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From the * Department of Andrology and
Dermatology, Faculty of Medicine, Alexandria University, Alexandria, Egypt;
and Center for Medical and Urological
Andrology, Ghent University, Ghent, Belgium.
| Correspondence to: Prof Dr Frank H. Comhaire, Ghent University Hospital, Center for Medical and Urological Andrology, De Pintelaan, 185, B-9000 Ghent, Belgium. |
| Received for publication December 19, 2001; accepted for publication May 29, 2002. |
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Abstract |
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Key words: Male infertility, Sertoli cell, testis biopsy, testicular sperm extraction
and either ßA or ßB),
that has an inhibitory effect on pituitary gonadotropin production
(Burger and Igarashi, 1988).
Recent studies have reported a strong negative correlation between serum
follicle-stimulating hormone (FSH) and serum inhibin B in both fertile and
subfertile men (Anderson et al,
1997). Serum inhibin B is considered a marker of Sertoli cell
function and of spermatogenesis (Anawalt et
al, 1996; Mahmoud et al,
1998a). It has been shown that inhibin B, similar to other Sertoli cell products, is secreted bidirectionally (ie, into the seminiferous tubular lumen via the apex of Sertoli cells, and via the base of these cells into testicular interstitial fluid). It has been generally assumed that the latter is the main route by which inhibin reaches the blood stream because of the proximity of interstitial fluid to the vasculature (Maddocks and Sharpe, 1989).
Cultured Sertoli cells secrete bioactive inhibin, and the majority of secretion is into the adluminal compartment (Handelsman et al, 1990). The level of inhibin B in seminal plasma can reach 10 times the level in serum. This suggests that elevated levels are caused by secretion of inhibin B by Sertoli cells into the adluminal space (Byrd et al, 1998). A significant correlation has been reported between inhibin B concentrations in seminal plasma and in serum (Anderson et al, 1998).
The main action of inhibin B secreted into the circulation is to regulate serum FSH levels. The precise role of inhibin secreted in seminiferous tubules is unknown. Impure preparations have been demonstrated to inhibit spermatogonial mitosis in rat testes (de Kretser and McFarlane, 1996). Blocking the inhibin effect increases spermatogenesis in animals (Schanbacher, 1991; Bame, 1999).
The objective of this work was to study the relationship between seminal plasma inhibin B and spermatogenesis and the outcome of testicular sperm extraction (TESE) in men with azoospermia.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Semen Analysis
Semen samples were produced by masturbation and left for 30 minutes to
liquefy, after which conventional semen analysis was performed according to
WHO (1999) recommendations.
Seminal plasma glucosidase was measured by the colorimetric method
using a commercial kit (EpiScreen, Fertipro, Beernem, Belgium;
Mahmoud et al, 1998b).
Measurement of Inhibin B in Seminal Plasma
Dimeric inhibin B was measured in seminal plasma with a solid phase
sandwich enzyme-linked immunosorbent assay kit (Serotec, Oxford, United
Kingdom) as described previously (Mahmoud
et al, 1998a). In normozoospermic controls, the measurement of
inhibin B in undiluted seminal plasma revealed values in excess of the maximum
level of the standard curve. Therefore, the samples were diluted up to 1:4 to
obtain a reading. A possible effect of a matrix factor was excluded by
assessing serial dilutions. The lower detection value of the method is 15
pg/mL; intraassay and interassay coefficients of variation were 11.0% and
10.7%, respectively.
Testicular Biopsy
Under general anesthesia, a bilateral transverse incision was carried out
across the layers of the scrotum over the midanterior surface of both testes.
The testicular tissue was protruded from a small incision in the tunica
albuginea. A biopsy was taken and submerged in 1 mL of Ham F10 medium with
Hepes (N1387; Sigma Chemical Company, St Louis, Mo) for testicular sperm
extraction. Another biopsy was fixed in Bouin fixative for histopathological
examination. The incisions were closed in layers after adequate
hemostasis.
Testicular biopsies were interpreted quantitatively according to a modified Johnsen score (Glander et al, 2000), and qualitatively according to the following 5 groups: normal spermatogenesis, hypospermatogenesis, maturation arrest, Sertoli cells only (SCO), and mixed patterns. Biopsies were classified into those with spermatogenic activity (ie, showing at least spermatocytes), and those without spermatogenic activity (presenting as SCO).
Testicular Sperm Extraction
After mincing and shredding testicular tissue in a Petri dish (BD
Biosciences, Erembodegem, Belgium), the samples were examined with an inverted
microscope (Hoffman Optics Zeiss Axiovert 135, Zeiss, Zaventem, Belgium) using
400x magnification. If no spermatozoa were detected, a chemical
digestion of the testicular tissue was performed using collagenase type 1A
(C2674; Sigma). Briefly, testicular tissue was incubated with collagenase for
2 hours at 37°C, and then transferred to a Falcon tube (BD Biosciences)
and centrifuged for 10 minutes at 3000 x g. The supernatant was
removed and the pellet was resuspended. One drop was reexamined with a
microscope using a Neubauer chamber (Laboroptik GmbH, Friedrichsdorf,
Germany). If no spermatozoa were detected, 5 µL of the suspension was
dispersed on a Petri dish and examined with the inverted microscope. The
result was interpreted as positive or negative TESE according to the presence
or absence of spermatozoa (Salzbrunn et
al, 1996).
Serum Hormones
Serum concentrations of luteinizing hormone (LH) and FSH were measured by
electrochemiluminescence immunoassay using Elecsys-LH and Elecsys-FSH kits
(Roche, Germany), respectively. Testosterone was measured using the Orion
Diagnostica (Spectria, Finland) radioimmunoassay test.
Statistics
Statistical analysis was performed using the MedCalc program (MedCalc
Software, Mariakerke, Belgium) (Schoonjans
et al, 1995). Significance of differences was assessed using the
Kruskal-Wallis and Wilcoxon tests. Rank correlation was calculated to detect
the relation between 2 variables.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Table 1 shows the results of qualitative testicular biopsy and their corresponding levels of seminal plasma inhibin B. Inhibin B was detectable in the seminal plasma in all but 8 subjects with azoospermia (group 3). Of the latter group, 2 research subjects showed maturation arrest at the spermatocyte stage, 4 exhibited SCO, and 2 had obstructive azoospermia. Inhibin B was undetectable in 5 of 10 postvasectomy subjects.
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The Figure shows that inhibin B in seminal plasma is significantly higher
in normozoospermic samples than in postvasectomy and azoospermic samples
(P < .0001). There was no significant difference in seminal
inhibin B levels between obstructive azoospermic and postvasectomy samples
(P > .1), nor between obstructive and nonobstructive (primary
testicular) azoospermic samples (P = .4, data not shown). There was a
tendency toward higher levels of inhibin B in azoospermic samples with
spermatogenic activity than in those with SCO (P = .1). No difference
was noticed between inhibin B levels in different qualitative pathological
patterns of testicular biopsy (Table
1), nor was there a significant correlation between seminal
inhibin B levels and testis biopsy score (r = .17, P =
.2).
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Seminal inhibin B showed a significant inverse correlation with serum FSH (P = -.58, P < .001), but no correlation with serum LH levels (P = .1).
Seminal -glucosidase was significantly higher in group 1 subjects
than in the other groups (P < .001), but the difference between
samples of subjects with obstructive or nonobstructive azoospermia did not
reach significance (P = .1). A strong positive correlation was found
between seminal inhibin B and seminal plasma alpha glucosidase (r =
.37, P = .002). There was a weak correlation between seminal inhibin
B and serum testosterone (r = .29, P = .03).
There was no difference in seminal inhibin B concentration in azoospermic samples with positive or negative outcome of TESE (Table 2).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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When subjects with obstructive azoospermia were compared to those without obstruction, there was no difference in seminal inhibin B concentrations. This finding contradicts the results of inhibin B measurement in serum, in which the level of inhibin B is higher in obstructive samples than in those without obstruction (von Eckardstein et al, 1999). Our finding can be explained by the fact that in subjects with obstructive azoospermia, the inhibin B secreted in seminiferous tubular fluid does not reach the ejaculated semen. The secretion of inhibin B in the circulation is independent of passage through the deferent ducts. At the other hand, the amount of inhibin B secreted may be lower in both serum and seminal plasma in subjects with nonobstructive azoospermia, due to Sertoli cell dysfunction.
We found a relatively high level of seminal inhibin B (100 pg/mL) in one patient with SCO with negative TESE and elevated serum FSH (51 IU/mL). In this patient, the presence of a high level of inhibin B and an absence of spermatozoa in testicular tissue could be explained by autonomous secretion of inhibin B by the Sertoli cells. This is in agreement with the report by Foresta et al (1999), who detected inhibin B in serum of 19% of cases with SCO. This autonomous secretion is either devoid of bioactivity, or there is peripheral resistance to its action, which explains the high level of FSH in these cases. The factors affecting autonomous inhibin B secretion are largely unknown (Raivio et al, 2000). Another possible explanation is that secretion of inhibin B from Sertoli cells may be under the regulation of factors other than germ cells, especially spermatids, that are proposed to exert paracrine control of inhibin B secretion (Andersson, 2000).
When comparing seminal inhibin B concentrations in subjects with azoospermia with or without spermatogenic activity, we found the level to be slightly higher in the former than in the latter. However, there was no correlation between inhibin B concentrations in seminal plasma and biopsy score. This contrasts with the results of Pierik et al (1998), who found a significant correlation between inhibin B in serum and biopsy score. Our data showed no difference in levels of inhibin B in seminal plasma related to the different qualitative pathological patterns, nor with the outcome of TESE. Similarly, Bohring and Krause (1999) and Von Eckardstein et al (1999) found that inhibin B in serum did not predict spermatogenic activity. The degree of overlap between different testicular pathologies appeared to limit the usefulness of inhibin measurement in predicting the result of TESE (Anderson, 2001). Indeed, in 2 of our subjects, spermatozoa were present in testicular tissue despite undetectable inhibin B in seminal plasma, whereas 2 subjects with seminal inhibin B values >100 pg/mL showed no spermatozoa upon TESE. These findings suggest that inhibin B in seminal plasma is not a useful marker for TESE. However, Ballesca et al (2000) and Brugo-Olmeda et al (2001) came to the opposite conclusion, suggesting that inhibin measurement in seminal plasma is a useful, noninvasive predictor of spermatogenesis and TESE outcome.
Anderson and Sharpe (2000) reported high FSH serum concentrations to be associated with lower seminal plasma inhibin B concentrations in men with oligoazoospermia, but there was no correlation between seminal plasma inhibin B and serum FSH concentration (Anderson et al, 1998). In contrast, our results revealed a significant inverse correlation between seminal inhibin B concentration and serum FSH, which is in agreement with the report by Scott and Burger (1981), who measured bioactive inhibin in seminal plasma.
We found a significant, albeit weak positive correlation between serum testosterone concentration and seminal inhibin B. This may suggest a possible role for androgens in the regulation of seminal inhibin B. Recent studies provide evidence for the role of steroids in the control of inhibin B secretion from the Sertoli cells. Carlsen et al (1999) found a significant diurnal variation in serum inhibin B levels in normal men, lacking evidence for a role of FSH. However, the covariation with serum concentrations of testosterone and estradiol suggested that these hormones might play a role in the diurnal rhythm of inhibin B, although some other common influences could not be excluded (Sanford et al, 2002). Comhaire et al (1995), using an alpha subunit assay, recorded a similar diurnal variability in serum inhibin, and so did Kamischke et al (2001). The former authors noticed a general parallelism between changes in testosterone concentration and dihydrotestosterone on one hand, and serum inhibin on the other. Also, a greater inhibin secretion occurred after injection of human chorionic gonadotropin, in parallel with higher testosterone levels.
In vitro experiments revealed that the addition of testosterone to cultured Sertoli cells inhibits inhibin B production, whereas estrogen stimulates inhibin production (Depuydt et al, 1999). Martin et al (2000) produced evidence for the differential regulation of the 2 components of the bidirectional secretion of inhibin by Sertoli cells. They observed a rapid decline in seminal plasma inhibin B level with little change in serum inhibin B following administration of the combination of desogestrel and testosterone for contraceptive purposes. In agreement with data from animal studies (Handelsman et al, 1990), the available data suggest a possible role for sex steroids in the control of the (bidirectional) secretion of inhibin B the cells of Sertoli.
There are reasons to believe that inhibin in seminal plasma can originate
from the accessory sex glands. In agreement with Anderson et al
(1998), who found detectable
values of seminal inhibin B in 2 of 20 postvasectomy samples, we detected
inhibin in seminal plasma of 5 of 10 men who had successful vasectomy, and in
8 men with obstructive azoospermia. Scott and Burger
(1981), using a bioactivity
assay, found detectable amounts of seminal inhibin B in all postvasectomy
samples and in 16 of 17 cases with obstructive azoospermia. This suggests that
inhibin may be secreted into the prostatic fluid, the seminal vesicular fluid,
or both (Vaze et al, 1980).
Expression of messenger RNA for inhibin and ß subunits was
detected in the human prostate, indicating the capacity to produce all the
known inhibins and activins (Thomas et al,
1998; Dowling and Risbridger,
2000). The accessory sex glands, however, appear unlikely primary
sources of inhibin bioactivity because subjects with azoospermia with elevated
plasma FSH levels had lower seminal plasma inhibin concentrations than those
who had had vasectomy (Scott and Burger,
1981). Le Lannou et al
(1979) have speculated that
most of the inhibin in the rete testis fluid would be reabsorbed in the caput
of the epididymis. However, we found a positive correlation between the
concentrations of inhibin B and levels of glucosidase in seminal
plasma. This suggests a relation between seminal plasma inhibin B and the
function of the epididymis, which seems contradictory to reabsorption of
inhibin B from the epididymis. Our results rather suggest a possible role of
inhibin B on epididymal function or a parallel effect of testosterone on both
the epididymis and the Sertoli cells.
Conclusion
Seminal plasma inhibin B does not reflect the functional state of
seminiferous epithelium, because there is no relation between seminal plasma
inhibin B levels on the one hand, and spermatogenesis and TESE outcome on the
other. This may be due to the contribution of accessory sex glands to the
amount of inhibin secreted in the seminal plasma, to autonomous secretion of
inhibin by the Sertoli cells, or to the differential regulation of inhibin B
secretion into the adluminal and interstitial testicular compartments.
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Footnotes |
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