AMH as predictor of premature ovarian insufficiency:

A longitudinal study of 120 Turner Syndrome patients Stine Aa. Lunding1, Lise Aksglaede2, Richard A. Anderson3, Katharina M. Main4,5, Anders Juul4,5, Casper P.

Hagen4,5,6, Anette T. Pedersen1,5,6

1Department of Gynaecology / Fertility Clinic, Rigshospitalet, University of Copenhagen, Denmark2Department of Clinical Genetics, Rigshospitalet, University of Copenhagen, Denmark3Medical Research Council Centre for Reproductive Health, University of Edinburgh, United Kingdom4Department of Growth and Reproduction and EDMaRC, Rigshospitalet, University of Copenhagen,

Denmark5The Paed-Gyn Endo Transition Clinic at Rigshospitalet, University of Copenhagen, Denmark6These authors contributed equally to the supervision of this study

Abstract: 231

Word count: 3434

Tables: 1

Figures: 4

References: 42

Abbreviated Title: AMH as a predictor of POI in TS patients.

Key terms: Anti-Müllerian hormone, AMH, MIS, Turner Syndrome, premature ovarian insufficiency, POI,

POF, fertility, puberty.

Disclosure statement: The authors have nothing to disclose.

Corresponding author:

Stine Aagaard Lunding

University of Copenhagen

Fertility Clinic, Rigshospitalet

Blegdamsvej 9

DK-2100 Copenhagen O

Denmark

Email: stinelunding@gmail.com

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Abstract

Context: The majority of Turner syndrome (TS) patients suffer from accelerated loss of primordial follicles.

Low circulating levels of anti-Müllerian Hormone (AMH) may predict lack of spontaneous puberty in

prepubertal girls and imminent premature ovarian insufficiency (POI) in TS women with preserved ovarian

function.

Objectives: To evaluate the association between circulating AMH and ovarian status in TS patient.

Design: Longitudinal observational cohort study.

Setting: Tertiary referral centre for paediatric and gynaecologic endocrinology.

Participants: 120 Turner syndrome patients, aged 0 to 48 years.

Main outcome measures: Longitudinal measurements of AMH, FSH, LH, estradiol and inhibin B according

to age, karyotype (45,X; 45,X/46,XX mosaicism; miscellaneous karyotypes) and ovarian status (group 0:

prepubertal; group 1: never ovarian function; group 2: ongoing ovarian function; group 3: loss of ovarian

function).

Results: Ovarian status was highly associated with the TS karyotype; spontaneous puberty: 45,X (3/44

patients), 45,X/46,XX (15/17), miscellaneous (17/42) and POI: 45,X (3/3 patients), 45,X/46,XX (1/15),

miscellaneous (8/17). AMH was associated with ovarian status (e.g. group 1: < 2 pmol/L vs. group 2: 19

pmol/L, p<0.001). AMH < 4 pmol/L (corresponding to < -2SD) predicted absent puberty in prepubertal girls

and POI in adolescent and adult patients.

Conclusion: The majority of women with mosaic karyotype 45,X/46,XX had on-going ovarian function in

early adulthood. AMH < -2SD predicted failure to enter puberty in young TS girls and imminent POI in

adolescent and adult TS patients.

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Introduction

Turner Syndrome (TS) affects 1/2000 new-born females (1,2). The syndrome is caused by X-chromosome

abnormalities with or without mosaicism and is characterized by an increased risk of primary or premature

ovarian insufficiency (POI) due to accelerated loss of germ cells before or after puberty (3–6).

Counselling TS patients about their reproductive function is challenging and depends on the age of the

patient. In prepubertal girls, the hypothalamic-pituitary-gonadal (HPG) axis is centrally inhibited, making the

biochemical assessment of ovarian activity difficult as circulating levels of reproductive hormones are

extremely low or undetectable (7). In adolescents and in adults, POI can be evaluated clinically and

biochemically with the classic combination of amenorrhea and elevated FSH concentrations

(hypergonadotropic hypogonadism) (8,9). However, in postpubertal adolescents and adult women,

reproductive hormones may remain within the normal range before POI is clinically evident, despite

significant depletion of the ovarian reserve (10–12). Very little is known about the timing and onset of POI

in TS patients with initially normal ovarian function, which makes it difficult to predict their fertility

prognosis.

The specific TS karyotype can to some degree predict the potential ovarian function in Turner patients, as

monosomic patients (45,X) usually are born with streak gonads, whereas those with 45,X/46,XX mosaicisms

have the best chance of spontaneous puberty and fertility (5,13,14). However, the karyotypes determined

from peripheral blood lymphocytes or skin biopsies lack specificity and sensitivity due to the unknown

degree of gonadal mosaicism (15).

In both prepubertal girls and adult women, anti-Müllerian hormone (AMH) is produced by small antral

follicles (16,17). In healthy adult women, circulating AMH reflects the number of primordial follicles (18)

and is therefore predictive of the reproductive lifespan (19–21). In girls and adolescents, AMH varies

markedly between individuals (reference ranging from 5 to 60 pmol/L), but overall each girl maintains her

AMH level through childhood and adolescence (22,23). In cross sectional studies, serum AMH is a sensitive

and specific marker of ovarian function in adolescent Turner syndrome patients (22,24,25).

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In this longitudinal study of 120 Turner Syndrome patients, we aimed to assess the predictive value of AMH

for spontaneous puberty in prepubertal TS girls and imminent POI in adolescent and adult TS patients.

Material and Method

TS patients

All TS patients (n=137) identified by ICD-10 codes DQ96-DQ96.9 were recruited from the Department of

Gynaecology, the Department of Growth and Reproduction and the Paed-Gyn Endo Transition Clinic at

Rigshospitalet, University of Copenhagen, Denmark. Seventeen of 137 patients were excluded from the

study due to gonadectomy (n=7), age above 50 years (n=4), or lack of medical record information (n= 6),

leaving 120 patients for further evaluation.

Clinical and biochemical information were retrieved from patient files. Circulating AMH measurements

were available from 2009-2014 in a total of 101 TS patients (81 %). Longitudinal samples ( 2 samples)

were available in 73 patients (median 3 samples, range 2-13 per patient). Data from 71 patients including

longitudinal FSH, LH, inhibin B and estradiol and single measurement of AMH, have been reported

previously (7,22)

Karyotypes

Diagnosis of TS was confirmed by G-band karyotyping of blood lymphocytes, including counting of at least

10 metaphases, three of which were fully analysed. All karyotypes in the present study were validated by the

same clinician (LA). According to their karyotype, the TS patients were divided into three groups: 45,X (n=

48), 45,X/46,XX (n= 26) and miscellaneous without Y chromosome material (n= 46)

Ovarian status

Regardless of karyotype and AMH, the 101 TS patients with AMH measurements were categorized into four

ovarian function status groups, evaluated by pubertal breast stage (Tanner’s classification (26)), occurrence

of spontaneous menarche, menstrual bleeding pattern, serum levels of estradiol, FSH and LH, and hormone

replacement therapy (HRT):

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Group 0. Prepubertal (n = 11): No spontaneous puberty (Tanner = 1) and age < 12 years. No HRT.

Group 1. Never ovarian function (n = 55): Age > 12 years and no spontaneous puberty. Puberty and

menarche induced by HRT.

Group 2. Ongoing ovarian function (n = 23): Spontaneous pubertal onset (Breast Tanner ≥ 2) and/or

spontaneous menarche and/or regular menstruation. No indications for HRT. Two patients with uterine

agenesis (both 45,X/46,XX) were categorized in this group as FSH, LH, inhibin B and estradiol were in the

reference range.

Group 3. Loss of ovarian function (n = 12): Spontaneous puberty and/or menarche followed by cessation of

pubertal development and/or secondary amenorrhea and hypergonadotropic hypogonadism. HRT indicated.

Hormone assays

Blood samples were taken as part of the clinical follow-up on the patients. The AMH assay has been

available for clinical use at Rigshospitalet, Copenhagen University Hospital since January 2009. All samples

were withdrawn from an antecubital vein, clotted, centrifuged and analysed. Serum AMH levels were

determined using the Beckman Coulter enzyme immunometric assay (IOT, Immunotech, Beckman Coulter

Ltd., Marseilles, France) with a detection limit of 2.0 pmol/l. The intra-assay coefficients of variation (CVs)

were less than 7.8% and 5.4% at 13 and 123 pmol/l, respectively. Inter-assay CV was less that 11.6% and

10.9% at 19 and 99 pmol/L, respectively.

The age specific reference range of AMH was based on serum samples from 926 healthy females aged 0-69

years (22).

Serum FSH and LH were measured by time-resolved immunofluorometric assays (Delfia; PerkinElmer,

Boston, MA) and by electrochemiluminescence immunoassays (Roche Diagnostics, Mannheim, Germany),

respectively .The detection limits from both assays were; FSH < 0.1 IU/L and LH < 0.1 IU/L. Intra-assay and

inter-assay CVs from both assays were < 5% for FSH and LH.

Between 1990 and 2010, serum inhibin B was measured using one of two double antibody enzyme

immunometric assays (Inhibin B DSL or Oxford Bio-Innovation Inhibin B), both with a detection limit of 20

pg/ml and intra- and interassay CV < 16%. From 2010, inhibin B was measured using the Beckman Coulter

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Inhibin B genII assay, with a detection limit of 3 pg/ml and intra- and interassay CV < 11%. The old and

new inhibin B methods were compared extensively before changing assays, and showed similar results and

no correction factor was needed. Estradiol was measured by RIA (Pantex, Santa Monica, CA; before 1998

distributed by Immuno Diagnostic Systems, Bolton, UK) with a detection of 18 pmol/L, and intra- and

interassay CVs < 8% and <13%, respectively. Testosterone was measured by radioimmunoassay (RIA)

(Coat-a-count, Diagnostic Products, Los Angeles, CA) with a detection limit of 0.23 nmol/L and intra- and

interassay CV both < 10%.

Statistics

Pearson’s chi-squared test was used to assess if the prevalence of spontaneous puberty depended on

karyotype.

Differences in AMH levels between groups of TS patients (according to karyotype or ovarian status) were

assessed by Mann-Whitney U test (2 groups) and Kruskall Wallis test (3 groups). Each patient contributed

with a single AMH value, using the mean value if repeated measurements were available.

Intra-individual coefficients of variation (CV = SD /mean x 100%) were calculated.

To evaluate AMH as a predictor of spontaneous puberty, we described repeated AMH measurements from

15 patients prior to pubertal onset; spontaneous puberty (n=5) vs. HRT induced puberty (n=10). To evaluate

AMH as a predictor of POI, we used the last AMH measurement prior to POI in adolescents and adults who

later experienced loss of ovarian function. In addition, we visualized all individual AMH values before and

after POI. We further evaluated AMH as a marker of POI in patients > 12 years. Each patient contributed

with a single AMH value, using the mean value if repeated measurements were available. A receiver

operating characteristic (ROC) curve was established, providing the AMH cut-off with the best combination

of sensitivity and specificity. Patients were divided in two groups; A) POI: patients with “Never ovarian

function” (Group 1) or “Loss of ovarian function” (Group 3) using AMH values measured after onset of POI;

B) Ovarian function: patients with “Ongoing ovarian function” (Group 2).

The study was approved by The Danish Data Protection Agency (2014-41-2699).

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Results

Description of the TS cohort

Descriptive characteristics of the TS cohort according to karyotypes and ovarian function are shown in Table

1. The prevalence of spontaneous puberty was significantly higher in patients with miscellaneous karyotypes

(17/42) than with karyotype 45,X (3/44) and highest in patients with 45,X/46,XX mosaicism (15/17), p <

0.001.

AMH levels and association with karyotype

45,X

AMH was measured in 39 out of 48 (81%) TS patients with karyotype 45,X (Figure 1A). twenty-seven of

these (69 %) had exclusively undetectable levels of AMH (average of 2 measurements, range 1 – 5). The

four prepubertal girls with 45,X had AMH levels below the normal reference range (max 2 pmol/L in all

samples)

45,X/46,XX mosaicism

AMH was measured in 21 out of 26 (80%) TS patients with 45,X/46,XX mosaicism (Figure 1B), median 16

pmol/L (< 2 – 92). Only three patients had undetectable AMH values. Three of the four prepubertal girls

with 45,X/46,XX had AMH levels within the reference range; median AMH 6 pmol/L (< 2 – 11).

Miscellaneous karyotypes

AMH was measured in 41 out of 46 (89%) TS patients with miscellaneous karyotypes (Figure 1C). Eight

patients (20 %) had AMH within the normal reference range; karyotypes; 45,X/46,XX/47,XXX (n = 2),

45,X/47,XX, 45,X/46,X,r(X), 46,X,del(X) (n =2), 45,X/46,X,del(X) (n = 2).

The three prepubertal girls with miscellaneous karyotypes had AMH levels below the reference range (max

2 pmol/L in all samples).

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AMH was higher in TS patients with mosaicism (45,X/46,XX) compared with patients with 45,X (16 pmol/L

(<2 – 92) vs. <2 pmol/L (<2 – 2), p<0.001) Figure 1). In the majority of TS patients, AMH levels remained

relatively stable during follow up: i.e. the median intra-individual CV was 21% (0 – 172%).

Eight TS patients were taking oral contraceptives at the time of AMH measurements (marked with black dots

in figure 1).

AMH levels and association with ovarian status

There were 55 patients who never had any clinical signs of ovarian function (group 1) and all but three

patients had AMH levels below the reference range (Figure 2A). The three patients with a single AMH value

in the reference range had elevated FSH serum levels at 102, 69 and 42 IU/L, respectively.

Twenty-three out of 35 TS patients with spontaneous puberty had ongoing ovarian function (66%) (group 2)

during the study period (Figure 2B), and one TS patient conceived naturally (45,X/46,XX). The karyotype

distribution for the 23 patients with ongoing ovarian function was 45,X: 4%, 45,X/46,XX: 65% and

miscellaneous 31%. All but two of these 23 patients had AMH within the reference range; median 19 pmol/L

(<2 – 92) (Figure 2B). In 17/23 patients, AMH was stable or slightly increasing. AMH declined in the

remaining 5 patients; median (range) decline of AMH was 20 (3 – 79) pmol/L during a median time period

of 1.8 years (0.2 – 3.6).

AMH was markedly lower in women who never had ovarian function compared with women with ongoing

ovarian function; AMH < 2 pmol/L (range <2 – 8) vs. 19 pmol/L (range < 2 – 92), p<0.001 (Figure 2A vs.

2B).

AMH as a predictor of pubertal onset

AMH prior to pubertal onset was available in 15 patients. In 10 of these 15 patients puberty was induced

with HRT, and AMH was detectable in 3 of these 10 girls, median (range) < 2 (< 2 – 4) pmol/L. AMH was

detectable in 4 of the 5 girls who later had spontaneous pubertal development, mean of repeated individual

AMH measurements 4, 6, 20 and 58 pmol/L, respectively.

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AMH as a predictor of POI

During follow up, 12 TS patients developed POI (group 3) (Figure 2C). Four of these patients lost ovarian

function after onset of spontaneous puberty but before menarche at a median age of 15.6 years (12.7 - 23.1),

while 8 patients had secondary amenorrhea at a median age of 19 years (15.2 – 32.9).

The karyotype distribution for these 12 patients was 25 % (3/12) 45,X, 8 % (1/12) 45,X/46,XX and 67 %

(8/12) miscellaneous karyotypes, respectively

AMH was measured in 5 of the 12 patients prior to cessation of pubertal development or secondary

amenorrhea (POI); median of last AMH value prior to POI: < 2 pmol/L (< 2 – 5) (Figure 3A).

Measurements of serum levels of FSH, LH, estradiol and inhibin B are shown only prior to onset of POI (and

hence prior to HRT) (Figure 3B – E). FSH, LH and estradiol levels prior to POI was 30.6 IU/L (5.86 – 93.7),

9 IU/L (0.5 – 47) and 20 pmol/L (< 18 – 54), respectively. Inhibin B was available in four patients prior to

POI; median < 20 pg/ml (< 20 – 55). Inhibin B decreased to undetectable levels within one year prior to POI

(Figure 3E).

AMH as a marker of POI

Based on the mean of individual AMH measurements from each adolescent and adult patient, the best cut-off

value for AMH as a marker of POI was 3 pmol/L. Both the sensitivity (probability of having POI when

AMH ≤ 3 pmol/L) and the specificity (probability of having ovarian function when AMH > 3 pmol/L) were

95 % (Supplementary Figure 1).

TS patients with high AMH

Seven TS patients had AMH > +2SD. Five of these patients had regular menstrual cycles, one patient

suffered from metrorrhagia and one was born with no uterus.

Ultrasound of the ovaries was described in 5 of the seven patients. Three of the patients had slightly enlarged

ovaries with up to 12 small follicles (< 9 mm diameter) per ovary, but morphology was not classically

PCOS-like with follicles located close to the ovarian cortex. Two patients had normal ovarian morphology.

Serum testosterone was measured in 5 of the 7 patients and found normal with a range from 0.02 to 1.9

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nmol/L. Median (range) LH/FSH ratio was 1.34 (0.25 – 4.22). Pubic hair from the symphysis to the

umbilicus was described in one patient.

Discussion

This unique longitudinal cohort of 120 Turner syndrome patients in a transition setting from paediatrics to

gynaecology allowed us for the first time to evaluate AMH as a predictor of absent puberty and imminent

premature ovarian insufficiency (POI). No prepubertal girls with AMH < 4 pmol/L entered puberty

spontaneously, and in adolescents, AMH < 5 pmol/L (< -2SD) was indicative of imminent POI. ROC

analysis of cross sectional data from adolescent and adult patients confirms that low AMH is indicative of

POI (AMH ≤ 3 pmol/L was a sensitive (95%) and specific (95%) marker of POI). Our present ROC analysis

does not asses the diagnostic value of AMH to predict future POI in adult patients with ongoing ovarian

function, but suggests that, AMH is a valuable predictive tool when counselling prepubertal Turner

Syndrome patients as well as adolescents regarding remaining ovarian function and their future reproductive

lifespan.

Supporting previous studies, we observed that the TS karyotype was a strong predictor for spontaneous

pubertal onset and menarche (15,22,24,25). Due to a potential hidden grade of mosaicism, which is

frequently found in TS patients when analysing extended number of metaphases (27), the predictive value of

karyotypes for POI is poor (28,29).

A subset of TS patients – predominantly those with mosaicism 45,X/46,XX – had ongoing ovarian function

well into early adulthood. The majority of TS women with spontaneous menarche and continuous ovarian

function had stable AMH levels within the reference range during the 5 years of follow up. As in adult

women, AMH seems to reflect the number of small growing follicles in both healthy girls and Turner

Syndrome patients (17,25). Thus, as in healthy adult women, AMH in girls and in TS patients most likely

reflects the number of resting primordial follicles, thus determining the reproductive lifespan.

In our longitudinal analysis, AMH below the reference range was associated with absence of spontaneous

puberty onset in prepubertal girls as well as with imminent POI in adolescents and adults . To explore further

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how AMH reflects ovarian function, we evaluated AMH as a marker (not a predictor) of POI in adolescent

and adult patients. This information is useful when interpreting AMH in prepubertal patients when the

central inhibition of the HPG axis prevents assessment of ovarian function with traditional reproductive

hormones (7). The highly sensitive and specific AMH cut-off (< 3 pmol/L) as a marker of POI in adolescent

TS patients supports that low AMH is reflecting compromised ovarian activity in prepubertal girls. Our

findings are in line with a large European Turner study reporting that the chance of spontaneous pubertal

onset was increased 19 times if AMH was detectable (24). This is also consistent with data from girls with

cancer treated with gonadotoxic chemotherapy and irradiation (30).

Compared with the AMH cut-off reported in a subgroup of TS patients from the present study (8 pmol/L)

(22), the new cut-off based on a larger number of patients is close to the detection limit of the AMH assay (2

pmol/L) and below -2SD. This is reassuring for normal girls with AMH in the low part of the reference

range, suggesting that they are not facing imminent POI. However, prospective studies are needed for

clarification.

Not all TS women with no ovarian function had undetectable AMH, and two patients even had single

measurements within the reference range. Thus, a limited number of small AMH producing follicles may be

present in patients who fulfil the criteria for the definition of POI. However, these small follicles may not

produce sufficient estradiol to initiate pubertal development (25).

When we evaluated other reproductive hormone levels as predictors of POI, particularly inhibin B appeared

to be of potential predictive value with levels dropping markedly during the year preceding POI, however,

data are sparse. In many of the patients, gonadotropins and estradiol were not affected before clinical

manifestation of POI. The finding of declining inhibin B is consistent with data indicating the predictive

value of inhibin B concerning the natural menopause (30,31). However, the predictive value of inhibin B is

hampered by its variation through the menstrual cycle, resulting in undetectable values in samples from some

healthy women during the luteal phase (33).

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In experimental settings, cryopreservation of ovarian tissue is offered to TS girls and adolescents with the

potential chance of preserving remaining ovarian follicles. However, there are no reports of follicle survival

after thawing, and no pregnancies have yet been reported after auto-transplantation of tissue in this patient

group (34,35). Therefore, ovarian cryopreservation introduces a risk of loss of precious ovarian tissue that

may have maintained ovarian function including endogenous hormone production and potentially

spontaneous pregnancy. Ovarian stimulation, aspiration and vitrification of mature oocytes, is an alternative

method of fertility preservation, however, not always feasible in young TS patients. As AMH is a

quantitative rather than a qualitative marker of primordial follicles, i.e. low AMH is associated with early

menopause (19,21), it is not predictive of fecundability in young adult women (36). Thus, it is not yet

possible to determine, which patients will benefit from fertility preservation strategies. Our data indicate, that

potential candidates may be found among patients with 45,X/46XX mosaicism and miscellaneous karyotypes

with relatively preserved AMH levels.

We were puzzled by the few patients with high AMH values, similar to what is observed in PCOS patients

(37). The etiology of PCOS is still not elucidated, however, early exposure to androgens may predispose to

PCOS (38,39). Premature adrenarche and increased levels of DHEAS have been observed in TS girls (40).

However, only one TS patient in this study had polycystic ovarian morphology (> 12 follicles size 2-9 mm)

and none had clinical or biochemical hyperandrogenism. Thus it is highly speculative if TS patients with

remaining ovarian function have increased risk of developing PCOS.

A higher number of Turner patients with a longer clinical follow-up of absent spontaneous puberty or

secondary amenorrhea are required to conclude firmly on the predictive value of absolute AMH levels.

AMH measurements cannot be directly compared between commercially available assays, and reports of

different converting factors suggest that the assays have been modified continuously (41). Therefore, the

absolute AMH levels reported in this study should be used with caution for other AMH assays. Nevertheless,

the relative levels based on a well-defined reference range are clinically useful as AMH < -2SD is predictive

of absent pubertal onset (prepubertal patients) and imminent POI (adolescents and adults). The introduction

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of more sensitive AMH assays may improve further the accuracy of interpretation of low AMH

concentrations. This has recently been demonstrated in the context of assessment of ovarian function after

chemotherapy for early breast cancer (42).

In conclusion, the majority of women with mosaic karyotype 45,X/46,XX had on-going ovarian function in

early adulthood and AMH levels within the reference range in early adulthood. AMH < -2SD predicted

failure to enter puberty in prepubertal TS girls and imminent POI in adolescent and adult TS patients. Longer

follow up is required to assess the value of AMH levels in predicting the reproductive lifespan in patients

with Turner syndrome.

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Table 1. Descriptive characteristics of the Turner syndrome patients according to karyotype and ovarian status (n = 117). In those with

45,X/46,XX three patients were not included in the table; two women had uterus agenesis and in one woman information on ovarian status

was missing.

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Figure Legends

Figure 1

Serum AMH levels in girls, adolescents and women with Turner syndrome according to age and karyotypes;

45,X (A), 45,X/46,XX (B) and miscellaneous karyotypes (C). Red: prepubertal girls; Green: patients with

ongoing ovarian function; Black: hormonal contraceptive treatment at the time of AMH measurement; Grey:

patients with no ovarian function. Note the logarithmic Y-scale. Blue lines indicate the reference range

(median, 2.5th and 97.5th percentile) based on 926 samples from healthy Danish infants, girls, adolescents and

adults (22).

Figure 2

Serum AMH levels in girls, adolescents and women with Turner syndrome according to age, reference range

and their ovarian status: never ovarian function (A), ongoing ovarian function (B), and loss of ovarian

function (C). Green: patients with ongoing ovarian function. Grey/black: patients with no ovarian function.

Note the logarithmic Y-scale. Blue lines indicate the reference range (median, 2.5 th and 97.5th percentile)

based on 926 samples from healthy Danish infants, girls, adolescents and adults (22).

Figure 3

Serum AMH levels (A) in women with Turner syndrome before and after POI, the 12 women are represented

by different colours. FSH (B), LH (C), estradiol (D) and inhibin B (E) levels in women with Turner

syndrome in the years preceding development of POI or initiation of HRT (red line). The blue line represents

the 2.5th percentile corresponding to -2SD of the reference range. The blue dotted line represents the

detection limit of 2 pmol/L.

Supplementary Figure 1

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Receiver operative characteristic (ROC) curve visualizing a high sensitivity and specificity for AMH as a

marker of POI in adolescent TS patients.

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