Human Reproduction, Vol. 14, No. 8, 2020-2024,
August 1999
© 1999 European Society of Human Reproduction and Embryology
Anti-Müllerian hormone as a seminal marker for spermatogenesis in non-obstructive azoospermia
1 Groupe de Recherche sur l'Interaction Gamétique CJF INSERM 95–04, Institut Fédératif de Recherche 50, Faculté de Médecine, 06107 Nice, and 2 Unité de Recherches sur l'Endocrinologie du Développement, INSERM U493, Ecole Normale Supérieure, Département de Biologie, 92120 Montrouge, France
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Abstract |
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Anti-Müllerian hormone (AMH) also known as Müllerian inhibiting substance or factor, is a Sertoli cell-secreted glycoprotein responsible in male embryos for Müllerian duct regression. However, its role in adults remains unknown. AMH seminal concentrations have been evaluated using an enzyme-linked immunoassay in three groups of young men: group 1, fertile donors (n = 18); group 2, obstructive azoospermia (n = 9) after vasectomy or associated with deferent duct agenesia; and group 3, non-obstructive azoospermia with spermatogenesis deficiency and normal karyotype (n = 23). AMH was present in seminal plasma of most fertile donors at concentrations ranging from undetectable (<3.5 pmol/l) up to 543 pmol/l (geometric mean: 153 pmol/l), higher than the serum level (range <3.5 up to 67 pmol/l, geometric mean: 10.7 pmol/l, n = 13). Seminal AMH concentrations were undetectable in all obstructive azoospermic patients, confirming its testicular origin. In non-obstructive azoospermia (group 3), seminal AMH concentration was lower (range <3.5–68.5 pmol/l, geometric mean: 17 pmol/l) than in fertile donors (P < 0.003) without correlation with plasma follicle stimulating hormone values. In group 3, comparison of seminal AMH concentration and the results of histological analysis of testicular biopsies revealed that undetectable AMH found in 14 cases was associated in 11 of them with lack of spermatozoa, while detectable concentrations of AMH (10–68.5 pmol/l) found in nine cases were associated in seven of them with persistent spermatogenesis. In the adult, AMH is secreted preferentially towards the seminiferous lumen. Although its relationship with spermatogenesis requires further investigation, our results suggest that seminal AMH may represent a non-invasive marker of persistent hypospermatogenesis in cases of non-obstructive azoospermia which may indicate the likely success of testicular spermatozoa recovery before intracytoplasmic sperm injection.
Key words: anti-Müllerian hormone/non-obstructive azoospermia/seminal marker/spermatogenesis/testicular sperm injection
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Anti-Müllerian hormone (AMH) is a glycoprotein dimer structurally related to inhibin and transforming growth factor ß, which is specifically secreted in the male by the Sertoli cells (for review see Josso et al., 1993
). Its major function identified so far is to allow regression of Müllerian duct in the male fetus. AMH blood concentration decreases dramatically during puberty (Josso et al., 1990) and persists at very low values in adults (Lee et al., 1996). However, it has been detected in the rete testis of bulls (Vigier et al., 1983), boars (Josso et al., 1979) and rams (Cazorla et al., 1998), and in seminal plasma of normal fertile men (Fallat et al., 1996). Very little is known about the function of AMH in postnatal life; it has recently been shown that it controls Leydig cell proliferation and steroidogenic function (Racine et al., 1998) and that it may be related to germinal cell proliferation (Cazorla et al., 1998). In order to better understand its possible physiological role in the adult male and to verify its potential value as a seminal marker of spermatogenesis in cases of infertility, we have analysed seminal AMH concentrations in three groups of patients: fertile donors and infertile patients with obstructive or non-obstructive azoospermia. |
Materials and methods |
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Patients
Samples of seminal plasma were collected after informed consent in three groups of young men (age = 28–38 years). Group 1 consisted of 18 fertile donors with normal sperm parameters following WHO criteria (WHO, 1992). Groups 2 and 3 consisted of a consecutive series of azoospermic patients attending the infertility department of the Centre Hospitalo-Universitaire of Nice between 1997 and 1998. Azoospermia was assessed following centrifugation and analysis of spermatozoa in at least two samples from each patient. Surgical exploration was indicated for diagnosis of obstructive or non-obstructive azoospermia. In each case, complete surgical exploration was conducted including each deferent duct, epididymis with analysis and immediate verification of spermatozoa in the epididymal fluid, and one open testicular biopsy performed on each testis for histological analysis. Group 2 consisted of four patients with deferens duct agenesia and five with vasectomy. Group 3 consisted of 23 cases of non-obstructive azoospermia with normal 46 XY karyotype and variable spermatogenesis deficiency. For group 3, clinical, biological and histological features are indicated in Table I
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Seminal plasma preparation and storage
Semen specimens were obtained by masturbation after sexual abstinence for 2–4 days and were allowed to liquefy at room temperature for 30 min. Seminal plasma was obtained by centrifugation at 600 g at 4°C for 15 min and then aliquotted and rapidly stored at –80°C until assayed.
AMH assay
AMH was measured in seminal plasma and in serum in duplicate using the AMH/MIS ELISA kit (Immunotech-Coulter, Marseille, France) as described (Rey et al., 1999). A preparation of purified recombinant human AMH was used to construct a standard curve. The limit of sensitivity of the assay was 3.5 pmol/l.
Histological analysis
Testicular biopsies were fixed by immersion in Bouin's solution and embedded in paraffin wax. The slides were stained with haematoxylin and eosin for evidence of spermatogenesis, which was classified according to Levin (Levin, 1979): normal spermatogenesis, germ-cell hypoplasia or hypospermatogenesis, complete or incomplete maturation arrest, complete or incomplete germ-cell aplasia (Sertoli cell-only syndrome) and tubular sclerosis. Serial sections in different regions of each biopsy were studied and qualitative analysis of >15 seminiferous tubules was performed.
Statistical analysis
The non-parametric Mann–Whitney test was used for comparison between groups of patients concerning follicle stimulating hormone (FSH) and AMH values. The correlation coefficient (r) between FSH and AMH was evaluated by analysing for Spearman's rank correlation test. Significance was defined as P < 0.05.
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Results |
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In 18 fertile men (group 1) AMH was present in seminal plasma (Figure 1
) at concentrations ranging from undetectable (<3.5 pmol/l) to 543 pmol/l (geometric mean: 153 pmol/l), significantly higher than the serum concentration (range <3.5–67 pmol/l, geometric mean 10.7 pmol/l) measured at the same time for 13 of the 18 men (P < 0.005). AMH was undetectable in three cases out of 18 in seminal plasma and five cases out of 13 in serum.
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AMH was not detectable in seminal plasma (Figure 1
) in any case of obstructive azoospermia after vasectomy or with deferent ducts agenesia, suggesting its testicular origin. This point was confirmed by one fertile patient who had been evaluated before (493 pmol/l) and after vasectomy (<3.5 pmol/l) and by another patient with bilateral anorchidism who did not present any detectable seminal AMH (data not shown).
Seminal AMH in non-obstructive azoospermia (Figure 1) was undetectable in 14 cases out of 23, including five cases of cancer treated by radio- and chemotherapy. In the remaining nine cases AMH was low (range: 3.5–68.5 pmol/l, geometric mean 17 pmol/l) compared to the control group (P < 0.003). No correlation could be found between seminal AMH and plasma FSH (r = 0.107, P = 0.12) (Figure 2) or with plasma testosterone (results not shown). Histological findings for this group of patients are shown in Table I. In some cases iterative spermograms obtained after centrifugation revealed a few spermatozoa in the pellet, indicating persistent hypospermatogenesis. Spermatozoa were found in pellets and/or in testicular biopsies in nine out of 23 cases from group 3. Complete lack of spermatozoa was diagnosed in 14 cases. Figure 3 represents FSH values in the non-obstructive azoospermic group according to positive or negative sperm identification. FSH values (means ± SD) were lower (11.4 ± 1.9 IU/l versus 20.9 ± 3.9 IU/l: P < 0.05) in the case of presence of spermatozoa in pellets and/ or in biopsies. However, this was a poor individual predictor because several patients with a low FSH value did not present any detectable spermatozoa in the biopsies. Plasma testosterone concentrations were not different between the two groups (data not shown). Seminal AMH seemed to be related to persistent hypospermatogenesis. As indicated in Figure 4, seminal AMH concentrations were significantly higher in patients with detectable spermatozoa in their biopsies as compared to patients without spermatozoa (P < 0.003). In addition, detectable AMH concentrations were found in 10 cases and in seven was associated with the presence of spermatozoa in the analysed biopsies; in 13 cases AMH was undetectable and in 11 cases was associated with the absence of spermatozoa. The positive predictive value of seminal AMH for sperm identification was 70% and the negative predictive value was 83%.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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This work demonstrates that high concentrations of AMH of testicular origin are detectable in seminal fluid of fertile adult men. These results are in agreement with several studies performed on the rete testis fluid in mammals (Josso et al., 1979
; Vigier et al., 1983; Cazorla et al., 1998) and on seminal plasma in men (Fallat et al., 1996). It has been estimated (Purvis et al., 1975) that the testicular/epididymal contribution to the volume of the ejaculate represents 5%. This may suggest that higher concentrations of AMH are present in the seminiferous tubule fluid as compared to seminal plasma. Comparison of seminal and serum AMH concentrations suggests that, after puberty, AMH is preferentially secreted by the apical pole of the Sertoli cell towards the seminiferous lumen. This may be related to the testicular barrier, which becomes functional during puberty. Whether it also suggests a functional role for AMH in the adult gonad needs to be elucidated. In adult rams, it has been reported (Cazorla et al., 1998) that there is a negative correlation between daily production of leptotene primary spermatocytes, expressed per testis or per Sertoli cell, and AMH concentration in the rete testis fluid, suggesting a relationship between meiotic entry on AMH expression in pubertal man and mouse (Rey, 1998). However, the presence of developmentally more advanced spermatogenic cells may enhance AMH secretion, relating to specific stages of the seminiferous epithelium cycle (Baarends et al., 1995). The fact that in the present study a dramatic decrease in seminal AMH concentration was associated with spermatogenic failure may also suggest a link between AMH and spermatogenesis. However, we cannot rule out the possibility that the decrease in AMH concentration reflects a primary alteration in Sertoli cell function that also leads to spermatogenic arrest.
AMH may represent a non-invasive marker of persistent spermatogenesis in non-obstructive azoospermia. Male infertility with testicular failure remains in most cases poorly understood. Azoospermia can be either of excretory or secretory origin. In the latter case, partial or complete lack of testicular production of normal spermatozoa is usually associated with a reduced testicular volume and an elevated FSH plasma concentration. Histological analysis of the testicular biopsies may show abnormal or lack of spermatogenesis and no therapeutic solution was, until recently, available. However, intracytoplasmic sperm injection (ICSI), which allows fertilization with very few spermatozoa, has recently provided an extraordinary therapeutic advance. Even in the case of azoospermia, epididymal puncture or testicular biopsies (Devroey et al., 1995) can yield sufficient spermatozoa to allow MESI (microsurgery sperm injection) or TESI (testicular sperm injection).
Recent reports have suggested that recovery of testicular spermatozoa may be possible in >50% of cases of true `non-obstructive' azoospermia regardless of clinical parameters concerning size of testes or plasma FSH concentrations (Tournaye et al., 1997). The presence of spermatozoa in a unique randomly taken testicular biopsy appears to be for the present the only predictive factor. Even lack of spermatozoa in one testicular biopsy does not guarantee a complete lack of spermatozoa in the testes (Gottschalk-Sabag and Weiss, 1995). In 43% of cases of secretory azoospermia, repetitive multiple biopsies enabled recovery of sufficient spermatozoa for microinjection, despite a negative preliminary biopsy, suggesting focal hypospermatogenesis (Tournaye et al., 1997). However, ICSI requires tight co-ordination with treatment of the female, repetitive surgery has psychological and financial implications and long-term consequences of multiple biopsies have not so far been well evaluated. Therefore objective markers of focal hypospermatogenesis are urgently needed to indicate whether biopsies should be repeated or not. FSH has been for a long time considered to be an efficient marker of azoospermia: elevated FSH represents an altered spermatogenesis (Martin-Du-Pan and Bishop, 1995) and normal FSH is associated with obstructive azoospermia. In fact, the increasing use of testicular screening has clearly demonstrated that plasma FSH can no longer be considered to be a definitive marker for azoospermia (Chen et al., 1996; Novero et al., 1997; Ezeh et al., 1998). Seminal markers have therefore been proposed to improve understanding of germinal deficiency and to allow discrimination between total lack of, incomplete or reduced spermatogenesis. Such markers must be of testicular origin, disappear after vasectomy, be specifically secreted by the Sertoli or the germinal cells at high concentrations, be associated with spermatogenesis and be still present in seminal plasma in a detectable concentration. Several have already been proposed, e.g. transferrin (Barthelemy et al., 1988), lactate dehydrogenase (LDH) (Orlando et al., 1988), insulin-like growth factor (IGF)-1 and 
2 macroglobulin (Glander et al., 1996) and inhibin B (Anderson et al., 1998). However, none appears to be a convenient testicular marker either for diagnosis or for prognosis. Seminal AMH may be such a marker with an individual predictive value, since in this study, testicular spermatozoa appear to be mainly associated with detectable seminal AMH. However, some fertile donors had undetectable AMH concentrations; it is possible that the period of time prior to freezing the samples may influence the results due to the presence of seminal proteases. These preliminary results must be confirmed on a wider scale and compared to other predictive factors such as testicular volume, plasma FSH and seminal inhibin B in a multivariate analysis before AMH is used to indicate repeated biopsies after an initial negative result.
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Acknowledgments |
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We are grateful to Mrs Liliane Nakache from the CECOS laboratory of Nice for her technical assistance and to Dr C.Pradier for statistical analysis.
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Notes |
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3 To whom correspondence should be addressed
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Submitted on December 29, 1998; accepted on April 19, 1999.
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