00-10:00 after 10-12 hours of fasting during the early follicular phase (days 3-5 of the menarcheal period) for examination of fasting blood glucose, lipid profile, prolactin, LH, FSH, E2, testosterone, 17-OH progesterone, and DHEAS. Biochemical assessments were made in the biochemistry and endocrinology laboratories of Ankara University School of Medicine. Total testosterone over 55 ng/mL, and free testosterone over 3 ng/mL was evaluated as hyperandrogenism.13,19
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Serum AMH was measured using a double antibody enzyme linked immunosorbent assay kit. Following collection and processing, serum samples for AMH were stored at -20° C until hormone analyses were performed. All serum AMH was performed in the same laboratory using
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the same assay. The results were expressed as ng/mL. The lower limit of detectability for this assay is 0.08 ng/mL (range 0.08 ng/mL to 20ng/mL). The intra and interassay coefficients of variation were 10% and 12% respectively. The sensitivity of this assay was defined as the lowest
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protein concentration that could be differentiated from zero. It was determined by subtracting two standard deviations to the standard replicates and calculating the corresponding
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concentration.
Ultrasonography was performed in a single center by a single expert. Ovarian and uterine size and polycystic ovarian morphology (PCOM) were evaluated. PCOM was accepted as the following conditions: 2-9 mm in diameter, with 12 or more follicles and/or increased ovarian
Statistical Method
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volume (>10 mL).20 Ovarian volume was calculated as 0.52 x length x width x thickness.
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Data analysis was applied using SPSS Windows 15 software. Descriptive statistics were stated as
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mean ±standard deviation (SD) and percentage (%). The Mann-Whitney U test was used to assess the significance of differences between means in two groups. When there were more than two groups, the significance of difference between the means was analyzed with ANOVA variance analysis while the significance of medians was analyzed with the Kruskal Wallis test. Nominal variables were analyzed with Pearson Chi-square or Fisher Exact tests.
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The correlation between continuous variables was analyzed with the Spearman correlation test for variables not showing normal distribution while the Pearson correlation test was used for
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normally distributed variables. A value of p<0.05 was accepted as statistically significant. Results:
The study was conducted in the adolescent clinic of Pediatric Endocrinology between December
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2013 and November 2014. A total of 53 cases were diagnosed with complete or incomplete PCOS and were compared with 55 healthy, age-matched adolescent girls as a control group.
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Within the study groups, Group 1 comprised 29 cases (complete PCOS); Group 2a, 16 cases, and Group 2b, 8 cases (incomplete PCOS). All the patients in the study groups had complaints of irregular menstruation. The second most frequent complaint was hirsutism (n: 27/53, 50.9%). Acne was reported in 2 cases (3.7%), and hair loss in 1 case (1.8%). The mean post-menarche
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period was 3.1±1.08 years for all cases. No statistically significant difference was determined between the study and control groups in respect of the post-menarche period (p: 0.301). Family history revealed that 28% (15/53) of first degree relatives of the patients had obesity,
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24% (13/53) had PCOS, 5.6% (3/53) had Type 2 DM and 12% (7/53) had dyslipidemia,
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hypertension or cardiovascular disease. The mean age on admission was 15.54±1.32 years in the study group, 15.55±1.33 years in complete PCOS group, 15.79±0.97 years in incomplete PCOS group, and 15.4±1.45 years in the control group. No significant difference was determined between the groups in respect of mean age (p: 0.485). The mean height SD of the whole study group was 0.25±0.83 SD. There was no significant difference in height SDS between the groups (p: 0.393) (Table 2). The frequency of obesity and overweight was determined as 51% (27/53) and 11.3% (6/53) respectively in the
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PCOS groups. In the control group, 43.6% (24/55) of the cases were of normal weight, 25.4% (14/55) were overweight, and 30.9% (17/55) were obese. There was no significant difference
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between RBMI between the groups (p: 0.557). Physical examination revealed that 22 (41%) cases in the study group had acanthosis nigricans. Serum DHEAS and free testosterone levels within the PCOS groups were similar. The total testosterone level of Group 1 was higher than that of Group 2a (p: 0.03), and Group 2b (p: 0.001)
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(Table 4). Serum 17-OH progesterone level was higher in Group 1 compared to Group 2a, with
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no significant difference between the other groups. FSH, LH, estradiol, fasting blood glucose, fasting insulin, total testosterone, HDL, LDL, and VLDL levels were similar in all the groups. No correlation was determined between AMH level and other laboratory parameters or between AMH level and RBMI (Table 3). There was no significant difference between the control group
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and all the PCOS groups in respect of AHM levels and no significant difference in AMH levels between the PCOS groups (Table 4). Discussion:
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In adults, the diagnosis of PCOS can be made easily with clinical and laboratory parameters and
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AMH levels are accepted as a good indicator. However, in the adolescent age group, there are still controversies about PCOS diagnosis. Using adult diagnostic procedures for adolescents may result in under-diagnosis, fail to make a diagnosis or over-diagnosis. During the first years following menarche in adolescent girls, anovulatory cycles are a physiological occurrence and polycystic ovary morphology may be seen on ultrasound and considered as signs of PCOS. Another error at this time is seeing acne as a sign of PCOS rather than a physiological finding. Over-diagnosis of PCOS can have negative effects both physiologically and psychologically.
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The aim of this study was to determine the AMH levels in patients with complete PCOS where all 3 diagnostic criteria are met and incomplete PCOS with 2 of the 3 criteria and to compare these with the levels in a control group. The results showed no statistically significant differences
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between either PCOS groups and the control group or between the different PCOS groups in respect of serum AMH levels.
Serum AMH levels can be affected by some parameters. The relationship between serum AMH
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levels and androgen levels is a matter of controversy. While some studies have shown a positive
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relationship between serum AMH levels and androgen levels in cases with PCOS, others have not.21-23 Serum androgen levels are reported to be more related with AMH levels and not with intraovarian androgen levels because of the inhibition of aromatase by AMH.24 In the current study no relationship was found between these parameters. This can probably be attributed to milder hyperandrogenism in the current study cases or that the hyperandrogenism period was
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still short and the FG score might not have reached 8. The laboratory values for hyperandrogenism have been derived from studies based on adults. There are no reliable assays with well-defined normal ranges for the documentation of hyperandrogenism in adolescence and
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accurate determinations of testosterone levels are often problematic.7
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As a significant number of PCOS cases are obese, it may not always be possible to obtain efficient results from suprapubic ultrasound. Transvaginal ultrasound cannot be applied to virgin adolescents. Furthermore, there is no exact value for the volume of normal ovaries during adolescence. In adults, either 12 or more follicles measuring 2-9 mm in diameter, and/or increased ovarian volume (greater than 10 mL) are considered as PCOM.8 To date, some studies have accepted different volumes between 7.8-9.6 mL as maximum volume in the adolescent
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period.6,25 Therefore, during adolescence, the diagnostic increase of ovarian volume for PCOS could be >10 mL.6
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In many studies a positive correlation has been found between serum AMH levels and ovarian volume.21,26 The reason behind this is the increased ovarian volume which is one of the criteria of PCOS. In the current study, ovarian volume of ≥10 mL was accepted as PCO. Thus controversial evaluations were excluded. PCO morphology in the current study was evaluated by follicle
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number and ovary volume but no relationship was found with either. This could be because
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many patients in this study were obese and could not be evaluated clearly with ultrasound or because of an insufficient number of cases.
Obesity, which is frequently associated with PCOS, is another factor that may have an effect on serum AMH levels27. There may be a negative correlation between AMH levels and obesity, but
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in the current study, no such relationship was found. Furthermore, there was no difference between obese cases with PCOS and obese cases without PCOS in respect of serum AMH levels. This situation might be attributable to the equal number of obese and overweight cases in both
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the study and control groups.
A positive correlation has been previously determined between cases with PCOS and serum
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AMH levels28, although this difference has not been observed in some studies. It has been considered that the reason for different results could originate from differences in age and ethnicity of the cases in the studies. The current study cases were in the early menarcheal period which could explain why there were no differences between the groups and the control patients. The differences may appear over a longer time.
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The cutoff value for AMH in the diagnosis of PCOS is controversial, as different kits have been used for AMH measurements. Many studies have been conducted to make PCOS diagnosis with precise AMH levels, and many authors think AMH levels are useful for the diagnosis of
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PCOS.11,21,28,29 Cutoff values for AMH ranging from 2.8 ng/mL-10.7 ng/mL have been suggested in those studies.11,30,31 In a recent meta-analysis, the cutoff value for AMH levels was accepted as 4.7 ng/mL.32 In some studies, it has been shown that there can be an overlap of AMH levels in
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PCOS cases and control group patients and has therefore been claimed that it cannot be used as an independent marker. In a study of 207 PCOS cases, it was shown that the AMH value had low
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sensitivity, specificity and a positive predictive value according to both NIH and the Rotterdam criteria.33 Explanations for these low values included selection bias, and increased prevalence of menstrual irregularities in adolescents.
In a study by Pigny et al, the cutoff value was accepted as 8.4 ng/mL with 92% specificity and
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67% sensitivity.34,35 In another study, the cutoff value was accepted as 8 ng/mL with 70% specificity and 61.7% sensitivity.29 In the current study, serum AMH was measured using a double antibody enzyme linked immunosorbent assay kit. However, no cut off value was
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determined as there was no significant difference between the groups in respect of AMH levels. Previously reported differences may have arisen from ethnic differences between study
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groups.11,29-31
There may be a correlation between serum AMH level and age, as is thought that AMH level is the marker of ovarian age36,37 and serum AMH levels are known to decrease with age. However, this decrease is slower in cases with PCOS compared to cases without PCOS.29 In the current study; the mean age of patients was 15.54 years, so no aging effect on serum AMH levels was expected.
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The reason for the low specificity and sensitivity of the results of AMH levels for PCOS diagnosis is the menstrual irregularity that can be frequently seen in adolescents. During the early reproductive years, menstrual irregularity may be a normal reflection of anovulatory
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cycles.38 Over-diagnosis of PCOS has been attributed to inadequate identification of adolescent polycystic ovarian morphology on ultrasound images even though it is clearly identifiable for adults. A large multi-follicular ovarian structure in adolescence has been indicated as normal in
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many studies.39
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To date, AMH levels have been reported to not change with the menstrual cycle.31,40 However, it has been recently reported that estradiol expresses AMH secretion within the in vitro environment.41 It can be thought that AMH levels may be low in the middle of the cycle. Low and similar AMH levels within the current study groups may have been due to the collection of blood specimens in the middle of the cycles and the number of cases may not have been
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sufficient to show a difference.
In conclusion, the serum AMH levels were found to be similar in the PCOS groups and the
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control group. During adolescence, especially at an early post-menarcheal age, the use of AMH levels as a diagnostic tool for PCOS is still controversial and more studies on this topic are
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References 1. Witchel SF: Hirsutizm and polycstic ovary syndrome. In: Pediatric Endocrinology, (5th ed.). Edited by Lifshitz F. New York, Informa Healthcare USA Inc, 2007, pp 325-348.
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2. Stein IF, Leventhal ML: Amenorrhea associated with bilateral polycystic ovaries. Am J Obstet Gynecol 1935; 29:181. 3. Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril. 2004; 81(1):19-25. 4. Azziz R, Carmina E, Dewailly D, Diamanti-Kandarakis E, et al: Task Force on the Phenotype of the Polycystic Ovary Syndrome of The Androgen Excess and PCOS Society. The Androgen Excess and PCOS Society criteria for the polycystic ovary syndrome: the complete task force report. Fertil Steril. 2009;91(2):456-88.
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5. Amsterdam ESHRE/ASRM-Sponsored 3rd PCOS Consensus Workshop Group. Consensus on women's health aspects of polycystic ovary syndrome (PCOS). Hum Reprod 2012; 27:14. 6. Carmina E, Oberfield SE, Lobo RA: The diagnosis of polycystic ovary syndrome in adolescent. Am J Obstet Gynecol 2010; 203:201. 7. Rosenfield RL. The Diagnosis of Polycystic Ovary Syndrome in Adolescents. Pediatrics. 2015; 136(6):1154-65. 8. Balen AH, Laven JS, Tan SL, et al: Ultrasound assessment of the polycystic ovary: international consensus definitions. Hum Reprod Update 2003; 9:505. 9. Fauser BC, Bouchard P: Uncertainty remains in women with PCOS regarding the increased incidence of cardiovascular disease later in life, despite the indisputable presence of multiple cardiovascular risk factors at a young age. J Clin Endocrinol Metab 2011; 96:3675. 10. Weenen C, Laven JS, Von Bergh AR, et al: Anti-müllerian hormone expression pattern in human ovary: potential implications for initial and cyclic follicle recruitment. Mol Hum Reprod 2004; 10:77. 11. Eilertsen TB, Vanky E, Carlsen SM: Anti-mullerian hormone in the diagnosis of polycystic ovary syndrome: can morphologic description be replaced? Hum Reprod 2012; 27:2494. 12. Weenen C, Laven JS, Von Bergh AR, et al: Anti-müllerian hormone expression pattern in human ovary:potential implications for initial and cyclic follicle recruitment. Mol Hum Reprod 2004; 10:77. 13. Roe AH, Prochaska E, Smith M, et al: Using the androgen excess-PCOS society criteria to diagnose polycystic ovary syndrome and the risk of metabolic syndrome in adolescents. J Pediatr 2013; 162:937. 14. Matthews DR, Hosker JP, Rudenski AS, et al: Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985; 28:412. 15. ACOG Committee Opinion. Menstruation in girls and adolescents: using the menstrual cycle as a vital sign. Obstet Gynecol 2006; 108:1323. 16. Kuczmarski RJ, Ogden CL, Grummer-Strawn LM, et al: 2000 CDC growth charts for the United States: methods and development. Vital Health Stat 2002; 11:1. 17. Poskitt EM: Defining childhood obesity; the relative body mass index (RBMI). European Childhood Obesity Group. Acta Paediatr 1995; 84:961.
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18. National High Blood Pressure Education Program Working Group on High Blood Pressure in Children and Adolescents: The fourth report on the diagnosis, evaluation, and treatment of high blood pressure in children and adolescents. Pediatrics 2004; 114:555. 19. Ferriman D, Gallwey J: Clinical assessment of body hair growth in women. Clin Endocrinol 1961; 21:1440. 20. Barber TM, Alvey C, Greenslade T, et al: Patterns of ovarian morphology in polycystic ovary syndrome: a study utilising magnetic resonance imaging. Eur Radiol 2010; 20:1207. 21. Pigny P, Merlen E, Robert Y, et al: Elevated serum level of anti mullarian hormone in patients with polycystic ovary syndrome: relationship to the ovarian follicle excess and to the follicular arrest. J Clin Endocrinol Metab 2003; 88:5957. 22. Siow Y, Kives S, Hertweck P, et al: Serum mullerian inhibiting substance levels in adolescent girls with normal menstrual cycles or polycystic ovary syndrome. Fertil Steril 2005; 84:938. 23. La Marca A, Orvieto R, Giulini S, et al: Mullerian inhibiting substance in women with polycystic ovary syndrome: relationship with hormonal and metabolic characteristics. Fertil Steril 2004; 82:970. 24. Park AS, Lawson MA, Chuan SS, et al: Serum anti Mullerian hormone concentrations are elevated in oligomenorrheic girls without evidence of hyperandrogenism. J Clin Endocrinol Metab 2010; 95:1786. 25. Chen Y, Yang D, Li L, et al: The role of ovarian volüme as a diagnostic criterion for Chinese adolescents with polycystic ovary syndrome. J Pediatr Adolesc Gynecol 2008; 21:347. 26. Van Rooij IA, Broekmans FJ, te Velde ER, et al: Serum anti Mullerian hormone levels: a novel measure of ovarian reserve. Hum Reprod 2002; 17:3065. 27. Nicandri KF, Hoeger K: Diagnosis and treatment of polycystic ovarian syndrome in adolescent. Curr Opin Endocrinol Diabetes Obes 2012; 19:497. 28. Laven JS, Mulders AG, Visser JA, et al: Anti müllerian hormone serum concentration in normoovulatory and anovulatory women of reproductive age. J Clin Endocrinol Metab 2004; 89:318. 29. Lin Li, Xiaoli Chen, Yaqin Mo, et al: Elevated serum anti müllerian hormone in adolescent and young adult Chinese patients with polycystic ovary syndrome. Wien Klin Wochenschr 2010; 122:519. 30. Acian P, Quereda F, Matallin P, et al: Insulin, androgens, and obesity in women with and without polycytic ovary syndrome: a heterogeneus group of disorders. Fertil Steril 1999; 72:32. 31. Hart R, Doherty DA, Norman RJ, et al: Serum antimullarian hormone (AMH) levels are elevated in adolescent girls with polycystic ovaries and polycystic ovarian syndrome (PCOS). Fertil Steril 2010; 94:1118. 32. Iliodromiti S, Kelsey TW, Anderson RA, et al: Can anti Mullerian hormone predict the diagnosis of polycystic ovary syndrome? A systematic review and meta- analysis of extracted data. J Clin Endocrinol Metab.2013; 98:3332. 33. Acian P, Quereda F, Matallin P, et al: Insulin, androgens, and obesity in women with and without polycytic ovary syndrome: a heterogeneus group of disorders. Fertil Steril 1999; 72:32. 34. Lin L, Yang DZ, Chen XL, et al: Clinical and metabolic features of polycystic ovary syndrome. Int J Gynecol Obstet 2007; 97:129.
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35. Al-Qahtani A, Muttukrishna S, Appasamy M, et al: Development of a sensitive enzyme immunoassay for anti-mullarian hormone and evaluation of pottential clinical applications in males and females. Clin Endocrinol (Oxf) 2005; 63:267. 36. Visser JA, De Jong FH, Laven JS, et al: Anti-Müllerian hormone: a new marker for ovary function. Reproduction 2006; 131:1. 37. de Vet A, Laven JS, de Jong FH, et al: Antimüllerian hormone serum levels: a putative marker for ovarian aging. Fertil Steril 2002; 77:357. 38. Adams Hillard PJ: Adolescent menstrual health. Pediatr Endocrinol Rev 2006; 3:138. 39. Sampalo P, Livieri C, Montanari L, et al: Precocious signs of polycystic ovaries in obese girls. Ultrasound Obstet Gynecol 1994; 4:310. 40. Hagen CP, Aksglaede L, Sorensen K, et al: Serum levels of anti-Mullerian hormone as a marker of ovarian function in 926 healty females from birth to adulthood and in 172 Turner syndrome patients. J Clin Endocrinol Metab 2010; 95:5003. 41. Grynber M, Pierre A, Rey R, et al: Differential regulation of ovarian anti müllerian hormone (AMH) by estradiol through α and β estrogen receptors. J Clin Endocrinol Metab 2012; 97:1649.
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Contributions: Pınar Kocaay made substantial contributions to the conception, design, analysis and
and gave final approval of the version to be published.
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interpretation of data, drafted the article, revised it critically for important intellectual content
Zeynep Siklar made substantial contributions to the conception, design, analysis and
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interpretation of data, drafted the article and gave final approval of the version to be published. Sema Buyukfirat made substantial contributions to the conception, design, analysis and
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interpretation of data.
Merih Berberoglu made substantial contributions to the conception, design, analysis and
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interpretation of data, drafted the article and gave final approval of the version to be published.
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Table Legends Table 1. Diagnostic criteria for PCOS
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Table 2. Clinical characteristics of the cases Table 3. Correlations between serum AMH levels and other laboratory parameters
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Table 4. Laboratory findings of the cases
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Table 1. Diagnostic criteria for PCOS PCOS DEFINITION
HYPERANDROGENISM
+
ROTTERDAM (2003)
+
First ESHRE/ASRM PCOS consensus workshops
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+
+
2 of 3 criteria
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AE-PCOS SOCIETY (2009)
POLYCYSTIC OVARY MORPHOLOGY
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NIH CRITERIA (1990)
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Clinical or Biochemical
OLIGOMENORRHEA
+
+
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AMSTERDAM (2012) Third ESHRE/ASRM PCOS consensus workshops
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1 of 2 criteria +
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All criteria for adolescent
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*Adult Phenotypes: Phenotype 1: “Hyperandrogenism+ Oligomenorrhea+ Polycystic ovary morphology”, Phenotype 2: “Hyperandrogenism+ Oligomenorrhea”, Phenotype 3: “Hyperandrogenism+ Polycystic ovary morphology”, Phenotype 4: “Oligomenorrhea+ Polycystic ovary morphology”
ACCEPTED MANUSCRIPT Table 2. Clinical findings of cases
Incomplete
Control
29
24
55
Age (year)
15.55±1.3
15.79±0.97
15.4±1.45
0.485
Height SDS
0.33±0,95
0.38±0.80
0.14±0.76
0.393
RBMI (%)
121±21.2
122.6±32.6
115±23.5
0.394
3.2±1.1
3.08±1.07
2.8±1.07
0.301
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Post-Menarcheal Age (%)
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Number of patient (n)
p
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Complete
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p value
Age
0.168
0.083
RBMI
-0,108
0.442
LH (mIU/mL)
-0.104
0.457
FSH (mIU/mL)
0.008
0.957
Estradiol (pg/mL)
-0,067
0.635
Total testesterone (ng/mL)
0.144
0.487
Freetestesterone (ng/mL)
0.122
0.745
DHEAS (mcg/dL)
0.07
0.657
0.104
0.432
0.144
0.302
-0.136
0.331
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HDL-kolesterol (mg/dL)
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17 OH progesteron (ng/dL)
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Correlation coefficient
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Variable
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Table 3. The Correlations between serum AMH levels and other laboratory parameters
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Triglyceride (mg/dL)
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Table 4. Laboratory findings of cases
Group 2b
AMH (ng/dL)
7.05±4.72
8.4±4.7
5.3±5.9
LH (mIU/mL)
9.8±7.8
10.9±9.5
12.9±7.4
FSH (mIU/mL)
5.2±1.96
4.9±1.6
4.93±2.1
17 OHP (ng/mL)
1.47±0.77 0.87±0.37
Control
p
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Group 2a
7.0±3.84
0.33
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0.48
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Group 1
0.82
1.03±0.34
-
0.01
1.65±0.6
1.76±0.77
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0.18
Total Testosterone (ng/mL) 69.1±25.1 46.3±18.5
46.7±21.2
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0.04
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Free Testosterone (ng/mL) 2.6±1.57
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