The Sertoli cell is important for sexual differentiation in

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1 ORIGINAL Endocrine ARTICLE Research Changes in Anti-Müllerian Hormone (AMH) throughout the Life Span: A Population-Based Study of 1027 Healthy Males from Birth (Cord Blood) to the Age of 69 Years L. Aksglaede, K. Sørensen, M. Boas, A. Mouritsen, C. P. Hagen, R. B. Jensen, J. H. Petersen, A. Linneberg, A.-M. Andersson, K. M. Main, N. E. Skakkebæk, and A. Juul University Department of Growth and Reproduction (L.A., K.S., M.B., A.M., C.P.H., R.B.J., J.H.P., A.-M.A., K.M.M., N.E.S., A.J.), Rigshospitalet, DK-2100 Copenhagen, Denmark; Department of Biostatistics (J.H.P.), University of Copenhagen, DK-1014 Copenhagen, Denmark; and Research Centre for Prevention and Health (A.L.), Glostrup University Hospital, DK-2600 Glostrup, Denmark Context: Anti-Müllerian hormone (AMH), which is secreted by immature Sertoli cells, triggers the involution of the fetal Müllerian ducts. AMH is a testis-specific marker used for diagnosis in infants with ambiguous genitalia or bilateral cryptorchidism. Aim: The aim of the study was to describe the ontogeny of AMH secretion through life in healthy males. Setting:This was a population-based study of healthy volunteers. Participants: Participants included 1027 healthy males from birth (cord blood) to 69 yr. A subgroup was followed up longitudinally through the infantile minipuberty [(in cord blood, and at 3 and 12 months), n 55] and another group through puberty [(biannual measurements), n 83]. Main Outcome Measures: Serum AMH was determined by a sensitive immunoassay. Serum testosterone, LH, and FSH were measured, and pubertal staging was performed in boys aged 6 to 20 yr (n 616). Results: Serum AMH was above the detection limit in all samples with a marked variation according to age and pubertal status. The median AMH level in cord blood was 148 pmol/liter and increased significantly to the highest observed levels at 3 months (P ). AMH declined at 12 months (P ) and remained at a relatively stable level throughout childhood until puberty, when AMH declined progressively with adults exhibiting 3 4% of infant levels. Conclusion: Based on this extensive data set, we found detectable AMH serum levels at all ages, with the highest measured levels during infancy. At the time of puberty, AMH concentrations declined and remained relatively stable throughout adulthood. The potential physiological role of AMH and clinical applicability of AMH measurements remain to be determined. (J Clin Endocrinol Metab 95: , 2010) ISSN Print X ISSN Online Printed in U.S.A. Copyright 2010 by The Endocrine Society doi: /jc Received May 28, Accepted August 16, First Published Online September 15, 2010 The Sertoli cell is important for sexual differentiation in the fetus and spermatogenesis in the adult and thereby for the expression of a normal male phenotype. In the fetal and early postnatal testis, Sertoli cells are morphologically and functionally immature. The main function of the immature Sertoli cells is to secrete anti-müllerian hormone (AMH), previously termed Müllerianinhibiting substance, that triggers the involution of the Abbreviations: AMH, Anti-Müllerian hormone; AR, androgen receptor; CV, coefficient of variation; T, testosterone. J Clin Endocrinol Metab, December 2010, 95(12): jcem.endojournals.org 5357

2 5358 Aksglaede et al. AMH through a Life Span in Males J Clin Endocrinol Metab, December 2010, 95(12): FIG. 1. Serum concentrations of AMH in healthy males from birth (cord blood) to the age of 69 yr as indicated by open black dots. The curves represent the median, the 2.5th percentile, and the 97.5th percentile corresponding to the mean, the mean 1.96 SD, and the mean 1.96 SD, respectively. In boys with longitudinal measurements during puberty, only the first measurement was included in this figure. As noted, no data on boys in the age interval between 1.5 and 4 yr are available, and the dotted curves are extrapolated from the endings of the existing data set. Males with anorchia are indicated by red dots. Müllerian ducts in the male fetus (1). AMH is expressed by the immature Sertoli cells from the time of sexual differentiation at approximately 8 wk gestation (2, 3), and accordingly, with the onset of puberty and increasing secretion of testosterone from the Leydig cells, the secretion of AMH declines in parallel with Sertoli cell maturation. From the time of puberty, the function of the Sertoli cells is mainly to support spermatogenesis. AMH is a 140-kDa dimeric glycoprotein hormone belonging to the TGF- superfamily (4, 5). Measurement of serum AMH does not require prior gonadal stimulation with gonadotropins, and it is a testis-specific marker in newborn infants with ambiguous genitalia or nonpalpable gonads (6). A measurable value in a boy or adult man with bilateral cryptorchidism is predictive of undescended testes, whereas an undetectable value is highly suggestive of anorchia. However, to date little is known about the biological role of postnatal AMH in males. The aim of this study was to describe the ontogeny of AMH secretion from birth to late adulthood in a crosssectional study of 1027 healthy male infants, boys, adolescents, and adult males to study the impact of puberty and to correlate the serum levels of AMH with the corresponding levels of LH, FSH, and testosterone (T). In addition, we describe in detail the AMH secretion longitudinally through the first year of life (n 55) and during puberty (n 83) in a subgroup of these healthy boys. Subjects and Methods Study population Blood was obtained from a total of 1027 healthy male infants, children, adolescents, and adult males from birth (cord blood) to age 69 yr. The participants were recruited as part of three different population-based studies of healthy subjects as previously described in detail (7 10). Children aged 0 6 yr were included from an ongoing longitudinal cohort study of healthy pregnant women and their offspring with clinical examination and blood sampling at 0 (n 82), 3(n 35), and 12 months of age (n 73) and at 4 6 yr of age (n 116) (8, 11). Boys aged 6 20 yr (n 616) were recruited from schools in the Copenhagen area as part of the COPENHAGEN Puberty Study (7). Pubertal staging according to Tanner (12) was performed in these boys. Testicular volume was estimated by using Prader s orchidometer (13). If the sizes of the two testes were not identical, the larger was chosen to determine testicular volume. Onset of puberty was defined as a testicular volume of more than 3 ml (13). At the onset of puberty, hormonal changes are likely to precede physical changes. We therefore divided the prepubertal boys (genital stage G1) into boys younger than 9.0 yr (G1a) and presumably truly prepubertal; and boys 9.0 yr or older (G1b), in which some of the boys might have an activated pituitary-gonadal axis despite lack of clinical signs of puberty. Adult males (n 150) were part of the Health2006 study, which aimed at investigating the prevalence and risk factors for chronic diseases, such as asthma, allergies, cardiovascular disease, and diabetes. A random sample of the general population aged yr and living in the southwestern area of Copenhagen was drawn from the Civil Registration System. All participants in this study underwent clinical examination and fulfilled a questionnaire (9). Only boys with no history of genital abnormalities or congenital or acquired cryptorchidism were included in the study, and no boys were cryptorchid at the time of evaluation. Blood sampling was performed between 0800 and 1800 h in the children aged 0 6 yr (except cord blood) between 0830 and 1300 h in school children aged 6 20 yr and between 0730 and 1300 h in the adults. A subgroup of boys was followed up longitudinally during the first year of life through the so-called minipuberty (n 55) and another group were followed up during puberty (n 83). The median number of measurements during minipuberty was three (range two to three) and during puberty five (range two to eight). In pubertal children with longitudinal measurements, only the first determination of AMH was used for construction of reference ranges (Fig. 1 and Table 1, and for the analyses on the correlation between AMH and LH, FSH, and T. AMH was measured repeatedly in eight healthy males (aged yr) who were recruited from a larger study on fetal growth performed on their mothers during Only young males with normal birth weights and normal fetal growth in the third trimester were included in the present study as described previously (14). From 2000 to 0800 h, blood samples (2 ml) were collected at 20-min intervals, and AMH was measured with 1-h intervals in these subjects. A group of 16 anorchid men aged yr served as a reference group. Two had congenital anorchia due to intrauterine testicular torsion, 13 were orchiectomized bilaterally for testicular cancer, and one was a 46, XY female who was orchiectomized because of complete androgen insensitivity.

3 J Clin Endocrinol Metab, December 2010, 95(12): jcem.endojournals.org 5359 TABLE 1. Median age and AMH concentrations in the different age/puberty intervals n Age range (yr) Median AMH (pmol/liter) (5 95%) Cord blood (53 340) Minipuberty ( ) 12 months ( ) Childhood ( ) G1a ( ) G1b ( ) G ( ) G (22 734) G (15 112) G (23 128) Adulthood (13 98) G1 G5, Genital stage according to Tanner; G1a, prepubertal younger than 9 yr; G1b, prepubertal older than 9 yr. Blood samples were withdrawn from an antecubital vein and were clotted, centrifuged, and serum stored at minus 20 C until hormone analyses were performed. Laboratory analysis AMH was measured by a sensitive immunoassay [Immunotech, Beckman Coulter Ltd., Marseilles, France (A16507)] with a detection limit of 2 pmol/liter. The intraassay coefficients of variation (CVs) were less than 7.8, 5.4, and 6.4% at 13, 123, and 231 pmol/liter, respectively. The interassay CVs were less than 11.6, 10.9, and 9.1% at 19, 99, and 209 pmol/liter, respectively, based on results from the first 152 assays (corresponding to three batches). In the two following batches, several of the low and medium controls were above 2 SD. All samples analyzed within these batches were therefore adjusted using batch-specific correction factors. Serum FSH and LH were measured by timeresolved immunofluorometric assays (Delfia; PerkinElmer, Boston, MA) with detection limits of 0.06 and 0.05 IU/liter for FSH and LH, respectively. Intra- and interassay CVs were less than 5% in both gonadotropin assays. T was measured with the DPC Coat-A-Count RIA kit (Diagnostic Products, Los Angeles, CA). The detection limit was 0.23 nmol/liter and the intra- and interassay CVs were 7.6 and 8.6%, respectively. Ethics The study of infants and adolescents was approved by the local ethics committee (no. KF , V /90, KF /97). The Healthy2006 study was registered at www. clinical.trials.com (unique ID: KA ). All participants or their parents (when below the age of 18 yr) signed informed consents. Statistics Reference curves for the skewed distribution of AMH as a function of age were calculated. The curves represent the median, the 2.5th percentile, and the 97.5th percentile corresponding to the mean, the mean 1.96 SD and the mean 1.96 SD, respectively. The curves were obtained using generalized additive models for location, scale, and shape (15). This technique takes into account both the dependence on age and the highly skewed distributions of the hormone. The results were verified using locally weighted regression quantiles, a completely nonparametric technique. The age-adjusted interdependence between AMH, FSH, LH, and T were described using partial correlations. Changes in AMH from one age to another during the minipuberty were assessed using a paired t test. When assessing AMH changes after pubertal onset as defined by genital stage G2, the age at pubertal onset was defined as the midpoint between the previous visit and the visit at which onset was first noted. FIG. 2. A, Longitudinal measurements of AMH concentrations at 0 (cord blood), 3, and 12 months of age. A line connects all measures from each individual child. B, Longitudinal measure of circulating AMH according to testicular volume. A line connects all measures from each individual child.

4 5360 Aksglaede et al. AMH through a Life Span in Males J Clin Endocrinol Metab, December 2010, 95(12): Results Serum AMH, FSH, LH, and T in relation to age and stage of puberty The serum concentration of AMH was above the detection limit in all samples throughout life with a marked variation according to age and pubertal status (Fig. 1 and Table 1). Thus, median AMH level in cord blood was 148 pmol/liter and increased significantly to 1047 pmol/liter at 3 months of age (P ). AMH declined at 12 months of age (P 0.001) and remained at a relatively stable level through childhood until puberty. AMH levels did not differ after the age of 12 months until 9 yr of age (G1b), after which AMH declined to a significantly lower level (P 0.005). AMH levels remained stable throughout late adolescence and adulthood at a level of 3 4% of infant levels. Correlation of AMH to gonadotropins and T at different stages of puberty (data not shown) In stage G1a, AMH correlated positively with LH, whereas no correlation to the other hormones was found. In stage G1b, there was no correlation between AMH and the other three hormones. In stages G2 and G3, a significant negative correlation was found between AMH and all measured hormones (the correlation with LH in stage G3 was borderline significant). In contrast, during stages G4 and G5, no correlations were found. Longitudinal serum AMH levels through minipuberty AMH increased in 38 of 38 (100%) infants from birth to 3 months of age during the minipuberty (Fig. 2A) and decreased in 27 of 35 (77%) infants from 3 to 12 months of age. The increase at 3 months of age and the decline from 3 to 12 months were highly significant (paired t test, both P ). Longitudinal biochemical evaluation during puberty During puberty circulating AMH declined with increasing age (Fig. 3) and more evidently declined with advancing puberty as defined by testicular growth in all subjects (Fig. 2B). By contrast, LH, FSH, and T increased with increasing age, although fluctuations were seen in some boys (Fig. 3). In addition, the decrease in AMH was already observed before any clinical sign of puberty was FIG. 3. Longitudinal measurements of circulating AMH (A), FSH (B), T (C), and LH (D) in relation to chronological age. A line connects all measures from each individual child.

5 J Clin Endocrinol Metab, December 2010, 95(12): jcem.endojournals.org 5361 observed (Fig. 4). Likewise, the increase in T, LH, and FSH was evident before clinical signs of pubertal onset (Fig. 4). Nocturnal variation in AMH secretion There was very limited nocturnal variation with a median CV of 5.3% (range %) (Fig. 5). AMH levels in men with anorchia AMH was below the detection limit in all samples from anorchid patients (Fig. 1). Discussion In this population-based cross-sectional study in 1027 healthy males from birth to the age of 69 yr of age, we found detectable serum concentrations of AMH in all samples throughout life with a marked variation according to age and pubertal status. In cord blood, the median AMH concentration was 148 pmol/liter, whereas serum AMH increased significantly during the infantile minipuberty with a median peak level of 1047 pmol/liter in our longitudinal part of the study. Hereafter AMH declined at 1 yr and remained at a relatively stable level until the time of puberty. During puberty, AMH declined with advancing pubertal development and remained at a relatively stable level throughout late adolescence and adulthood. In contrast, AMH was unmeasurable in orchiectomized individuals. FIG. 5. Nocturnal variation in AMH secretion in healthy males between 2000 and 0800 h. Each line represents the variation in each individual. It is well established that the hypothalamic-pituitarygonadal axis is transiently activated during the first months of postnatal life. In the healthy male infant, gonadotropins, sex steroids, and inhibin B increase to pubertal or even adult levels at approximately 3 months of age, followed by a relatively quiescent period until reactivation of the hypothalamic-pituitary-gonadal axis in puberty (16, 17). Sertoli cells do not express androgen receptors (ARs) during fetal life and in the early postnatal period. Sertoli cells therefore do not respond to the high levels of T during fetal life and the first months after birth, when AMH expression remains at its highest levels (18, 19). In accordance, we found significantly higher AMH FIG. 4. Longitudinal measurements of circulating AMH (A), FSH (B), T (C), and LH (D) in relation to time from pubertal onset defined as genital stage G2. A line connects all measures from each individual child.

6 5362 Aksglaede et al. AMH through a Life Span in Males J Clin Endocrinol Metab, December 2010, 95(12): levels during the minipuberty at the age of 3 months as compared with any other stages of life. In our longitudinal study during minipuberty, AMH levels exhibited a dramatic increase at 3 months of age followed by a decline at 12 months in all subjects. To our knowledge, longitudinal measurements of AMH during minipuberty have been evaluated in only one previous study (20). In that study on five infant boys, the authors proposed an increase in circulating AMH from birth with a peak between 6 and 12 months of age followed by a subsequent decline. However, in three of these five infants, no obvious increase during the first 2 yr of life was presented, and the later peak may be a consequence of the low number of boys included in that study, although we do not have any measurements at 6 months of age for comparison. The exact time of the peak therefore remains to be determined. The Sertoli cells remain immature until puberty during which maturation is induced by the high intratesticular T levels. The concentration of intratesticular T increases before circulating T (21), and our reported decline in AMH could therefore be the result of the elevation of intratesticular T. At this point in time Sertoli cells stop proliferating, AMH expression is down-regulated (3, 18), and the secretion of AMH decreases (4). AR expression is found in only a low proportion of Sertoli cell nuclei from 6 to 12 months of life, and a progressive increase is observed between ages 4 and 8 yr. By the age of puberty, all Sertoli cell nuclei are AR positive and respond to the increase of local androgen concentration with cytological signs of Sertoli cell maturation, AMH down-regulation, and massive entry of germ cells into meiosis (22, 23). AMH is negatively regulated by T from the time of puberty and in adulthood (20, 24). In addition to negative regulation of AMH by androgens, there is evidence for a positive regulation by FSH on postnatal AMH secretion (25, 26). In accordance, we found a significant negative correlation between AMH and T, and despite the known positive regulation of AMH by FSH, we found a negative correlation between AMH and FSH in pubertal stages G2 to G3 after correction for age. This discrepancy is most likely explained by the fact that the negative effect of T prevails over the positive effect of FSH on AMH secretion. Thus, serum AMH concentrations are elevated in patients with androgen insensitivity (27). Such cross-sectional comparisons may be hampered by the fact that some diurnal variation exists for T. However, all samples were collected at a relatively narrow interval. In addition, no diurnal variations in AMH levels were found. One of the strengths of the present study is the use of the same ultrasensitive AMH assay on this extensive normative material covering the entire life span. A limitation, however, is the lack of inhibin B measurements as a supplementary Sertoli cell marker for comparison. The uniqueness of the study is also the inclusion of longitudinal measurement of AMH during minipuberty and puberty, and the thorough clinical examination including Tanner staging and determination of testicular volume during puberty. These longitudinal measures of circulating AMH allowed us to describe the AMH secretion in detail and confirmed previous studies as well as our present crosssectional findings of a marked increase during the first year of life in minipuberty (20, 28 31) and a marked decline with advancing puberty (20, 24, 29, 30). In accordance with our findings during puberty, high AMH levels relative to chronological age have been noted in boys with delayed puberty (32), whereas boys with precocious puberty had lower AMH levels relative to chronological age than normally developed boys of the same age (24, 29). The clinical diagnostic use of AMH determinations is well known in pediatric endocrinology. However, the interest for identifying its clinical use in adulthood has been increasing during recent years. In females, the measurement of circulating AMH has been acknowledged as a marker of ovarian reserve and prediction of the success of in vitro fertilization treatment (33, 34). In males, most studies on the relationship between AMH and spermatogenesis have been performed on determination of AMH in seminal fluid (35 39), and the results are contradictory at present. However, in one study significantly lower serum AMH levels were found in infertile males as compared with normal control men (38). In conclusion, based on this extensive normative data set, we found detectable AMH serum levels at all ages, with the highest measured levels postnatally during the minipuberty. At the time of puberty, AMH concentrations declined and remained relatively stable throughout adulthood, with adults exhibiting 3 4% of infant levels. The potential physiological role of AMH for testicular function, including spermatogenesis, and thereby the clinical applicability of AMH measurements remain to be determined. Acknowledgments We thank the members of the Nordic Cryptorchidism Study Group, who performed all the examinations of the infant boys, and the laboratory technicians for their excellent work with the hormone analyses. Address all correspondence and requests for reprints to: Lise Aksglaede, Department of Growth and Reproduction GR, Rigshospitalet, Section 5064, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark. lise.aksglaede@rh.regionh.dk. The COPENHAGEN Puberty Study was funded by the Kirsten and Freddy Johansen Foundation. A.J. received financial

7 J Clin Endocrinol Metab, December 2010, 95(12): jcem.endojournals.org 5363 support from The Danish Medical Research council (Grant ) and L.A. from Dronning Louises Børnehospitals Foundation. The infant cohort study was supported by European Union Grants QLK4-CT , QLK , QLK ), and Dan-ED ; Medical Research Council Grants and ; and the Novo Nordisk Foundation. Beckmann-Coulter kindly supplied the AMH kits free of charge for the study. Disclosure Summary: The authors have nothing to disclose. References 1. Lee MM, Donahoe PK 1993 Mullerian inhibiting substance: a gonadal hormone with multiple functions. Endocr Rev 14: Josso N, Lamarre I, Picard JY, Berta P, Davies N, Morichon N, Peschanski M, Jeny R 1993 Anti-mullerian hormone in early human development. 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