1 Groupe de Recherche sur l'Interaction Gamétique CJF INSERM 9504, 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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Key words: anti-Müllerian hormone/non-obstructive azoospermia/seminal marker/spermatogenesis/testicular sperm injection
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Introduction |
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Materials and methods |
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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 MannWhitney 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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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: 3.568.5 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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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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Notes |
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References |
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Submitted on December 29, 1998; accepted on April 19, 1999.