Centre For Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan, Belgium
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: ICSI/inhibin B/male infertility/non-obstructive azoospermia/TESE
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Serum inhibin is a glycoprotein of gonadal origin which has an inhibitory effect on gonadotrophin secretion. It is known to originate from the Sertoli cells, and its secretion is regulated by interaction with germ cells (Pineau et al., 1990). This glycoprotein is a heterodimer consisting of an
-subunit and one of two different ß-subunits: ßA (inhibin A) and ßB (inhibin B). Inhibin B is the most important form in human males and may serve as a marker of spermatogenesis (Anawalt et al., 1996
; Illingworth et al., 1996
; Jensen et al., 1997
; Kolb et al., 2000
). The normal range of serum concentration in proven fathers is 94 to 327 pg/ml (Von Eckardstein et al., 1999
).
One report (Ballesca et al., 2000) suggested that inhibin B might also be a good non-invasive predictor for retrieving sperm by TESE, though another study failed to corroborate this finding (Von Eckardstein et al., 1999
). The aim of the present study was to assess the predictive value of inhibin B before testicular sperm retrieval in a large population of patients with NOA.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hormone analyses
Serum inhibin B was measured using a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) (Inhibin B, Oxford Bio-Innovation Ltd, Oxford, UK). The samples were pre-treated with detergent (sodium dodecyl sulphate), heated to 100°C, and exposed to hydrogen peroxide. Pre-treated samples were incubated in microtitre plates, coated with a monoclonal antibody to the ßB-subunit of inhibin. The detection antibody was a monoclonal antibody specific for the -subunit of inhibin and coupled to alkaline phosphatase. The kit was calibrated against recombinant 32 kDa inhibin B. The detection limit of the assay was 8 pg/ml, and the between-assay coefficient of variation was 7.5 at 197 pg/ml (n = 8). A cross-reactivity of <1% with inhibin A was apparent, but no cross-reactivity was found with pro-alpha C subunits or activins. Patients with inhibin B concentrations below the detection level were included in the analysis and assigned a value of 8 pg/ml.
FSH assessment was performed during the initial work-up for each man before embarking on a TESE procedure. Serum FSH was measured using a commercially available monoclonal immunoradiometric assay (bioMérieux, France). The FSH assay, calibrated against the 2nd IRP 78/549, had an analytical sensitivity of 0.25 IU/l and within- and between-run coefficients of variation of<4 and <9% respectively.
Testicular sperm recovery
Open excisional testicular biopsies were taken under general anaesthesia (Tournaye et al., 1997) on the day of oocyte retrieval. Surgery was discontinued when sperm or late elongated spermatids (stage Sd2) (Clermont, 1963
) were found on wet preparations of the biopsies, or when a representative number of samples (at least four biopsies on each side) were taken randomly. Wet preparation of testicular tissue was performed by mechanical shredding as described previously (Jow et al., 1993
; Verheyen et al., 1995
). Since 1998, enzymatic digestion of the testicular tissue with collagenase type IV has been performed if no sperm were found after mechanical shredding (Crabbé et al., 1998
). During surgery, a randomly taken single small biopsy was sent for histopathological examination.
Statistical analysis
The two groups of patients with either successful or unsuccessful sperm recovery were evaluated by receiver operating characteristics (ROC) analysis (Zweig and Campbell, 1993) using Medcalc (Medcalc Software, Ghent, Belgium). ROC graphs are plots of all the sensitivity (y-axis) and specificity (x-axis) pairs which are possible for all levels of a particular parameter, for example serum inhibin B concentration. Calculation of the area under the curve provides a quantitative measure of accuracy, namely the ability of this parameter to discriminate between two conditions. A test with perfect discrimination has an ROC plot that passes through the upper left corner, where the true positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity). The theoretical plot for a test with no discrimination (identical distributions of results for the two groups) is a 45° diagonal line from the lower left corner (0% true-positive rate and 0% false-positive rate) to the upper right corner (100% true-positive rate and 100% false-positive rate) with an area under the curve of 0.5 (Zweig and Campbell, 1993
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As shown in Figure 1, there was also a wide distribution of inhibin B concentrations, with an important overlap between successful and unsuccessful sperm retrievals. Many patients also had an inhibin B concentration below the detectable limit.
Despite the fact that the role of inhibin B as a marker for spermatogenesis has been established, the inability to detect inhibin B concentrations <8 pg/ml with the currently available assay might be a cause of failure to predict sperm retrieval before TESE.
Inhibin B has been reported to be associated with late stages of spermatogenesis (Pineau et al., 1990; Andersson et al., 1998
). Because only a few men in the present study population may have had spermatogenesis progressing beyond the spermatocyte stage in a representative number of their seminiferous tubules, inhibin B might have a limited predictive power.
The difference with the above-mentioned studies may have been be due to the number of patients included, and also to differences in patient selection. Prediction based on inhibin B in men appears to be better in those with a higher spermatogenetic output (Figure 4). In the present study, 73% of azoospermic patients had severely disturbed spermatogenesis (germ-cell aplasia and tubular sclerosis and atrophy), whereas in studies reported earlier (Pierik et al., 1998
; Von Eckardstein et al., 1999
; Ballesca et al., 2000
) it was mainly men with a better spermatogenetic function who were included.
The difference in endpoint in the different studies might also be a reason for the discrepancy in results. In one study (Pierik et al., 1998) a quantitative endpoint (the Johnsen score) was used, while others (Von Eckardstein et al., 1999
) used the histological subtypes (semi-quantitative). In the present study and one other study (Ballesca et al., 2000
), sperm retrieval was used as an endpoint (qualitative). As for the other previously examined predictors, namely testicular size, serum FSH level and testicular histology, the poor correlation of successful sperm retrieval with inhibin B might also have been due to the non-quantitative nature of this endpoint. Standardization of sperm retrieval is difficult in a clinical setting, and consequently the amount of testicular tissue was not uniform for all patients. However, an attempt was made to standardize this endpoint as well as possible by taking at least four biopsies from each side if no sperm were directly found at wet preparation. These biopsies were taken randomly because of the heterogeneously distributed spermatogenesis throughout the testis (Gottschalk-Sabag et al., 1995
; Tournaye et al., 1996
).
The predictive power of assessing inhibin B combined with serum FSH level has also been assessed (Von Eckardstein et al., 1999). Although the combination showed a better predictive value, these authors nevertheless concluded that inhibin B alone or in combination with FSH was of limited predictive value for TESE. The present results also indicate that combining FSH and inhibin B, either by using a ratio or by creating subgroups of patients on the basis of their serum FSH levels, does not improve the predictive power of inhibin B. Although the role of inhibin B is clear as a marker for spermatogenesis, the present data indicate that it does not have any role in predicting the presence of sperm before TESE.
In the future, other markersboth genetical and/or biochemicalwill likely be investigated in order to improve the prediction of successful TESE. One study (Brandell et al., 1998) has outlined results from a limited series of patients in whom the presence of AZFb micro-deletions of the Y chromosome indicated an unsuccessful TESE.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anawalt, B.D., Bebb, R.A., Matsumoto, Groome, N.P., Illingworth P.J., McNeilly, A.S. and Bremner W.J. (1996) Serum inhibin B levels reflect Sertoli cell function in normal men and men with testicular dysfunction. J. Clin. Endocrinol. Metab., 81, 33413345.[Abstract]
Ballesca, J.L., Balasch, J., Calafell, J.M., Alvarez, R., Fabregues, F., Martinez de Osaba, M.J., Ascaso, C. and Vanrell, J.A. (2000) Serum inhibin B determination is predictive of successful testicular sperm extraction in men with non-obstructive azoospermia. Hum. Reprod., 15, 17341738.
Brandell, R.A., Mielnik, A., Liotta, D., Ye, Z., Veeck, L.L., Palermo, G.D. and Schlegel, P.N. (1998) AZFb deletions predict the absence of sperm with testicular sperm extraction: preliminary report of a prognostic genetic test. Hum. Reprod., 13, 28122815.
Clermont, Y. (1963) The cycle of the seminiferous epithelium in men. Am. J. Anat., 112, 3552.[ISI]
Crabbé, E., Verheyen, G., Silber, S., Tournaye, H., Van de Velde, H., Goossens, A. and Van Steirteghem, A. (1998) Enzymatic digestion of testicular tissue may rescue the intracytoplasmic sperm injection cycle in some patients with non-obstructive azoospermia. Hum. Reprod., 13, 27912796.
Devroey, P., Liu, J., Nagy, Z., Goossens, A., Tournaye, H., Camus, M., Van Steirteghem, A. and Silber, S. (1995) Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum. Reprod., 10, 14571460.[Abstract]
Ezeh, U.I.O., Taub, N.A., Moore, H.D.M. and Cooke, I.D. (1999) Establishment of predictive variables associated with testicular sperm retrieval in men with non-obstructive azoospermia. Hum. Reprod., 14, 10051012.
Friedler, S., Raziel, A., Strassburger, D., Soffer, Y., Komarovsky, D. and Ron-El, R. (1997) Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia. Hum. Reprod., 12, 14881493.[Abstract]
Gottschalk-Sabag, S., Weiss, D.B., Folb-Zacharow, N. and Zukerman, Z. (1995) Is one testicular specimen sufficient for quantitative evaluation of spermatogenesis? Fertil. Steril., 64, 399402.[ISI][Medline]
Illingworth, P.J., Groome, N.P., Byrd, W., Rainey, W.E., McNeilly, A.S., Mather, J.P. and Bremner, W.J. (1996) Inhibin-B: a likely candidate for the physiologically important form of inhibin in men. J. Clin. Endocrinol. Metab., 81, 13211325.[Abstract]
Jensen, T.K., Andersson, A.-M., Hjollund, N.H.I., Scheike, T., Kolstad, H., Giwercman, A., Henriksen, T.B., Ernst, E., Bonde, J.P., Olsen, J. et al. (1997) Inhibin B as a serum marker of spermatogenesis: correlation to differences in sperm concentration and follicle-stimulating hormone levels. A study of 349 Danish men. J. Clin. Endocrinol. Metab., 82, 40594063.
Jow, W.W., Steckel, J., Schlegel, P., Magid, M.S. and Goldstein, M. (1993) Motile sperm in human testis biopsy specimens. J. Androl., 14, 194198.[Abstract]
Kolb, B.A., Stanczyk, F.Z. and Sokol, R.Z. (2000) Serum inhibin B levels in males with gonadal dysfunction. Fertil. Steril., 74, 234238.[ISI][Medline]
Pierik, F.H., Vreeburg, J.T.M., Stijnen, T., De Jong, F.H. and Weber, R.F.A. (1998) Serum inhibin B as a marker of spermatogenesis. J. Clin. Endocrinol. Metab., 83, 31103114.
Pineau, C., Sharpe, R.M., Saunders, P.T.K., Gerard, N. and Jegou, B. (1990) Regulation of Sertoli cell inhibin production and of inhibin -subunit mRNA levels by specific germ cell types. Mol. Cell. Endocrinol., 72, 1322.[ISI][Medline]
Ron-El, R., Strauss, S., Friedler, S., Strassburger, D., Komarovsky, D. and Raziel, A. (1998) Serial sonography and colour flow Doppler imaging following testicular and epididymal sperm extraction. Hum. Reprod., 13, 33903393.[Abstract]
Schlegel, P.N. and Su, L. (1997) Physiological consequences of testicular sperm extraction. Hum. Reprod., 12, 16881692.[Abstract]
Tournaye, H., Camus, M., Goossens, A., Nagy, Z., Silber, S., Van Steirteghem, A.C. and Devroey, P. (1995) Recent concepts in the management of infertility because of non-obstructive azoospermia. Hum. Reprod., 10 (Suppl. 1), 115119.[ISI][Medline]
Tournaye, H., Liu, J., Nagy, P.Z., Camus, M., Goossens, A., Silber, S., Van Steirteghem, A.C. and Devroey, P. (1996) Correlation between testicular histology and outcome after intracytoplasmic sperm injection using testicular sperm. Hum. Reprod., 11, 127132.[Abstract]
Tournaye, H., Verheyen, G., Nagy, P., Goossens, A., Ubaldi, F., Silber, S., Van Steirteghem, A. and Devroey, P. (1997) Are there any predictive factors for successful testicular sperm recovery in azoospermic patients? Hum. Reprod., 12, 8086.[ISI][Medline]
Verheyen, G., De Croo, I., Tournaye, H., Pletinckx, I., Devroey, P. and Van Steirteghem, A. (1995) Comparison of four mechanical methods to retrieve sperm from testicular tissue. Hum. Reprod., 10, 29562959.[Abstract]
Von Eckardstein, S., Simoni, M., Bergmann, M., Weinbauer, G.F., Gassner, P., Schepers, A.G. and Nieschlag, E. (1999) Serum inhibin B in combination with serum follicle-stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesis in men, but cannot predict the presence of sperm in testicular tissue samples. J. Clin. Endocrinol. Metab., 84, 24962501.
Zweig, M.H. and Campbell, G. (1993) Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clin. Chem., 39, 561577.
Submitted on April 24, 2001; resubmitted on October 2, 2001; accepted on December 5, 2001.