madman
Super Moderator
Introduction
Two primary functions of mature male testis are spermatogenesis and androgen production. Both these functions are under the control of the hypothalamic-pituitary-testicular (HPT) axis. Gonadotropin-releasing hormone (GnRH) secreted from the hypothalamus acts on the pituitary to stimulate the secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH), the 2 main endocrine stimulators of the testis. LH causes the maturation of interstitial Leydig cells, which are the primary source of testosterone (T) in males. FSH causes the multiplication of immature Sertoli cells, which release the anti-mullerian hormone(AMH).1 Under the influence of intratesticular testosterone (ITT), immature Sertoli cells mature and release growth factors required for germ cell survival. Therefore, an optimal level of ITT is a prerequisite for adequate spermatogenesis. Mature Sertoli cells also produce Inhibin B (Inh B), which regulates FSH secretion via negative feedback on the HPT axis.2,3 Low AMH and high Inh B levels indicate the presence of mature Sertoli cells and thus are an indirect functional marker of optimal IIT and spermatogenesis.1,4
In males with congenital hypogonadotropic hypogonadism(CHH), the HPT axis fails to get activated during adolescence, resulting in low gonadotropin and androgen levels. This results in the absence of secondary sexual characters, loss of libido, infertility etc.5 Hormonal replacement is the first-line option in the management of hypogonadotropic hypogonadism (HH). The choice of hormone to be administered primarily depends on whether fertility is desired or not. Testosterone replacement therapy is the standard of care for those seeking HH treatment for manifestations other than infertility. On the other hand, if the patient’s main treatment objective is to gain fertility, gonadotropin therapy or pulsatile GnRH therapy needs to be started. Due to the cumbersome nature of the GnRH portable pump, gonadotropin therapy seems more feasible.6,7
The regime of gonadotropin therapy has evolved over the years, from human chorionic gonadotropin (hCG) monotherapy to initial hCG followed by a combination of hCG and FSH. Currently, FSH and hCG are administrated from the outset of treatment as many studies have shown better results of this regimen in the induction of spermatogenesis.8 Usually, high-dose hCG is used in standard gonadotropin therapies. The rationale behind using such doses is to achieve normal plasma total T level, which is traditionally used as a surrogate marker of optimal IIT levels required for spermatogenesis. However, recently, it has been observed that adequate ITT levels essential for sperm production could be achieved even at hCG doses lower than that required for normalizing plasma total T levels.9,10 Thus, administration of high-dose hCG might not be obligatory for fertility induction in HH patients.
Apart from infertility, eunuchoid body habitus, lack of libido, sexual infantilism, absence of secondary sexual characters, etc, are the major manifestations of HH and can lead to anxiety, depression,and poor quality of life (QoL) in such patients.11 Low plasma total T level is responsible for these presentations, and hence its normalization is also an essential aspect of HH management. This can be achieved by either high dose hCG or exogenous T therapy. Though high-dose hCG will normalize plasma total T levels, few studies have reported that long-term usage of high-dose hCG can have a detrimental effect on testicular functioning.12,13 On the other hand ,exogenous T will not impair sperm production in HH as the HPT axis of these patients is already nonfunctional
With this background, we postulated that low-dose hCG with FSH could be an alternative approach to standard high-dose hCG based therapy for the treatment of infertility in HH patients, while concomitant administration of exogenous T could be given for triggering virilization and improvement of other hypogonadal related symptoms. Titration of hCG dose could be done based on plasma AMH levels as a proxy marker of ITT, instead of plasma T.
Results
A total of 33 patients with HH were reviewed for inclusion in the study. Out of this, 1 patient had postpubertal HH, 1 refused to consent, and 1 had cryptorchidism. Therefore, 30 patients fulfilling the eligibility criteria were randomized into 2 groups (GroupA = 14, Group B = 16) and were analyzed (Fig. 1)
The overall mean (SD) age of the patients was 22.6 (5.3) years.Mean (SD) weight and body mass index were 63.5 (14.9) kg and.22.4 (4) kg/m2 respectively. Of the 30 patients, 46.7% had some form of pituitary defects, most common was agenesis/hypoplasia (7 patients), 16.7% had Kallman syndrome, while in rest, no identifiable abnormalities could be found. Three (10%) subjects had multiple pituitary hormone deficiencies and required corticosteroids and thyroxine replacement. There were no statistically significant differences in the baseline parameters of the 2 groups, as shown in Table 1.
The median weekly doses of hCG at the time of spermatogenesis in groups A and B were 4000 (4000-8000) U/week and 9500 (6000-15000) U/week, respectively (P = 0.013). All patients in both groups achieved the target FSH level (4-8 mIU/ml) with a constant FSH dose of 150 U/thrice per week. The plasma level was measured within 24 h of FSH injection
Between group A [64.3% (9 out of 14)] and group B [87.5% (14 out of 16)] (P = 0.204) (Table 2), the overall median (interquartile range) time for spermatogenesis was 12 (9-14.9) months. The median time for initial spermatogenesis in group A [15 (12-17.9) months] was not statistically different from that in group B [12 (9-14.9) months] (X2(1) = 3.376, P = 0.066) (Table 2, Fig. 2). Comparison of median sperm concentrations between group A and B [8.5 (2.5-18.0) million/ml vs 13.0 (2.5-30.0) million/ml] was statistically not significant (P = 0.569). Patients achieving spermatogenesis had higher basal FSH, T and Inh B at follow up.(Supplementary Table 1). The median time to spermatogenesis was shorter in patients receiving prior T therapy (Supplementary Table 2).
Hormonal Concentrations
There was a significant increase in T with both regimens. Group A patients were having significantly lower T compared to Group B till 12 months (P < 0.05) (Table 3). Spermatogenesis was initiated at a much lower total T level in Group A compared to Group B (8.42 (5.84-11.25) nmol/l vs 16.72 (12.78-24.26) nmol/l, P < .001).
A remarkable and statistically significant reduction from baseline to the last follow-up AMH levels was observed, dropping from 18.69 (11.14-29.9) to 3.83 (1.85-5.78) ng/mL (P = 0.005). No substantial differences in AMH levels were noted between the two groups during serial follow-up assessments (Table 3). Both groups reached similar median plasma AMH levels at the induction of spermatogenesis, (6.6 (3.3-9.76) ng/ml vs 4.41 (2.3-6.47) ng/ml, P = 0.298).
A notable and statistically significant increase in Inh B levels,rising from 38.88 (10.5-58.63) pg/mL to 161.57 (93.93-161.57) pg/mL (P =.007). Interestingly, there were no significant differences in Inh B levels between the groups during the serial follow-up, as highlighted in Table 3. Furthermore, both groups achieved similar Median plasma Inh B at spermatogenesis (152.4 pg/ml (101.7-198.0) vs 149.1 pg/ml (128.7-237.3), P = 0.488).
Mean Testicular Volume
There was a consistent and noteworthy increase in mean TV with the administered treatment, which was statistically significant (P = .01) (Table 3). During the initiation of spermatogenesis, there was no significant difference in mean TV between groups A and B (3.31 (2.6-3.98) cc vs 3.67 (3.38-5.06) cc, P = 0.122). (Table 2). Although group B exhibited significantly higher TV measurements up to 9 months, the difference was no longer significant from 12 months onward.
Virilization
Subjects in both groups achieved adequate virilization with similar improvement in the Tanner stage, as shown in Table 2. In addition, facial hair growth and voice changes were observed in all subjects in both groups.
A substantial increase in median FG scores from 10 (9-11) to 16 (13-21) (P < .001). This change was uniform across both groups, with no statistically significant discrepancies between them (P = .806).
A statistically significant increase in PDS from baseline of 8 (7-10) and 9 (7.25-10) to 14 (12.5-16) and 16 (13-18) in Groups A and B, respectively, was achieved (P < 0.001). No statistically significant difference was found between the groups in PDS (P = 0.272).(Supplementary Table 3).
QoL
There was a statistically significant improvement in the QoL in both groups. After treatment, AMS scores at 9 months in Group A ,32 (24.5-40) and B, 38 (31-40) were significantly lower than pretreatment (A, 70 (61.2-75.7) and B, 72 (65-77)) (P < 0.001) (Supplementary Appendix). AMS scores were not significantly different when comparing post-treatment scores between the groups (P = 0.132). All patients were successful in giving semen samples at the final follow-up.
Adverse Effects
During the follow-up, gynecomastia occurred in 13.3% (4/30) of subjects overall, and all were in group B (25%, 4/16 of patients in Group B). Acne occurred in 16.7% (5/30) patients overall and 7.1% (1/14) and 25% (4/16) in Groups A and B, respectively. (P = 0.336) Treatment-related adverse effects were higher in Group B than in Group A; however, none required treatment discontinuation and were managed carefully. No allergic reaction to the highly purified urinary gonadotropins was noted in either Group.
Predictors for Spermatogenesis
We applied logistic regression to ascertain the effect of various baseline parameters on the likelihood of successful spermatogenesis induction. Age (P = 0.005), baseline FSH (P = 0.012), and presence of microlithiasis (P = 0.001) were found to significantly impact the initiation of sperm production, while presence of panhypopituitarism or Kallman syndrome, prior T treatment, baselineT, AMH, Inh B, and TV had no significant effect on likelihood of successful spermatogenesis. However, on multivariate analysis, none of the above baseline parameters were found to be significant. Cox regression showed that a lower FG score (OR = 3.311, 95% confiCI 1.299-8.442, P = 0.012) was a statistically significant predictor of longer duration of therapy required for spermatogenesis (Supplementary Tables 4 and 5).
During follow-up, T (P < 0=001), AMH (P = 0.009), Inh B (p - 0.007), TV (P < 0.001), the dose of hCG (P = 0.004), duration of therapy (P < 0.001) and percent change in AMH from baseline (P < 0.001) significantly predicted successful spermatogenesis. A multivariate logistics regression was performed to ascertain the effects of afore said variables on the likelihood that participants have successful spermatogenesis. The logistic regression model was statistically significant, x2 (8) = 40.358, P < 0.001. The model explained 58.6% (Nagelkerke R2) of the variance in spermatogenesis outcome and correctly classified 86% of the cases. Higher basal T (OR 1.326, 95% CI 1.02-1.73, P = 0.038), longer duration of therapy (OR 1.38, 95% CI 1.03-1.85, P = 0.033), and higher percentage change in AMH from baseline (OR 1.06, 95% CI 1.01-1.12,P = 0.034), were associated with an increased likelihood of spermatogenesis, while high AMH (OR 0.675, 95% CI 0.474-0.961, P = 0.029) during follow-up was associated with a decreased likelihood of successful spermatogenesis (Supplementary Tables 6 and 7).
We performed ROC curve analysis (Supplementary Fig. 1) on the follow-up parameters, the cut-off values derived predicting successful spermatogenesis were AMH <6,9 ng/ml [Sensitivity (Sn) 81.8%, specificity (Sp) 75.5%, Area under curve (AUC) 0.837, P < 0.001], Inh B of 92.4 pg/ml (Sn 94.7%, Sp 67.1%, AUC- 0.794, P < 0.001) and T of 6.96 nmol/L (Sn 87%, Sp 60.8%, AUC- 0.782, P < 0.001) respectively. The cumulative hCG dose of 3000 IU/week predicted successful spermatogenesis (Sn 95.7%, Sp 35.5%, AUC- 0.678, P < 0.001). The median time to spermatogenesis had a cut-off of >4-5 months (Sn 82.4%, Sp 77%, AUC-0.877, P < 0.001), with an average ultrasonographic mean TV of 3.015 cc (Sn 82.4%, Sp 77%, AUC-0.817,P < 0.001). Age <24 years was found to have a favourable outcome (Sn-78.3%, Sp 85.7%, P < 0.001, AUC- 0.851, P < 0.001) (Supplementary Table 8).
The increase in sperm count demonstrated a relatively weak, but positive association with Inh B (r = 0.393, 95% CI 0.209-0.550,P < 0.05), total T (r = 0.315, 95% CI 0.176-0.442, P < .05), the duration of therapy (r = 0.422, 95% CI 0.298-0.533, P < .05), and TV (r = 0.392, 95% CI 0.225-0.537, P < .05). . Conversely, as anticipated ,albeit weak, there was a negative correlation with AMH (r = -0.385, 95% CI -0.511 to -0.242, P < .05). The correlation between sperm concentration and hCG dose was very weak (r = 0.194, 95% CI 0.053-0.327, P < .05).
On the other hand, the increase in TV exhibited a moderate positive correlation with plasma Inh B (r = 0.738, 95% CI 0.625 to 0.821, P < .05), total T values (r = 0.600, 95% CI 0.468 to 0.706,P < .05), the duration of therapy (r = 0.720, 95% CI 0.618 to 0.798, P < .05), and a moderate negative association with AMH (r = -0.505, 95% CI -0.631 to -0.353, P < .05) (Supplementary Table 9).
Discussion
A logical approach to induce fertility in HH males is to initiate combination therapy with FSH (targeting FSH in the range of 4-8 mIU/ml), hCG (primarily titrating to achieve target AMH levels),and exogenous T (targeting normal systemic total T). Under these conditions, Sertoli cells will be exposed to the full paracrine ITT levels required for spermatogenesis as evidenced by a decrease in AMH and an increase in Inh B. Exogenous T will provide sufficient virilization and improve the QoL of these subjects. The starting dose of hCG of 3000 IU per week, divided into at least 2 injections reasonable approach to start for induction of spermatogenesis. AMH, Inh B, and T concentrations should be measured every 4-6 weeks, and the dose should be adjusted, if necessary, until the steady target state is reached. This proposed regimen reduces the cost of more hCG doses, prevents the testis from the detrimental effects of high hCG doses, and helps maintain a good QoL.
The strength of our study lies in the fact that it is the first randomized study comparing gonadotropin regimens that followed strict eligibility criteria for only patients with CHH, excluding patients with partial hypogonadism, along with long-term follow-up, and its ability to deliver multiple predictors of successful spermatogenesis, few of which have not been previously reported. Applying the ROC-derived cut-off value and its application during follow-up make the results more robust. Such approaches not only enhance our understanding of normal development but may also be a way to maximize the effectiveness of treatment for patients in the future. The major limitation of the study was its small sample size leading to an underpowered study. Consequently, the significance of the regression analysis was questionable though the trend may remain the same. One factor for the small sample size can be partly attributed to the rarity of the disease. A study with larger sample sizes to evaluate fertility outcomes, with varying dose adjustments of hCG, FSH and T therapy, is required to optimize gonadotropin therapy in individuals with CHH.
Conclusion
The study indicated that initiation of spermatogenesis is both gonadotropin dose and duration-dependent. LFT regimen is noninferior to conventional treatment for CHH. Moreover, systemic total T normalization is not a prerequisite for achieving spermatogenesis, as sperm production can occur much before total Tl evels normalize. Furthermore, our study demonstrates the potential of AMH and inh B levels as proxy indicators for the likelihood of spermatogenesis.
