Department of Urology, Department of Organs Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, Kobe, Japan
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Abstract |
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Key words: inhibin B/spermatogenesis/sperm count/varicocele testis/varicocelectomy
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Introduction |
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Inhibins, which are glycoprotein members of the transforming growth factor-ß family, participate in the control of spermatogenesis by a negative feedback influence on the secretion of FSH (De Kretser and McFarlane, 1966; de Jong, 1988
; Maddocks et al., 1992
; Spiteri-Grech and Nieschlag, 1993
). Earlier immunoassays for inhibin were limited by cross-reaction with inactive monomeric precursor forms present in plasma, by lack of sensitivity, or by an inability to discriminate between inhibin A and B (Robertson et al., 1996
). Recently assays able to distinguish bioactive dimeric inhibins from unprocessed higher molecular weight inhibin precursors and free subunits of inhibin have been developed, and hence the physiological role of inhibins in men can be investigated in greater detail. Biologically active forms include two dimers, each comprising a common
subunit linked by disulphide bonds to either a ßA subunit (inhibin A) or a ßB subunit (inhibin B). While men have measurable amounts of circulating inhibin B, circulating inhibin A concentrations are below the limits of detection of current assays (Illingworth et al., 1996
). Several reports have documented a strong negative correlation between FSH and inhibin B in fertile and subfertile men (Su et al., 1995
; Anwalt et al., 1996
; Illingworth et al., 1996
; Klingmuller and Haidl, 1997
; Foresta et al., 1999
). In addition, inhibin B concentrations are closely related to both sperm concentration (Jensen et al., 1998
) and testicular volume (Klingmuller and Haidl, 1997
; Pierik et al., 1998
). Inhibin B has also been found to be slightly more sensitive than FSH as an index of spermatogenic status, showing a significant correlation (Von Eckardstein et al., 1999
), and has also been proposed as a marker for spermatogenesis (Leifke et al., 1997
; Pierik et al., 1998
).
The correlation of serum concentrations of inhibin B with recovery of spermatogenesis induced by correction of a varicocele has not been investigated. In the current study, the relationship of inhibin B to serum concentrations of FSH, LH, prolactin, testosterone and oestradiol, as well as sperm concentration and testicular volumes, was examined in infertile men with varicocele. In addition, serum concentrations of inhibin B and FSH, LH, prolactin, testosterone and oestradiol were compared before and after varicocelectomy in order to determine whether inhibin B measurements might predict an improvement in the seminogram, as well as changes in reproductive hormonal function after surgery.
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Materials and methods |
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Semen samples were obtained by masturbation after at least 5 days of abstinence; semen analyses were performed according to published procedures (WHO, 1992). Only patients followed-up for more than 6 months were included in this study. Semen analysis was performed preoperatively and postoperatively at 3-month intervals. Preoperative and postoperative semen analysis after
6 months were compared in order to identify any improvement according to published criteria (Fujisawa et al., 1994
). Patients were regarded as responders when preoperative sperm counts of <1x106/ml, from 15x106/ml, from 510x106/ml, and from 1020x106/ml were increased to >5x106/ml, 10x106/ml, 20x106/ml, and twice the preoperative value respectively.
Serum hormones and inhibin B assay
Measurements of serum hormones and inhibin B were performed preoperatively and 6 months postoperatively. Serum samples were obtained from all patients and stored at 20°C until the assays were performed. FSH, LH and prolactin were analysed using chemiluminescence assays (Bayer Corp., Germany). Testosterone and oestradiol were measured by radioimmunoassay (Yatoron, Japan). The detection limits were: FSH, 0.3 mIU/ml; LH, 0.1 mIU/ml; prolactin, 0.3 ng/ml; testosterone, 0.05 ng/ml; and oestradiol, 10 pg/ml. Inhibin B was measured using a commercially available, double-antibody, enzyme-linked immunoassay (Serotec, Oxford, UK). Intra- and inter-assay coefficients of variation (CV) for inhibin B were 6% and 15%, respectively. The lowest detectable inhibin B concentration was 15 pg/ml.
GnRH stimulation test
A gonadotrophin-releasing hormone (GnRH) stimulation test was performed in each subject using an i.v. injection of 200 µg GnRH. FSH and LH responses in serial blood samples, obtained at 0, 15, 30, 60, 90 and 120 min, were examined. This test was carried out before varicocelectomy, and repeated at 6 months after surgery.
Data analysis
All results were expressed as mean ± SD. Statistical analyses were performed using the MannWhitney test and 2 test. Correlation was assessed using linear regression analysis. A P-value < 0.05 was considered significant.
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Results |
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Effect of varicocelectomy on seminogram, clinical variables, and serum hormones
The seminograms of 47 oligozoospermic patients, excluding the five patients with a normal preoperative seminogram, were evaluated. On the basis of published criteria (Fujisawa et al., 1994), the sperm concentration was increased in 22 patients (responders, 46.8%). Neither responders nor non-responders showed any significant difference in age, duration of infertility, testicular volume, or preoperative sperm concentration (Table I
). Furthermore, no significant difference was observed in concentrations of LH, testosterone or oestradiol between responders and non-responders (Table II
). In contrast, concentrations of FSH and prolactin were significantly higher in non-responders than in responders (P < 0.05). No apparent changes were observed in concentrations of FSH, LH, testosterone, prolactin or oestradiol before and after varicocelectomy (Table II
).
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However, 15 out of 25 patients (60%) with increased inhibin B showed an improvement in the seminogram, while only five out of 22 patients (23%) with unchanged or decreased concentrations of inhibin B showed any such improvement. Those patients with an increased inhibin B concentration after varicocelectomy showed a significantly higher rate of the improvement in their seminogram than did those with no change or even reduced inhibin B (P < 0.05).
Inhibin B concentration and the GnRH test before and after varicocelectomy
Peak concentrations of FSH in response to GnRH correlated significantly with inhibin B concentrations (Figure 1; r = 0.577, P < 0.0001). The patients were classified into three groups on the basis of their pre- and postoperative peak FSH and LH concentrations. For FSH, group I included patients with normal pre- and postoperative peaks, while groups II and III included patients with a high preoperative peak that either decreased (group II) or did not decrease (group III) postoperatively. Both pre- and postoperative inhibin B concentrations in groups II and III were significantly lower than those in group I. No significant differences were observed between pre- and postoperative inhibin concentrations in any group. Even when high peak FSH concentrations before surgery were reduced after surgery (group II), inhibin B concentrations did not change significantly.
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Discussion |
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Sperm concentration and testicular volume have been reported to correlate with the inhibin B concentration in serum (Bohring and Krause, 1999), and the current study confirmed these relationships. In addition, several studies have shown a correlation of inhibin B with the quality of spermatogenesis (Klaiji et al., 1994
; Anwalt et al., 1996
; Illingworth et al., 1996
; Klingmuller and Haidl, 1997
; Andersson et al., 1998
; Jensen et al., 1998
; Pierik et al., 1998
; Von Eckardstein et al., 1999
). One group (Andersson et al., 1998
) reported that inhibin B concentrations correlated directly with testicular status based on concurrent biopsy specimens; serum inhibin B concentrations in adult men with normal spermatogenesis were higher than in subjects with maturation arrest (MA) or Sertoli cell only (SCO). Serum inhibin B concentrations in postpubertal men were closely related to the presence of germ cells from pachytene spermatocytes to early spermatids. It was reported (Von Eckardstein et al., 1999
) that the percentages of SCO tubuli or tubules with elongated spermatids were closely related to serum inhibin B concentration, indicating the role of late-stage germ cells in the regulation of inhibin B. Circulating inhibin B concentrations may also reflect the extent of interaction between Sertoli cells and germ cells (Pineau et al., 1990
; Von Eckardstein et al., 1999
), and particularly elongated spermatids (Allenby et al., 1991
). However, the current present study disclosed no significant correlation between the percentage of any stage of germ cell and inhibin B concentrations. In addition, no significant correlation was observed between the Johnsen score and inhibin B, probably because most of the patients in this small study had a Johnsen score of 5 to 6. Others (Bohring and Krause, 1999
) found that there was no significant difference between inhibin B concentrations in normal spermatogenesis and hypospermatogenesis, and concluded that inhibin B cannot reliably predict spermatogenic failure. Thus, no specific type of germ cell appeared to be involved in the regulation of inhibin B secretion.
Although major cytotoxic damage to the testis reduces the overall circulating inhibin B concentration, the effect of minor damage to Sertoli cells or germ cells is unknown. However, inhibin B is considered a potentially useful marker of Sertoli cell and/or germ cell function, for the clinical assessment of individual patients as well as for future clinical and toxicological investigation of male infertility. In the current study, the pre- and postoperative serum inhibin B concentrations were examined in an effort to evaluate testicular function after varicocelectomy. Previously, a small but statistically insignificant difference was reported in mean inhibin B concentrations between patients with and without a varicocele and who had an identical sperm count (40x106/ml) (Bohring and Krause, 1999). However, others (Plymate et al., 1992
), by considering all of their varicocele patients together, reported no abnormalities in either sperm concentration, inhibin B or FSH. Therefore, varicocele itself may not affect inhibin B concentrations. In contrast, it was reported that inhibin was more abundant in sera from a homogeneous group of sterile patients with varicocele and FSH abnormalities than in sera from normal fertile men (Baccetti et al., 1991
, 1993
). In observing that rat Sertoli cells in vitro produced more inhibin at 37°C than at 32°C, it was proposed that an increased temperature resulting from the varicocele directly stimulated Sertoli cells to increase secretion of inhibin, hence causing a reduction in FSH and a consequent alteration in spermatogenesis (Ultee-van Gessel et al., 1986
). However, these studies were based on an assay that was unable to discriminate between the various inhibins, and so the results might have been different had current, highly specific assays been used. In the current study, inhibin B concentrations were lower in severe, than in mild, oligozoospermia. In addition, inhibin B concentrations in varicocele patients were much lower than the range reported for 356 normal Japanese men (Baba et al., 2000
). Therefore, Sertoli cell function as reflected by inhibin B production was considered to be reduced in varicocele patients. Overall, no significant difference was found between pre- and postoperative inhibin B concentration, and the change in inhibin B related to surgery was not significant in either the responder or non-responder subgroups. However, patients with increased inhibin B showed significantly more improvement than those with no change or a decrease in inhibin B. Therefore, an increased concentration of inhibin B after surgery may be associated with an increase in sperm production. Although postoperative improvement in testosterone production in Leydig cells and in spermatogenesis may occur simultaneously, improvements in hormonal regulation may be independent of any improvement in spermatogenesis. The effect of varicocelectomy on spermatogenesis may be related to changes in venous pressure, temperature or intratesticular interstitial fluid volume, as opposed to purely hormonal changes. It is also possible that increased inhibin B secretion and improved spermatogenesis after varicocelectomy may not occur simultaneously in all patients.
With regard to the GnRH stimulation test, it has been reported previously that exaggerated gonadotrophin (LH and FSH) responses can normalize after surgical correction of the varicocele, supporting the view that varicocele can induce reversible testicular hormonal dysfunction (Fujisawa et al., 1994). Reduction of the LH peak after surgery is significantly related to an improvement in testicular function and fertility. In the current study, a postoperative decrease from the exaggerated preoperative FSH peak was not necessarily associated with a postoperative increase in inhibin B. Although it was not possible to demonstrate a change of testosterone concentration after varicocelectomy, a significant increase has been reported in mean serum testosterone following microsurgical inguinal varicocelectomy (Su et al., 1995
). Therefore, normalization of the FSH peak may be related to small changes in testosterone and oestradiol concentrations.
In conclusion, it was found that mean concentrations of inhibin B and other hormones changed little between pre- and postvaricocelectomy sera. Sertoli cell function, as estimated by inhibin B concentrations, did not normalize during the early period after surgery, even if the sperm count increased. However, the increase in inhibin B concentration after varicocelectomy might suggest an improvement in testicular function.
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Notes |
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References |
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Submitted on February 1, 2001; accepted on April 19, 2001.