Testicular Growth and Spermatogenesis: New Goals for Pubertal Hormone Replacement in Boys With Hypogonadotropic Hypogonadism?

A Multicentre Prospective Study of hCH/rFSH Treatment Outcomes During Adolescence

Julia Rohayem; Berthold P. Hauffa; Margaret Zacharin; Sabine Kliesch; Michael Zitzmann

Disclosures

Clin Endocrinol. 2017;86(1):75-87. 

In This Article

Patients and Methods

The study was performed over 4 years, between 3/2011 and 3/2015, in 26 centres for paediatric endocrinology throughout Germany and co-ordinated by the first author at the Department for Clinical Andrology, Centre for Reproductive Medicine, University of Münster/Germany.

Ethics

Informed written consent by majors and assent by minors with consent of their parents was obtained for all procedures. The study was approved by the Ethics Committee of the State Medical Board of Westfalen-Lippe (approval number: 2010-427-f-S).

Inclusion Criteria

Males aged 14–22 years with hypogonadotropic hypogonadism (HH) were enrolled in the study. Subsets of participants were as follows: boys/adolescents with:

  • absent puberty by age 14, confirmed by testicular volumes <4 ml each side, prepubertal levels of LH, FSH and testosterone, and failure of GnRH agonist (buserelin, 10 μg/kg s.c) to stimulate LH >4 U/l after 4 h and/or absent pubertal response to 'priming' with low-dose (50–100 mg) testosterone-enanthate i.m. over 3–6 months.

  • early pubertal arrest after age 14, confirmed by arrested testicular growth (with volumes >4 ≤ 8 ml each side) and prepubertal levels of LH, FSH and testosterone.

  • Kallmann syndrome, confirmed by presence of anosmia or severe hyposmia (by 'Sniffin-sticks', Burghart Messtechnik GmbH, Wedel, Germany) or KAL1 mutation.

  • congenital or acquired multiple pituitary hormone deficiencies (MPHDs)

  • CHARGE syndrome.

Exclusion Criteria

Patients with constitutional delay of growth and puberty (CDGP), testicular disorders (primary hypogonadism), Prader–Willi syndrome, functional hypogonadism (due to eating disorders or chronic diseases) or with HH due to untreated abnormalities of the hypothalamic–pituitary region were excluded.

Primary Study End-points

  • Final bi-testicular volumes (BTVs= the sum of both testicular volumes) by Prader orchiometry.

  • Final sperm concentrations (SCs), according to WHO 2010 criteria[13]

Secondary Study End-points

QoL post-gonadotropin treatment, compared to pretreatment, assessed by three standardized questionnaires: 'Inventory for assessment of quality of life in children and adolescents' ('ILK'),[14] 'Inventory for assessment of depression in children and adolescents' ('DIKJ')[15] and 'Questionnaire for assessment of emotion regulation strategies in children and adolescents' ('FEEL-KJ'),[16] and by two questions on self-perceived satisfaction with testis size and masculinity.

Treatment Protocols

Patients Were Divided Into Two Groups:. Group A included prepubertal HH boys (Tanner stage G1 with testicular volumes (TVs) <4 ml each side or HH boys with early pubertal arrest (Tanner G2–3 with TVs >4 ≤ 8 ml).

Group B comprised fully virilized (Tanner G4–5, TVs <4 ml each side) HH adolescents who had received at least 1·5 years of full (250 mg i.m. every 3–4 weeks) testosterone-enanthate replacement.

Highly purified urinary-derived hCG (Brevactid®), followed by combined hCG/rFSH (Gonal f®), was self-administered with or without parental help via subcutaneous injections, two injections/week (on Mondays and Fridays) for hCG and three injections/week for rFSH (on Mondays, Wednesdays and Fridays).

The end of gonadotropin replacement was defined by cessation of testicular growth and plateauing of sperm concentrations in the ejaculate over at least two observations at 3-month intervals.

Protocol Group A. For testosterone-naïve prepubertal boys (or early pubertal boys with arrested puberty), the following gonadotropin replacement protocol was recommended: as prepubertal boys had not yet attained their adult height, a relatively low starting dose of (250-) 500 IU hCG was injected subcutaneously on Mondays and Fridays, and incremental increases of 250–500 IU hCG per injection every 6 months to a maximum of 3 × 2500 IU hCG s.c./week were recommended. The aim was to achieve pubertal levels (serum testosterone ≥1·5 ng/ml, [5·2 nmol/l]) after around 6 months and levels in the mid-normal adult range (testosterone >3·5 ng/ml, [12 nmol/l]) by one year. rFSH (follitropin alpha) 3 x (75-)150 IU s.c/week (injected Mondays, Wednesdays and Fridays) was added when pubertal serum testosterone levels (>5·2 nmol/l) were reached, without subsequent rFSH dosage modifications above 150 IU per injection, aiming at physiologic serum FSH target levels between 1–7 U/l.

Protocol Group B. In those adolescents previously treated with testosterone who had completed pubertal virilization and linear growth (documented by left hand digital epiphyseal fusion), the following gonadotropin replacement protocol was used: a full (adult) hCG starting dose of 1·500 IU s.c. was initially applied twice weekly (injected subcutaneously on Mondays and Fridays). HCG dose reduction was recommended if erythrocytosis, gynaecomastia or excessive acne occurred. If testosterone levels remained below the normal adult range (<12 nmol/l) after 6–9 months, the hCG dose was increased by increments of 500(−1000) IU per injection every 6 months until achievement of serum testosterone levels ≥ 12 nmol/l, but not above a maximum of 3 × 2500 IU hCG s.c./week. In all patients, rFSH (follitropin alpha) 150 IU was additionally injected thrice weekly (on Mondays, Wednesdays and Fridays) after 3 months of hCG, without subsequent dose modifications, aiming at physiologic serum FSH target levels between 1 and 7 U/l.

Baseline, Follow-up and Outcome Measurements

All boys were examined at baseline and during 3-month follow-up visits while undergoing gonadotropin substitution; annual bone age estimations were performed in group A.

Individual clinical details at baseline (including underlying causes of HH, and presence or absence of previous cryptorchidism) and outcome data (primary study end-points) are described in Table 1. Testicular volumes were measured using a Prader orchidometer; summated left plus right testicular volumes (BTVs) were calculated. To confirm testicular growth and to rule out intratesticular pathologies, ultrasound investigations were additionally performed (using the formula: length x width x depth/2 for baseline and follow-up volumes and the ellipsoid method[17] for final volumes). AMH and inhibin B levels were assessed at baseline, at initiation of rFSH replacement and on final assessment. LH levels were measured to rule out spontaneous activation of the hypothalamo–pituitary–gonadal (HPG) axis on gonadotropin substitution; serum testosterone levels were measured every 3 months to monitor Leydig cell response and, along with FSH levels, adherence to treatment. Once psycho-sexual maturity was attained, ejaculates were collected (by masturbation) after at least 48 h of sexual abstinence and thereafter repeated every 3 months until plateauing of sperm concentrations in the ejaculate was documented in two follow-up visits. All samples were analysed for volume, sperm concentrations, progressive motility and morphology;[13] total sperm counts were calculated. By the end of gonadotropin substitution, all patients able to provide a semen sample had a final centralized assessment by one experienced physician (JR), comprising primary and secondary end-points and final height.

Laboratory Methods

Inhibin B and AMH levels were analysed in frozen blood serum samples in the central study centre:

Inhibin B (solid phase sandwich assays, Beckman-Coulter; intra-assay coefficient of variation (CV): 3·3%; (high control: 4·9%); detection limit (DL): 10 pg/ml; cross-reactivity with inhibin A: 1%); AMH (ELISA, DRG Instruments GmbH; CV: 5·7% (high control: 9·0%); DL: 0·14 pg/ml).

The other standard hormone investigations (LH, FSH, testosterone) were performed by the participating centres during hCG/rFSH substitution and again in the study centre at final assessment.

QoL Assessment

All patients were asked to fill in four questionnaires at baseline and by the end of gonadotropin replacement. The ILK questionnaire included 9 rating items on the adolescent's perception of his situation in life, involving school, family, personal interests and leisure activities, physical fitness, mental fitness, disease burden and burden by therapeutic interventions. Evaluation was performed by calculation of health-related QoL scores (normal: 70–100%) and problem scores (on severity scale from 1–7). The DIKJ questionnaire included 26 items on the adolescent's emotional and somatic state, including negative feelings and consequences of depressive mood. Evaluation was performed by calculation of t-scores (with significant depression defined as a score >60%). The FEEL-KJ questionnaire included 24 items assessing adaptive and maladaptive strategies for emotional regulation. Two additional questions evaluated 'satisfaction with testis size' and 'satisfaction with masculinity' on a scale from −2 to +2.

Statistics

Analysis and drafting of figures were performed using Graph Pad Prism 5·0 (GraphPad Software Inc. La Jolla, USA). All results are expressed as the mean ± SD and additional median (range) for QoL data. Where normality of distribution was determined, t-tests for independent samples were conducted; otherwise, the Mann–Whitney U-test was used. Dependence between two variables was assessed using Spearman's rank correlation coefficient. Significance was defined as P-value <0·05.

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