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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 9 4128-4136
Copyright © 2002 by The Endocrine Society

Original Article

Predictors of Outcome of Long-Term GnRH Therapy in Men with Idiopathic Hypogonadotropic Hypogonadism

Nelly Pitteloud, Frances J. Hayes, Andrew Dwyer, Paul A. Boepple, Hang Lee and William F. Crowley, Jr.

Reproductive Endocrine Unit of the Department of Medicine, National Center for Infertility Research (N.P., F.J.H., A.D., P.A.B., W.F.C.), and Biostatistic Center (H.L.), General Clinical Research Center, Massachusetts General Hospital, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Nelly Pitteloud, M.D., Reproductive Endocrine Unit and National Center for Infertility Research, Bartlett Hall Extension 5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: . npitteloud{at}partners.org

Abstract

GnRH treatment is successful in inducing virilization and spermatogenesis in men with idiopathic hypogonadotropic hypogonadism (IHH). However, a small subset of IHH men, poorly characterized to date, fail to reach a normal testicular volume (TV) and produce sperm on this therapy. To determine predictors of outcome in terms of TV and sperm count, we studied 76 IHH men (38% with anosmia) undergoing GnRH therapy for 12–24 months.

The population was stratified according to the baseline degree of prior pubertal development: absent (group 1, n = 52), partial (group 2, n = 18), or complete (adult onset HH; group 3, n = 6). Cryptorchidism was recorded in 40% of group 1, 5% of group 2, and none in group 3. Pulsatile GnRH therapy was initiated at 5–25 ng/kg per pulse sc and titrated to attain normal adult male testosterone (T) levels. LH, FSH, T, and inhibin B (IB) levels were measured serially, and maximum sperm count was recorded. A longitudinal mixed effects model was used to determine predictors of final TV.

LH (97%) and T (93%) levels were normalized in the majority of IHH men. Groups 2 and 3 achieved a normal adult testicular size (92%), FSH (96%), IB levels (93%), and sperm in their ejaculate (100%). However, given their prior complete puberty and thus primed gonadotropes and testes, group 3 responded faster, normalizing androgen production by 2 months and completing spermatogenesis by 6 months. In contrast, group 1 failed to normalize TV (11 ± 0.4 ml) and IB levels (92 ± 6 pg/ml) by 24 months, despite normalization of their FSH levels (11 ± 2 IU/liter). Similarly, sperm counts of group 1 plateaued well below the normal range (median of 3 x 106/ml) with 18% remaining azoospermic. The independent predictors of outcome of long-term GnRH therapy were: 1) the presence of some prior pubertal development (positive predictor; group effect (ß) = 4.3; P = 0.003); 2) a baseline IB less than 60 pg/ml (negative predictor; ß = -3.7; P = 0.009); and 3) prior cryptorchidism (negative predictor; ß = -1.8; P = 0.05). Notably, anosmia was not an independent predictor of outcome when adjusted for other baseline variables.

Our conclusions are: 1) pulsatile GnRH therapy in IHH men is very successful in inducing androgen production and spermatogenesis; 2) normalization of the LH-Leydig cell-T axis is achieved more uniformly than the FSH-Sertoli cell-IB axis during GnRH therapy; and 3) favorable predictors for achieving an adult testicular size and consequently optimizing spermatogenesis are prior history of sexual maturation, a baseline IB greater than 60 pg/ml, and absence of cryptorchidism.

ALTHOUGH IDIOPATHIC HYPOGONADOTROPIC hypogonadism (IHH) is an uncommon cause of male infertility, its clinical importance lies in the fact that it is very responsive to hormonal therapy, i.e. exogenous gonadotropins (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) or pulsatile GnRH (8, 11, 12, 13, 14, 15, 16, 17, 18). Thus, IHH as a disease model provides important insights into the function of the reproductive axis in the human. IHH is a disorder that selectively affects the secretion or function of GnRH (11). When the disease occurs in association with an impaired sense of smell, it is referred to as Kallmann syndrome (19). Although androgen administration is successful in inducing and maintaining normal secondary sexual characteristics and sexual function in IHH men, testicular maturation and stimulation of spermatogenesis requires therapy with exogenous gonadotropins or pulsatile GnRH (1, 11, 20, 21). The key requirement for pulsatile release of GnRH for normal gonadotrope function was demonstrated in 1978 (22), and the first human studies of pulsatile GnRH therapy were performed in 1982 (11). Although both gonadotropins and GnRH are effective in the treatment of IHH men, a subset of patients, poorly defined to date, fails to reach a normal testicular size and produce sperm by either therapy (1, 2, 10, 11, 20). Additionally, the impact of cryptorchidism or small testes on the efficacy of GnRH or gonadotropins with respect to testicular growth and fertility is still disputed in the literature (1, 2, 10, 12, 20).

We recently refined the clinical and biochemical phenotypic features of a large cohort of IHH men studied before GnRH therapy (23). We introduced a classification based on the degree of their spontaneous pubertal development rather than the presence or absence of anosmia that allowed the population to be distinguished more clearly in terms of other clinical (testicular size, cryptorchidism, microphallus) and biochemical parameters [gonadotropin levels, inhibin B (IB), and Mullerian Inhibitory Substance]. Moreover, we believe this classification provides insight into the time of onset and severity of GnRH deficiency.

Thus, the aims of the present study were 2-fold: 1) to determine the efficacy of 2 yr of pulsatile GnRH therapy in this same cohort of IHH men in terms of normalization of T secretion, stimulation of testicular growth, and spermatogenesis; and 2) to define the predictors of outcome of long-term GnRH therapy.

Subjects and Methods

Study population: men with IHH

The cohort was composed of IHH men, aged 18–55 yr, recruited from the Reproductive Endocrine Clinic of the Massachussetts General Hospital between 1979 and 1999 who underwent at least 12 months of pulsatile GnRH therapy. Criteria for the diagnosis of IHH uniformly included: 1) age greater than 18 yr; 2) clinical signs or symptoms of hypogonadism; 3) T levels in the hypogonadal range (serum T < 3.4 nmol/liter) in the presence of low or normal gonadotropins; 4) normal thyroid, adrenal, and GH axes as assessed by TRH and insulin tolerance tests and normal serum prolactin and ferritin concentrations; and 5) normal radiological imaging of the hypothalamic and pituitary area. Reproductive hormone therapy [T, human chorionic gonadotropin (hCG) alone, or hCG and FSH therapy] was discontinued for at least 3 months before baseline evaluation.

Of the 78 IHH men who underwent long-term pulsatile GnRH therapy for at least 12 months, 2 patients were withdrawn from this analysis because they were found to exhibit a normal hypothalamo-pituitary-gonadal (HPG) axis after the discontinuation of GnRH therapy. Both patients were 18 yr old when studied initially. One had evidence of some testicular growth (TV, 6 ml) but had hypogonadal T levels and a flat LH pulse pattern; the other had no signs of pubertal development, hypogonadal T level, yet a normal LH pulse pattern. This observation suggests that their initial diagnosis may have been delayed puberty, which is 12 times more common in families with IHH than in the general population (24).

Study protocol

Baseline clinical assessment of IHH. A detailed history and physical examination were obtained from each patient to evaluate any evidence of spontaneous sexual maturation. All patients were specifically questioned about historical evidence of the spontaneous occurrence of puberty, such as: 1) a marked increase in the number of erections, ejaculations, and nocturnal emissions; 2) the occurrence of a growth spurt; 3) initiation of shaving; and 4) marked increase in libido. The presence of at least two of these historical points or a TV greater than 4 ml at presentation in the absence of prior gonadotropin therapy was interpreted as evidence of partial spontaneous sexual maturation. The subjective report of anosmia was noted because few had formal smell testing. A complete family history was recorded for each individual with specific reference to the occurrence of delayed puberty, hypogonadism, and anosmia, as well as previous therapy with androgens or gonadotropins.

This large cohort has been previously studied in detail regarding its baseline clinical and biochemical characteristics (23). Following this report, we decided to further stratify the population based upon the degree of prior pubertal development: group 1, absent puberty (n = 52); group 2, partial puberty (n = 18); and group 3, complete puberty (adult onset HH, n = 6; Ref. 25).

Baseline biochemical assessment. Subjects were admitted to the General Clinical Research Center of the Massachusetts General Hospital for an overnight frequent blood sampling study (every 10 min x 12 h) to assess the LH secretion pattern. Serum LH, FSH, T, and IB were measured in pooled samples, constituted from equal aliquots of the 10-min samples. Pulsatile hormone secretion was analyzed using a modification of the Santen and Bardin method (26). Semen samples were obtained in those patients with an ejaculate. The men were asked to abstain from ejaculation for at least 48 h before the sample was collected and analyzed, according to standard World Health Organization criteria (27).

Long-term GnRH therapy. After baseline evaluation, patients were discharged on a regimen of pulsatile GnRH therapy. GnRH (Salk Institute, La Jolla, CA) was administered sc via a portable infusion pump (Ferring Laboratories, Kiel, Germany) at 2-h intervals to mimic the LH pulse frequency observed in normal men (11, 28, 29). The doses of GnRH varied from 5–25 ng/kg and were increased progressively for each individual to reach and maintain T levels in the mid-normal adult male range as previously described (11, 14). All patients were treated with GnRH therapy for at least 12 months, with more than 70% of the cohort receiving treatment for 24 months.

Responses to GnRH therapy were monitored by bimonthly visits during which a physical examination was performed and blood samples were drawn for serum LH, FSH, T, and IB. At each visit, the time of the blood draw was standardized to 20 min after the last GnRH dose. Testicular volume (TV) was assessed at each time-point using a Prader orchidometer. A normal testicular size is defined as 15–25 ml (30). Once an ejaculate was present, semen specimens were requested at each visit and analyzed according to World Health Organization guidelines (27).

To define more closely the relationship between FSH, IB, and T during the early stages of GnRH therapy, we also studied a subset of patients (n = 10) in group 1 who were evaluated every 2 wk for the first 8 wk of therapy (31). Blood sampling was performed every 10 min for 6 h at each visit. LH, FSH, T, and IB were measured in a pool comprising equal aliquots of each sample obtained during the study.

These studies were approved by the Ethics Committee of Massachusetts General Hospital, and all subjects provided written, informed consent.

Assays. Because this study spanned a 20-yr experience, two immunoassay systems were used for the measurements of FSH and LH and are described in detail in our previous manuscript (23). Serum T concentrations were measured using the DPC Coat-A-Count RIA kit (Diagnostic Products Corp., Los Angeles, CA), which has an intra- and interassay coefficient of variation of less than 10% for all samples. IB was measured using a commercially available double-antibody enzyme-linked immunosorbent assay (Serotec, Oxford, UK) as previously described (32). In our use, the clinical detection limit of this assay is 15.6 pg/ml, with a coefficient of variation of 4–6% within plate and 15–18% between plates.

Statistical methods

The primary efficacy end points were T levels, testicular size as measured using a Prader orchidometer, and the spermatogenic response in terms of sperm concentrations. The secondary endpoints were LH, FSH, and IB levels. Because missing sperm count data were recorded in approximately 30% of subjects at each time period, the median of the two highest sperm counts for each subject during GnRH therapy was used to compare the different subsets of IHH patients.

The data are expressed as mean ± SEM unless otherwise indicated. The overall comparison between group means for T, gonadotropins and plasma IB concentrations as well as TV were conducted using ANOVA. Post hoc comparisons between individual groups were performed by Newman-Keuls test when appropriate. Mann-Whitney rank sum test was used to compare maximal sperm concentration between the groups during therapy. A P value less than 0.05 was considered significant.

To determine the prognostic factors for testicular growth during long-term GnRH therapy, we used longitudinal random effects regression analysis (33). The longitudinal random effects analysis provides the estimated effects of putative prognostic factors on the mean outcome variable (i.e. mean TV), whereas the time trend and other covariate effects on the outcome are controlled. The longitudinal observation y for a subject i at time t is expressed by a linear combination of fixed regression coefficients (b0, b1, ... ) and a random effect (si), i.e. yit = b0 + b1 · t + b2 · t2 + b3 · predictor + b4 · (t x predictor interaction) + si + eit, where b0 is the fixed intercept, b1 and b2 are the regression coefficients for the linear and quadratic time trends (in months), b3 is the predictor effect, and b4 is the incremental linear time trend among the subjects with the treatment (t x predictor interaction); si is the measure of the subject level random effect with mean 0; and eij is the error term with mean 0 (this error is assumed to have compound symmetry such that any pair of data points over time share the same amount of covariability).

The covariates (history of spontaneous pubertal development, history of cryptorchidism, Kallmann syndrome, baseline LH, baseline FSH, LH pulse pattern, and initial IB) were chosen as indices of the severity of GnRH deficiency. We hypothesized that the more severe cases would respond less well to therapy. In addition, prior gonadotropin or T therapy was assessed as a prognostic factor. We used prior degree of pubertal development instead of baseline TV as a prognostic variable, because one third of our cohort has undergone gonadotropin treatment before GnRH therapy.

To determine the association between the covariates and testicular growth among the 76 longitudinal datasets, we fitted an individual model for each prognostic factor while controlling for the time trends. Then, each covariate was tested again while controlling for prognostic factors simultaneously. We dichotomized each of the prognostic factors to allow practical interpretation of the effects of these variables on the mean.

Results

Baseline clinical and biochemical assessments of IHH

The clinical and biochemical characteristics of the entire population have been described in detail previously (23) and are summarized in Table 1Go.


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  		Table 1. Baseline characteristics of 76 men with IHH

 
GnRH dosing for IHH

The individual doses of sc GnRH necessary to normalize LH, FSH, and T varied from 25–600 ng/kg per pulse. The doses of GnRH at the time of T normalization (T >= 10 nmol/liter) for each patient were significantly higher for group 1 compared with groups 2 and 3 (176 ± 15, 105 ± 23, and 63 ± 17 ng/kg per pulse, respectively; P < 0.05). Moreover, for the duration of GnRH therapy, the dose required to maintain T levels in the normal adult range remained significantly higher in those with no prior pubertal development compared with groups 2 and 3 (185 ± 16 ng/kg vs. 121 ± 28 vs. 65 ± 33 ng/kg at 18 months; P < 0.002).

Response to GnRH therapy: clinical parameters

Secondary sexual characteristics. Complete virilization was observed in all patients who normalized their T levels (n = 70). Gynecomastia was observed in 30% of the cases at baseline and did not increase significantly on GnRH therapy. Among the patients with gynecomastia at presentation (all in groups 1 and 2), a third had received no prior therapy, a third had received T, and the remainder had received hCG therapy.

TV. GnRH induced a significant rise in TV that was most pronounced during the first year of therapy (6.8–12.9 ml; P < 0.0001). Further testicular growth occurred by 24 months (TV, 13.9 ml), but was not statistically significant. In group 1, TV increased predominantly during the first 12 months but failed to reach an optimal adult size even by 24 months of treatment (3.2 ± 0.3 to 11.3 ± 0.4 ml; Fig. 1Go). Within group 1, final TV was significantly smaller among those with a history of cryptorchidism (10 ± 0.7 vs. 12.3 ± 0.8 ml; P < 0.05). In group 2, TV normalized by 6 months and showed no significant change thereafter (11.8 ± 1.2 to 18 ± 2 ml; Fig. 1Go). Group 3 had a normal TV at baseline, and although a small increase in TV was recorded during the first 2 months of therapy, it was not statistically significant (19 ± 2 to 22 ± 1 ml; P = 0.3).



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  		Figure 1. TV changes during 24 months of GnRH therapy in IHH men without prior puberty (group 1, n = 52), with partial puberty (group 2, n = 18), and with complete puberty (group 3, n = 6). The shaded area represents the mean (±2 SD) in 36 normal men.

 
Hormonal profile

GnRH therapy was successful in the great majority of cases in normalizing LH and T concentrations in the IHH population. Gonadotropins reached the normal adult range by 6 months (LH, 2.2 ± 0.2 to 10 ± 0.9 IU/liter; FSH, 2.6 ± 0.2 to 9.1 ± 0.7 IU/liter). Ninety-three percent attained normal T levels on GnRH therapy (499 ± 37 ng/dl at 24 months). In contrast, IB increased but stabilized at a level significantly lower than the normal adult range at 100.9 ± 6.5 pg/ml (normal adult range, 170 ± 40 pg/ml; Ref. 23). Although LH normalization proceeded similarly in all three groups, T normalized by 2 months in group 3 and by 6 months in groups 1 and 2 (Fig. 2Go). In contrast, FSH and IB differed considerably between the groups. In patients with no prior pubertal development (group 1), IB and FSH increased initially but plateaued after 2 months (42 ± 2 to 92 ± 6 pg/ml and 2.20.2 to 11 ± 1.7 IU/liter, respectively; Fig. 2Go). In contrast, in group 2, IB was already in the normal adult range at baseline and did not change significantly despite a gradual, persistent increase in FSH (from 2.8 ± 0.3 IU/liter at baseline to 5.9 ± 1 IU/liter at 6 months; P < 0.05; Fig. 2Go). In group 3, baseline IB levels were already at the low end of the normal range (96 ± 17 pg/ml), reflecting their prior pubertal development and testicular size. Baseline IB levels increased further with the rise in FSH (2.6 ± 0.3 to 9.2 ± 1 IU/liter; P < 0.05) consistent with a recovery of spermatogenesis (34), although this increase did not reach significance (115 ± 17 pg/ml). After 24 months of GnRH therapy, IB levels differed between those with and without a history of cryptorchidism (76.7 ± 8.1 vs. 106 ± 8.3 pg/ml, respectively; P < 0.05) across both groups 1 and 2. In the subset of group 1 who were evaluated every 2 wk for 2 months, IB initially increased dramatically but surprisingly plateaued by 4 wk at a value below the normal adult male range (87 ± 13 pg/ml) despite a further increase in FSH (Fig. 3Go). Meanwhile, serum T remained at midpubertal levels for the first month of therapy (2.4 ± 0.4 nmol/liter), and then increased sharply to normal adult male levels.



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  		Figure 2. LH and T responses (left panels) and FSH and IB responses (right panels) to GnRH therapy in IHH men without (group 1, n = 52) and with (group 2, n = 18) a history of some degree of spontaneous puberty. To convert values of serum T from nanomoles per liter to nanograms per decaliter, multiply by 28.84.

 


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  		Figure 3. LH, T, FSH, and IB responses to short-term GnRH therapy in 10 IHH men with no prior puberty. To convert values of serum T from nanomoles per liter to nanograms per decaliter, multiply by 28.84. Asterisks represent a significant difference from the previous time point. *, P < 0.05.

 
Spermatogenesis

There was considerable variability in the time course and success in inducing spermatogenesis. At baseline, only one subject had sperm present in his ejaculate (0.1 x 106/ml). He had an initial TV of 20 ml, but decreased virilization, eunuchoidal proportions, and hypogonadal T levels, thereby meeting the criteria for a fertile eunuch (35, 36). During 2 yr of therapy, two thirds of the IHH men provided semen samples for analysis. The larger group having semen analysis parameters was compared with the group lacking semen analysis to assess for potential selection bias. No differences were evident between the two groups in terms of baseline characteristics such as TV (6.6 ± 0.9 vs. 7.4 ± 1.6 ml), absence of puberty (71 vs. 62%), history of cryptorchidism (31 vs. 32%), initial IB levels (65 ± 9.8 vs. 81 ± 12.8 pg/ml), and final TV (14 ± 1 vs. 13.6 ± 1.5 ml). Thus, we are confident that the data presented is an accurate reflection of this cohort of IHH men. Spermatogenesis was successfully induced in 24 of 31 patients (77%) by 12 months and in 42 of 51 (82%) by 24 months of GnRH therapy. IHH men who remained azoospermic were all in group 1 (n = 9, 18%). Of these men, five had cryptorchidism, and in two of them it was bilateral. Furthermore, in all but one of these cases, azoospermia occurred in the presence of normal T levels. Sperm counts were significantly higher in groups 3 and 2 compared with group 1 (median of 37, 21, and 3 x 106/ml, respectively; P = 0.05). However, variability was observed within each group. Nine IHH men with unilateral cryptorchidism provided semen specimens, two remained azoospermic (20%), five exhibited sperm counts less than 5 x 106/ml, and two had a sperm count greater than 5 x 106/ml. Three of six IHH men with bilateral cryptorchidism remained azoospermic, and two displayed a sperm count below 5 x 106/ml. The time interval between initiation of GnRH therapy and the appearance of sperm in the ejaculate was quite variable (2–24 months of therapy), depending largely on the initial stage of pubertal development. Indeed, all patients in groups 2 and 3 had sperm by 6 months, compared with less than 50% of patients in group 1.

Suboptimal hormonal outcome on GnRH therapy

Only two IHH subjects, both fertile eunuchs, failed to exhibit a gonadotropin response to increasing doses of GnRH. Five patients (7%) failed to normalize T levels after 12–18 months of GnRH therapy despite increasing the dose to 600 ng/kg GnRH. Among them were the two fertile eunuchs described above. One patient with adult onset HH developed an anti-GnRH antibody after 2–4 months of GnRH therapy; his gonadotropins decreased, and he was thus unable to normalize T levels. The remaining two nonresponders had Kallmann syndrome and complete absence of pubertal development. The first, who had bilateral cryptorchidism, failed to normalize T levels by 1 yr of therapy despite high gonadotropin levels. The second had variable gonadotropin levels in response to GnRH and was able to normalize his T after only 1 month of hCG, consistent with a lack of compliance with pulsatile GnRH therapy. Finally, six IHH men who initially responded adequately to pulsatile GnRH, attaining normal gonadotropins and T levels, were subsequently removed from the study because of noncompliance with the GnRH pump as evidenced by declining gonadotropin and T levels in the absence of GnRH antibodies.

Predictors of outcome

Analysis of these data showed a statistically significant effect of the following initial parameters on TV after 24 months of GnRH therapy: 1) prior pubertal development [group effect (ß) = 4.3; P = 0.003]; 2) baseline IB less than 60 pg/ml (ß = -3.7; P = 0.009); and 3) prior cryptorchidism (ß = -1.9; P = 0.05; Table 2Go and Fig. 4Go).


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  		Table 2. Impact of prognostic factors on testicular growth during long-term GnRH therapy

 


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  		Figure 4. The left panel represents the final TV after 24 months of GnRH therapy in IHH men with (group 1, Yes; n = 52) and without (group 2, No; n = 18) spontaneous pubertal development. Center panel displays baseline IB level vs. final TV. A cut-off of 60 pg/ml is the best discriminator of testicular growth on GnRH therapy. The right panel illustrates the effect of cryptorchidism upon final TV.

 
Discussion

Prior studies have demonstrated the effectiveness of both pulsatile GnRH and gonadotropins as a therapy for IHH men (1, 2, 3, 4, 5, 6, 7, 12, 13, 15, 20, 21). However, most studies were limited by having a small sample size (1, 3, 4, 8, 9, 13, 16, 17, 37) and/or including heterogeneous groups of hypogonadotropic patients, such as men with delayed puberty and hypopituitarism (1, 2, 18, 20). Furthermore, several studies used only one gonadotropin (hCG) (4, 9, 38), a choice that precludes an optimal response in those IHH men with no prior pubertal development (4, 21, 39). The present study is unique for its large cohort of IHH men in whom detailed clinical and biochemical evaluation was performed before therapy. This detailed phenotyping permitted clear stratification according to baseline characteristics and the identification of predictors of outcome on long-term GnRH therapy.

Long-term pulsatile GnRH therapy in this large cohort of IHH men proved effective in stimulating normal gonadotropin and T secretion. However, although testicular growth occurred in most IHH men, a significant spectrum of responses was apparent, depending largely on the history of prior pubertal development. Moreover, FSH stimulation of IB production from Sertoli cells and spermatogenesis also differed according to the degree of pubertal development. In addition to a prior history of pubertal development, baseline IB levels greater than 60 pg/ml and absence of cryptorchidism were strong positive predictors of testicular growth on GnRH therapy.

The dose of GnRH needed to achieve a normal adult male T level varied according to prior pubertal development. This observation is consistent with our prior study reporting an increased pituitary-gonadal responsiveness as sexual maturation occurs in IHH men (14). However, maintenance doses for those with no prior pubertal development remained significantly higher than those with partial or complete puberty, suggesting either a decreased responsiveness to GnRH at the pituitary and/or to LH at the level of the testis in those with the most severe GnRH deficiency. Although the precise mechanism is unclear, evidence from animal models supports the critical role of HPG activation for Leydig cell development during the neonatal window of gonadal development (40). First, GnRH antagonist therapy prevents neonatal Leydig cell maturation in subhuman primates (41). Second, hpg mice have only 10% of normal adult Leydig cells resulting from an absence of postnatal proliferation (d 5–20; Ref. 42). Although the dynamic of the waves of Leydig cell maturation across life remains unclear, it is possible that some neonatal Leydig cells do not degenerate but contribute to the adult Leydig cell population (43).

The failure of gonadotropins to increase on high doses of GnRH in two IHH men with the fertile eunuch syndrome raised the possibility of a GnRH receptor mutation (44). However, a genetic analysis of the coding sequence of the GnRH receptor gene was negative in both patients. An unusual form of adrenal hypoplasia congenita characterized by a predominantly hypogonadal phenotype (45) was also considered but is unlikely, given the completely normal adrenal reserve during the ITT performed at baseline. Included among the patients who failed to normalize their T levels was a man with Kallmann syndrome and bilateral cryptorchidism who harbored a point mutation in the KAL gene. He became hypergonadotropic during GnRH therapy. The development of high gonadotropins levels on pulsatile GnRH suggests a concomitant gonadal defect, but the KAL gene is not expressed in the testes and cannot explain this phenotype (46). However, an impairment of Leydig cell function has been described in patients with bilateral cryptorchidism (28).

FSH, IB, sperm counts, and TV exhibit differential response to GnRH depending upon the stage of pubertal development. IHH men with no prior puberty, despite normal maturation of the neuroendocrine axis, including normal adult male T levels, fail to reach normal IB levels, sperm counts, and adult testicular size. Furthermore, IB levels stabilize after only 4 wk of GnRH therapy, whereas FSH is still rising and T levels are not yet at the midpubertal range. In contrast, in men with prior spontaneous pubertal development, GnRH therapy induces a normal testicular size and spermatogenesis but fails to stimulate IB secretion further despite an increase in FSH levels. These observations are consistent with a cross-sectional study in healthy pubertal boys showing that adult IB levels are attained as early as Tanner stage II (47). Interestingly, spermarche is also achieved early in puberty, before the complete appearance of secondary sexual characteristics (48). Thus, the physiological change in FSH regulation of IB secretion seems to appear at a time when Sertoli cell maturation takes place and spermatogenesis is initiated. It is therefore plausible to hypothesize that terminal differentiation of Sertoli cells may be one factor that limits the ability of FSH to further stimulate IB secretion after the early stage of puberty. In support of this hypothesis, the administration of physiological doses of recombinant FSH to normal men fails to induce a further increase in IB concentrations (49).

Prior studies have reported a predictive role of initial TV for achieving optimal testicular size on gonadotropin or pulsatile GnRH therapy (6, 7, 14, 18, 21, 50). These studies are limited by the unclear delineation of the confounding roles of cryptorchidism and prior therapy. Other investigators did not find a significant correlation between initial and maximal TV in IHH men during therapy (1, 7, 38). In contrast, our study clearly demonstrates that the degree of prior pubertal development is the strongest predictor of testicular growth on GnRH therapy after adjusting for other baseline characteristics. This is significant for the impact on spermatogenesis and therefore potential fertility. Those patients with no prior pubertal development harbor some limitation on seminiferous tubule growth that is not present in men with partial or complete puberty and that results in failure to achieve a normal adult TV.

A major determinant of TV and sperm count in several species, including the human, is the Sertoli cell population (51, 52, 53, 54, 55). Two main surges of Sertoli cell proliferation occur in the neonatal period and early puberty (56, 57, 58, 59) coincidental with waves of activation of the neuroendocrine axis (60, 61). Thereafter, terminal differentiation of Sertoli cells, an androgen-dependent process (59, 62, 63, 64), coincides with the establishment of the blood-testis barrier, maturation of Sertoli cell histology, and initiation of spermatogenesis (65). Thereby, premature achievement of adult male T levels through simultaneous administration of LH and FSH may limit the potential for maximal proliferation of Sertoli cells in those men with the most severe form of IHH (66). Indeed, these patients appear to lack FSH-stimulated Sertoli cell proliferation during the neonatal period and early puberty (23). In addition, the failure of activation of gonadal genes during the neonatal window (i.e. for gonocyte maturation) may be critical for subsequent testicular growth.

Our study indicates that baseline IB level is a strong predictor of testicular growth on GnRH therapy. A cut-off value of 60 pg/ml allows the best discrimination for subsequent testicular growth. This is in agreement with a prior study from our group reporting a significantly higher fertility rate among a small group of IHH men with a baseline IB greater than 60 pg/ml (67). In animal models, IB has proven a reliable marker of Sertoli cell number (68, 69). Furthermore, coincident with neuroendocrine axis activation, the ontogeny of IB secretion in the human mimics Sertoli cell proliferation with a neonatal and early pubertal rise (47, 70) before stabilization at adult levels unless spermatogenesis is disrupted (71). Serum IB may therefore serve as a potential surrogate marker of Sertoli cell number in the human male both in the prepubertal period and in adulthood, as long as spermatogenesis is maintained at the level of the spermatid stage (23, 72). Thus, it is not surprising that both prior degree of pubertal development and IB were found to be good predictors of outcome of GnRH therapy.

A history of cryptorchidism is generally considered a negative prognostic factor for testicular growth and spermatogenesis in IHH men, although controversy still exists in the literature (1, 5, 16, 20). Our data demonstrates that cryptorchidism is a negative predictor of testicular growth in IHH men. We have reported previously a high incidence of cryptorchidism (40%) among IHH men with no prior pubertal development, likely caused by the lack of androgen production during the fetal-neonatal period (23). This high frequency of cryptorchidism is relevant given the association between cryptorchidism and reduced fertility. In agreement with the literature, 50% of IHH men with bilateral cryptorchidism remained azoospermic (73). The impaired fertility seems to be caused by secondary degeneration of germ cells and possible Leydig cell dysfunction. Early orchidopexy is advocated to prevent loss of germ cells (74). Maturation of gonocytes into Ad spermatogonia during the neonatal window seems critical for male fertility (75). More precisely, it is this transformation, rather than the total number of germ cells at the time of surgery that has been reported to be the best indicator of fertility in patients who undergo orchidopexy before 2 yr of age (76). The trigger of this transformation of gonocytes to Ad spermatogonia is unclear and awaits further investigation. However, it is interesting to note that this maturation does not occur in patients with complete androgen insensitivity syndrome (76) who, like the most severely affected IHH men, lack HPG activation during the neonatal period (77).

Although this study represents the largest cohort of IHH men in whom the response to GnRH therapy was evaluated, fertility per se as an outcome was not assessed. Indeed, most of our patients initiated GnRH therapy at a time when induction of sexual maturation rather than fertility was their primary objective. Furthermore, serial semen analyses were not consistently reported in a subset of our population. Although TV and sperm counts are generally considered good indicators of fertility, the IHH population is unique in that relatively high fertility rates have been reported with low sperm counts (78).

In conclusion, administration of pulsatile GnRH to IHH men represents a unique human model to investigate male reproductive physiology. Our large cohort provides a cross-section of pubertal development affording insight into testicular physiology. GnRH therapy was very successful in inducing sexual maturation. Although normalization of LH/Leydig cell/T production was achieved in most IHH men, a limitation in seminiferous tubule growth was encountered in those patients with no prior puberty as evidenced by failure to achieve normal IB levels, sperm count, and testicular size. The cause of this suboptimal response is still unclear but points to the critical neonatal window for normal gonadal development. Our analysis further identifies prior history of sexual maturation, a baseline IB greater than 60 pg/ml, and absence of cryptorchidism as favorable predictors of outcome of GnRH therapy.

Acknowledgments

We gratefully acknowledge the nurses of the General Clinical Research Center (M01-RR-01066) for their excellent clinical care and the technicians of the Reproductive Endocrine Sciences Center RIA Core (P30-HD-28138) for their superb technical contributions to this study.

Footnotes

This work was supported by Grants R01 HD15788 and M01-RR-01066, by the National Institute of Child Health and Human Development/National Institutes of Health through cooperative agreement (U54-HD-28138, U54 DK07028-24) as part of the Specialized Cooperative Centers Program in Reproduction Research, and the Swiss Roche Foundation for medical research.

Abbreviations: hCG, Human chorionic gonadotropin; HPG, hypothalamo-pituitary-gonadal; IB, inhibin B; IHH, idiopathic hypogonadotropic hypogonadism; T, testosterone; TV, testicular volume.

Received April 1, 2002.

Accepted May 16, 2002.

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