Congenital hypogonadotropic hypogonadism, functional hypogonadotropism or constitutional delay of growth and puberty? An analysis of a large patient series from a single tertiary center

STUDY QUESTION

What diagnoses underlie delayed puberty (DP) and predict its outcome?

SUMMARY ANSWER

A multitude of different diagnoses underlie DP, and in boys a history of cryptorchidism, small testicular size and slow growth velocity (GV) predict its clinical course.

WHAT IS KNOWN ALREADY

DP is caused by a variety of underlying etiologies. Hormonal markers can be used in the differential diagnosis of DP but none of them have shown complete diagnostic accuracy.

STUDY DESIGN, SIZE, DURATION

Medical records of 589 patients evaluated for DP in a single tertiary care center between 2004 and 2014 were retrospectively reviewed.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Clinical and biochemical data of 174 boys and 70 girls who fulfilled the criteria of DP were included in the analyses. We characterized the frequencies of underlying conditions and evaluated the predictive efficacy of selected clinical and hormonal markers.

MAIN RESULTS AND THE ROLE OF CHANCE

Thirty etiologies that underlie DP were identified. No markers of clinical value could be identified in the girls, whereas a history of cryptorchidism in the boys was associated with an increase in the risk of permanent hypogonadism (odds ratio 17.2 (95% CI; 3.4-85.4, P < 0.001)). The conditions that cause functional hypogonadotropic hypogonadism were more frequent in boys with a GV below 3 cm/yr than in those growing faster (19% vs 4%, P < 0.05). In this series, the most effective markers to discriminate the prepubertal boys with constitutional delay of growth and puberty (CDGP) from those with congenital hypogonadotropic hypogonadism (CHH) were testicular volume (cut-off 1.1 ml with a sensitivity of 100% and a specificity of 91%), GnRH-induced maximal LH (cut-off 4.3 IU/L; 100%, 75%) and basal inhibin B (INHB) level (cut-off 61 ng/L; 90%, 83%).

LIMITATIONS, REASONS FOR CAUTION

The main limitation of the study is the retrospective design.

WIDER IMPLICATIONS OF THE FINDINGS

Prior cryptorchidism and slow GV are two important clinical cues that may help clinicians to predict the clinical course of DP in boys, whereas markers of similar value could not be identified in girls. In prepubertal boys, testicular size appeared as effective as INHB and GnRH-induced LH levels in the differential diagnosis between CHH and CDGP.

STUDY FUNDING/COMPETING INTEREST(S)

This study was supported by the Academy of Finland (268356), Foundation for Pediatric Research (7495), Sigrid Juselius Foundation (2613) and the Finnish Medical Foundation (011115). The authors have no competing interests to report.

TRIAL REGISTRATION NUMBER

Not applicable.

delayed puberty, hypogonadism, testicular volume, cryptorchidism, INHB

Introduction

Delayed puberty (DP), defined as the absence of clinical signs of puberty at the age of 2–2.5 years SD above the mean of the general population (Marshall and Tanner, 1969; Marshall and Tanner, 1970), most frequently results from constitutional delay of growth and puberty (CDGP), which is a late variant of the normal spectrum in pubertal timing (Sedlmeyer and Palmert, 2002). CDGP is diagnosed after the exclusion of pathological causes of DP, which can be categorized into permanent hypogonadotropic hypogonadism (PHH), functional hypogonadotropic hypogonadism (FHH) and hypergonadotropic hypogonadism (Hyper H) (Sedlmeyer and Palmert, 2002). Three of the four categories appear to show a gender-dependent variation: CDGP and FHH are male-predominant, whereas Hyper H appears to be more frequent in females (Toublanc et al., 1991; Sedlmeyer and Palmert, 2002). Despite its relative frequency, remarkably few studies have investigated the underlying causes and the clinical characteristics of DP (Reindollar and McDonough, 1981; Toublanc et al., 1991; Sedlmeyer and Palmert, 2002; Lawaetz et al., 2015). Moreover, the interpretation of the findings in these studies is complicated by their focus on a single gender or the risk of tertiary center referral bias, which may have increased the frequency of pathological causes of DP. The most comprehensive series of DP was published over 10 years ago (Sedlmeyer and Palmert, 2002).

Clinicians who evaluate patients with DP would greatly benefit from evidence-based clinical and biochemical markers that could predict the clinical course of DP. Moreover, such markers could aid in the differentiation between CDGP and the pathological forms of DP, particularly between CDGP and congenital hypogonadotropic hypogonadism (CHH) (Palmert and Dunkel, 2012). In fact, despite some recent promising advances (Wei and Crowne, 2015), there is not a single clinical feature or a simple biochemical test that would reliably discriminate CDGP from (normosmic) CHH (Dunkel et al., 1985; Segal et al., 2009; Adan et al., 2010; Coutant et al., 2010; Grinspon et al., 2010; Palmert and Dunkel, 2012).

We investigated the underlying causes of DP in a large series of boys and girls evaluated in a single tertiary care center, which serves as a direct referral center for the primary care. In addition, we evaluated which clinical and biochemical markers were the most effective ones to predict the course of DP and to discriminate between the four DP categories and their subclasses (Sequera et al., 2002; Coutant et al., 2010; Grinspon et al., 2010; Palmert and Dunkel, 2012).

Materials and Methods

We performed an International Classification of Diseases (ICD)-10 code-based inquiry (ICD-10 codes: E28, E28.3, E28.8, E28.9, E29, E29.0, E29.1, E29.8, E29.9, E30.00, E30.09, E30.8 and E30.9) to the electronic patient information system and identified 408 boys and 181 girls who were evaluated for DP and visited the Pediatric Endocrine Outpatient Clinic of Helsinki University Hospital between 2004 and 2014 (Supplementary Fig. SI). The eligibility criteria for DP were defined in boys as pubertal stage G1 and/or testicular volume (TV) <3 ml at the age of 14.0 years (Ojajärvi, 1982), or G2 at the age of 15.0 years (Ojajärvi, 1982). In girls, the inclusion criteria were pubertal stage B1 at the age of 13.0 years or B2 at the age of 14.3 years (Ojajärvi, 1982). A total of 244 (174 boys and 70 girls) patients met the inclusion criteria (Supplementary Fig. SI).

Blood samples for hormone measurements were routinely obtained before 10 am and assayed by the clinical laboratory of the Helsinki University Central Hospital according to their procedures. Tanner stage was documented in all patients. The first-line investigations of DP, which included bone age (determined by pediatric endocrinologist, radiologist or BoneXpert (Thodberg et al., 2009)), basal LH and FSH levels, and sex steroids were available in 227 (93%), 216 (88%), 218 (89%) and 217 (89%) patients, respectively. The family history of pubertal timing of each patient was reviewed by a pediatric endocrinologist and was available for all patients. It was considered positive if the mother or a sister of the patient was reported to have menarche after the age of 14 years or the father or a brother delayed the timing of puberty or the adolescent growth spurt (Supplementary Table SI). A GnRH stimulation test was performed in 104 (43%) patients, and inhibin B (INHB) levels were available in 126 (72%) boys. A growth hormone (GH) stimulation test was performed in eight boys and seven girls, and only two of them (panhypopituitarism and juvenile rheumatoid arthritis) had a result consistent with GH deficiency. The length and the width of the testes, measured with a ruler, were documented in 120 (69%) boys. TV was calculated by using the formula: length × width² × 0.52 (Behre et al., 1989). Annual growth velocity (GV), available for 230 (94%) patients, was determined from the height measurements carried out by nurses trained in auxology and was taken at least 6 months apart. The latest measurements were obtained before the initial evaluation in the tertiary clinic. We used an age- and sex-adjusted BMI (ISO-BMI) to describe the weight progression (Saari et al., 2011).

The classification of patients with DP into the four main groups (CDGP, PHH, FHH, Hyper H) is presented in Supplementary Fig. SI. We considered the underlying cause to be occult if at the time of initial evaluation there were no clinical cues (suspicious symptoms or clinical findings, ISO-BMI <16 kg/m², exceptionally slow GV (<2 cm/yr), or history of cryptorchidism) that should have raised a suspicion of a chronic disease or permanent hypogonadism. The diagnosis of CDGP (in 142 boys and in 39 girls) was based on the clinical, biochemical and imaging data that ruled out pathological underlying causes for DP. Of note, three CDGP patients had a history of unilateral cryptorchidism. The progression of spontaneous puberty was confirmed in the hospital outpatient clinic in 118 (83%) boys and in 36 (89%) girls with CDGP. The remaining cases with CDGP were referred to the school healthcare for the follow-up, and were not re-referred back to the tertiary center for further evaluation of puberty.

The patients were classified into the PHH group if they had a confirmed pathology in the central nervous system or an organic cause for their condition, and/or low gonadotrophin and sex steroid levels, and no spontaneous progression of puberty during the follow-up until the age of 18 years. The most common subgroup of PHH is congenital isolated gonadotrophin deficiency (i.e. CHH) (Sedlmeyer and Palmert, 2002). Four boys with CHH reported a history of cryptorchidism (two unilateral and two bilateral) and one had anosmia, whereas none had a history of micropenis. Overall, 11 boys and 5 girls were diagnosed with CHH and 12 of them were screened for CHH genes (Laitinen et al., 2011, 2012; Hero et al., 2012, 2015; Varimo et al., 2016). Four boys and three girls had a genetically verified diagnosis of CHH. Two girls had an fibroblast growth factor receptor 1 (FGFR1) mutation (c.1305_1306dupAT p.(Ser436TyrfsTer3) and c.246_247delAG p.(Glu84GlyfsTer26)) (Laitinen et al., 2011), and one with CHARGE syndrome carried a mutation in chromodomain helicase DNA binding protein 7 (CHD7) (c.5273A > G p.(Asp1758Gly)), whereas the four boys had either an FGFR1 mutation ((c.1009 G > C p.(Gly337Arg) and c.1305_1306dupAT p.(Ser436TyrfsTer3)), a hemizygous anosmin 1 (ANOS1) (c.571 C > T p.(Arg191Ter)) mutation, or a homozygotic GnRH receptor (c.416 G > A p.(Arg139His)) mutation. In addition, one boy with PHH had a hypothalamic hamartoma.

The diagnosis of Hyper H was confirmed with gonadal failure and high basal LH and FSH levels. The patients were classified into the FHH category if they had an identified underlying disease that likely delayed pubertal maturation, such as celiac disease, a spontaneous progression of puberty during the follow-up, and no biochemical and clinical signs that suggested the presence of a permanent reproductive endocrine disorder (i.e. PHH or Hyper H) (Sedlmeyer and Palmert, 2002). Remarkably, the spontaneous progression of puberty was confirmed in 29 (97%) patients with FHH (14 girls and 15 boys) within 3 years after the initial evaluation. Furthermore, the patients diagnosed with anorexia nervosa and those with severe underweight (ISO-BMI <16 kg/m²) prior to presentation were considered to have FHH as a result of nutritional deficiency (Saari et al., 2011).

Estradiol levels were determined with radioimmunoassay (RIA) (DiaSorin S.p.A, 103040 Saluggia, Vercelli, Italy) until September 2009, with a different RIA (AutoDELFIA, Perkin-Elmer, Turku, Finland) between October 2009 and February 2014, and, thereafter, with a immunoluminometric assay (Immulite 2000 Xpi, Siemens Healthcare Diagnostics, NY, USA) or with a mass spectrometer. Similarly, testosterone was measured with RIA (Lipidex-5000) (Apter et al., 1976) until March 2005, when a mass spectrometer method was utilized (API 2000 tandem mass spectrometer, AB Sciex, Foster City, CA, USA). Gonadotropin levels were measured with immunofluorometric assay (AutoDELFIA, Wallac, Turku, Finland) until June 2011, and then changed to electrochemiluminescence immunoassay (Roche Diagnostics Gmbh, Penzberg, Germany). Before December 2010, INHB levels were determined with OBI INHB ELISA (MCA1312KZZ, OBI-DSL, Upper Heyford, UK), and, thereafter, with Beckman Coulter INHB Gen II ELISA (A81303) and INHB Gen II Calibrators and Controls (A81304, Beckman Coulter, Inc., CA, USA). However, the newest INHB assay showed only 7% lower INHB levels than the OBI ELISA.

Informed consent and ethical approval

Since the study is entirely based on health record data, no ethical permission was required according to the Finnish Medical Research Act. The Helsinki University Central Hospital approved the study.

Statistical analysis

The data are presented with a mean (SD) unless otherwise stated. We used SPSS statistical software, release 22.0 (SPSS, Chicago, IL, USA), for statistical analyses. Since the data contained small subgroups, the Mann–Whitney U-test was used for the comparison of two groups, and with more than two groups we used the Kruskal–Wallis N-test. Categorical variables were analyzed with the Fisher's exact test, and odds ratios (OR) with 95% CI were calculated. We constructed receiver operating characteristic (ROC) curves and calculated the AUC with 95% CI to evaluate the diagnostic performance of different hormonal and clinical markers of puberty. Based on the ROC curves, cut-off values that maximized sensitivity and specificity were determined. As INHB levels show an individual sample variation particularly at low levels (Kalra et al., 2010), we fitted the quality control measures of INHB, obtained between 2004 and 2014, to a linear model that predicted the dispersion for each mean. Consequently, INHB levels were divided into three categories by using mean ± 2 SE of the quality control measurements (10–49, 49–111 and 111–212 ng/l). Finally, a logistic regression model was used to predict the disease risk for combinations of testis size and INHB levels. All tests were two-sided and the statistically significant level was set to P < 0.05.

Results

Underlying causes of DP

Overall, 30 different underlying causes of DP were found (Table I). As expected, the single most common cause was CDGP in both sexes (n = 181), and it was more frequent in the boys than in the girls (82% vs 56%, P < 0.001, respectively). A similar proportion of the boys and the girls with CDGP had a first-degree relative with DP (63% vs 69%, respectively) (Supplementary Table SI). In addition, the patients with CDGP had a positive family history of DP more frequently than the subjects in the FHH, PHH and Hyper H groups (P < 0.05) (Supplementary Table SI). The second most frequent cause in both sexes was FHH, but a sex-dependent difference was evident in the FHH and Hyper H categories, as they both affected the girls more frequently than the boys (20% vs 9%, P < 0.05; 16% vs 2%, P < 0.001, respectively). Importantly, six girls with Hyper H were diagnosed with an idiopathic ovarian dysfunction. They had tested negative for the common Finnish FSH receptor founder mutation (c.566 C > T p.(Ala189Val)) (Aittomaki et al., 1995), and were devoid of circulating ovarian antibodies, had normal karyotype, and had either normal pelvic ultrasound or MRI. Besides, the girls were not related to each other. At the initial evaluation, clinical cues, which could have been interpreted as a sign of a chronic disease or permanent hypogonadism, were present in 90% (95% CI: 74–97%) of patients with FHH, in 7% (1–31%) of patients with Hyper H, and in 37% (19–59%) of patients with PHH.

Table I

The causes of DP categorized into the four main diagnostic groups.

	Male, n (%)	Female n (%)
CDGP	142 (82)	39 (56)
PHH	13 (7)	6 (9)
CHH	11	5
Hypopituitarism	1
Craniopharyngioma		1
Prader-Willi syndrome	1
FHH	16 (9)	14 (20)
Severe underweight	4	3
Anorexia	1	2
Severe developmental delay	1	2
Crohn's disease	2
Clinical CHARGE syndrome without CHD7 mutation	1	1
VATER association	1
Cornelia de Lange syndrome		1
Cartillage-hair hypoplasia		1
Celiac disease		1
Intense exercise		1
Jacobsen syndrome and hypothyroidism		1
Hydrocephalus	1
Pulmonary fibrosis	1
Juvenile idiopathic arthritis	1
Systemic lupus erythematosus		1
Radiation therapy for malignancy	1
Central nervous system disorders (Small Rathke´s cleft cyst or empty cella)	2
Hyper H	3 (2)	11 (16)
Anorchia	1
Idiopathic testicular failure	1
Idiopathic ovarian failure		6
Chemotherapy for malignancy (Askin tumor)		1
Turner syndrome		3
FSH receptor gene mutation		1
Sex-determining region Y (SRY)-positive XX male	1

CDGP, constitutional delay of growth and puberty; PHH, permanent hypogonadotropic hypogonadism; FHH, functional hypogonadotropic hypogonadism; Hyper H, hypergonadotropic hypogonadism; CHH, congenital hypogonadotropic hypogonadism; CHD7, chromodomain helicase DNA binding protein 7; DP, delayed puberty.

Predictive markers of diagnoses underlying DP

A history of prior cryptorchidism was more frequent in those with Hyper H or PHH than in those with FHH or CDGP (P < 0.05) (Supplementary Table SI) and in those with CHH than in those with CDGP (36% vs 2%, respectively, P < 0.05). The history of prior cryptorchidism was associated with an increased risk of permanent hypogonadism (OR 17.2, 95% CI: 3.4–85.4, P < 0.001). In contrast, a boy with normally descended testes and a positive family history of DP had very low probability of permanent hypogonadism (OR 0.02, 95% CI: 0.002–0.2, P < 0.001).

Subsequently, different clinical and hormonal parameters of puberty were evaluated by using the ROC analysis. Based on the AUCs, TV (cut-off level of 1.1 ml, sensitivity 100%, specificity 91%), GnRH-induced LH (4.3 IU/l, 100%, 75%, respectively) and baseline INHB level (61 ng/L, 90%, 83%, respectively) appeared as the most effective markers to discriminate the prepubertal boys with CHH from those with CDGP (Fig. 1A). After we combined INHB or GnRH-induced LH levels with TV, a slightly more accurate level of discrimination was reached (Fig. 1B). Then we tested the efficacy of an earlier reported cut-off level of INHB (<35 ng/l) as it has been suggested to completely differentiate between the prepubertal boys with CDGP and CHH (Coutant et al., 2010). The OR of INHB below 35 ng/l to detect CHH in the prepubertal boys with DP was 10.0 (95% CI: 2.16–46.3, P < 0.01), but four prepubertal CHH patients (40%) had INHB levels above, and four CDGP patients (7%) below, 35 ng/L.

Figure 1

The performance of clinical and hormonal parameters in the differential diagnosis between prepubertal boys with CDGP and CHH.

(A) ROC curves and AUCs with 95% CIs for TV, GnRH-induced LH and inhibin B (INHB). TV available in 65 (56 CDGP and 9 CHH), GnRH-induced LH in 47 (37 and 10) and INHB in 70 (60 and 10) patients. (B) ROCs for the combination of TV and INHB, which was available in 47 patients with CDGP and 7 with CHH patients, or TV and GnRH-induced LH (available in 28 and 7, respectively). CDGP, constitutional delay of growth and puberty; CHH, congenital hypogonadotropic hypogonadism; TV, testicular volume.

Rather than relying on a single cut-off value, we constructed a clinical decision-making tool that takes into account the performance of the INHB assay at different analyte concentrations and TV. We calculated risk estimates for CHH in prepubertal boys with CDGP or CHH presenting with DP. These analyses showed that the mean risk of CHH was the highest (0.9, range 0.5–1.0) in those boys with low INHB (10–49 ng/l) and small testes (<1 ml) (Table II). The risk estimates for other combinations of INHB and TVs are presented in Table II.

Table II

The risk estimates for CHH calculated from a set of prepubertal boys with either CHH or CDGP presenting with DP.

Inhibin B (ng/L)	TV
	<1.0 ml	1.1–2 ml
10–49	0.9 (0.5–1)	0.2 (0–0.8)	Probability of CHH. Mean (range)
49–111	0.6 (0.1–1)	0.1 (0–0.4)
111–212	0.2 (0–0.9)	0 (0–0.1)

The estimates are reported as combinations of three different inhibin B categories (which reflect the assay performance at different hormone concentrations) and two different categories of testicular volumes. CHH, congenital hypogonadotropic hypogonadism; CDGP, constitutional delay of growth and puberty; DP, delayed puberty; TV, testicular volume.

Growth velocity in the prediction of an underlying disease or a hypothalamic-pituitary lesion

At presentation, the mean height SD score did not differ significantly between the four diagnostic categories in either sex (P = NS) (Supplementary Table SI). However, the boys with FHH (n = 13) had a significantly lower mean GV (3.2 ± 1.3 cm/yr) than those with CDGP or CHH (n = 142) (4.1 ± 1.7 cm/yr, P < 0.05). As a recent diagnostic algorithm suggests that annual GV <3 cm would help to discriminate patients with a chronic disease (i.e. FHH) from those with CDGP or GnRH deficiency (Palmert and Dunkel, 2012), we divided the boys and the girls into two groups: those who grew slowly (<3 cm/yr) and those with the GV more than 3 cm/yr. In boys, the frequency of FHH was higher in those with GV <3 cm/yr than in those with GV >3 cm/yr (19% vs 4%, P < 0.05). In the ROC analysis, the previously suggested (Palmert and Dunkel, 2012) GV cut-off level of 3 cm/yr discriminated the boys with FHH from those with CDGP or CHH with a sensitivity of 50% and a specificity of 80%. The best GV cut-off level was 3.6 cm/yr with a sensitivity of 71% and a specificity of 64%. In girls, the distributions of the four underlying diagnostic subclasses did not differ between those growing slower or faster than 3 cm/yr (Fig. 2).

Figure 2

The frequencies of CDGP, PHH, FHH and Hyper H in boys (left panel) and in girls (right panel) with DP and with growth velocity (GV) less or more than 3 cm/yr (Palmert and Dunkel, 2012). *P < 0.05, FHH versus CDGP. CDGP, constitutional delay of growth and puberty; PHH, permanent hypogonadotropic hypogonadism; FHH, functional hypogonadotropic hypogonadism; Hyper H, hypergonadotropic hypogonadism; DP, delayed puberty.

The most frequent reason for the clinician to order an MRI was a suspicion of CHH (n = 31), followed by headache (n = 5) and other neurological symptoms (n = 3). We tested the effectiveness of GV to detect the patients with an abnormal brain MRI scan, performed in 39 patients (24 boys and 15 girls). One girl (craniopharyngioma) and five boys (two with Rathke's cleft cyst, one with empty cella and two with hypothalamic hamartoma) showed an abnormal finding in the MRI scan (i.e. 6/39). The girl with craniopharyngioma grew slowly (2 cm/yr), whereas the mean GV of the boys with an abnormal MRI scan (3.5 ± 0.9 cm/yr) did not differ significantly from those with a normal MRI scan (4.2 ± 2.4 cm/yr, n = 18, P = NS).

At the time of the initial evaluation, the boys with PHH showed significantly higher ISO-BMI values (25.6 ± 6.0 kg/m²) than those with CDGP (22.6 ± 5.5 kg/m², P < 0.05) or FHH (16.8 ± 2.8 kg/m², P < 0.05), whereas the ISO-BMI values of the girls did not differ between the categories (P = NS) (Supplementary Table SI).

Discussion

We described the underlying causes and the prognostic factors of DP in the largest patient series to date including both sexes and a thorough clinical and biochemical evaluation of DP. Furthermore, this is the first work that has evaluated the diagnostic value of GV in patients with DP. Our results showed that CDGP, the most common cause of DP in both sexes, was more frequent in boys, whereas the proportion of Hyper H was higher in girls. These results are in agreement with Sedlmeyer's series (Sedlmeyer and Palmert, 2002). However, the proportion of CDGP in both sexes was higher in our series than reported before (Reindollar and McDonough, 1981; Toublanc et al., 1991; Sedlmeyer and Palmert, 2002). This probably reflects the fact that our tertiary center serves as an exclusive public referral center for general practitioners who screen schoolchildren for pubertal and growth disorders in Helsinki. This, in our opinion, suggests that our case series reflects the general pediatric population well.

The boys who had DP and a history of cryptorchidism showed a clearly elevated risk for permanent hypogonadism, and GV emerged as another parameter that associated with the underlying cause of DP. We found out that FHH diagnoses were enriched among the boys with the GV <3 cm/yr, and, on average, the boys with FHH grew slower than the boys with CDGP or CHH. This likely reflects the influence of undernutrition and inflammatory conditions on the GH-insulin-like growth factor I axis (Misra et al., 2003; Wong et al., 2010). Although slow growth was associated with an underlying pathology in boys, as suggested before (Palmert and Dunkel, 2012), GV showed only moderate sensitivity and specificity in identifying patients with a pathological cause for DP. In addition, the previously suggested GV cut-off level did not show discriminatory efficacy in the girls with DP (Palmert and Dunkel, 2012). GV did not appear helpful in selecting patients for a brain MRI scan, as it did not differ between those with and those without hypothalamic-pituitary abnormalities. On the other hand, our series included a low number of patients with significant hypothalamic-pituitary lesions, such as craniopharyngioma that unequivocally compromise pituitary function. Patients with such lesions commonly present a reduced GV even years prior to the neurologic or the ophthalmic symptoms (Taylor et al., 2012), and therefore poor GV in a patient with DP should always lower the threshold for a brain MRI scan. Currently, there is no evidence-based algorithm, which would guide the use of second-line investigations of boys with DP, such as GnRH stimulation test and INHB that are targeted to distinguish patients with CHH from those with CDGP. Based on our findings, these studies may be reserved to those with a history of undescended testis, reproductive or non-reproductive features of CHH, exceptionally small testicular size, or a failure to show progression of puberty during follow-up.

The differential diagnosis between CDGP and CHH in a boy with DP and no non-reproductive features of CHH remains extremely challenging (Palmert and Dunkel, 2012). The effectiveness of Sertoli cell markers, and GnRH and hCG stimulation tests and their combination in the differential diagnostics of DP have been investigated before, but none of the tests have shown a complete diagnostic accuracy (Dunkel et al., 1985; Segal et al., 2009; Adan et al., 2010; Coutant et al., 2010; Grinspon et al., 2010). Interestingly, first morning-voided urine gonadotropins appear to detect the onset and progression of puberty with equal accuracy compared with the GnRH stimulation test, but, to our knowledge, it has not been evaluated specifically in the differential diagnosis of CDGP and CHH (Demir et al., 2016). In our series, testicular size emerged as a marker with similar discriminatory effectiveness to that of INHB or GnRH-induced LH level to identify the prepubertal boys with CHH from those with CDGP. Although ultrasound reaches a more accurate estimate of testis size than a clinical assessment of testis size (Diamond et al., 2000), its use in clinical practice is limited by costs and availability, especially outside tertiary centers. On the other hand, the study by Taskinen et al. (1996) showed that assessment of testicular size with ultrasound, orchidometer or ruler all gave comparable results and none of the methods were superior to any other (Taskinen et al., 1996). Surprisingly, the INHB cut-off level of 35 ng/l, proposed to differentiate the boys with CHH from those with CDGP (Coutant et al., 2010), performed only moderately in our series.

This study has limitations that mainly stem from the retrospective design. Our series included only a limited number of patients with CHH and PHH and subjects with a history of cryptorchidism, which may have influenced the results. While our findings ideally should be confirmed in a prospective research setting, conducting a prospective study of this extent is practically impossible, since approximately only one patient with CHH is born in Finland every year (Laitinen et al., 2011). In the child welfare clinics and school healthcare, all Finnish children are examined frequently before the age of 14 years by a nurse or a primary care physician to identify early subjects with symptoms or growth disturbance that suggest the presence of a chronic disease. This may have resulted in a lower frequency of FHH in our series, and caution is required when interpreting our results in the absence of such a screening system. In addition, testicular measurements were not standardized since several pediatric endocrinologists measured the testis sizes over the years. Despite this, TV still emerged as a marker with similar effectiveness to INHB and GnRH-induced LH to discriminate between CDGP and CHH.

Conclusion

A variety of causes underlie DP, which makes it a diagnostic challenge. In boys, however, physical examination and a careful review of medical and family history appear to aid in the process. In particular, the history of cryptorchidism serves as a risk factor for permanent hypogonadism, and TV appeared to be as effective as INHB and GnRH-induced LH levels to differentiate between CHH and CDGP in prepubertal boys. Furthermore, our results suggest that the GV has value in the differential diagnosis of DP in boys, but less in girls.

Supplementary data

Supplementary data are available at http://humrep.oxfordjournals.org/.

Acknowledgements

We thank M.Sc. Mitja Lääperi for statistical support and M.A. Annika Tarkkanen for linguistic guidance.

Authors’ roles

All authors provided a substantial contribution to drafting of the manuscript. M.H. and T.R. designed the study protocol. T.V. collected and analyzed the data and wrote first version of the manuscript. M.H., T.R., P.J.M. and J.K. redrafted the manuscript. All authors approved the final version of the manuscript.

Funding

The Academy of Finland (268356), Foundation for Pediatric Research (7495), Sigrid Juselius Foundation (2613) and the Finnish Medical Foundation (011115).

Conflict of interest

None declared.