Pharmacological treatment for pubertal progression in boys with delayed or slow progression of puberty: A small-scale randomized study with testosterone enanthate and testosterone undecanoate treatment

Context The use of testosterone enanthate (TE), 50–75 mg intramuscularly (i.m.)/month, for the treatment of boys with delayed puberty or slow progression to induce puberty is the standard of care (SoC) in Sweden. This treatment is empirical and has not been scientifically evaluated. Replacement therapy in hypogonadal boys/young men in Sweden after induction is mainly performed with testosterone undecanoate (TU), 1,000 mg/3 months. TE is only available on license. TE was deregistered in Sweden in 2006. Therefore, this study was initiated to compare the two products. Objective To clinically evaluate pubertal progression with six injections of TE, 75 mg i.m./month (1/3–1/5 of adult dose), compared with two injections of TU, 250 mg i.m./3 months (1/4 of adult dose). Trial design In the Pubertal Replacement in Boys Study (PRIBS), boys aged 14–16 years in West Sweden with pubertal delay were randomized in a parallel study to TE or TU for pubertal progression. Inclusion criteria were morning testosterone levels of 0.5–3 nmol/L and testicular volume ≤6 ml. Between June 2014 and Nov 2019, 27 boys were included. Methods The primary outcome was testicular enlargement ≥8 ml after 12 months. TU treatment was considered clinically similar if the number of boys with testicular enlargement ≥8 ml was 80%–125% of the number of boys with TE. Fisher’s exact chi-square test was used for this analysis. Results Both treatments were well tolerated. Twelve of 14 (86%) TU-treated boys reached the primary outcome and 12/12 in the TE group. Fisher’s exact chi-square testing indicated a one-sided p-value of 0.28 (the two-sided p-value was 0.483). The TU treatment was considered not clinically different from SoC. A post-hoc study showed 25% power. Therefore, no evidence-based conclusion can be drawn from the results even if the clinical data support a similar effect of the treatments. Conclusion The present small-scale study supports that both TE and TU had similar effects in terms of pubertal progression. Clinical Trial Registration https://www.clinicaltrials.gov/ct2/home, identifier NCT05417035; https://www.clinicaltrialsregister.eu/ctrsearch/search, identifier EUDRACTEudraCT nr 2012-002337-11.

Patient recruitment for PRIBS started on 1 June 2014 and ended on 1 August 2019. Forty boys were recruited based on intention to treat (20 TU and 20 TE). The study was initially approved for three years, by which time only 18 patients had been recruited, so the protocol was amended to allow another two years of recruitment. After five years, 27 patients had been randomized, 12 in the TE group and 15 in the TU group. One patient in the TU group was lost to follow up due to septicemia 11 months after study start.
During this period, 146 boys were referred for delayed puberty. All eligible patients were invited. The majority could not participate because they did not fulfill the inclusion criteria, either having morning testosterone levels >3 nmol/L or growth acceleration. Fear of injections and long distance from the clinic were other reasons for not participating.

Interim Analysis
The aim of TU treatment was to achieve testosterone levels in the lower range found in mid puberty (4-9 nmol/L). Two of five patients in the TU-group had testosterone levels <4 nmol/L and none over 9 nmol/L after 2 months of treatment: their values were 2.4, 2.6, 4.1, 4.5, and 5.8 nmol/L, so the dosage was not changed.

Futility Analysis
After 20 patients were recruited (10 in each group), a futility analysis was conducted. Two of 10 patients (80%) in the TU group did not have a TV ≥8 mL (both patients had a TV of 6 mL) after 12 months. The new treatment was 80% effective, as 8/10 patients reached the primary outcome, whereas 10/10 reached the primary outcome in the traditional treatment group. Fisher's exact chi-square test showed that our hypothesis could not be rejected. A one-sided mid-p-value of 0.237 indicates that there is no evidence of a statistically significant difference between the treatment groups.

DHEAS and Inhibin B
The mean DHEAS level for all boys in PRIBS was 3.2 µmol/L (range 1.4-5.6) at study start, 3.14 (1.0-5.8) after 6 months, and 4.2 (1.0-6.9) after 12 months. The mean DHEAS level in the TE group was 3.3 µmol/L at study start and 4.4 after 12 months, while in the TU group it was 3.4 and 4.1 µmol/L at study start and 12 months, respectively.
The Inhibin B range in all boys was 65-341 pg/mL (mean 186 pg/mL) at study start and 62-311 pg/mL (mean 191 pg/mL) after 12 months. At study start, the mean Inhibin B level was 194 pg/mL in TE-treated patients and 178 pg/mL in TU-treated patients. At 12 months, the mean Inhibin B level was 208 and 193 pg/mL in the TE-and TU-treated patients, respectively. The two boys who did not reach a TV of 8 mL at 12 months had Inhibin B levels of 86 and 65 pg/mL at start and 122 and 62 pg/mL at 12 months. Replicates were analyzed for 10 patients and the CV% varied between 0.6 and 7.1 with a mean of 3.4%.

Long-Term Safety
Blood pressure, pulse, liver enzymes, and Hb were measured four times during the study. Adverse events and severe adverse events were recorded. One patient was lost to follow up due to septicemia 11 months after study start: he had an underlying disorder that predisposed him to septicemia and could not attend the 12-month visit. No other severe adverse events were noted during the study.
The liver enzymes ALAT (normal range 0.36-1.1µkat/L) and ASAT (0,4-0,8) were measured at study start and after 3, 6, and 12 months. No patients had elevated liver enzymes at start. Three patients had transient increases in liver enzymes: one had an elevated ALAT value of 1.6 µkat/L at 3 months, two had values of 1.1 and 1.8 µkat/L at 6 months, and no patients had elevated values at 12 months. Two patients had elevated ASAT values of 1.1 and 2.2 µkat/L at 3 months and three had elevated values of 0.92, 0.99, and 1.4 µkat/L at 6 months; otherwise, the ASAT values were normal. The mean Hb was 135 g/L at study start, 134 after 3 months, 135 after 6 months, and 137 after 12 months; the maximum change in Hb level was a 13 g/L rise.

Bone Age
After PRIBS started, there was a change in methodology from manual to computerized bone age estimation using BoneXpert software, and for the rest of the study bone age was assessed using BoneXpert (with the TW3 model). Bone age was therefore not used as an inclusion criterion, and it was up to the investigator whether or not to consider bone age. 23/27 patients were investigated with Bone Xpert and the mean skeleton age was 11.8 years in the TE group and 11.3 years in the TU group.

Growth
The mean height for the whole group was 153.9 cm before treatment, 156.4 cm after 3 months, 159.4 cm after 6 months, and 163.2 cm after 12 months, for a mean gain of 9.3 cm (range 6.9-12.8). The TU-treated boys had a mean height at study start of 153.4 cm with a height gain of 9.4 cm during the study; in the TE group the mean starting height was 154.5 cm and the height gain was 9.2 cm.
The mean weight for all boys at study start was 43.9 kg (range 32-74 kg). The mean weights for the TE and TU groups were 47.6 and 40.8 kg, respectively, at study start, 50.6 and 43.2 after 3 months, 52.2 and TU 44.9 after 6 months, and 54.6 and 48.4 after 12 months. The mean weight for all boys was 51.3 kg (range 39.5-79.7 kg) after 12 months.
In the TE group the mean height was -2,76 SD at study start and 12 months after treatment start it was -1,97, an increase of 0,78 SD and in the TU treated group the mean was -2,56 at study start and 12 months later -1,96 SD an increase of 0,61 SD

Testicular Growth and Signs of Masculinization
The mean left and right TVs at study start were 4.3 and 4.4 mL (range 2-6 mL). The TE and TU groups had mean TVs of 4.6 and 4.2 mL (range 2-6 mL), respectively, at study start, 7.1 and 7.2 mL (range 3-11 mL) at 6 months, and 10.6 and 10.6 mL (range 6-15 mL) at 12 months.

Testosterone levels
In figure 2b the mean testosterone levels for the the TE and TU group are shown at studystart(day0) twice before study start, 2, 7 ,30,60,90,180 and 360 days after studystart.

Legends and Figures
Supplementary Figure 1 Testosterone levels for the TE (Testoviron Depot®) and TU(Nebido®) treated boys. Day 0 is study start.