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Hum. Reprod.-1997-Klingm黮ler-2376-8 by wuzhengqin


									Human Reproduction vol.12 no.ll pp.2376-2378, 1997

Inhibin B in men with normal and disturbed

D.Klingmiiller1'3 and G.Haidl2                                       chain: A and B. Correspondingly we differentiate inhibin A
                                                                     with a PA- and inhibin B with a (3B-chain. Besides these
department of Clinical Biochemistry, Division of Endocrinology
and 2Department of Dermatology, Division of Andrology,
                                                                     dimeric proteins, biologically inactive precursor and fragments
University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn,            (free cc-chain) of inhibin are secreted into the blood (Illingworth
Germany                                                              et al, 1996).
To whom correspondence should be addressed
                                                                        Under the influence of FSH, inhibin is formed in the Sertoli
                                                                     cells, which interact closely with the gonadal cells. The inhibin
Inhibin, a dimeric gonadal glycoprotein, inhibits the pro-           concentration in the serum probably reflects the extent of this
duction and/or secretion of follicle stimulating hormone             interaction. Until now only assays using antibodies against the

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(FSH). The major species currently recognized are inhibin            oc-chain were available. A differentiation between inhibins A
A (ccp* A subunit) and inhibin B (a|3 B subunit). In men,            and B and free a-chain was therefore not possible (Brennemann
inhibin B seems to be the physiologically important form             et al, 1992; Comhaire et al, 1995; Meriggiola et al, 1996).
of inhibin. Therefore we measured serum inhibin B using              The clinical value of these assays was questionable (de Kretser
a new two-site immunoenzymatic assay in 14 men (mean                 et al, 1989). Now, because of a recently developed hormone
 ± SEM age, 34.5 ± 0.7 years) with sperm counts >20 x 106/           analysis for detecting inhibin B (Groome et al, 1996), we are
ml, in 35 men (mean ± SEM age, 36.4 ± 1.3 years) with                able to compare the inhibin B concentration with sperm
oligozoospermia (sperm count <20 x 106/ml) and in men                cell concentration in men with or without spermatogenic
with azoospermia (three orchidectomized men, three men               disturbances. The results suggest a renaissance of inhibin.
with Klinefelter's syndrome, 10 men with Kallmann's
syndrome). We compared inhibin B concentrations with                 Materials and methods
serum FSH and sperm concentrations. In men with normal               Patients
sperm concentrations (44.7 ± 6.4 x 106/ml), the concentra-           A total of 14 patients with a normal spermiogram (sperm concentration
tion of inhibin was 223 ± 18 pg/ml and of FSH 5.0 ± 0.7              >20Xl0 6 /ml, mean ± SEM age 34.4 ± 1.0 years), 35 patients with
IU/1; in patients with low sperm concentrations (3.7 ±               oligoteratoasthenozoospermia (sperm density <20Xl0 6 /ml, mean ±
0.8 x 106/ml), the concentration of inhibin B was 107 ± 12           SEM age 36.7 ± 1.7 years), three patients with anorchia (mean
pg/ml and of FSH 12.2 ± 1.5 IU/1. In all patients, except            ± SEM age 29.3 ± 3.5 years), 10 patients with hypothalamic
those with hypogonadotrophic hypogonadism, the relation-             hypogonadism (Kallmann's syndrome; mean ± SEM age 30.2 ± 3.8
                                                                     years) and three patients with Klinefelter's syndrome (mean ± SEM
ship between inhibin B and FSH concentrations was inverse
                                                                     age 34.3 ± 1 . 6 years) were investigated.
(r = -0.69, P < 0.0001). In all patients the sperm concentra-
                                                                        Blood was taken between 08.00 and 11.00 h and centrifuged at
tion was positively correlated with inhibin B concentrations         3000 g for 15 min. The serum was separated and stored at -20°C
(r = 0.70, P < 0.0001) and negatively correlated with FSH            until assay.
concentrations (r = -0.37, P < 0.01). We conclude that
inhibin B may be a marker of exocrine testicular function            Hormone analysis
and could offer improved diagnosis and treatment modalit-            Luteinizing hormone (LH), FSH and testosterone were measured
ies for male infertility.                                            using commercially available enzyme immunoassays (Boehringer-
                                                                     Mannheim, Mannheim, Germany). The normal range of LH is 1.8-
Key words: FSH/inhibin B/spermatogenesis/testicular volume           9.2 IU/1, of FSH 1.6-9.7 IU/1 and of testosterone 10.4-34.7 nmol/1.
                                                                     The detection limits of LH, FSH and testosterone are 0.2 IU/1, 0.2
                                                                     IU/1 and 0.1 nmol/1 respectively.
                                                                        Inhibin B concentrations were measured using a new enzyme-
Introduction                                                         linked immunosorbent assay (Serotec Limited, Kidlington, UK) which
                                                                     utilizes two distinct antibodies directed against distinct epitopes on
In the 1920s it was postulated that the testes secrete a substance   the a- and P-subunits (Groome et al, 1996). The detection limit was
that intervenes in the regulation of the function of the pituitary   15 pg/ml. The interassay variation was 14.2%; the within-assay
cells (Mottram and Cramer, 1923). McCullagh (1932) called            variation was 4.3%.
this unknown substance 'inhibin'. Later it was assumed that
                                                                     Testicular volume
inhibin inhibits follicle stimulating hormone (FSH) secretion
and thus decisively influences the regulation of gonadal             Testicular volume was measured using a Prader orchidometer.
function.                                                            Semen analysis
   Inhibin has been found to be a glycoprotein, consisting of        Semen analyses were performed using conventional methods. In each
two units, an a- and a |3-chain. There are two types of (3-          case the abstinence interval varied between 3 and 5 days.
2376                                                                               European Society for Human Reproduction and Embryology
                                                                                                Inhibin B in men with normal and disturbed spermatogenesis

Data analysis                                                                    negatively correlated with FSH concentrations (r = -0.37, P
All results are expressed as mean ± SEM. Statistical analyses were               < 0.01), as shown in Figure 1A and B. In all patients, except
performed using the Mann-Whitney test. Correlations were assessed                those with hypogonadotrophic hypogonadism, the relationship
using a linear regression analysis. A P value <0.05 was considered               between inhibin B and FSH concentrations was inverse (r =
to be significant.                                                               -0.69, P < 0.0001; Figure 1C).
                                                                                   Inhibin B concentrations were highly significantly correlated
Results                                                                          with testicular volume in all 65 men (r = 0.79, P < 0.0001;
In men with normal sperm concentrations (44.7 ± 6.4 X106/                        Figure ID).
ml), the concentration of inhibin was significantly higher
(P < 0.0001) than in those with low sperm concentrations
(3.7 ± 0.8X106/ml) (223 ± 18 versus 107 ± 12 pg/ml). In                          Discussion
patients with Kallmann's syndrome (hypothalamic hypogonad-                       An important result of this study is the correlation between
ism; without spermatogenesis), the inhibin concentration was                     inhibin B and testis size, which reflects the number of inhibin-
49 ± 8 pg/ml. Patients with anorchia and Klinefelter's syn-                      secreting Sertoli cells. Patients with a large testicular volume
drome had no detectable inhibin B (Table I).                                     have higher inhibin B concentrations than patients with small
   In all patients sperm concentration was positively correlated                 testicular volumes. In all of our patients there is a highly
with concentrations of inhibin B (r = 0.70, P < 0.0001) and                      significant positive correlation between inhibin B concentration

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Table I. Clinical and biochemical features of patients with normal or impaired spermatogenesis, anorchia or Kallmann's or Klinefelter's syndrome

Patients («)                       Sperm concentration     FSH (IU/1)                Inhibin B (pg/ml)              Age (years)                     Testis size (ml) left/right
                                   (X 106/ml)

Normal spermatogenesis (14)        44.7 ± 6.4               5.0 ± 0.7                223 ± 18                       34.4   ±   1.0                  19.1 ± 1.4/19.4 ± 1.3
Impaired spermatogenesis (35)       3.7 ± 0.8              12.2 ± 1.5                107 ± 12                       36.7   ±   1.7                  13.7 ± 0.8/14.0 ± 0.8
Anorchia (3)                       ND                      54 ± 16                   ND                             29.7   ±   3.5                           -/-
Kallmann's syndrome (10)           ND                       0.4 ± 0.3                 49 ± 8                        30.2   ±   3.8                   4.0 ± 1.1/4.3 :t 1.1
Klinefelter's syndrome (3)         ND                      28.0 ± 6.0                ND                             34.3   ±   1.6                   2.0 ± 0.6/2.0 :t 0.6

FSH = follicle stimulating hormone; ND = not detectable.

                              A                                                            C
                        400   -|                                                     400   -|

                  •i 300 -\
                                                                               •i    300 -\
                  CQ    200   -                                                1
                   C                                                           ffl   200 -
                  !5                                                            C
                  2     100 -                                                  n                           o°
                  tz                                                           IE    100 -
                          o -                                                                                                             >/ <?CO
                                           I         I                                 o -                      r          I
                                          25        50        75        100                                 10         15            20    25        100

                                   Sperm concentration (x10 )                                                FSH (IU/I)
                        100 -

                         80 -
                                                                               ffi   200    -
                  X      60 -                                                   C
                  CO                                                           'E
                         40 -                                                  'E    100 H

                         20 -      » ° % °&>° o°o
                                           I         i         1
                                                                                       o - 3oo8             I                                           I
                          0 -                                           100                                                     20
                                          26        50        76                                           10                                         30

                                    Sperm concentration (x10)                                        Testicular volume (ml)

Figure 1. Serum inhibin B (A) and follicle stimulating hormone (FSH; B) concentrations plotted against sperm concentration for 65 patients
with normal or impaired spermatogenesis. Serum inhibin B (C) concentrations plotted against FSH for 55 patients with normal or impaired
spermatogenesis (O) and 10 patients with Kallmann's syndrome ( • ) , and against testicular volume for all patients (D).

D.Klingmiiller and G.Haidl

and testicular volume (Figure ID) — nearly the same as in            the criteria of judging new therapeutic methods for male
gonadotrophin-releasing hormone-deficient patients with or           infertility.
without a history of puberty (Nachtigall et al, 1996). Patients
with anorchia, as well as those with Klinefelter's syndrome,
have no detectable inhibin B concentrations. This confirms
                                                                     We thank Mrs I.Horn for skilled technical assistance. We are grateful
that inhibin B is produced in the testes (Anawalt et al, 1996).
                                                                     to Dr N.P.Groome for technical help. This work was presented in
   All patients with a normal sperm concentration have an            part at the 41 st Symposium of the German Society of Endocrinology,
inhibin B concentration >112 pg/ml; all patients with lower          Liibeck, Germany, 6-8 May, 1997. This paper is dedicated to Prof.
inhibin B concentrations (<112 pg/ml) have a reduced sperm           Dr F.Bidlingmaier on the occasion of his 60th birthday.
concentration. Inhibin B serum concentrations are positively
correlated with sperm concentration —more closely than the           References
negative correlation with FSH (Figure 1A and B). This would          Anawalt, B.D., Bebb, R.A., Matsumoto, A.M. et al. (1996) Inhibin-B levels
be expected because inhibin is produced in the testes, whereas          reflect Sertoli cell function in normal men and men with testicular
FSH is produced in the pituitary gland. Therefore an increase           dysfunction. J. Clin. Endocrinol. Metab., 81, 3341-3345.
                                                                     Brennemann, W., Stoffel-Wagner, B., Bidlingmaier, F. et al. (1992)
in FSH reflects only indirectly a testicular dysfunction.               Immunoreactive plasma inhibin levels in men after polyvalent chemotherapy
   There are, however, two subgroups of patients whose sperm            of germinal cell cancer. Ada Endocrinol., 126, 224-228.
concentration and inhibin B concentration are not correlated         Comhaire, F.H., Rombauts, L., Vereecken, A. et al. (1995) Inhibin and steroid
                                                                        responses to testicular stimulation in normal men. Hum. Reprod., 10,
closely (Figure 1A):

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   (i) patients with an inhibin B concentration in the lower         de Kretser, D.M., McLachlan, R.I., Robertson, D.M. et al. (1989) Serum inhibin
normal range but with a markedly subnormal sperm concentra-             levels in normal men and men with testicular disorders. J. Endocrinol, 120,
tion. Obviously their Sertoli cells secrete normal amounts of           517-523.
inhibin. Therefore the disturbance must be located distal to         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.
the Sertoli cells. In some of these patients signs of chronic           Metab., 81, 1401-1405.
epididymal inflammation were observed (data not shown).              Illingworth, P.J., Groome, N.P., Byrd, W. et al. (1996) Inhibin-B: a likely
Thus, sperm transport could be impaired by a partial obstruction        candidate for the physiologically important form of inhibin in men. J. Clin.
of the efferent ducts due to infections or trauma.                      Endocrinol. Metab., 81, 1321-1325.
                                                                     McCullagh, G.R. (1932) Dual endocrine activity of the testes. Science, 76,
   (ii) patients with azoospermia and mostly a low detectable            19-20.
inhibin B concentration. Patients with hypogonadotrophic             Meriggiola, M.C., Noonan, E.A., Paulsen, C.A. et al. (1996) Annual patterns
hypogonadism, especially those with Kallmann's syndrome,                of luteinizing hormone, follicle stimulating hormone, testosterone and
                                                                        inhibin in normal men. Hum. Reprod., 11, 248-252.
belong to this group. They have a low inhibin concentration and      Mottram, J.C. and Cramer, W. (1923) On the general effects of exposure to
a usually undetectable FSH concentration. There is evidently a          radium on metabolism and tumour growth in the rat and the special effects
basal gonadotrophin-independent secretion of inhibin B. In              on testis and pituitary. Quart. J. Exp. Physiol, 13, 209-228.
                                                                     Nachtigall, L.B., Boepple, P.A., Seminara, S.B. et al. (1996) Inhibin B
patients with hypergonadotrophic hypogonadism, inhibin secre-           secretion in males with gonadotropin-releasing hormone (GnRH) deficiency
tion is also reduced. Obviously the Sertoli cells are so impaired       before and during long-term GnRH replacement: relationship to spontaneous
that their inhibin secretion is reduced, despite the increased          puberty, testicular volume, and prior treatment — a clinical research center
FSH.                                                                    study. J. Clin. Endocrinol. Metab., 81, 3520-3525.

   In cases of hypogonadism the measurement of both inhibin          Received on April 2, 1997; accepted on August 18, 1997
B and FSH concentrations in serum helps us to localize the
defect: low FSH with low inhibin concentrations is typical of
a central disturbance, whereas high FSH with reduced inhibin
concentrations is typical of impairment of testicular function.
   In patients whose hypothalamo-pituitary function is intact,
but whose Sertoli cell function is impaired, the secretion of
inhibin B is decreased and that of FSH is increased. Therefore
we find an inverse relationship between inhibin and FSH
concentrations (Figure 1C). This suggests that inhibin B may
be important in the regulation of FSH (Illingworth et al, 1996).
   Until now the increase in pituitary FSH allowed us to
diagnose testicular dysfunction (impairment of sperm cell
formation) indirectly. However, we now have the ability to
measure inhibin B (a product of the testes) directly. Inhibin B
therefore seems to be a better indicator of exocrine testicular
function than FSH. In particular, in cases with normal FSH
and impaired sperm parameters, it seems possible now to
differentiate more accurately between disturbances of sperma-
togenesis and those of peripheral origin, e.g. fertility disorders
such as epididymal dysfunction. This opens new perspectives
for the diagnosis of fertility disturbances and especially for

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