1 Clinica Medica 3, University of Padova, Padova, 2 Centro per la Diagnosi e la Terapia della sterilità di coppia, Divisione di Ostetricia e Ginecologia, Arcispedale S. Maria Nuova, Reggio Emilia and 3 Servizio di Medicina di Laboratorio, Azienda Ospedaliera di Padova, Padova, Italy
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Abstract |
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Key words: cytology/fine needle aspiration/FSH therapy/inhibin B/oligozoospermia
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Introduction |
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In this study we evaluated the reciprocal behaviour of inhibin B and FSH secretion in a large group of oligozoospermic subjects with well-defined testicular alterations in order to investigate which maturational stage of spermatogenesis was able to influence the production of inhibin B by Sertoli cells. Furthermore, to clarify the clinical meaning of inhibin B concentrations in oligozoospermic subjects, the behaviour of this hormone was evaluated after FSH treatment in patients affected by different degrees of testicular damage.
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Materials and methods |
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The study was approved by the Hospital Ethical Committee and informed consent was obtained from each subject.
Hormone assays
FSH and LH plasma concentrations were measured in each subject by radioimmunoassay using 125I-labelled FSH and LH (Ares-Serono, Milan, Italy). Inter-assay and intra-assay coefficients of variation were 2.8 and 3.7% respectively for LH and 2.6 and 3.6% respectively for FSH.
Testosterone plasma concentrations were determined in all subjects using the double antibody radioimmunoassay utilizing commercial kits (Radim, Rome, Italy). All specimens were measured in duplicate in the same assay. Intra- and inter-assay coefficients of variation were 7.8 and 7.0% respectively.
Inhibin plasma concentrations were measured by a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) specific for the dimeric inhibin-B form (Serotec, Oxford, UK) (Groome et al., 1996; Illingworth et al., 1996
). The first antibody is directed to the ßB-subunit, and the second antibody to the
-subunit and conjugated to alkaline phosphatase. The assay has a cross-reactivity of <0.1% with activins and ~1% with inhibin A. Assay sensitivity was 15 pg/ml and the inter- and intra-plate coefficients of variation were 6.4 and 6.8% respectively.
Fine needle aspiration cytology
The testicular structure was analysed in all patients by means of bilateral fine needle aspiration cytology (FNAC), in order to evaluate the tubular status related to oligozoospermia. The methods of aspiration and cytological analysis have been described previously in detail (Foresta and Varotto, 1992; Foresta et al., 1992
, 1995
).
Briefly, bilateral FNAC was performed aspirating by a 23-G (0.6 mm) butterfly needle attached to a 20 ml syringe. The retrieved material was placed on two or more microscope slides for each testis, allowed to air-dry for 24 h, stained with MayGrünwald and Giemsa stains, and examined under a light Orthoplan microscope (Wild-Leitz, Wetzlar, Germany) at magnifications of x125, x400 and x1250, counting at least 200 cells per smear. Tubular cells were recognized because of their staining and morphological aspects, such as cell diameter, chromatin pattern and cytoplasm size, as described previously (Foresta et al., 1992). Cytological analysis allowed identification of the following spermatogenetic cell types: spermatogonia, primary and secondary spermatocytes, early and late spermatids (corresponding to SaSb and ScSd steps of spermatogenesis respectively) and spermatozoa. The relative number of each cell type was expressed as a percentage. Sertoli cells were expressed as the Sertoli index (SEI; the number of Sertoli cells/100 spermatogenic cells), which has been found to be a reliable index of the tubular germ cell potential, and constitutes an attempt to simplify the interpretation of cytological results. Since the number of Sertoli cells is constant per unit of tubular length, an increase in the SEI indicates tubular germ cell depopulation (hypospermatogenesis).
The proportion of spermatozoa was expressed as the spermatic index (SI; the number of spermatozoa/100 spermatogenic cells) that reflects the final maturational step and the spermiogenic process.
Forty normozoospermic infertile subjects, whose characteristics have been reported in previous studies (16 autoimmune, 24 idiopathic), were considered as controls for testicular cytology analysis (Foresta et al., 1992). Studies on the reproducibility of the cytological analysis have been performed previously, and have reported very low coefficient of variation between differential counts (Foresta et al., 1992
).
On the basis of the testicular cytological findings, all considered subjects were divided into four different groups as follows: (i) severe hypospermatogenesis, defined as a strong quantitative reduction of germ cell line with respect to Sertoli cells, as indicated by a higher SEI (>300, where normal value is <50); (ii) spermatogonia and/or spermatocytic arrest, characterized by a significant increase of spermatogonia and/or spermatocytes (>40%, where normal value is <20%), with very low percentages of spermatids and spermatozoa; (iii) spermatid arrest, characterized by a significant increase of both early and late spermatids (>65%, where normal value is <45%), with a strong reduction of spermatozoa; and (iv) mild hypospermatogenesis, defined as a moderate reduction of germ cell line with respect to Sertoli cells, with a slight increase of SEI (100300, where normal value is <50).
Treatment with FSH
Twenty-five of the 135 patients were treated with human highly purified FSH (Metrodin HP; Serono, Milan, Italy) at the dose of 75 IU i.m. on alternate days for 3 months. All these patients were affected by idiopathic oligozoospermia and showed a sperm count <10x106/ml. Seminal parameters, inhibin B, FSH, LH and testosterone plasma concentrations were measured weekly for the first month, and then monthly.
Statistical analysis
The results are given as mean ± SD. Statistical analyses were performed using the Statview statistical package (ABACUS, Berkeley, CA, USA). Statistical analysis was performed using an analysis of variance (ANOVA). Correlations were performed after logarithmic transformation of the data. Probability (P) values < 0.05 were regarded as statistically significant.
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Results |
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Discussion |
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These results may justify the lack of any correlation between concentrations of spermatozoa and plasma inhibin B observed in this study, since in a large number of patients oligozoospermia was related to a maturational disturbance at the spermatidic level.
Circulating FSH has long been considered a reliable marker of Sertoli cell function and spermatogenesis, but represents an indirect parameter of tubular function, since FSH is produced by the pituitary and is influenced by hypothalamic regulation as well as by testicular factors and steroids. Since inhibin B is produced directly and solely from testis, the investigation has been made recently as to whether plasma concentrations of inhibin may be considered a better indicator of exocrine testicular function than FSH (Jensen et al., 1997; Klingmuller and Haidl, 1997
). At present, this aspect has not been completely clarified, although some authors have suggested that seminal plasma inhibin B concentrations could be a marker of the functional state of the seminiferous epithelium (Anderson et al., 1998
). The results of this study demonstrate a significant inverse relationship between the plasma concentrations of inhibin B and FSH in subjects with disturbed spermatogenesis, confirming the supposed role of inhibin B in the mechanisms regulating FSH secretion. This relationship, at first sight, may suggest that both hormones express the same evaluation of tubular dysfunction, but a careful analysis of results shows that FSH and inhibin B plasma concentrations do not always behave similarly. In fact, when inhibin B serum concentrations are low (<80 pg/ml), plasma concentrations of FSH are inevitably high, while high plasma FSH concentrations (>7 IU/l) may be associated with low or normal inhibin B concentrations.
The presence of high FSH plasma concentrations despite normal inhibin B concentrations remains to be clarified, but shows that elevated FSH is not always a marker of decreased inhibin B production; it also suggests that in these cases a residual function of tubular cells exists. One hypothesis could explain the elevated plasma FSH concentrations as being secondary to a partial alteration of testicular tubular function determining a decrease of inhibin B negative feed-back, and thus inducing a compensatory Sertoli cell response, resetting the pituitarytesticular axis and maintaining normal inhibin B production. In these patients, cytological analysis of tubular status showed that the number of spermatids was higher than that observed in patients with elevated FSH and low inhibin B plasma concentrations. Furthermore, it is important to underline that in these patients the testicular volume was not different from that in controls.
On the basis of these findings, inhibin B appears to represent a more specific marker of tubular function than FSH, and permits the identification among oligozoospermic subjects with high FSH plasma concentrations of two sub-populations, each characterized by different degrees of testicular damage and spermatogenetic cell content.
To clarify further the relationship between FSH and inhibin B and the meaning of inhibin B evaluation, three groups of patients characterized by: (i) normal FSH and normal inhibin B plasma concentrations (group A); (ii) high FSH and normal inhibin B plasma concentrations (group B); and (iii) high FSH and low inhibin B plasma concentrations (group C) were treated with FSH. In this study, doses of FSH were used that had been shown previously to influence human Sertoli cell functions and human spermatogenesis in vivo (Foresta and Varotto, 1994; Foresta et al., 1998
). The findings of this study provide considerable insight into the dynamics of testicular inhibin B secretion in response to FSH administration. In fact, when FSH plasma concentrations were high, independently from those of inhibin B, FSH treatment did not induce any modification of inhibin B production. In contrast, in subjects with normal FSH and normal inhibin B plasma concentrations, FSH treatment stimulated inhibin B production. The lack of any increase of inhibin B after FSH treatment in subjects with high basal FSH and low inhibin B plasma concentrations is not surprising, and possibly reflects an incompetence of Sertoli cells in the production of this hormone, even if it is impossible to distinguish an intrinsic alteration of these cells from their dysfunctional status related to the lack or reduction of germ cells. In subjects with high plasma FSH and normal inhibin B concentrations, the absence of any increase in plasma inhibin B after FSH treatment is unclear. In these patients the high endogenous plasma FSH concentrations may have induced a maximal functional activation of Sertoli cells which cannot be further stimulated. Another explanation for the lack of inhibin B production in these subjects following FSH administration could lie in the germ cell depopulation seen in subjects not responding to FSH in terms of inhibin B production, thus strengthening the hypothesis that the presence of differentiated germ cells is necessary for Sertoli cells to produce inhibin B. This hypothesis seems to be confirmed by the observation that the higher the Sertoli cell index, the poorer the response to FSH in terms of inhibin B production.
In subjects with normal plasma FSH concentrations, the increase in inhibin B was evident in the second week of treatment, although circulating FSH was raised significantly after the first week of treatment, and declinedas did plasma FSH concentrationsafter 4 weeks of therapy. The decline in plasma concentrations of inhibin B and FSH after 8 and 12 weeks of treatment may be due to the negative influence of increased inhibin B plasma concentrations on endogenous FSH production, leading to a decline in FSH secretion and, in turn, of inhibin B production. This hypothesis seems to be confirmed by the persistently increased plasma FSH concentrations seen throughout the treatment period in patients with high FSH concentrations but who showed no increase in inhibin B production.
The reason for the delayed response of inhibin B production to exogenous FSH administration is unknown, but confirms previous observations in monkeys during chronic treatment with FSH (Arslan et al., 1992) and in humans after acute administration of recombinant FSH (Kamischke et al., 1998
). One possible explanation could lie in the requirement of germ cell replication for inhibin B production to occur after FSH administration. In respect of this, Anderson et al. (1997) observed that the recovery of inhibin B secretion following gonadotrophin suppression was related to the delay in reinitiation of spermatogenesis. Recently, it has been reported that inhibin B concentrations remained unchanged after treatment with recombinant FSH in a large group of oligozoospermic subjects (Kamischke et al., 1998
). However, in that study the evaluation of inhibin B was performed at 6 and 12 weeks after the initiation of treatment, and therefore the initial increase in inhibin B production may have not been observed.
The ability of Sertoli cells to increase inhibin B production after FSH treatment may allow the identification of those oligozoospermic patients responsive to FSH treatment. To verify this hypothesis, treated patients were distinguished as those who doubled sperm concentrations after FSH treatment (responder), and those who did not (non-responder), as reported recently (Foresta et al., 1998). The similar behaviour of inhibin B production in responder and non-responder subjects excludes the possibility that inhibin B production after FSH administration could be utilized as a marker for the response to this treatment in terms of an increase in sperm production. On the other hand, in both cases the spermatid population evaluated by cytological analysis of the testis was well represented, thus justifying these findings.
In conclusion, the results of this study demonstrate that: (i) in humans, the production of inhibin B by Sertoli cells reflects interactions between these cells and spermatids; (ii) in oligozoospermic subjects, low plasma concentrations of inhibin B as well as high plasma FSH concentration identify pathological conditions characterized by a severe depopulation of seminiferous tubules; (iii) elevated plasma FSH concentrations are not always indicative of a decrease of inhibin B production, and in this case a normal inhibin B concentration demonstrates the presence of a residual function of tubular cells related to the presence of spermatids; (iv) FSH treatment causes an increase in inhibin B production only when plasma FSH concentrations are in the normal range; and (v) an increase of inhibin B production after FSH treatment may occur both in responders and non-responders, thus not allowing distinction between patients responsive to this treatment in terms of an increase in the production of spermatozoa.
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Notes |
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References |
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Anawalt, B.D., Bebb, R.A., Matsumoto, A.M. et al. (1996) Serum inhibin-B levels reflect Sertoli cell function in normal men and men with testicular dysfunction. J. Clin. Endocrinol. Metab., 81, 33413345.[Abstract]
Anderson, R.A., Wallace, E.M., Groome, N.P. et al. (1997) Physiological relationship between inhibin B, follicle stimulating hormone secretion and spermatogenesis in normal men and response to gonadotrophin suppression by exogenous testosterone. Hum. Reprod., 12, 746751.[Abstract]
Anderson, R.A., Irvine, D.S., Balfour, C. et al. (1998) Inhibin B in seminal plasma: testicular origin and relationship to spermatogenesis. Hum. Reprod., 13, 920926.[Abstract]
Arslan, M., Weinbauer, G.F., Schlatt, S. et al. (1992) FSH and testosterone, alone or in combination, initiate testicular growth and increase the number of spermatogonia and Sertoli cells in a juvenile non-human primate (Macaca mulatta). J. Endocrinol., 136, 235243.[ISI]
Bartlett, J.M.S., Kerr, J.B. and Sharpe, R.M. (1988) The selective removal of pachytene spermatocytes using methoxy acetic acid as an approach to the study in vivo of paracrine interactions in the testis. J. Androl., 9, 3140.[Abstract]
Bhasin, S., Krummen, L.A., Swerdloff, R.S. et al. (1989) Stage dependent expression of inhibin alpha- and beta-B subunits during the cycle of the rat seminiferous epithelium. Endocrinology, 124, 987991.[Abstract]
Foresta, C. and Varotto, A. (1992) Assessment of testicular cytology by fine needle aspiration as a diagnostic parameter in the evaluation of the oligospermic subject. Fertil. Steril., 58, 10281033.[ISI][Medline]
Foresta, C. and Varotto, A. (1994) Immunocytochemical localization of epidermal growth factor receptors in human testis from infertile subjects. Fertil. Steril., 61, 941948.[ISI][Medline]
Foresta, C., Varotto, A. and Scandellari, C. (1992) Assessment of testicular cytology by fine needle aspiration as a diagnostic parameter in the evaluation of the azoospermic subject. Fertil. Steril., 57, 858865.[ISI][Medline]
Foresta, C., Ferlin, A., Bettella, A. et al. (1995) Diagnostic and clinical features in azoospermia. Clin. Endocrinol., 43, 537543.[ISI][Medline]
Foresta, C., Bettella, A., Ferlin, A. et al. (1998) Evidence for a stimulatory role of FSH on spermatogonial population in adult man. Fertil. Steril., 69, 636642.[ISI][Medline]
Groome, N.P., Illingworth, P.J., O'Brien, M. et al. (1996) Measurement of dimeric inhibin-B throughout the human menstrual cycle. J. Clin. Endocrinol. Metab., 81, 14001405.
Illingworth, P.J., Groome, N.P., Byrd, W. et al. (1996) Inhibin-B: a likely candidate for the physiologically important form of inhibin in men. J. Clin. Endocrinol. Metab., 81, 13211325.[Abstract]
Jegou, B., Laws, A.O. and de Kretser, D.M. (1984) Changes in testicular function induced by short-term exposure of the rat testis to heat: further evidence for interaction of germ cells, Sertoli cells and Leydig cells. Int. J. Androl., 7, 244257.[ISI][Medline]
Jensen, T.K., Andersson, A.M., Hjollund, N.H.I. 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.
Kamischke, A., Behre, H.M., Bergmann, M. et al. (1998) Recombinant human follicle stimulating hormone for treatment of male idiopathic infertility: a randomized, double-blind, placebo-controlled, clinical trial. Hum. Reprod., 13, 596603.[Abstract]
Klingmuller, D. and Haidl, G. (1997) Inhibin B in men with normal and disturbed spermatogenesis. Hum. Reprod., 12, 23762378.[Abstract]
Nachtigall, L.B., Boepple, P.A., Seminara, S.B. et al. (1996) Inhibin B secretion in males with gonadotropin-releasing hormone (GnRH) deficiency before and during long-term GnRH replacement: relationship to spontaneous puberty, testicular volume and prior treatment A clinical research center study. J. Clin. Endocrinol. Metab., 81, 35203525.[Abstract]
Pineau, C., Sharpe, R.M., Saunders, P.T.K. et al. (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]
Pinon-Lataillade, G., Velez de la Calle, J.F., Vignier-Martinez, M.C. et al. (1988) Influence of germ cells upon Sertoli cells during continuous low-dose rate gamma-irradiation of adult rats. Mol. Cell. Endocrinol., 58, 5163.[ISI][Medline]
Wallace, E.M., Riley, S.C., Crossley, J.A. et al. (1997) Dimeric inhibins in amniotic fluid and maternal and fetal serum in human pregnancy. J. Clin. Endocrinol. Metab., 82, 218222.
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction. Cambridge University Press, Cambridge, UK.
Submitted on September 7, 1998; accepted on December 16, 1998.