Circulating antim ullerian hormone levels in boys decline during early puberty and correlate with inhibin B

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1 ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY Circulating antim ullerian hormone levels in boys decline during early puberty and correlate with inhibin B Matti Hero, M.D., Ph.D., a Johanna Tommiska, Ph.D., a,b Kirsi Vaaralahti, M.Sc., a,b Eeva-Maria Laitinen, M.D., a,b Ilkka Sipil a, M.D., Ph.D., a Lea Puhakka, b Leo Dunkel, M.D., Ph.D., c and Taneli Raivio, M.D., Ph.D. a,b a Children's Hospital, Helsinki University Central Hospital (HUCH), and b Institute of Biomedicine/Physiology, University of Helsinki, Helsinki, Finland; and c William Harvey Research Institute, Queen Mary University of London and Centre for Endocrinology, Barts and the London School of Medicine and Dentistry, London, United Kingdom Objective: To investigate peripheral levels of inhibin B and antim ullerian hormone (AMH) in boys during peripuberty and in patients with congenital hypogonadotropic hypogonadism (HH). Design: Randomized, placebo-controlled trial (peripubertal boys); and cross-sectional clinical study (males with HH). Setting: University central hospital. Patient(s): Twenty-eight peripubertal boys with idiopathic short stature (ISS), 19 males with Kallmann syndrome. Intervention(s): Letrozole (2.5 mg/day) or placebo in boys with ISS for 2 years. Main Outcome Measure(s): Longitudinal follow-up observation of serum AMH and its relationship with inhibin B during early puberty and the influence of high (letrozole-treated boys) and low (males with HH) gonadotropin exposure on circulating AMH. Result(s): In boys with ISS receiving placebo, the decrease in AMH levels and the increase in inhibin B levels were correlated. The serum AMH level had already declined before a clinically significant increase in testis volume or serum testosterone occurred. Letrozole did not appear to modulate the decline in AMH. The AMH levels were lower in boys and young adults with Kallmann syndrome and prepubertal testes (mean: ng/ml, n ¼ 6) as compared with prepubertal ISS boys ( ng/ml). Conclusion(s): The gonadotropin-mediated early pubertal increase in inhibin B is tightly coupled to decrease in AMH levels and may reflect androgenmediated differentiation of Sertoli cells. Profound gonadotropin deficiency is associated with low AMH levels, suggesting impaired development of the Sertoli cell population. (Fertil Steril Ò 212;97: Ó212 by American Society for Reproductive Medicine.) Key Words: AMH, aromatase inhibitors, idiopathic hypogonadotropic hypogonadism, inhibin B, ISS, letrozole, puberty In males, Sertoli cell-derived antim ullerian hormone (AMH) may serve as an index of gonadal integrity (1). It may be of value in differentiating constitutional delay of growth and puberty from congenital hypogonadotropic hypogonadism (2), and may indicate the presence or absence of testes in patients with bilateral cryptorchidism (3). During the transient postnatal surge of gonadotropins and testosterone (minipuberty of infancy) and early puberty, important changes occur in the Sertoli cell population, and these changes are paralleled by changes in the peripheral levels of AMH and inhibin B, another Sertoli cell derived hormone (4 6). During the minipuberty of infancy, for example, immature Sertoli cells secrete both inhibin B and AMH abundantly. During the prepubertal hiatus of low gonadotropin secretion, inhibin B levels are low but AMH remains elevated and only declines during early puberty, probably reflecting Sertoli cell maturation (7, 8). Early puberty is also associated with a rapid Received November 28, 211; revised February 1, 212; accepted February 14, 212; published online March 9, 212. M.H. has nothing to disclose. J.T. has nothing to disclose. K.V. has nothing to disclose. E.-M.L. has nothing to disclose. I.S. has nothing to disclose. L.P. has nothing to disclose. L.D. has nothing to disclose. T.R. has nothing to disclose. Supported by the Academy of Finland, the Foundation for Pediatric Research, the Helsinki University Central Hospital Research Funds, the Sigrid Juselius Foundation, Helsinki University Research Funds, the Emil Aaltonen Foundation, and the Jalmari and Rauha Ahokas Foundation. Reprint requests: Taneli Raivio, M.D., Ph.D., Institute of Biomedicine/Physiology, University of Helsinki, Biomedicum Helsinki, P.O. Box 63 (Haartmaninkatu 8), FI-14 University of Helsinki, Helsinki, Finland ( taneli.raivio@helsinki.fi). Fertility and Sterility Vol. 97, No. 5, May /$36. Copyright 212 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:1.116/j.fertnstert increase in circulating inhibin B levels, but the interrelationship between the longitudinal changes in AMH and inhibin B yet to be fully elucidated. Especially, longitudinal data of simultaneous follow-up measurements of both Sertoli cell products are lacking. There are very few studies available that have reported the impact of altered gonadotropin secretion during early puberty on peripheral AMH levels. Congenital hypogonadotropic hypogonadism (HH), with an incidence approximately 1 in 1, to 3, in men (9, 1), represents a rare human disease model for investigating the relationship between inappropriately low gonadotropin secretion and Sertoli cell development. However, the impact of increased gonadotropin secretion on maturation and function of Sertoli cells is much more difficult to investigate because of the lack of a suitable experimental paradigm in humans. However, we have previously shown that boys treated with the 1242 VOL. 97 NO. 5 / MAY 212

2 Fertility and Sterility aromatase inhibitor letrozole display elevated gonadotropin and sex steroid levels after entering puberty (11), and this model thus represents a unique opportunity to investigate subsequent dynamic changes in Sertoli cell maturation. We studied the longitudinal changes in inhibin B and AMH levels in boys during normal puberty. We compared these values to those of boys treated with the aromatase inhibitor letrozole (11) and to those in males with congenital HH and variable degrees of endogenous puberty. MATERIALS AND METHODS Patients Boys with idiopathic short stature. The idiopathic short stature (ISS) group comprised 28 boys who participated in a double-blind, randomized, placebo-controlled trial investigating the efficacy and safety of aromatase inhibitor therapy in the treatment of ISS (11). Fourteen boys were randomized to receive placebo and 14 to receive letrozole (2.5 mg/day), a potent and specific third-generation aromatase inhibitor; the protocol together with gonadotropin, testosterone, and inhibin B concentrations were previously reported elsewhere (11). These boys were examined every 6 months for 2 years, and finally at 3 years after the start of the study. Follow-up visits included a physical examination with evaluation of the stage of puberty according to the Tanner scale (12) and estimation of testicular volume by using this formula: Length Width 2.52 (13). In addition, the baseline (obtained before the onset of letrozole therapy) clinical and hormone data of prepubertal boys randomized to receive aromatase inhibitor were included in the analyses, resulting in total number of 24 patients. As evaluated by medical history, clinical examination, and routine laboratory tests, none of the boys showed signs of chronic or endocrine illness to account for their short stature. In boys with ISS, venous blood samples were obtained between 7:3 A.M. and 1: A.M. after an overnight fast, and sera were stored at 8 C until analyzed. Boys and men with congenital HH. The clinical features and molecular genetic diagnoses of the 19 males with Kallmann syndrome (KS; congenital HH and anosmia) have been reported elsewhere (1). All patients were initially diagnosed based on [1] absent or incomplete pubertal development by the age of 18 years; [2] low circulating basal sex steroid levels in association with inappropriately low or normal gonadotropin levels, and subnormal or normal response to a gonadotropin-releasing hormone (GnRH) stimulation test; [3] otherwise normal anterior pituitary function; [4] no organic cause for their condition; and [5] defective sense of smell, as defined by the 4-item University of Pennsylvania Smell Identification Test (UPSIT; Sensonic Inc.), with a test score <5th percentile of age and/or absent or rudimentary olfactory bulbs in magnetic resonance imagining (MRI). In addition, one 13-year-old boy with unequivocal signs of severe congenital HH (history of cryptorchism and micropenis) and anosmia was included (1), and a clinical follow-up evaluation since has confirmed the diagnosis of congenital HH. Thirteen patients had a history of cryptorchidism and/or micropenis consistent with deficient hypothalamicpituitary-gonadal axis activation in fetal life and/or during the minipuberty of infancy. Two patients displayed partial pubertal development, whereas puberty was absent in the remaining 17 cases. All participants with KS were clinically examined, and their testicular volume was measured with a ruler; the volume was calculated as already described. At participation, seven KS patients were not receiving any hormone replacement therapy, and 12 were on testosterone replacement therapy (TRT). A single blood sample was obtained for DNA extraction and for serum hormone measurements. For further hormonal analyses, sera were stored at 8 C. The molecular genetic diagnoses and phenotypic features of males with KS have been reported previously elsewhere. In brief, the participants were screened for mutations in the seven genes known to underlie KS (1), and six males carried putative loss-of-function mutations in KAL1 (n ¼ 3) or FGFR1 (n ¼ 3). The KAL1 mutations were the nonsense mutation c.784c>t (p.r262x) and the frameshift mutation c.471_472delct (p.s158wfsx45) leading to premature stop codons, and the four base-pair deletion g.2357_236delagta that abolishes the splice site and most likely results in incorrect splicing. Loss-of-function is also evident for FGFR1 mutations c.11g>a (p.w4x) and c.961_962delaa (p.k321rfsx13) as they lead to premature stop codons. One proband carried a missense FGFR1 mutation, c.626g>a (p.r29h), not present in 1 controls. In addition, we included in the study a rare patient with normosmic HH. This 15-year-old boy had a homozygous GNRHR mutation c.416g>a (p.r139h) that allowed definite diagnosis of congenital HH at an early age. Moreover, this boy had not been exposed to exogenous gonadotropins, which potentially could confound the interpretation of AMH levels. Laboratory Analyses Serum inhibin B was measured as previously reported elsewhere (11). The inhibin B interassay coefficient of variation (CV) was 21% at 5 ng/l and <13% at concentrations above. The sera for AMH analyses were stored at 8 C, and AMH was measured with a sensitive immunoassay [Immunotech, Beckman Coulter Ltd. (A79765)] according to the manufacturer's instructions. The detection limit of this assay was.8 ng/ml, and the intra-assay CV was 3.3% at 12 ng/ml. The interassay CVs were % between concentrations 3.1 and 13.8 ng/ml. Serum testosterone in the ISS participants was determined using a modified radioimmunoassay, as previously described elsewhere (11). The sensitivity of the assay was.3 nmol/l. The interassay CV was 16% at.2 nmol/l and below 1% at concentrations above.8 nmol/l. The intraassay CV was 11% at.2 nmol/l and below 7% above.4 nmol/l. In men with congenital HH, the serum testosterone concentrations were measured with an API 2 tandem mass spectrometer (AB Sciex), with a limit of detection.5 nmol/l (14). Interassay CVs were 4.2% to 7.6% at mean testosterone concentrations of nmol/l. The serum luteinizing hormone (LH) and folliclestimulating hormone (FSH) levels were quantified with VOL. 97 NO. 5 / MAY

3 ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY ultrasensitive time-resolved immunofluorometric assays (AutoDELFIA) (15). The detection limit of the LH assay was.5 IU/L, and the interassay coefficient CV was less than 4% in the concentration range.3 42 IU/L. For FSH, the detection limit was.5 IU/L, and the interassay CV was 5% or less in the concentration range 2 78 IU/L. The mutation screening has been described previously elsewhere (1). Briefly, the coding exons and exon-intron boundaries of these genes were polymerase chain reaction (PCR) amplified, and the PCR products were purified with ExoSAP-IT treatment (Amersham Biosciences); they were bidirectly sequenced by use of the ABI BigDyeTerminator Cycle Sequencing Kit (v3.1) and ABI Prism 373xl DNA Analyzer automated sequencer (Applied Biosystems). The sequences were aligned and read with Sequencher 4.9 software (Gene Codes Corporation). All primer sequences and PCR conditions are available upon request. Written informed consent was obtained from all participants and/or their guardians. The study protocols were approved by the ethics committee of the Hospital District of Helsinki and Uusimaa, Finland. Statistical Analyses The longitudinal changes in serum AMH and inhibin B levels were investigated by employing summary measures (16). In short, a linear regression line was fitted for each subject with age as an independent variable and AMH or inhibin B as a dependent variable. The regression coefficients obtained were subsequently tested with t-test or subjected to correlation analysis. The difference in AMH levels across the groups (ISS boys, patients with congenital HH) was assessed by t-test or Mann-Whitney U test. The relationship between variables with skewed distributions was investigated with Spearman's rank correlation. Data are reported as mean standard error of the mean (SEM), unless otherwise stated. P<.5 was considered statistically significant. RESULTS Longitudinal Changes in AMH and Inhibin B in Boys During Early Puberty Longitudinal profiles of circulating inhibin B and AMH levels in healthy boys with ISS receiving placebo are shown in Figure 1. It is evident from these data that the steep decline in circulating AMH starts already in late prepuberty (with testis volume <2 ml) (especially in boys 1, 2, 8, 13, 14, 16, 19, and 23). The boys who remained prepubertal throughout the follow-up period (i.e., boys 6, 1, and 22) did not display such a decline. In early puberty, serum AMH and inhibin B levels correlated negatively (Fig. 2A), as did the changes in these hormones in individual boys (r ¼.77, P<.1, n ¼ 14) (see Fig. 2B). This decline in AMH has started already before the serum testosterone has reached 1 nm (see Fig. 3B). The decline in AMH levels in relation to the growth in testicular size was similar in the majority of letrozole- and placebotreated boys (Fig. 3A). One boy receiving placebo and two boys receiving letrozole showed some delay in AMH decline in relation to increasing serum testosterone (see Fig. 3B). AMH Levels in Patients with Congenital HH Among the 19 KS males (age range: 13 to 61 years), the AMH levels correlated positively with inhibin B (rs ¼.6, P<.5, n ¼ 19) but not with age, testis volume, or serum testosterone, LH, or FSH levels (data not shown). Twelve men with KS were on testosterone replacement therapy, and seven were off therapy. Accordingly, their testosterone levels differed ( nm vs..7.3 nm, respectively; P<.1), whereas the mean AMH levels did not ( vs ng/ml; P¼.6). The levels of AMH did not differ between patients with KAL1 or FGFR1 mutations (n ¼ 3 in each group; 4 17 vs ng/ml; not statistically significant). The KS patients with prepubertal testis size (<2 ml; n ¼ 6) had statistically significantly lower AMH levels (mean: 2.9 ng/ml; range: ng/ml) as compared with the prepubertal (testis size <2 ml; n ¼ 24) ISS boys (12.3 ng/ml, ng/ml) (P<.5). However, these KS patients comprised boys and young adults (age range: 13 to 28 years) and thus were older than the prepubertal ISS boys (age range: 9.1 to 13.9 years). We therefore further compared AMH levels between 24 prepubertal ISS boys and two adolescents with HH. The first boy was a 13-year-old with KS (testis volume.3 ml). The other adolescent boy with HH was 15 years old, had small testes (.3 ml), had no history of cryptorchidism or microphallus, and had no LH response (<.1 IU/L) in the GnRH test. He had a homozygous GNRHR mutation c.416g>a (p.r139h), and his parents were heterozygous carriers for this mutation. These two adolescent boys with severe congenital HH had lower AMH levels (13.2 ng/ml and 15.7 ng/ml, respectively) than any of the prepubertal boys with ISS (P<.5). DISCUSSION Our results show that AMH decreases during very early puberty in healthy boys, before any notable increase in testis volume or serum testosterone has occurred. Thus, longitudinal assessment of AMH for example, sampling 3 to 4 months apart might predict the clinical onset of puberty without the need for repeated clinical examination or GnRH testing. Such a prognostic marker would be of clinical value in patients with constitutional delay of puberty. Few studies have addressed the longitudinal changes in AMH levels during puberty in healthy boys, and none of these studies have reported on the concomitant changes in circulating AMH and inhibin B. Follicle-stimulating hormone stimulates proliferation of immature Sertoli cells (17, 18) as well as secretion of both inhibin B and AMH (19). However, our results show that, in healthy boys, the increase in inhibin B and decrease in AMH levels correlate inversely during early puberty, suggesting that factors other than FSH contribute to these reciprocal changes. It has been previously proposed that the pubertal decline in AMH results from gradual (re)activation of the hypothalamic-pituitary-gonadal axis (and subsequent increase in intratesticular testosterone) rather than from the interaction between Sertoli cells and spermatogenic cells (8, 2). Indeed, functional androgen receptors (ARs) appear to be essential for intratesticular testosterone-mediated 1244 VOL. 97 NO. 5 / MAY 212

4 Fertility and Sterility FIGURE 1 Inhi bin B (ng/l) AM MH (ng/ml) 3 25 #1 # # #8 #1 # #14 #16 # #22 #23 # #28 # Age (yrs) Serum antim ullerian hormone (AMH) and inhibin B levels in 14 healthy boys with idiopathic short stature during early puberty. The numbers within the panel denote testicular volumes (ml). Hero. AMH and inhibin B in early puberty. Fertil Steril 212. AMH repression to occur in human Sertoli cells (21). Against this background, our results, and those of Aksglaede et al. (5) showing an early decrease in AMH during puberty, suggest that immature Sertoli cells begin to express AR before the clinical onset of puberty. However, we cannot exclude the possibility that other paracrine mechanisms (i.e., independent of Sertoli cell AR expression) participate in mediating these changes. For example, the decline in AMH in mice with the Sertoli cell specific knockout of AR may be mediated by peritubular myoid cells that abundantly express AR (2). On the other hand, although fetal and newborn testis peritubular myoid cells (but not Sertoli cells) in the human express AR, the peripheral AMH levels still do not decline during the minipuberty of infancy (21). The close relationship between AMH and inhibin B suggests that inhibin B is an indirect indicator of AR-mediated Sertoli cell maturation. Third-generation aromatase inhibitors, potent and selective blockers of estrogen biosynthesis, delay bone maturation and increase the predicted adult height in some growth disorders. Thus, they have been used as an experimental treatment VOL. 97 NO. 5 / MAY

5 ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY FIGURE 2 FIGURE 3 3 /ml) log AMH (ng log inhibin B (ng/l) 14 ibin B (ng/l)/yr ΔInh ΔAMH (ng/ml)/yr (A) Relationship between serum antim ullerian hormone (AMH) and inhibin B levels and (B) their individual changes in healthy boys with idiopathic short stature during 3 years of follow-up evaluations. Hero. AMH and inhibin B in early puberty. Fertil Steril 212. for short stature in boys (22). As estrogen is crucial for the sex steroid mediated central negative feedback effect on gonadotropin secretion, letrozole treatment after the onset of central puberty results in enhanced gonadotropin secretion and a rapid increase in testosterone levels, without concomitant increase in circulating estradiol (11). This may, in theory, shorten the peripubertal time window for proliferation of immature Sertoli cells. It is not known whether increased intratesticular testosterone concentration influences the final Sertoli cell number, but, according to a recent report, aromatase inhibitor treatment of male rats during peripuberty resulted in a decreased number of Sertoli cells (23). For ethics reasons, we did not perform biopsies of the testes, and thus only indirect evidence is available on the effect of letrozole on Sertoli cell number. Our AMH data suggest, however, that Sertoli cell maturation in boys treated with the aromatase inhibitor letrozole or given placebo do not differ. The intratesticular testosterone concentrations are many-fold higher than circulating levels (24), and the Kd (dissociation constant) of AR for testosterone is relatively low (25). Thus, it is possible that saturating testosterone levels Relationship between (A) testis volume and serum antim ullerian hormone (AMH) and (B) serum testosterone and serum AMH in healthy boys with idiopathic short stature during 3 years of followup evaluations. Boys received either placebo (open circles) or letrozole (asterisks). Dotted lines indicate serial measurements of individuals with delayed decline in AMH level as compared with testicular growth. Hero. AMH and inhibin B in early puberty. Fertil Steril 212. prevail in the testis already during early puberty; a further letrozole-induced increase in intratesticular testosterone may have a minor impact, if any, on Sertoli cell maturation. Also, testis growth is not impaired in pubertal boys receiving aromatase inhibitor treatment, but in fact increases more rapidly than in untreated boys (11). These data suggest that aromatase inhibitor treatment during adolescence does not impair Sertoli cell proliferation and maturing spermatogenesis. However, posttreatment sperm parameters in this patient group have only been reported for anastrozole, a nonsteroidal aromatase inhibitor with less potency compared with letrozole (26). In that report, anastrozole treatment during adolescence in combination with growth hormone did not influence 1246 VOL. 97 NO. 5 / MAY 212

6 Fertility and Sterility posttreatment sperm parameters when compared with controls and with a group who had received growth hormone only. Patients with KS had variable AMH levels. The levels in patients with small testes were clearly lower when compared with prepubertal (testis volume <2 ml) boys with ISS, suggesting that congenital HH affects the development of the Sertoli cell population. We are aware that there were various factors that may confound direct comparison of adult HH patients with small testis size to prepubertal boys with ISS. However, the two adolescent boys with congenital HH had lower AMH levels than any of the prepubertal ISS boys. This finding suggests that severe gonadotropin deficiency leads to a decreased number of Sertoli cells and therefore low AMH (current work) and inhibin B (27) levels. Indeed, our findings and those of Coutant et al. (2) suggest that circulating AMH provides a potentially useful tool for differentiating congenital HH from constitutional delay of growth and puberty (CDGP) in males with delayed puberty (2). This study, however, was not primarily designed to address this issue, and further research is clearly needed. The AMH levels did not differ between patients with KAL1 or FGFR1 mutations, although the former had the more severe reproductive phenotype (1). This may be attributable to the small number of participants and the confounding effects of differences in age and treatments. Our data show that the early pubertal increase in inhibin B is tightly coupled to a decrease in AMH and therefore may reflect androgen-mediated differentiation of Sertoli cells. Modulation of the hormonal milieu with an aromatase inhibitor has no apparent influence on early pubertal decline in circulating AMH. 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