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,
From the * Department of Medicine, University of
Washington, Seattle, Washington; the
Department of Medicine, Education and Clinical
Center, Veterans Affairs Puget Sound Health Care System, Seattle, Washington;
the Department of Medicine, Boston University,
Boston, Massachusetts; and the Department of
Geriatric Research, Education and Clinical Center, Veterans Affairs Puget
Sound Health Care System, Seattle, Washington.
| Correspondence to: Dr John K Amory, University of Washington, Box 356429, 1959 NE Pacific, Seattle, WA 98195 (e-mail: jamory{at}u.washington.edu). |
| Received for publication December 4, 2006; accepted for publication February 12, 2007. |
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Abstract |
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1 million sperm/mL). However, 10% to 20% of men have persistent sperm
production despite profound gonadotropin suppression. Since insulin-like
factor 3 (INSL3) has been shown to prevent germ cell apoptosis in mice, we
hypothesized that INSL3 might be higher in men with persistent spermatogenesis
during treatment with male hormonal contraceptives. In a retrospective
analysis, we measured serum INSL3 in 107 men from 3 recent male hormonal
contraceptive studies and determined the relationship between suppression of
spermatogenesis and serum INSL3. At the end of treatment 63 men (59%) were
azoospermic and 44 men (41%) had detectable sperm in their ejaculates.
Baseline INSL3 did not predict azoospermia; however, end of treatment serum
INSL3 was significantly higher in nonazoospermic men compared with those with
azoospermia (median [interquartile range]: 95 [73–127] pg/mL vs 80
[67–101] pg/mL; P = .03). Furthermore, serum INSL3 was
positively correlated with sperm concentration (r = .25; P =
.009) at the end of treatment and was significantly associated with
nonazoospermia by multivariate logistic regression (P = .03). After 6
months of treatment with a hormonal male contraceptive regimen, higher serum
INSL3 concentrations were associated with persistent sperm production. INSL3
may play a role in preventing complete suppression of spermatogenesis in some
men on hormonal contraceptive regimens. This finding suggests that INSL3 may
be a potential target for male contraceptive development.
Key words: Azoospermia, oligospermia
The major mystery in the field of male hormonal contraceptive research is
why some men fail to suppress spermatogenesis completely despite suppression
of serum gonadotropins to extremely low levels. Since there are no apparent
differences in gonadotropin concentrations among men who suppress to
azoospermia and those who do not, the degree of gonadotropin suppression alone
does not explain this difference (Wallace
et al, 1993; Handelsman et al,
1995; Amory et al,
2001; McLachlan et al,
2004). Alternative explanations include the hypothesis that
nonazoospermic men might have greater 5
-reductase activity and hence
higher intratesticular concentrations of the potent androgen
dihydrotestosterone (DHT) (Anderson et al,
1996). However, 2 studies have demonstrated that the addition of
the 5-reductase inhibitor finasteride did not enhance suppression of
spermatogenesis beyond that achieved by T alone or T plus a progestogen, as
would be expected if higher intratesticular DHT were the reason for persistent
sperm production (McLachlan et al,
2000; Kinniburgh et al,
2001). Lastly, genetic polymorphisms known to impact androgen
action do not appear to influence individual responsiveness to hormonal
suppression (Yu and Handelsman,
2001). Clearly, further investigation is required into the
differences in the intratesticular environments that allow some men to
continue to produce sperm in the extremely low gonadotropin environment of a
male hormonal contraceptive regimen.
Insulin-like factor 3 (INSL3) is a recently described peptide hormone produced almost exclusively by Leydig cells and released into the serum where it can be measured by immunoassay (Ivell et al, 1997). Serum INSL3 levels are greatly diminished in men after orchiectomy or in the setting of testicular dysfunction, suggesting that it is a sensitive marker of Leydig cell function (Foresta et al, 2004; Bay et al, 2005). LH stimulates INSL3 secretion; however, in contrast to T production, prolonged exposure to LH is required for normal levels of synthesis (Bay et al, 2006; Ferlin et al, 2006). During fetal development, INSL3 appears to play a role in testicular descent since mice lacking INSL3 have cryptorchidism (Zimmermann et al, 1999; Nef and Parada, 1999; Kumagi et al, 2002), but its role in adult men is not well delineated. The receptor for INSL3 is the transmembrane leucine-rich G protein–coupled receptor LGR8, which is expressed in developing male germ cells (Anand-Ivell et al, 2006). Administration of INSL3 to mice was recently shown to prevent the apoptosis of male germ cells in the setting of induced gonadotropin deficiency (Kawamura et al, 2004), suggesting that INSL3/LGR8 paracrine interactions may support germ cell survival.
If INSL3 supports germ cell survival in men, differential INSL3 production may explain persistent spermatogenesis in some men on male hormonal contraceptive regimens. We hypothesized that men with persistent spermatogenesis during treatment would exhibit higher serum INSL3 levels than those who achieved azoospermia. Therefore, we measured serum INSL3 levels in men who had successfully completed 1 of 3 male hormonal contraceptive studies at our research site and compared the levels to the degree of sperm suppression. In this way, we aimed to determine if differential production of INSL3 could partially explain why some men fail to completely suppress spermatogenesis despite the profound gonadotropin suppression mediated by male hormonal contraceptives.
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Methods |
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Measurements
Serum INSL3 was measured by a radioimmunoassay (RIA) (Phoenix
Pharmaceuticals Inc, Belmont, Calif). The normal range was 291 to 1132 pg/mL.
The lower limit of detection was 16 pg/mL; the intraassay coefficients of
variation were 7.6%, 5.2%, and 5.7% and the interassay coefficients of
variation were 30%, 13%, and 8.1% for low, mid, and high pools, respectively.
FSH and LH levels were measured by immunofluorometric assay (DELFIA; Wallac
Oy, Turku, Finland). The sensitivity of the assay for FSH and LH was 0.016
IU/L and 0.019 IU/L, respectively. For low-, mid-, and high-pooled values of
0.05, 1.0, and 21 IU/L FSH, the intraassay coefficients of variation were
5.9%, 3.0%, and 3.0% and the interassay coefficients of variation were 20.7%,
5.0%, and 6.2%, respectively. For low-, mid-, and high-pooled values of 0.06,
1, and 16 IU/L LH, the intraassay coefficients of variation were 12.6%, 5.6%,
and 4.1% and the interassay coefficients of variation were 16.5%, 13.9%, and
10.5%, respectively. T was measured by RIA (Diagnostic Products Corp, Los
Angeles, Calif), and the assay sensitivity for T was 0.5 nmol/L. For low-,
mid-, and high-pooled values of 3.8, 10.6, and 24.4 nmol/L T, the intraassay
coefficients of variation were 4.4%, 5.1%, and 6.0% and the interassay
coefficients of variation were 17.5%, 11.8%, and 12.9%, respectively.
Statistical Analyses
Azoospermia was defined as the absence of detectable sperm in the ejaculate
of 2 consecutive seminal fluid specimens by the end of the treatment phase,
while severe oligospermia was defined as a sperm concentration of 1
million sperm/mL ejaculate. All data were analyzed in a nonparametric fashion
due to a lack of normality. Comparisons of serum hormone concentrations
between groups (azoospermic vs nonazoospermic and severely oligospermic vs
nonseverely oligospermic) were performed by Wilcoxon rank-sum tests.
Correlations between variables were performed using Spearman's (nonparametric)
technique. The association between azoospermia or severe oligospermia and
hormone concentrations, age, and weight was analyzed by univariate and
multivariate logistic regression using robust standard errors. For all
comparisons, a 2-sided of <.05 was considered significant.
Statistical analyses were performed using Stata version 8.0 (StataCorp LP,
College Park, Tex).
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Results |
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Serum INSL3 significantly decreased with contraceptive hormone treatment (median [interquartile range]: baseline: 792 [626–1058] pg/mL vs end of treatment: 86 [68–109] pg/mL; P < .0001). Serum INSL3 decreased in all men during contraceptive treatment; however, INSL3 was significantly higher in men with persistent sperm production compared with those with azoospermia (95 [73–127] pg/mL vs 80 [67–101] pg/mL; P = .03) (Figure 1A). In addition, serum INSL3 levels were significantly higher in men with sperm concentrations >1 million/mL compared with those with severe oligospermia (106 [86–151] pg/mL vs 82 [68–102] pg/mL; P = .04) (Figure 1B). When the studies were considered individually, there was a significant difference in serum INSL3 levels in study 1 between azoospermic and nonazoospermic men (105 [86–152] pg/mL vs 80 [68–96] pg/mL; P = .03) (Figure 1A) and a similar, although nonsignificant, difference was apparent in the other 2 studies. Differences in serum INSL3 levels between severely oligospermic and nonseverely oligospermic men were similarly more profound in study 1 (108 [88–152] pg/mL vs 79 [68–95] pg/mL; P = .01) than those observed in the other 2 studies (Figure 1B). End of treatment LH, FSH, and T concentrations did not significantly differ between azoospermic and nonazoospermic or severely oligospermic and nonseverely oligospermic men (Table 2).
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Baseline serum INSL3 concentrations were not significantly correlated with baseline sperm concentrations or baseline serum LH, FSH, or T levels. In contrast, there was a significant correlation between each individual's sperm concentration and his corresponding serum INSL3 level at the end of treatment (r = .25, P = .009) (Figure 2A). This correlation persisted even when the individual with the highest sperm count and highest INSL3 concentration was omitted (r = .23, P = .02) (Figure 2B). In contrast, no such relationship was suggested between sperm concentration and end of treatment LH (r = .07, P = .48) or FSH (r = .01, P = .94) level. At the end of the treatment period, the concentration of serum INSL3 was significantly correlated with serum LH (r = .25; P = .009) but not serum FSH (r = .17, P = .08) level.
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Univariate logistic regression modeling of factors associated with azoospermia revealed a statistically significant association between end of treatment INSL3 level and azoospermia (odds ratio [OR], 0.987 [95% CI, 0.977, 0.995]; P = .004). This association remained statistically significant in multivariate logistic regression with adjustment for baseline INSL3, T, LH, FSH, age, and weight (OR, 0.984 [95% CI, 0.974, 0.998]; P = .03). There was no significant association between azoospermia and LH or FSH level by either univariate or multivariate regression.
Univariate logistic regression modeling of factors associated with severe oligospermia did not reveal a significant association between end of treatment INSL3 concentration and severe oligospermia (OR, 0.993 [95% CI, 0.985, 1.00]; P = .07). This association attained significance in multivariate modeling with adjustment for baseline INSL3, T, LH, FSH, age, and weight (OR, 0.983 [95% CI, 0.965, 0.998]; P = .03).
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Discussion |
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1 million sperm/mL ejaculate). Moreover, higher end of
treatment concentrations of serum INSL3 were significantly correlated with
higher end of treatment sperm concentrations, and higher end of treatment
INSL3 concentrations were significantly associated with nonazoospermia after
correction for other variables using logistic regression. The finding of a
relative preservation of serum INSL3 level is therefore the first description
of a significant hormonal difference during treatment between men who
completely suppress sperm production and those who do not with male hormonal
contraceptive treatment. It is unclear from the analyses presented here whether the relationship between serum INSL3 concentration and persistent spermatogenesis is merely an association or is causal. On the one hand, a higher serum INSL3 level could simply be a marker for individuals whose Leydig cells are more functional in the setting of low serum gonadotropins, or higher serum levels of INSL3 could result from factors secreted from the seminiferous epithelium in individuals with persistent spermatogenesis. Such individuals may maintain higher intratesticular testosterone concentrations, which could allow spermatogenesis to persist since intratesticular testosterone is known to be essential for spermatogenesis in animal models (Cunningham and Hickins, 1979; Turner et al, 1984; Zirkin et al, 1989). Moreover, because many of the measurements of the gonadotropins were at or below the lower limit of quantitation, a relationship between gonadotropin concentration and sperm concentration may have been missed.
On the other hand, a functional role for INSL3 in supporting spermatogenesis has been suggested in mice. In this work, the administration of INSL3 prevented apoptosis of developing germ cells in the mouse in the setting of experimentally induced gonadotropin suppression (Kawamura et al, 2004). As a result, it is tempting to speculate that in men who do not completely suppress spermatogenesis the relatively higher level of INSL3 is allowing developing germ cells to avoid the apoptosis normally mediated by gonadotropin withdrawal. If this were the case, agents directed at blocking INSL3 activity may have potential utility as male contraceptive agents, as has been suggested (Del Borgo et al, 2006), or may function as an adjunctive agent in a hormonal regimen. Moreover, since INSL3 appears to reduce cyclic AMP levels in developing sperm, it is conceivable that a germ cell–specific phosphodiesterase inhibitor might antagonize the function of INSL3 and increase germ cell apoptosis, thereby functioning as a male contraceptive.
The testes are known to be the primary source of INSL3 production; however, a small amount is synthesized in other parts of the male reproductive tract (Zarreh-Hoshyari-Khah et al, 1999), such that serum INSL3 concentrations in anorchid men are roughly 10% those of normal subjects (Foresta et al, 2004; Bay et al, 2005). It is notable then that serum INSL3 concentrations in the azoospermic subjects were very similar to the values reported for anorchid men, implying that most of the INSL3 in these men was of nontesticular origin. Therefore, the higher serum INSL3 concentrations seen in subjects with persistent spermatogenesis likely reflects testicular production of the hormone, and the difference in the intratesticular concentrations of INSL3 may be much greater than those observed in the serum. Since INSL3 is thought to exert its effect on developing germ cells in a paracrine fashion, the intratesticular concentration may be of greater importance than the serum concentration.
This study is limited in that it is retrospective and involves only men recruited and treated at 1 site. As a result, ethnic (eg, Asian vs non-Asian), nutritional, and geographic differences related to the degree of sperm suppression were not assessed. In addition, we were limited to the use of stored serum from studies with similar designs and durations. Moreover, the correlations between INSL3 and sperm concentration are not robust as several subjects with persistent spermatogenesis had relatively low serum INSL3 concentrations. In addition, while the serum INSL3 level was greater in subjects with persistent spermatogenesis in all 3 trials, this difference was only significant in 1 of the 3 studies when assessed individually. Since the treatments in each of the trials was different, it is possible that the ability of INSL3 to serve as a biomarker of persistent spermatogenesis depends somewhat on the trial regimen and the compounds administered to study subjects. Lastly, there is a fairly large variance in the INSL3 measurement that may bias the conclusions. Clearly, larger integrated analyses, such as that recently completed for recovery from hormonal suppression (Liu et al, 2006), and prospective studies of the relationship between INSL3 and contraceptive effect should be performed to corroborate these initial findings.
In conclusion, we demonstrated here an association between serum INSL3 levels and persistent spermatogenesis in the setting of experimental male hormonal contraception. Since INSL3 may play a role in preventing germ cell apoptosis, these findings suggest that the INSL3 pathway may represent a novel target for improving the efficacy of current experimental male contraceptive regimens, potentially bringing the dream of safe, effective, reversible male contraception to fruition.
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Acknowledgments |
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Footnotes |
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