Anti-M ullerian hormone in men with normal and reduced sperm concentration and men with maldescended testes

Transcription

1 Anti-M ullerian hormone in men with normal and reduced sperm concentration and men with maldescended testes Frank T uttelmann, M.D., a Nina Dykstra, Cand. Med., a Axel P. N. Themmen, Ph.D., b Jenny A. Visser, Ph.D., b Eberhard Nieschlag, M.D., a and Manuela Simoni, M.D., Ph.D. a a Institute of Reproductive Medicine of the University, M unster, Germany; and b Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands Objective: To evaluate serum anti-m ullerian hormone (AMH) in well-characterized men with normal and reduced sperm concentration and in men with a history of or persistent maldescended testes as a possible clinical marker of male factor infertility and/or maldescended testes. Design: Retrospective analysis of 199 men selected from our database (Androbase). Setting: The university-based Institute of Reproductive Medicine. Patient(s): One hundred eight men with normal and 60 men with reduced sperm concentration without known cause of infertility and additionally 31 infertile men with current or former maldescended testes were evaluated. Intervention(s): Serum AMH was analyzed by an in-house ELISA. Main Outcome Measure(s): Hormone and semen parameters were compared and correlated with AMH. Result(s): No significant differences were found in AMH levels. Only in men with maldescended testes did AMH correlate negatively with FSH and positively with testicular volume and sperm concentration. No correlations between AMH and LH or testosterone (T) were found. Conclusion(s): Anti-M ullerian hormone serum levels are not significantly affected by impaired spermatogenesis in general but are correlated with spermatogenic parameters in men with current or former maldescended testes. Therefore, AMH measurement does not improve clinical routine diagnostics but should be evaluated further in patients with maldescended testes. Anti-M ullerian hormone might serve as a marker of Sertoli cell number, function, and/or maturation in these men. (Fertil Steril Ò 2009;91: Ó2009 by American Society for Reproductive Medicine.) Key Words: AMH, MIS, spermatogenesis, male fertility, FSH, maldescended testis, cryptorchidism Anti-M ullerian hormone (AMH), also called M ullerian inhibiting substance (MIS), has been known for several decades to be responsible for establishing sexual dimorphism after destination of the bipotential gonad has been predefined by SRY. At the early stages of development, fetuses of both sexes have two pairs of M ullerian (forming the anlagen for fallopian tubes, uterus, and upper third of the vagina) and Wolffian ducts. Under the influence of AMH produced by Sertoli cells in the emerging testes the M ullerian ducts involute in male fetuses while androgens induce the Wolffian ducts to form epididymides, vasa deferentes, and seminal vesicles (1 4). In the male, AMH level rises rapidly after birth, is highest during late infancy, then gradually declines until puberty (5) and can therefore serve as a testis-specific indicator for diagnosis in newborn infants with ambiguous genitalia Received October 8, 2007; revised and accepted February 12, 2008; published online April 18, F.T. has nothing to disclose. N.D. has nothing to disclose. A.P.N.T. has nothing to disclose. J.A.V. has nothing to disclose. E.N. has nothing to disclose. M.S. has nothing to disclose. Reprint requests: Eberhard Nieschlag, M.D., Institute of Reproductive Medicine of the University, Domagkstrasse 11, D M unster, Germany (FAX: ; eberhard.nieschlag@ ukmuenster.de). or nonpalpable gonads (6). The decrease of AMH corresponds to the Sertoli cell maturation status: with the onset of puberty and spermatogenesis, AMH is expressed only by immature Sertoli cells and by immunohistochemistry found only in tubules with spermatogenic arrest or Sertoli-cell only syndrome (7 9). Maldescended testes (called cryptorchidism only if testes are not detectable by clinical examination) are the most common genital anomaly in newborn boys with an incidence of 2% to 5% for term infants and approximately 0.8% to 1% at 1 year of age (10). The condition is associated with a 10- fold risk of testicular malignancy and impaired fertility due to reduced number of spermatogonia and mature spermatocytes and, therefore, reduced sperm concentration and count in the ejaculate (11). A role for AMH during the first stage of testicular descent (abdominal phase) has been proposed (12), suggested by the clinical observation that the testes usually fail to descend in persistent M ullerian duct syndrome, in which AMH action is lacking (13, 14). On the other hand, the maldescended testes in these patients have been attributed to the retained ductal structures, which limit movement of the testes (15). Further investigations challenge the significance of AMH, because AMH-deficient male mice show normal testis descent (16). Insulin-like 1812 Fertility and Sterility â Vol. 91, No. 5, May /09/$36.00 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 hormone 3 seems to be the most important signal for testicular descent (17, 18), which is probably augmented by AMH and T (19). The regulation of AMH after birth is complex: basal levels of AMH are independent of gonadotropin regulation, for example, during childhood and in patients with hypogonadotropic hypogonadism (20). Throughout pubertal development, AMH correlates negatively with serum T. This correlation persists if androgen levels are abnormally high (e.g., activating mutation of the LH receptor) but gonadotropins are low (21). These findings suggest that AMH is down-regulated by androgens and not directly by gonadotropins. In patients with androgen insensitivity or deficient androgen production, serum AMH levels are extremely elevated indicating that, without the inhibitory androgen effect, FSH stimulates AMH production (1). Anti-M ullerian hormone is also produced by granulosa cells in the ovary after birth but is undetectable in most prepubertal female subjects (2, 5). In postpubertal women, it inhibits primordial follicle recruitment, may also inhibit FSH-dependent selection of follicles for dominance, and recently has been evaluated as a marker for ovarian follicle reserve useful for infertility treatment and for prognosis after cancer treatment (22 24). Two recent studies demonstrated the importance of seminal fluid AMH as a marker of spermatogenesis (25, 26), although this was not confirmed by a third study (27). The latter also found significantly reduced AMH serum concentrations in infertile men with oligozoospermia (n ¼ 15) as defined by the World Health Organization (WHO) (sperm concentration < /ml [28]) compared with control men (n ¼ 21) (27) and suggested its possible superiority to seminal plasma AMH in male factor infertility. The difference in serum AMH between men with normal and reduced sperm concentration was not confirmed by a second study by the same authors (29), who measured serum and seminal plasma AMH in parallel in a small cohort of uncharacterized infertile men. Although serum and semen AMH levels were not directly compared (no correlation shown), a correlation of serum AMH with sperm concentration, serum FSH, and free T was found. Finally, very recently two studies (30, 31) showed that seminal plasma AMH is of no advantage in predicting testicular sperm extraction results, again in a rather small group of men. To date, the two above-mentioned studies (27, 29) remain the only ones analyzing the diagnostic value of serum AMH concerning male fertility. They are limited by the low number of samples and probably heterogeneous study populations with varying causes for their reduced sperm concentrations (described as male factor infertility ). For this reason, we wanted first to verify, in a large number of well-characterized men with normal or reduced sperm concentration, whether serum AMH determination would add any diagnostic advantage over current endocrine diagnostics; second, given the role of AMH in Sertoli cell function, to assess whether serum AMH correlates with hormonal and clinical parameters in men with maldescended testes. MATERIALS AND METHODS Study Population From the database of the Institute of Reproductive Medicine, M unster (Androbase [32]), we retrospectively selected patients (n ¼ 110) with idiopathic infertility (no known cause) and thereby excluded all known genetic disorders including Klinefelter syndrome and Y-chromosomal deletions, testicular tumors, infections of the genital tract, history of or persistent maldescended testes, varicocele, or any major general disease. Furthermore, we selected volunteers from unrelated clinical trials (n ¼ 58). Only men with complete semen and hormone analysis and a sperm concentration of at least /ml were included; men with azoospermia were excluded. Because our patients visit for couple infertility, we suspect problems on the female side in patients with normal sperm concentration (R /ml, WHO laboratory handbook [28]), and the screened volunteers with reduced sperm concentration (< /ml) are suspected to be infertile. Because our parameters under investigation were related only to spermatogenesis (and not pregnancy), we pooled the selected men and divided them into groups solely by their sperm concentration (normal sperm concentration vs. oligozoospermia) irrespective of their patient or volunteer status. In addition, we selected patients (n ¼ 31) irrespective of their sperm concentrations with current or former unilateral or bilateral maldescended testes but no other discernible cause of infertility. All subjects gave informed consent for scientific evaluation of their clinical data and underwent a complete clinical and physical examination. Scrotal contents and testicular volumes were investigated by ultrasonography. Semen and Hormone Analyses Analysis of sperm concentration was performed with Neubauer improved chambers, and motility was assessed on a heated (37 C) microscope stage. Slides for morphology assessment were stained with the modified Papanicolaou method and examined with phase-contrast optics. All values were determined in accordance with current WHO criteria (28). Progressive motility comprises WHO categories a and b. Total sperm count was calculated by multiplying semen volume by sperm concentration. Semen analysis in our laboratory is under constant internal and external quality control (33). Serum concentrations of LH, FSH, sex hormone binding globulin (SHBG), PRL, E 2, and prostate-specific antigen (PSA) were determined by immunofluorometric assays (Auto- DELFIA; Perkin Elmer, Freiburg, Germany) and serum T by a commercial direct solid-phase enzyme immunoassay (DRG AURICA ELISA Testosterone Kit; DRG Instruments, Marburg, Germany). Free T was calculated from total T and SHBG concentrations (34). Anti-M ullerian hormone levels were measured with use of an in-house ELISA assay (35) with Fertility and Sterility â 1813

3 an interassay and intra-assay coefficient of variation of <5% and <10%, respectively. External quality control for hormones followed the Deutsche Gesellschaft f ur Klinische Chemie e.v. (renamed Deutsche Vereinte Gesellschaft f ur Klinische Chemie und Laboratoriumsmedizin e.v. in 2004 [ All hormone assays were continuously under internal quality control. Statistics Comparisons between groups were carried out with use of the nonparametric Mann-Whitney test (for two groups) or Kruskal-Wallis test for comparison of the three groups. If significant differences were found, Dunn s multiple-comparison post hoc test was used. For correlation analyses, data were log transformed to approximate normal distribution and detect linear correlations by Pearson s analysis. All calculations were performed with GraphPad Prism (version 5.00 for Windows; GraphPad Software, San Diego, CA) or the Statistical Package for the Social Sciences (version 14.0; SPSS Inc., Chicago, IL). For all comparisons, P values (two sided) <.05 were considered statistically significant. For the correlation analyses 10 statistical tests were performed in each group, and therefore the significance level was adjusted so that only P values <.005 were considered significant (Bonferroni correction). RESULTS Cohort Characteristics Sixty men had a sperm concentration < /ml, and 108 men had normal concentrations. The group with maldescended testes (n ¼ 31) comprised 5 men with current unilateral (n ¼ 4) or bilateral (n ¼ 1) maldescended testes and 26 men with former maldescensus (n ¼ 9 unilateral and n ¼ 7 bilateral, side unknown in 10 men). Ten men had a history of hcg treatment, 19 of surgery (orchidopexy), and 2 of failed hcg treatment and subsequent surgery. The anthropometric characteristics and results of semen analyses of the three groups are described in Table 1. Age, height, weight, and body mass index (BMI) were not different between the groups. Bitesticular volume was significantly lower in men with oligozoospermia and men with maldescended testes compared with the men with normal sperm concentration (45 and 45 vs. 57 ml). Additionally, men with unilateral were compared with men with bilateral maldescended testes (n ¼ 13 and n ¼ 8, respectively) in a subgroup analysis. No significant differences were found between these groups (data not shown). Semen Parameters Duration of abstinence and semen volume were not different between the groups (Table 1). Sperm concentration (by selection criteria) was lower in the group with oligozoospermia but also in the group with maldescended testes. Total sperm count, percentage of progressively motile sperm, and normal morphology exhibited the same pattern, in the men with oligozoospermia and men with maldescended testes having comparable values that were always significantly lower than in the men with normal sperm concentration (Table 1). All semen parameters were comparable between men with unilateral and bilateral maldescended testes. TABLE 1 Anthropometric characteristics and results of semen analysis of men with normal sperm concentration, oligozoospermia, or maldescended testes. Normal sperm concentration (n [ 108) Oligozoospermia (n [ 60) Maldescended testes (n [ 31) P value Age (y) 34 (20 53) 34 (18 57) 35 (22 42).880 Height (cm) 180 ( ) 180 ( ) 183 ( ).179 Weight (kg) 84 (57 121) 85 (62 160) 86 (62 142).312 BMI (kg/m 2 ) 25.7 ( ) 26.3 ( ) 25.4 ( ).675 Bitesticular volume (ml) a 57 (22 96) 45 (23 74) b 45 (5 76) b <.001 Duration of abstinence (d) 3 (2 20) 3 (2 10) 4 (2 28).177 Sperm concentration (10 6 /ml) 49 (20 205) 9 (0.2 19) b 4 (0.1 63) b <.001 Semen volume (ml) 3.3 ( ) 3.6 ( ) 3.7 ( ).577 Total sperm count (10 6 ) 158 (24 677) 32 ( ) b 9 ( ) b <.001 Progressive motility (a þ b) (%) 51 (23 70) 45 (7 58) b 46 (15 64) c <.001 Normal morphology (%) 12 (4 28) 7 (1 18) b 6 (0 20) b <.001 Note: Data presented as median (range). Overall P values calculated by Kruskal-Wallis test. a Sum of volume of right and left testis. b Significantly different from men with normal sperm concentration, P<.001. c Significantly different from men with normal sperm concentration, P<.05. T uttelmann. AMH in men with normal and reduced sperm concentration. Fertil Steril T uttelmann et al. AMH in men with normal and reduced sperm concentration Vol. 91, No. 5, May 2009

4 Hormone Levels The hormone values are presented in Table 2. Follicle-stimulating hormone level was significantly higher in men with oligozoospermia and maldescended testes compared with men with normal sperm concentration. The only other differences between hormone levels were found between men with maldescended testes and oligozoospermia with lower T and free T levels in the former group. Moreover, E 2 was lower in the group with maldescended testes compared with the group with normal sperm concentration. No significant differences were found between men with unilateral and bilateral maldescended testes. Anti-M ullerian hormone levels did not show significant differences, if all three groups were tested (overall P¼.084). If the men with normal sperm concentration were compared with both groups of infertile men together (either oligozoospermia or maldescended testes), AMH in the infertile men then was found to be slightly lower than in the men with normal sperm concentration (5.3, vs. 6.3, ng/ml, P¼.028). Correlations No correlations were found between AMH and age, height, body weight, or BMI. Anti-M ullerian hormone correlated positively with bitesticular volume (Table 3) only in the men with maldescended testes (r ¼ 0.58, P¼.001) but not in the men with normal and reduced sperm concentrations (r ¼ 0.06, P¼.564 and r ¼ 0.16, P¼.223, respectively). Concerning hormones, AMH correlated negatively with FSH (Table 3) in men with oligozoospermia (r ¼ 0.43, P¼.001) and more pronounced in men with maldescended testes (r ¼ 0.64, P<.001). The correlation between AMH and FSH was not found in men with normal sperm concentration (r ¼ 0.13, P¼.181). No correlation between AMH and T or free T or any other hormone (LH, SHBG, PRL, E 2,or PSA) was found. Anti-M ullerian hormone also correlated with sperm concentration (Table 3) and similarly with total sperm count in men with maldescended testes (r ¼ 0.60, P<.001 for sperm concentration and r ¼ 0.52, P¼.003 for total sperm count) but not in oligozoospermia (r ¼ 0.29, P¼.024 and r ¼ 0.30, P¼.021) or men with normal sperm concentration (r ¼ 0.13, P¼.175 and r ¼ 0.11, P¼.258). Because the correlations were found only in the men with oligozoospermia and/or men with maldescended testes, we repeated the analyses in newly formed subgroups: the complete cohort (N ¼ 199) was divided into quartiles according to their sperm concentration, and the results of correlation analyses of the first quartile (men with sperm concentration /ml, n ¼ 50) are presented in Table 4. No correlations were found in the other three quartile groups. Subsequently, only the men of the lowest sperm concentration group were analyzed individually (either idiopathic or with maldescended testes), and correlations were confirmed only in the men with maldescended testes (Table 4). Additionally, these two groups were compared concerning all available anthropometric, semen, and hormone parameters: a significant difference was found only for T serum levels, where the men with maldescended testes had lower values (15.5, 9 24 vs. 12.5, 7 18 nmol/l, P¼.003). TABLE 2 Results of hormone analysis of men with normal sperm concentration, oligozoospermia, or maldescended testes. Normal sperm concentration (n [ 108) Oligozoospermia (n [ 60) Maldescended testes (n [ 31) P value FSH (U/L) 3.5 ( ) 5.6 ( ) a 4.2 (1.4 47) b <.001 LH (U/L) 3.2 ( ) 3.7 ( ) 3.2 ( ).165 T (nmol/l) 14.7 ( ) 15.0 ( ) 12.8 ( ) c.030 SHBG (nmol/l) 32 (10 72) 30 (14 67) 32 (12 56).797 Free T (pmol/l) 297 ( ) 310 ( ) 268 ( ) c.041 E 2 (pmol/l) 79 (47 164) 74 (42 146) 71 (41 102) b.038 PRL (mu/l) 192 (73 573) 198 (63 623) 164 (88 315).205 PSA (mg/l) 0.6 ( ) 0.7 ( ) 0.7 (0.2 2).753 AMH (ng/ml) 6.3 ( ) 4.9 ( ) 5.8 ( ).084 Note: Data presented as median (range). Overall P value calculated by Kruskal-Wallis test. a Significantly different from men with normal sperm concentration, P<.001. b Significantly different from men with normal sperm concentration, P<.05. c Significantly different from men with oligozoospermia, P<.05. T uttelmann. AMH in men with normal and reduced sperm concentration. Fertil Steril Fertility and Sterility â 1815

5 TABLE 3 Correlation analyses in men grouped according to study protocol. TVol FSH Conc T Men with normal sperm concentration (n ¼ 108) AMH r P TVol r P FSH r P Men with reduced sperm concentration (n ¼ 60) AMH r 0.16 L P TVol r P FSH r P Men with maldescended testes (n ¼ 31) AMH r 0.58 L P.001 <.001 < TVol r L P < FSH r L P < Note: Statistically significant correlations (P<.005) are marked in bold. TVol ¼ bitesticular volume; Conc ¼ sperm concentration. Values for r and P were calculated from log-transformed data. T uttelmann. AMH in men with normal and reduced sperm concentration. Fertil Steril DISCUSSION This study shows that serum AMH is significantly correlated with serum FSH and sperm concentration only in men with current or former maldescended testes but is not of diagnostic significance in male idiopathic infertility. As expected, in the presented cohort not only sperm concentration (by selection criteria) but also the other sperm parameters (total sperm count, percentage progressive motility and normal morphology) and bitesticular volume were lower in the men with oligozoospermia because these usually decrease with reduced sperm output and form the triad of oligoasthenoteratozoospermia. Furthermore, sperm concentration and count, motility and morphology, and T and free T serum levels were lower in the men with maldescended testes compared with men with normal sperm concentration, although they were selected irrespective of sperm concentration. These findings agree with those of previous studies showing that maldescended testes lead to a distinct phenotype with reduced endocrine and exocrine testicular function (11, 36) that might be part of the testicular dysgenesis syndrome (37). Follicle-stimulating hormone level was higher in men with oligozoospermia and maldescended testes alike as an indication of defective spermatogenesis and a result of feedback control probably by inhibin B (see below) or maybe by a direct involvement of AMH. Estradiol level was slightly reduced in men with maldescended testes compared with men with normal sperm concentration, most likely because of lower T levels. Anti-M ullerian hormone levels were not found to be significantly different between the three groups 1816 T uttelmann et al. AMH in men with normal and reduced sperm concentration Vol. 91, No. 5, May 2009

6 TABLE 4 Correlation analyses in all men grouped by sperm concentration (quartiles) and in men of the lowest quartile of sperm concentration with idiopathic oligozoospermia and maldescended testes separately. TVol FSH Conc T Sperm concentration First quartile (n ¼ 50) AMH r 0.49 L P <.001 <.001 < TVol r L P <.001 < FSH r L P < Second quartile (n ¼ 50) NS NS NS NS Third quartile (n ¼ 50) NS NS NS NS Fourth quartile (n ¼ 49) NS NS NS NS Sperm concentration, first quartile only Men without cause (n ¼ 29) Men with maldescended testes (n ¼ 21) AMH r P TVol r P FSH r P AMH r 0.59 L P.005 <.001 < TVol r L P < FSH r L P < Note: Statistically significant correlations (P<.005) are marked in bold. TVol ¼ bitesticular volume; Conc ¼ sperm concentration; NS ¼ not significant. Values for r and P were calculated from log-transformed data. T uttelmann. AMH in men with normal and reduced sperm concentration. Fertil Steril of men. However, when the groups of men with oligozoospermia and maldescended testes were compared with men with normal sperm concentration, serum AMH was found to be slightly lower in the former. This is in accordance with results of a previous study (27), which was contradicted by a second study of the same group (29) and indicates heterogeneity of the study populations; the authors probably included patients with maldescended testes in their male factor infertility group. Anti-M ullerian hormone levels in men with maldescended testes were comparable to those in the other two groups, and therefore absolute levels do not seem to distinguish between these pathologic entities. Most likely, only a subgroup of men with oligozoospermia not distinguishable by current clinical testing shares the underlying testicular pathophysiology with men with maldescended testes, thus explaining the discordant results. A positive correlation between AMH and bitesticular volume was found in men with current or former maldescended testes but not in the other two groups. Thus, serum AMH levels seem not to reflect Sertoli cell number and function in otherwise healthy men, unlike in prepubertal boys (38), but potentially do so in men with maldescended testes. Anti- M ullerian hormone correlated negatively with FSH in oligozoospermia and positively with sperm concentration and count in men with maldescended testes, but not in men with normal sperm concentration. In a previous study, fertile and infertile men were analyzed together and similar correlations were described (29), a finding possibly due to the men with oligozoospermia. Because AMH correlated positively with sperm concentration in the men with maldescended testes, AMH might play a role as a marker of Sertoli cell function and maturation status in adult males when spermatogenesis is impaired. This is supported by the histologic finding that AMH is found only in tubules with incomplete spermatogenesis (7). All correlations with AMH were either detected only or found to be most distinct in men with maldescended testes, and the analysis of the subgroup of men with lowest sperm Fertility and Sterility â 1817

7 concentrations (first quartile) suggests that AMH might be a marker for spermatogenesis only if it is impaired and, most likely, the maldescended testis represents a discrete pathophysiologic situation. The shared pathophysiology of the maldescended testes (in relation to testicular dysgenesis syndrome) is underlined by the fact that these correlations were found, although men with different phenotypes (unilateral or bilateral maldescensus) and treatments (hcg, orchidopexy, none) were included. On the other hand, we did not study men with other known causes of their reduced spermatogenesis (e.g., varicocele, infections) and cannot extrapolate this conclusion to those conditions. The negative correlation between AMH and FSH either might reflect an involvement in the signaling and regulation of FSH (29) or most probably be a symptom of impaired or immature Sertoli cells. Overall, the characteristics of AMH closely resemble those of inhibin B as another product of Sertoli cells, which has been studied in adult men for fertility evaluation for a longer time. Like AMH, inhibin B correlates positively with testicular volume and sperm concentration and negatively with FSH, while, in the case of inhibin B, involvement in the regulation by negative feedback on the pituitary is well established (39 41). Whether the additional determination of AMH might augment the diagnostic value of inhibin B and vice versa in male factor infertility workup, outcome prognosis of assisted reproduction or likelihood of finding sperm by testicular sperm extraction remains to be determined. In conclusion, AMH serum levels are not affected by impaired spermatogenesis in general but are significantly correlated with spermatogenic parameters in men with current or former maldescended testes. Therefore, AMH measurement does not improve current clinical routine diagnostics. However, as a marker of Sertoli cell number, function, and/or maturation AMH should be carefully evaluated in patients with maldescended testes in the future but might also be worthwhile studying in patients with other diagnoses leading to reduced spermatogenesis. Acknowledgments: The authors thank Ms. Reinhild Sandhowe for her technical assistance. The language editing of Susan Nieschlag, M.A., is gratefully acknowledged. REFERENCES 1. Rey R, Lukas-Croisier C, Lasala C, Bedecarras P. AMH/MIS: what we know already about the gene, the protein and its regulation. Mol Cell Endocrinol 2003;211: Lee MM, Donahoe PK. Mullerian inhibiting substance: a gonadal hormone with multiple functions. Endocr Rev 1993;14: Josso N, Picard JY, Rey R, di Clemente N. Testicular anti-mullerian hormone: history, genetics, regulation and clinical applications. Pediatr Endocrinol Rev 2006;3: Wilhelm D, Palmer S, Koopman P. Sex determination and gonadal development in mammals. Physiol Rev 2007;87: Lee MM, Donahoe PK, Hasegawa T, Silverman B, Crist GB, Best S, et al. Mullerian inhibiting substance in humans: normal levels from infancy to adulthood. J Clin Endocrinol Metab 1996;81: Bergada I, Milani C, Bedecarras P, Andreone L, Ropelato MG, Gottlieb S, et al. Time course of the serum gonadotropin surge, inhibins, and anti-mullerian hormone in normal newborn males during the first month of life. J Clin Endocrinol Metab 2006;91: Maymon BB, Yogev L, Paz G, Kleiman SE, Schreiber L, Botchan A, et al. Sertoli cell maturation in men with azoospermia of different etiologies. Fertil Steril 2002;77: Steger K, Rey R, Kliesch S, Louis F, Schleicher G, Bergmann M. Immunohistochemical detection of immature Sertoli cell markers in testicular tissue of infertile adult men: a preliminary study. Int J Androl 1996;19: Steger K, Rey R, Louis F, Kliesch S, Behre HM, Nieschlag E, et al. Reversion of the differentiated phenotype and maturation block in Sertoli cells in pathological human testis. Hum Reprod 1999;14: Barthold JS, Gonzalez R. The epidemiology of congenital cryptorchidism, testicular ascent and orchiopexy. J Urol 2003;170: Murphy F, Paran TS, Puri P. Orchidopexy and its impact on fertility. Pediatr Surg Int 2007;23: Hutson JM, Donahoe PK. The hormonal control of testicular descent. Endocr Rev 1986;7: Guerrier D, Tran D, Vanderwinden JM, Hideux S, Van Outryve L, Legeai L, et al. The persistent Mullerian duct syndrome: a molecular approach. J Clin Endocrinol Metab 1989;68: Brook CG, Wagner H, Zachmann M, Prader A, Armendares S, Frenk S, et al. Familial occurrence of persistent Mullerian structures in otherwise normal males. Br Med J 1973;1: Josso N, Fekete C, Cachin O, Nezelof C, Rappaport R. Persistence of Mullerian ducts in male pseudohermaphroditism, and its relationship to cryptorchidism. Clin Endocrinol 1983;19: Behringer RR, Finegold MJ, Cate RL. Mullerian-inhibiting substance function during mammalian sexual development. Cell 1994;79: Adham IM, Agoulnik AI. Insulin-like 3 signalling in testicular descent. Int J Androl 2004;27: Nef S, Parada LF. Cryptorchidism in mice mutant for Insl3. Nat Genet 1999;22: Hutson JM, Hasthorpe S. Testicular descent and cryptorchidism: the state of the art in J Pediatr Surg 2005;40: Young J, Rey R, Couzinet B, Chanson P, Josso N, Schaison G. Antimullerian hormone in patients with hypogonadotropic hypogonadism. J Clin Endocrinol Metab 1999;84: Rey R, Lordereau-Richard I, Carel JC, Barbet P, Cate RL, Roger M, et al. Anti-mullerian hormone and testosterone serum levels are inversely related during normal and precocious pubertal development. J Clin Endocrinol Metab 1993;77: Themmen AP. Anti-Mullerian hormone: its role in follicular growth initiation and survival and as an ovarian reserve marker. J Natl Cancer Inst Monogr 2005;34: Visser JA, de Jong FH, Laven JS, Themmen AP. Anti-Mullerian hormone: a new marker for ovarian function. Reproduction 2006;131: Laven JS, Mulders AG, Visser JA, Themmen AP, De Jong FH, Fauser BC. Anti-Mullerian hormone serum concentrations in normoovulatory and anovulatory women of reproductive age. J Clin Endocrinol Metab 2004;89: Fenichel P, Rey R, Poggioli S, Donzeau M, Chevallier D, Pointis G. Anti-Mullerian hormone as a seminal marker for spermatogenesis in non-obstructive azoospermia. Hum Reprod 1999;14: Fujisawa M, Yamasaki T, Okada H, Kamidono S. The significance of anti-mullerian hormone concentration in seminal plasma for spermatogenesis. Hum Reprod 2002;17: Al-Qahtani A, Muttukrishna S, Appasamy M, Johns J, Cranfield M, Visser JA, et al. Development of a sensitive enzyme immunoassay for anti-mullerian hormone and the evaluation of potential clinical applications in males and females. Clin Endocrinol 2005;63: World Health Organization. Laboratory manual for the examination of human semen and semen cervical mucus interaction. 4th ed. New York: Cambridge University Press, 1999: Appendix IA, Appasamy M, Muttukrishna S, Pizzey AR, Ozturk O, Groome NP, Serhal P, et al. Relationship between male reproductive hormones, sperm DNA damage and markers of oxidative stress in infertility. Reprod Biomed Online 2007;14: T uttelmann et al. AMH in men with normal and reduced sperm concentration Vol. 91, No. 5, May 2009

8 30. Mostafa T, Amer MK, Abdel-Malak G, Nsser TA, Zohdy W, Ashour S, et al. Seminal plasma anti-mullerian hormone level correlates with semen parameters but does not predict success of testicular sperm extraction (TESE). Asian J Androl 2007;9: Duvilla E, Lejeune H, Trombert-Paviot B, Gentil-Perret A, Tostain J, Levy R. Significance of inhibin B and anti-mullerian hormone in seminal plasma: a preliminary study. Fertil Steril 2008;89: T uttelmann F, Luetjens CM, Nieschlag E. Optimising workflow in andrology: a new electronic patient record and database. Asian J Androl 2006;8: Cooper TG, Bjorndahl L, Vreeburg J, Nieschlag E. Semen analysis and external quality control schemes for semen analysis need global standardization. Int J Androl 2002;25: Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab 1999;84: Kevenaar ME, Meerasahib MF, Kramer P, van de Lang-Born BM, de Jong FH, Groome NP, et al. Serum anti-mullerian hormone levels reflect the size of the primordial follicle pool in mice. Endocrinology 2006;147: Hutson JM, Hasthorpe S. Abnormalities of testicular descent. Cell Tissue Res 2005;322: Kaleva M, Toppari J. Cryptorchidism: an indicator of testicular dysgenesis? Cell Tissue Res 2005;322: Rey R, al-attar L, Louis F, Jaubert F, Barbet P, Nihoul-Fekete C, et al. Testicular dysgenesis does not affect expression of anti-mullerian hormone by Sertoli cells in premeiotic seminiferous tubules. Am J Pathol 1996;148: Kumanov P, Nandipati KC, Tomova A, Robeva R, Agarwal A. Significance of inhibin in reproductive pathophysiology and current clinical applications. Reprod Biomed Online 2005;10: Lockwood G. The diagnostic value of inhibin in infertility evaluation. Semin Reprod Med 2004;22: Meachem SJ, Nieschlag E, Simoni M. Inhibin B in male reproduction: pathophysiology and clinical relevance. Eur J Endocrinol 2001;145: Fertility and Sterility â 1819