To Örebro University

Puberty is initiated by a series of complex mechanisms determining the age at pubertal onset. The aim of this thesis was to increase our knowledge of the role of ghrelin and kisspeptin in puberty, to describe the clinical management of delayed puberty and to study the long-term socioeconomic consequences of delayed male puberty.

Study I and II were both based on a population of 13 girls with suspected central precocious puberty, who underwent a modified gonadotropin-releasing hormone stimulation test twice in a randomized controlled setting. Neither ghrelin nor kisspeptin plasma levels differed between the two tests up to 150 minutes. In Study I, different methods for the preservation of acylated ghrelin were studied as well, and the addition of the protease inhibitor AEBSF to precooled bloodsampling tubes, and cooled centrifugation within 30 minutes were found to result in the highest levels of acylated ghrelin.

Study III was an observational study based on a review of the medical records of 91 boys with delayed puberty in central Sweden showing that puberty nomograms are useful diagnostic instruments, and that underlying pathology is rare but psychosocial distress is common.

Study IV was a longitudinal, retrospective national cohort study, which included 1,250 men previously diagnosed with delayed puberty and 12,500 unexposed men. There was a lower likelihood of marriage or cohabitation, but no negative effects on educational achievements or labour market outcomes through early adulthood among those having had delayed puberty.

Increased knowledge of the physiology and long-term consequences alterations in pubertal timing may improve the management of pubertal

OBJECTIVES: Kisspeptin plays a major role in the onset of puberty by stimulating the gonadotropin-releasing hormone (GnRH) neurons. The aim of this study was to investigate whether GnRH inhibits kisspeptin secretion via a negative feedback mechanism and potential associations between kisspeptin levels and other hormones of importance for pubertal onset.

METHODS: Thirteen girls with suspected central precocious puberty underwent a GnRH stimulation test twice in a randomized, placebo-controlled manner. Blood was sampled up to 150 min after an IV injection of either Relefact LHRH® or saline. The levels of kisspeptin, acylated ghrelin, ultrasensitive oestradiol, luteinizing hormone (LH), follicle-stimulating hormone (FSH), insulin and glucose were analysed.

RESULTS: Baseline kisspeptin levels ranged from 9.9 to 201.6 pg/mL. Neither area under the curve for kisspeptin levels nor peaks were significantly lower after the GnRH injection compared to placebo. Baseline kisspeptin and glucose levels tended to be associated (rho=0.55, p=0.051) but no other associations were found between kisspeptin and other hormones.

CONCLUSIONS: Basal levels of kisspeptin vary widely in young girls. We found no evidence of a negative feedback mechanism of GnRH on kisspeptin in this small pilot study. The suggested association between kisspeptin and glucose levels needs further investigations.

Introduction: Delayed puberty in boys, defined as lack of pubertal onset by the age of 14 years, is often constitutional and self-limited. However, it can lead to feelings of sadness and anxiety. The long-term health consequences of delayed puberty in males are not well understood.

Objective: To study the long-term morbidity in young men exposed to delayed puberty.

Methods: All Swedish men born between 1991 and 1993 who were diagnosed with delayed puberty at the ages of 14.0–17.9 years were identified in nation-wide registries. For each index person, 10 control individuals were randomly selected, matched for sex, year of birth, and county of residence. Swedish nation-wide registries were also used to determine outcomes, including inpatient and outpatient care, prescription of medications, and mortality. The outcomes were tracked annually from the age of 18 until the end of year 2022 (approximately 30 years of age).

Results: 1,245 men with delayed puberty and 12,450 control individuals were identified and included in the study. During the follow-up period, 32% of those with delayed puberty had at least one inpatient care occasion compared to 27% of those without delayed puberty (p < 0.001). Among those with at least one inpa-tient care occasion, men with delayed puberty had more inpatient stays compared to control individuals (median (25–75th percentile) 1 (1–3) vs. 1 (1–2), p = 0.016). A higher proportion of men with delayed puberty had a hospital-based outpatient visit compared to control individuals (90% vs. 86%, p < 0.001). The number of outpatient visits was higher in men with delayed puberty (6 (2–14) vs. 4 (1–10), p < 0.001). In addition, prescriptions of medications were provided more often to men with delayed puberty (16 (6–45) vs. 10 (4–25), p < 0.001). Ten men with delayed puberty and 95 control individuals died during the follow-up (0.80% vs. 0.76%, p = 0.879). Most deaths were due to injuries or intoxications. Among deceased men, those with delayed puberty were older when they died (27.5 years (26–29) vs. 24 years (21–26), p = 0.031).

Conclusion: Delayed puberty in boys is associated with a higher frequency of inpatient stays, outpatient visits, and prescription of medications in young adulthood. This indicates that male delayed puberty is not harmless, and careful follow-up of these patients is needed. Further investigations of the increased need of health care are warranted.

Introduction: Ghrelin concentrations decline during puberty by an unclear mechanism. Acylated ghrelin (AG) is unstable in sampling tubes, but no standardized sampling protocol exists. We hypothesized that ghrelin levels decrease as a consequence of increased gonadotropin-releasing hormone (GnRH) signalling and that the addition of a protease inhibitor to sampling tubes preserves the AG levels.

Methods: In this randomized, placebo-controlled, cross-over study, 13 girls with suspected central precocious puberty were included. They performed an adjusted GnRH stimulation test twice and were given Relefact LHRH (R)(100 mu g/m(2)) or saline in a randomized order. Blood was sampled repeatedly for 150 min for the analysis of hormone concentrations. Oestradiol levels were only measured at baseline. The protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) was added to the sampling tubes. Specific ELISA kits were used for the analysis of AG and desacylated ghrelin (DAG) levels.

Results: Neither AG nor DAG levels changed after GnRH analogue injection in comparison to saline. The addition of AEBSF preserved AG levels (650.1 +/- 257.1 vs. 247.6 +/- 123.4 pg/mL, p < 0.001) and decreased DAG levels (51.9 [12.5-115.7] vs. 143.5 [71.4-285.7] pg/mL, p < 0.001). Both AG and DAG levels were inversely associated with insulin levels (r = -0.73, p = 0.005, and r = -0.78, p = 0.002, respectively). AG levels were inversely associated with oestradiol levels (rho = -0.57, p = 0.041).

Conclusion: Ghrelin levels do not decrease following a pharmacological dose of a GnRH analogue in the short term in girls. Addition of a protease inhibitor to the sampling tubes decreases AG degradation, resulting in preserved AG and decreased DAG levels. (C) 2022 The Author(s). Published by S. Karger AG, Basel

OBJECTIVES: To compare the usefulness of the classical definition of delayed puberty (DP) in boys with puberty nomograms and to describe the management of DP in boys in a hospital-based setting.

STUDY DESIGN: Observational retrospective multicentre study with a short-term follow-up.

SETTING AND PARTICIPANTS: Boys diagnosed with DP during 2013-2015 at paediatric departments in four counties in central Sweden. The medical records of 165 boys were reviewed.

PRIMARY AND SECONDARY OUTCOME MEASURES: Number of boys with DP after re-evaluation of the diagnosis according to the classical definition in comparison with puberty nomograms. Description of investigations performed and treatment provided to boys with DP.

RESULTS: In total, 45 and 58 boys were found to have DP according to the classical definition and the nomograms, respectively. Biochemical and/or radiological testing was performed in 91% of the 58 boys, but an underlying disease was only found in 9% of them. Approximately 79% of the boys received testosterone treatment, either as injections of testosterone enanthate or as testosterone undecanoate.

CONCLUSIONS: Puberty nomograms may be helpful instruments when diagnosing pubertal disorders in boys as they are not limited to an age close to 14 years and also identify boys with pubertal arrest. The majority of boys with DP undergo biochemical or radiological examinations, but underlying diseases are unusual emphasising the need for structural clinical practice guidelines for this patient group.

Introduction: Kisspeptin stimulates the gonadotropin releasing hormone (GnRH) neurons in hypothalamus initiating puberty. However, it is not known whether GnRH inhibits kisspeptin secretion by negative feedback and whether there are any associations between circulating levels of kisspeptin and other hormones, like ghrelin, important for the onset of puberty.

Methods: Thirteen girls with suspected central precocious puberty performed an adjusted GnRH stimulation test twice, placebo-controlled in a randomized order, at Örebro or Uppsala University Hospital, Sweden. Blood was sampled 0, 30, 60, 90, 120 and 150 min after the iv injection of either Relefact LHRH® or saline. The protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) was added to the sampling tubes to a final concentration of 2 mg/ml. An ELISA kit from LifeSpan BioSciences, Inc. (No LS-F8231) was used for the analyses of Kisspeptin-1 levels. The levels of acylated ghrelin were analyzed with Millipore® Human Ghrelin (Active) ELISA kit (#EZGRA-88K). Serum ultrasensitive estradiol, luteinizing hormone (LH), follicle-stimulating hormone (FSH), insulin and glucose levels were analyzed using the usual clinical methods.

Results: The median Kisspeptin-1 level at baseline was 39 pg/ml (min–max: 0.1–221.3 pg/ml). The area-under-the curve for Kisspeptin-1 levels was not significantly lower after the GnRH injection as compared to the placebo injection. We did not find any significant correlations between the levels of kisspeptin-1 and acylated ghrelin, estradiol, LH, FSH, or insulin. However, we could see a positive correlation between kisspeptin-1 and glucose levels at baseline (Spearman’s rank test, rho = 0.63, p=0.021).

Discussion: We did not find evidence of a negative feedback mechanism between GnRH and kisspeptin in girls with suspected central precocious puberty since the circulating levels of kisspeptin-1 were unaffected by an intravenous injection of a GnRH analogue. However, paracrine actions in the hypothalamus cannot be ruled out by this study. The positive correlation found between kisspeptin-1 and glucose levels is in accordance with previous findings in both adults and children, suggesting a possible role for kisspeptin signaling in glucose metabolism.

Introduction: Ghrelin is a growth hormone-releasing acylated peptide stimulating the appetite, mainly produced in the stomach, and with an important role in pubertal development (1). Two ghrelin forms have been described, acylated (AG) and desacylated (DAG), but it is debated whether DAG is an active hormone or a degradation product of AG (2). Our aim was to evaluate the effects of adding the protease inhibitor 4-(2-aminoethyl) benzenesufonyl fluoride hydrochloride (AEBSF) to sampling tubes and acidification of plasma on levels of AG and DAG in girls with suspected central precocious puberty (CPP).

Methods: 13 girls aged 6.6 to 10.1 years with suspected CPP undergoing a gonadotropin-releasing hormone stimulation test during 2015-2017 at the Departments of Paediatrics, at Örebro or Uppsala University Hospital were included. Blood samples were collected at 0 min in precooled EDTA tubes with or without AEBSF at a final concentration of 2mg/ml. After cold centrifugation, HCl at a final concentration of 50 μmol/l, was added to 50% of the plasma tubes containing AEBSF. The AG and DAG concentrations were measured by ELISA kits. Comparison was performed using one-way ANOVA for repeated measurements.

Results: The mean plasma AG levels were significantly higher after the addition of AEBSF only (650.9 +/- 257.1 pg/ml) or AEBSF+HCl (681.2 +/- 299 pg/ml) compared to the concentrations without additives (247.6 +/- 123.4 pg/ml, p<0.01 for both comparisons). There was no significant difference between the AG levels after AEBSF and AEBSF+HCl addition. The plasma levels of DAG were significantly lower after the addition of AEBSF+HCl (69.3 +/- 30.6 pg/ml) and even further lowered after the addition of AEBSF only (56.3+/- 30.9 pg/ml) compared to the concentrations of DAG in tubes without any additives (149.9 +/- 73.7 pg/ml, p < 0.01 for both comparisons).

Discussion: Due to the unstable nature of AG, special procedures are required for accurate measurement of its plasma levels in children, including the use of a protease inhibitor like AEBSF. However, DAG was still measurable indicating that it may not only be a degradation product of AG. 1. Kojima M, Kangawa K. Ghrelin: structure and function. Physiol Rev. 2005;85(2):495-522.2. Blatnik M, Soderstrom CI, Dysinger M, Fraser SA. Prandial ghrelin attenuation provides evidence that des-acyl ghrelin may be an artifact of sample handling in human plasma. Bio-analysis. 2012;4(20):2447-55.

BACKGROUND: We found an increase in the incidence rate (IR) of childhood thyrotoxicosis (CT) during the 1990s in central Sweden. The optimal treatment method for CT is a subject that is still debated upon.

OBJECTIVES: To investigate whether the increase in IR of CT in Sweden persists and to study the treatment outcome.

METHOD: Children <16 years of age diagnosed with CT during 2000-2009 and living in 1 of 5 counties in central Sweden were identified retrospectively using hospital registers. Data on clinical and biochemical characteristics and outcomes of treatment were collected from medical records. The corresponding data from 1990 to 1999 were pooled with the new data.

RESULTS: In total, 113 children were diagnosed with CT during 1990-2009 in the study area. The overall IR was 2.2/100,000 person-years (95% CI 1.2-2.5/100,000 person-years). The IR was significantly higher during 2000-2009 than during 1990-1999 (2.8/100,000 [2.2-3.6] vs. 1.6/100,000 person-years [1.2-2.2], p = 0.006). The increase was significant for both sexes. Seventy percent of the patients who completed the planned initial treatment with antithyroid drugs (ATDs) and were not lost to follow-up relapsed within 3 years. Boys tended to relapse earlier than girls (6.0 months after drug withdrawal [95% CI 1.9-10.0] vs. 12.0 months [95% CI 6.8-17.3], p = 0.074).

CONCLUSIONS: The IR of CT is increasing in both girls and boys. Relapse rate after withdrawal of ATD treatment is 70%. Boys tend to relapse earlier than girls, and this needs to be further investigated.

Aim: To study the incidence of childhood thyrotoxicosis in five counties in central Sweden during 1990–2009 and to study the treatment outcome.

Methods: Children below the age of 16 years diagnosed with thyrotoxicosis during the 20-years period and living in the study area were identified retrospectively. Data on the total number of children below 16 years of age living in the area during the study period was collected from the National Board of Statistics, Sweden. Data regarding clinical and biochemical characteristics and the outcome of the treatment were collected from medical records.

Results: 113 patients were identified. The annual incidence was 2.2/100,000 children during the whole study period. The incidence was higher during the last ten studied years as compared to the first ten studied years (2.8 vs. 1.6/100,000, p = 0.006). The increase in incidence was seen in both girls and boys (p = 0.041 and p = 0.038, respectively). Treatment with antithyroid drugs (ATD) was the first hand choice, but 69% of the patients relapsed within three years after the planned discontinuation of the ATD treatment. Boys relapsed more often than girls (p = 0.013), but we could not identify any other significant predictor for relapse.

Conclusion: Thyrotoxicosis is uncommon in pediatric patients but the incidence seems to be increasing. The outcome of the initial treatment with ATD is poor with high relapse rates. Boys seems to have an increased risk for relapse compared to girls. More studies are needed to identify an optimal treatment protocol for each individual.