Spermatogonial quantity in human prepubertal testicular tissue collected for fertility preservation prior to potentially sterilizing therapy.

STUDY QUESTION: Does chemotherapy exposure (with or without alkylating agents) or primary diagnosis affect spermatogonial quantity in human prepubertal testicular tissue? SUMMARY ANSWER: Spermatogonial quantity is significantly reduced in testes of prepubertal boys treated with alkylating agent therapies or with hydroxyurea for sickle cell disease. WHAT IS KNOWN ALREADY: Cryopreservation of spermatogonial stem cells, followed by transplantation into the testis after treatment, is a proposed clinical option for fertility restoration in children. The key clinical consideration behind this approach is a sufficient quantity of healthy cryopreserved spermatogonia. However, since most boys with malignancies start therapy with agents that are not potentially sterilizing, they will have already received some chemotherapy before testicular tissue cryopreservation is considered. STUDY DESIGN, SIZE, DURATION: We examined histological sections of prepubertal testicular tissue to elucidate whether chemotherapy exposure or primary diagnosis affects spermatogonial quantity. Quantity of spermatogonia per transverse tubular cross-section (S/T) was assessed in relation to treatment characteristics and normative reference values in histological sections of paraffin embedded testicular tissue samples collected from 32 consecutive boy patients (aged 6.3 ± 3.8 [mean ± SD] years) between 2014 and 2017, as part of the NORDFERTIL study, and in 14 control samples (from boys aged 5.6 ± 5.0 [mean ± SD] years) from an internal biobank. PARTICIPANTS/MATERIALS, SETTING, METHODS: Prepubertal boys in Sweden, Finland and Iceland who were facing treatments associated with a very high risk of infertility, were offered the experimental procedure of testicular cryopreservation. Exclusion criteria were testicular volumes >10 ml and high bleeding or infection risk. There were 18 patients with a diagnosis of malignancy and 14 patients a non-malignant diagnosis. While 20 patients had the testicular biopsy performed 1-45 days after chemotherapy, 12 patients had not received any chemotherapy. In addition, 14 testicular tissue samples of patients with no reported testicular pathology, obtained from the internal biobank of the Department of Pathology at Karolinska University Hospital, were included as control samples in addition to reference values obtained from a recently published meta-analysis. The quantity of spermatogonia was assessed by both morphological and immunohistochemical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: The main finding was a significant reduction in spermatogonial cell counts in boys treated with alkylating agents or with hydroxyurea for sickle cell disease. The mean S/T values in boys exposed to alkylating agents (0.2 ± 0.3, n = 6) or in boys with sickle cell disease and exposed to hydroxyurea (0.3 ± 0.6, n = 6) were significantly lower (P = 0.003 and P = 0.008, respectively) than in a group exposed to non-alkylating agents or in biobank control samples (1.7 ± 1.0, n = 8 and 4.1 ± 4.6, n = 14, respectively). The mean S/T values of the testicular tissue samples included in the biobank control group and the patient group exposed to non-alkylating agents were within recently published normative reference values. LIMITATIONS, REASONS FOR CAUTION: Normal testicular tissue samples included in this study were obtained from the internal biobank of Karolinska University Hospital. Samples were considered normal and included in the study if no testicular pathology was reported in the analysed samples. However, detailed information regarding previous medical treatments and testicular volumes of patients included in this biobank were not available. WIDER IMPLICATIONS OF THE FINDINGS: This study summarizes, for the first time, spermatogonial quantity in a prepubertal patient cohort just before and after potentially sterilizing treatments. Boys facing cancer and cytotoxic therapies are regarded as the major group who will benefit from novel fertility preservation techniques. There are no previous reports correlating spermatogonial quantity to cumulative exposure to alkylating agents and anthracyclines (non-alkylating agents) and no information about the timing of cytotoxic exposures among this particular patient cohort. For prepubertal boys in whom fertility preservation is indicated, testicular tissue should be obtained before initiation of chemotherapy with alkylating agents, whilst for those with sickle cell disease and treated with hydroxyurea, this approach to fertility preservation may not be feasible. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from The Swedish Childhood Cancer Foundation (PR2016-0124; TJ2016-0093; PR2015-0073, TJ2015-0046) (J.-B.S. and K.J.), the Jane and Dan Olssons Foundation (2016-33) (J.-B.S.), the Finnish Cancer Society (K.J.), the Foundation for Paediatric Research (J.-B.S.), Kronprinsessan Lovisas Förening För Barnasjukvård/ Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation (J.-B.S.), the Väre Foundation for Paediatric Cancer Research (K.J.) and the Swedish Research Council (2012-6352) (O.S.). R.T.M. was supported by a Wellcome Trust Fellowship (09822). J.P.A.-L. and M.K. were supported by the ITN Marie Curie program 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Body mass index standard deviation score and obesity in children with type 1 diabetes in the Nordic countries. HbA1c and other predictors of increasing BMISDS.

BACKGROUND: Intensified insulin therapy may increase body weight and cause obesity. This study compared body mass index standard deviation score (BMISDS) and obesity rate in children with type 1 diabetes (T1D) in Denmark, Iceland, Norway and Sweden, and uncovered predictors for increasing BMISDS. METHODS: Data registered in the Nordic national childhood diabetes databases during the period 2008-2012 on children below 15 years with T1D for more than 3 months were compiled, including information on gender, age, diabetes duration, hemoglobin A1c (HbA1c ), insulin dose, severe hypoglycemia (SH), treatment modality, height and weight. The Swedish reference chart for BMI was used for calculating BMISDS. RESULTS: Totally, 11 025 children (48% females) (30 994 registrations) were included. Medians by the last recorded examination were: age, 13.5 years; diabetes duration, 4.3 years; HbA1c , 7.9% (63 mmol/mol); insulin dose, 0.8 IU/kg/d and BMISDS, 0.70. Obesity rate was 18.5%. Adjusted mean BMISDS (BMISDS adj) was inversely related to HbA1c and directly to diabetes duration. Higher BMISDS adj was found in those with an insulin dose above 0.6 IU/kg/d, and in girls above 10 years. Pump users had higher BMISDS adj than pen users, and patients with registered SH had higher BMISDS adj than patients without SH (both P < .001). CONCLUSION: Obesity rate in children with T1D in the Nordic countries is high, however, with country differences. Low HbA1c , long diabetes duration, higher insulin dose, pump treatment and experiencing a SH predicted higher BMISDS. Diabetes caregivers should balance the risk of obesity and the benefit of a very low HbA1c.

Teasing and social rejection among obese children enrolling in family-based behavioural treatment: effects on psychological adjustment and academic competencies.

OBJECTIVES: The first objective was to determine the prevalence of psychological maladjustment (emotional and behavioural problems), low academic competencies and teasing/social rejection among obese Icelandic children enrolling in a family-based behavioural treatment. A second objective was to explore the degree to which teasing/social rejection specifically contributes to children's psychological adjustment and academic competencies when controlling for other variables, including demographics, children's physical activity, parental depression and life-stress. METHODS: Participants were 84 obese children (mean body mass index-standard deviation score=3.11, age range=7.52-13.61 years). Height and weight, demographics and measures of children's psychological adjustment, academic competencies, teasing/social rejection and physical activity were collected from children, parents and teachers. Parental depression and life-stress was self-reported. RESULTS: Over half the children exceeded cutoffs indicating concern on at least one measure of behavioural or emotional difficulties. Children endorsed significant levels of teasing/social rejection, with almost half acknowledging they were not popular with same-gender peers. Parent reports of peer problems were even higher, with over 90% of both boys and girls being rated by their parents as having significant peer difficulties. However, rates of low academic competencies as reported by teachers were not different from those of the general population. In regression analyses controlling for other variables, self-reported teasing/social rejection emerged as a significant contributor to explaining both child psychological adjustment and academic competencies. CONCLUSIONS: The results indicate that among obese children enrolled in family-based treatment, self-reported teasing/social rejection is quite high and it is associated with poorer psychological adjustment as well as lower academic competencies. Parent reports corroborate the presence of substantial peer difficulties, supporting the need to address peer relations with overweight children both in clinical practice and in public health interventions.

Fertility preservation in boys: recent developments and new insights †.

BACKGROUND: Infertility is an important side effect of treatments used for cancer and other non-malignant conditions in males. This may be due to the loss of spermatogonial stem cells (SSCs) and/or altered functionality of testicular somatic cells (e.g. Sertoli cells, Leydig cells). Whereas sperm cryopreservation is the first-line procedure to preserve fertility in post-pubertal males, this option does not exist for prepubertal boys. For patients unable to produce sperm and at high risk of losing their fertility, testicular tissue freezing is now proposed as an alternative experimental option to safeguard their fertility. OBJECTIVE AND RATIONALE: With this review, we aim to provide an update on clinical practices and experimental methods, as well as to describe patient management inclusion strategies used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss. SEARCH METHODS: Based on the expertise of the participating centres and a literature search of the progress in clinical practices, patient management strategies and experimental methods used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss were identified. In addition, a survey was conducted amongst European and North American centres/networks that have published papers on their testicular tissue banking activity. OUTCOMES: Since the first publication on murine SSC transplantation in 1994, remarkable progress has been made towards clinical application: cryopreservation protocols for testicular tissue have been developed in animal models and are now offered to patients in clinics as a still experimental procedure. Transplantation methods have been adapted for human testis, and the efficiency and safety of the technique are being evaluated in mouse and primate models. However, important practical, medical and ethical issues must be resolved before fertility restoration can be applied in the clinic.Since the previous survey conducted in 2012, the implementation of testicular tissue cryopreservation as a means to preserve the fertility of prepubertal boys has increased. Data have been collected from 24 co-ordinating centres worldwide, which are actively offering testis tissue cryobanking to safeguard the future fertility of boys. More than 1033 young patients (age range 3 months to 18 years) have already undergone testicular tissue retrieval and storage for fertility preservation. LIMITATIONS REASONS FOR CAUTION: The review does not include the data of all reproductive centres worldwide. Other centres might be offering testicular tissue cryopreservation. Therefore, the numbers might be not representative for the entire field in reproductive medicine and biology worldwide. The key ethical issue regarding fertility preservation in prepubertal boys remains the experimental nature of the intervention. WIDER IMPLICATIONS: The revised procedures can be implemented by the multi-disciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss. STUDY FUNDING/COMPETING INTERESTS: The work was funded by ESHRE. None of the authors has a conflict of interest.

Decrease in adiponectin levels correlates to growth response in growth hormone-treated children.

BACKGROUND/AIMS: Adiponectin is secreted by adipose tissue and circulates in human plasma at high levels. Decreased adiponectin levels are associated with insulin resistance and obesity. The aim of this study was to investigate whether changes in serum adiponectin levels are related to the growth response, insulin levels and insulin resistance during growth hormone (GH) treatment. METHODS: The study included 94 short prepubertal children (19 girls and 75 boys). The mean age at the start of daily GH injections was 9.04 +/- 2.38 years. Adiponectin levels in serum were measured using an ELISA. RESULTS: At baseline, adiponectin correlated with the first-year growth response (r = 0.26, p = 0.012). Adiponectin decreased significantly after 1 week, 3 months and 1 year from 14.5 +/- 5.71 to 13.1 +/- 5.22 (p < 0.0001), 10.3 +/- 4.82 (p < 0.0001) and 12.5 +/- 5.34 microg/ml (p < 0.0001), respectively. There were significant correlations between the first-year growth response and the decrease in adiponectin levels after 3 months and 1 year (r = -0.38, p < 0.0001 and r = -0.47, p < 0.0001, respectively). No correlations between adiponectin, insulin and the homeostasis model assessment of insulin resistance were seen. CONCLUSIONS: GH treatment in prepubertal children decreases serum adiponectin levels, and the decrease is correlated to the growth response. No correlations between adiponectin and insulin levels or insulin resistance were found.

Incidence of severe hypoglycemia in children with type 1 diabetes in the Nordic countries in the period 2008-2012: association with hemoglobin A 1c and treatment modality.

OBJECTIVE: Treatment of type 1 diabetes has been intensified aiming at normalizing blood glucose, which may increase the risk of severe hypoglycemia (SH). We aimed to compare the incidence of SH events in the four Nordic countries Denmark, Iceland, Norway and Sweden, and to assess the influence of hemoglobin A1c (HbA1c) and treatment modalities on the frequency of SH; particularly, to explore if a HbA1c target ≤6.7% (50 mmol/mol) is feasible. RESEARCH DESIGN AND METHODS: Data on children below 15 years with a diabetes duration more than 1 year, registered in the national childhood diabetes databases in the four Nordic countries from 2008 to 2012, were compiled. Data completeness was more than 95%. RESULTS: Totally 8806 (48% females) patients with 29 715 person years were included, mean age and diabetes duration were 11 years and 5.1 years, respectively. The overall rate of SH was 6.0 per 100 patient-years, and did not change during the study period. The Swedish population constantly had the lowest SH incidence while it decreased significantly in the Danish population. HbA1c decreased significantly over time (p<0.01), while the number of pump users increased (p<0.01). Stratifying for HbA1c levels showed the lowest risk of SH in patients with HbA1c ≤6.7% (≤50 mmol/mol), but in the statistical models adjusting for possible confounders the difference between the HbA1c groups disappeared. Pump users had the lowest SH risk, also after adjusting for possible confounders. CONCLUSIONS: Risk of SH differs between the Nordic countries with the lowest risk in Sweden. Pump therapy was associated with decreased risk of SH. The low HbA1c group had the same or a lower risk of SH compared with the highest HbA1c groups. A target HbA1c ≤6.7% (≤50 mmol/mol) seems achievable without increasing the risk of SH.

Bone mineral density and bone turnover in young adult survivors of childhood acute lymphoblastic leukaemia.

OBJECTIVE: Treatment for childhood leukaemia induces many risk factors for development of decreased bone mineral density (BMD). Physical activity is also known to affect BMD. The aim was to study BMD and markers of bone turnover in a well-defined group of survivors of acute lymphoblastic leukaemia (ALL) who had all reached final height as well as peak bone mass, taking both previous treatment and physical activity into consideration. DESIGN: All patients treated for ALL before the onset of puberty in the region of western Sweden, between 1973 and 1985, in first remission were included. Thirty-five out of forty-seven patients aged 20-32 years participated. Nineteen patients had received cranial radiotherapy, and the median follow-up time was 20 years. METHODS: BMD was assessed using dual-energy X-ray absorptiometry (DEXA). Serum concentrations of markers of bone turnover were analysed. Physical performance was measured using a performance exercise capacity stress test. RESULTS: BMD was slightly reduced in lumbar spine (-0.4 SD), but not in femoral neck or total body. BMD in femoral neck was correlated to physical performance and dose of corticosteroid, but no correlation was found with spontaneous growth hormone (GH) secretion. Markers of bone turnover were also correlated to physical performance, but not to GH secretion. CONCLUSIONS: Physical fitness seems to be the most important factor in developing and preserving normal bone mineral density in ALL patients. We propose that lifestyle education promoting physical activity is encouraged from an early point in time for these patients.

Body composition in young adult survivors of childhood acute lymphoblastic leukaemia.

OBJECTIVE: Obesity is frequently reported in patients treated for childhood leukaemia. Obesity, particularly abdominal obesity, is one of the main characteristics of the metabolic syndrome and a risk factor for cardiovascular disease and non-insulin-dependent diabetes mellitus (NIDDM). DESIGN: All patients treated for acute lymphoblastic leukaemia (ALL) before the onset of puberty in the region of western Sweden, between 1973 and 1985, and in first remission, were included. 35 out of 47 patients aged 20-32 years participated. 19 patients had received cranial radiotherapy, and the median follow-up time was 20 years. The focus of this report was to study body composition and signs of the metabolic syndrome and correlate the findings to spontaneous growth hormone (GH) secretion. METHODS: Body composition was assessed using dual-energy X-ray absorbtiometry (DEXA). We analyzed serum concentrations of insulin, glucose, leptin and lipids. RESULTS: No patient was obese according to World Health Organization criteria (body mass index, BMI > or = 30 kg/m2) but one-third were overweight (BMI 25-29.9 kg/m2). The maximal GH peak during 24 h (GHmax) was correlated to percentage of total body fat (r = -0.42; P = 0.017), trunk fat (r = -0.5; P = 0.005) and fat-free mass (r = 0.42; P = 0.017). GHmax was also correlated to s-triglycerides (r = -0.54; P = 0.001), low-density lipoprotein-cholesterol (r = -0.382; P = 0.024) and high-density lipoprotein-cholesterol (r = 0.45; P = 0.007). CONCLUSIONS: We found little effect on BMI but an increased percentage of total body fat, especially trunk fat, and a tendency for an unfavourable lipid profile in adult survivors of childhood leukaemia. These findings were related to low endogenous GH secretion due to cranial irradiation.

No evidence for involvement of the growth hormone/insulin-like growth factor-1 axis in psoriasis.

We have examined whether alterations in the growth hormone/insulin-like growth factor-1 axis play a role in the pathogenesis of psoriasis. Serum, urine, full skin biopsies, and suction blister roofs were obtained from patients with psoriasis and from healthy controls. Serum concentrations of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 were measured by radioimmunoassay. Growth hormone-binding protein was measured by ligand-mediated immunofunctional assay. Growth hormone concentration in urine was measured by an immunometric assay, and growth hormone receptor-gene expression was measured by RNase protection assay or by quantitative reverse transcriptase polymerase chain reaction in total RNA isolated from epidermal suction blister roofs. Serum concentrations of insulin-like growth factor-1 (249 +/- 12 micrograms per liter, mean +/- SEM, n = 42, and 277 +/- 21 micrograms per liter, n = 9, for psoriatic patients and controls, respectively), insulin-like growth factor binding protein-3 (3.1 +/- 0.08 mg per liter, n = 42, and 3.3 +/- 0.22 mg per liter, n = 9), growth hormone-binding protein (344 +/- 65 pmol per liter, n = 10, and 311 +/- 83 pmol per liter, n = 9), urinary growth hormone excretion during 24 h (12.8 +/- 2.7 microIU per 24 h, n = 12, and 12.3 +/- 1.6 microIU per 24 h, n = 9), and epidermal growth hormone receptor gene expression [32 +/- 12 x 10(3) mRNA transcripts per microgram total RNA (involved skin), n = 11, and 47 +/- 14 x 10(3) mRNA transcripts per microgram total RNA, n = 9] were similar in patients and controls. For insulin-like growth factor-1 and insulin-like growth factor binding protein-3 the values in psoriatic patients were also similar to those in larger control groups, n = 195 and n = 400, respectively. In addition, we found no evidence of local expression of growth hormone or prolactin in full skin punch biopsies from psoriatic involved skin by reverse transcriptase polymerase chain reaction. In conclusion, our results suggest that alterations in the growth hormone/ insulin-like growth factor-1 axis do not play a major role in the pathogenesis of psoriasis.

Acute and chronic effects of subcutaneous growth hormone (GH) injections on plasma levels of GH binding protein in short children.

A ligand-mediated immunofunctional assay was used to measure levels of GH binding protein (GHBP) in plasma from children on GH treatment. Blood was drawn at the time of sc injection of GH and thereafter every 2 h for 16 or 24 h in the acute group. The samples were analyzed for GHBP and GH. To study changes in GHBP levels after onset of GH treatment, samples were taken at the start and after 10, 30, and 90 days of treatment (short-term group) and at the start and after 1 and 2 yr of treatment (long-term group). All children in the short-term and long-term groups were prepubertal throughout the study. For the study of short-term and long-term effects of GH treatment, the children were divided into two groups according to the maximal GH response (GHmax) in an arginine-insulin tolerance test and, if available, the maximum peak on 24-h secretion. Children with a GHmax below 20 mU/L(10 micrograms/L) were classified as GH deficient (GHD), and children with a GHmax above 20 mU/L were classified as children of short stature (SS). In the acute group, GH was injected either at 1800 h (n = 73) or at 1000 h (n = 12). In the group given GH at 1800 h there was a decrease in GHBP levels, with the lowest values occurring after 8-10 h, i.e. between 0200 and 0400 h. We have previously reported that there was no correlation between endogenous GH peaks and changes in GHBP levels, but there was a small but significant diurnal variation in GHBP levels (coefficient of variation of about 10%), with a nadir between 0000 and 0400 h. In the group given GH at 1000 h, GHBP levels decreased to a minimum at 10 h after injection (at 2000 h, P < 0.01), with a further decrease at 16 h after injection (P < 0.05), corresponding to the nadir seen in normal children. The mean GHBP levels fell to 71.2% of the baseline value after 90 days in children with GHD (P = 0.004), but was unchanged in children with SS (P = 0.92). In children with GHD, the GHBP levels were unchanged at 1 yr (100.5% +/- 10.7) and 2 yr (97.1% +/- 6.6) after start of treatment. In children with SS, there was no significant increase after 1 yr (125.6% +/- 10.9, P = 0.16) and 2 yr (120.3% +/- 6.4, P = 0.17).(ABSTRACT TRUNCATED AT 400 WORDS)

Growth hormone (GH)-binding protein in prepubertal short children born small for gestational age: effects of growth hormone treatment. Swedish Study Group for Growth Hormone Treatment.

This study was undertaken to characterize the serum levels of GH-binding protein (GHBP) before and during GH treatment in prepubertal short children born small for gestational age (SGA) and their relationship with growth parameters. Sixty-seven prepubertal short children (49 boys and 18 girls; height SD score, -5.4 to -2.0; age, 2.0-12.8 yr) born SGA, 8 of whom (6 boys and 2 girls) had signs of Silver-Russell syndrome, participated in the study. Total GHBP was measured by a ligand-mediated immunofunctional assay. The mean (SD) change in height SD score during the year before the start of GH treatment (0.1 IU/kg.day) was 0.11 (0.20) SD score, and this value increased to a 0.84 (0.43) SD score during the first year (P < 0.001) and to a 1.27 (0.63) SD score during the 2-yr period of therapy (P < 0.001). The baseline GHBP values ranged from 49-392 pmol/L, and no relationships were found among sex, chronological age, and maximal GH response to an arginine-insulin tolerance test. A positive correlation between GHBP and body composition, expressed as weight for height SD score, was found in the whole group (r = 0.28; P < 0.05) and in boys (r = 0.44; P < 0.01). No relationship was found between GHBP and spontaneous 24-h GH secretion, in terms of either GH secretion rate or pulsatile pattern, whereas GHBP was positively correlated with insulin-like growth factor I (IGF-I) SD score (r = 0.28; P < 0.05) and IGF-binding protein-3 SD score (r = 0.39; P < 0.01). Using a multiple stepwise linear regression analysis, the model using the IGF-binding protein-3 SD score and the weight for height SD score at the start of GH therapy accounted for 33% of the variance in the baseline GHBP values. A mean increase of 27 (51)% in GHBP levels was found after 1 yr of therapy. However, a high degree of variability in the response of individuals to GH treatment in terms of GHBP levels was observed: in some children GHBP levels increased, whereas in others they decreased. In conclusion, GHBP levels in short prepubertal children born SGA were mostly within the normal range previously reported and correlated directly with body composition. An increase in GHBP levels was observed during GH treatment in some SGA children. No correlation was found between pretreatment GHBP levels and growth response to GH treatment.

Serum leptin levels correlate with growth hormone secretion and body fat in children.

The aim of this study was to investigate the relationship among GH secretion, leptin concentrations, and body composition measured with x-ray absorptiometry (DXA) in children. In total, 71 children were investigated, 51 males and 20 females. Their mean chronological age was 10.8 yr (range, 6.2-17.7 ys), and their mean height (SD) was -2.1 (0.63) SD scores. Their mean weight for height SD scores (WH(SDS)) was 0.2 (1.18). Body composition was investigated using DXA. Blood samples were taken for analysis of leptin, insulin-like growth factor I (IGF-I), IGF-binding protein-3, and 24-h GH secretion. A positive correlation was found between leptin and total body fat (r = 0.83; P < 0.0001) and when fat was expressed as a percentage of body weight (r = 0.86; P < 0.0001). There were significant (P < 0.0001) relationships between leptin and WH(SDS) (r = 0.45) and between leptin and body mass index (r = 0.69). A significant gender difference in leptin levels was found, but this disappeared after adjustment for body fat, as measured by DXA. There were significant (P < 0.001) inverse correlations between leptin and the AUCb for GH (r = -0.41), leptin, and GHmax (r = -0.38), where AUCb is the area under the curve above the calculated baseline, and GHmax is the maximum peak during the 24-h GH profile (percent fat and AUCb for GH, r = -0.43; percent fat and GHmax, r = -0.39). In a multiple stepwise forward regression analysis with leptin as the dependent variable, the percent trunk fat accounted for 77.7% of the leptin variation. With AUCb for GH as the dependent variable, the percent trunk fat accounted for 20.3% of the variation. With GHmax as the dependent variable, the percent trunk fat accounted for 18.8% of the variation, IGF-binding protein-3 for another 8.5%, and the percentage of fat from arms and legs for another 4.4%. We demonstrated a strong positive correlation between leptin levels and body fat, a significant negative correlation between leptin levels and GH secretion, and a significant negative correlation between body fat and GH secretion. We have also shown that specific regional fat depots have different relationships with leptin and particular markers of GH secretion.

Growth hormone treatment prevents the decrease in insulin-like growth factor I gene expression in patients undergoing abdominal surgery.

Acquired GH resistance together with reduced skeletal muscle mass are found in patients with increased protein catabolism due, for example, to sepsis, trauma, or major surgery. Both administration of glutamine-containing parenteral nutrition and GH treatment have been found to diminish this catabolism. The effects of GH are mediated in part by insulin-like growth factor I (IGF-I) that is produced in the liver and locally in GH target tissues. The aim of this study was to investigate the effect of GH treatment on expression of the IGF-I gene and GH receptor (GHR) gene in skeletal muscle after major surgery. A new quantitative RT-PCR-based assay was established to measure IGF-I gene expression. Metabolically healthy patients, without significant preoperative weight loss, who were undergoing elective abdominal surgery were included in the study. Five patients (one woman and four men) were treated with daily injections of GH (0.3 IU/kg.day) in addition to being given total parenteral nutrition including glutamine (0.28 g/kg.day). The control group consisted of eight patients (three women and five men), who were given glutamine-enriched total parenteral nutrition but no GH. A muscle biopsy was taken from the lateral portion of the quadriceps femoris muscle preoperatively (day 0) after induction of anesthesia. A second biopsy was taken under local anesthesia on postoperative day 3. Total ribonucleic acid (RNA) was extracted from the muscle biopsies, and IGF-I messenger RNA (mRNA) and GHR mRNA were measured by competitive quantitative RT-PCR assays. IGF-I mRNA and GHR mRNA levels were related to the expression of a housekeeping gene (cyclophilin). In the control group, IGF-I mRNA levels decreased from 1505 +/- 265 (mean +/- SEM) transcripts/cpm cyclophilin on day 0 to 828 +/- 172 on day 3 (P < 0.05). In contrast, IGF-I mRNA levels did not change in the GH-treated group (1188 +/- 400 transcripts/cpm cyclophilin on day 0 vs. 1089 +/- 342 transcripts/cpm cyclophilin on day 3). No statistically significant changes were seen in GHR expression. We conclude that administration of GH prevents the reduction in IGF-I gene expression in skeletal muscle after abdominal surgery.

Cartilage oligomeric matrix protein increases in serum after the start of growth hormone treatment in prepubertal children.

Both GH and IGF-I stimulate bone growth, but the molecular mechanisms mediating their effects on the growth plate are not fully understood. We measured gene expression by microarray analysis in primary cultured human chondrocytes treated with either GH or IGF-I. One of the genes found to be up-regulated by both GH and IGF-I was that encoding cartilage oligomeric matrix protein (COMP). This protein is predominantly found in the extracellular matrix of cartilage. Mutations in the COMP gene have been associated with syndromes of short stature. To verify that COMP is regulated by GH in vivo, we measured COMP levels in serum in short children treated with GH. The study included 113 short prepubertal children (14 girls and 99 boys) with a mean (+/- sd) age of 8.84 +/- 2.76 yr, height sd score of -2.74 +/- 0.67, and IGF-I sd score of -1.21 +/- 1.07 at the start of GH administration. Serum levels of COMP were 1.58 +/- 0.28, 1.83 +/- 0.28 (P < 0.0001), 1.91 +/- 0.28 (P < 0.0001), 1.78 +/- 0.28 (P < 0.001), and 1.70 +/- 0.24 (P < 0.05) microg/ml at baseline and after 1 wk and 1, 3, and 12 months, respectively. In conclusion, we have demonstrated that COMP expression is up-regulated by both GH and IGF-I in primary cultured human chondrocytes. Furthermore, serum levels of COMP increase after the start of GH treatment in short children.

Measurement of human growth hormone receptor messenger ribonucleic acid by a quantitative polymerase chain reaction-based assay: demonstration of reduced expression after elective surgery.

Studies of GH receptor (GHR) gene expression in human tissues have been hampered by the limited amount of tissue available for analysis and the low sensitivity of conventional methods. We have developed a quantitative reverse transcriptase-PCR assay for measurement of GHR messenger ribonucleic acid levels in small human tissue biopsies. To compensate for sample to sample variation, an internal RNA standard, which differs from the wild-type GHR transcript by only a few nucleotides, was reverse transcribed and amplified together with the GHR transcripts. PCR was carried out using one biotinylated primer to permit the purification of single stranded PCR products on streptavidin-coated microtiter plates. The ratio between the wild-type and mutated transcripts was determined by two separate minisequence reactions in which a primer, annealed immediately 3' of a variable nucleotide, was extended by a single 3H-labeled nucleotide, complementary to either the wild-type or mutated sequence. The assay range was 0.125-8 x 10(5) transcripts/sample, the mean intraassay coefficient of variation was 8.7%, and the lower limit of detection was 0.125 x 10(5) transcripts/sample. GHR messenger ribonucleic acid levels were detectable in small amounts (10-100 ng) of total RNA extracted from adipose tissue, skeletal muscle, and liver. The GHR gene expression in liver was approximately 10-fold higher than that in skeletal muscle, whereas intermediate levels were found in adipose tissue. In nine patients undergoing elective abdominal surgery, GHR gene expression in skeletal muscle was reduced on day 3 after surgery compared to the baseline level. The decrease in GHR gene expression was accompanied by a decrease in skeletal muscle glutamine. This suggests that the postoperative protein catabolism may be caused at least partly by acquired GH insensitivity due to reduced expression of the GHR gene.

The individual responsiveness to growth hormone (GH) treatment in GH-deficient adults is dependent on the level of GH-binding protein, body mass index, age, and gender.

The aim of the present trial was to study the individual responsiveness to GH treatment in terms of body composition and to search for possible predictors of the response in GH-deficient adults. Sixty-eight patients (44 men and 24 women) with a mean age of 44.3 (1.2) yr and verified GH deficiency participated in a 2-phase treatment trial with an initial randomized, double blind, placebo-controlled, 6-month period, followed by an open treatment period, thereby ensuring all patients 12 months of GH treatment. Recombinant human GH was administered sc daily at bedtime, with a target dose of 12 micrograms/kg x day. GHBP was measured by ligand-mediated immunofunctional assay, and serum insulin-like growth factor I (IGF-I) was determined by RIA after acid-ethanol extraction, using a truncated IGF-I analog as the radioligand. Lean body mass (LBM) and body fat (BF) were determined by dual energy x-ray absorptiometry, and total body water (TBW) was determined by bioelectrical impedance. During the placebo control period, serum IGF-I,LBM, and TBW increased (P < 0.001), whereas BF decreased (P < 0.001) and serum GHBP was unchanged in the group treated with GH compared with the patients treated with placebo. After 12 months of GH treatment, the individual changes in BF ranged from -12.5 to 4.3 kg and from -4.5 to 10.1 kg in LBM. Age (P < 0.05) and baseline GHBP level (P < 0.01) were inversely correlated with the increase in LBM. The GH-induced increment in IGF-I and TBW was greater in men than in women (P < 0.01), whereas the decreases in BF were similar in men and women. This trial demonstrates the variability in responsiveness to GH administration in GH-deficient adults. The best response to GH was obtained in younger patients with low GHBP levels. Furthermore, men responded better than women.

Asthma and immunoglobulin E antibodies after respiratory syncytial virus bronchiolitis: a prospective cohort study with matched controls.

OBJECTIVE: To study the occurrence of bronchial obstructive symptoms and immunoglobulin (Ig) E antibodies after respiratory syncytial virus (RSV) bronchiolitis in infancy. Previous studies of this subject have mostly been retrospective or without controls, or the controls have not been followed prospectively. DESIGN: This was a prospective cohort study with matched controls. PARTICIPANTS: Forty-seven infants had experienced RSV bronchiolitis severe enough to cause hospitalization at a mean age of 3 1/2 months. For each child with RSV infection, two controls were acquired from the local Child Health Center and matched for date of birth, sex, and residence. Only one control was obtained for one RSV child, and the control group thus contained 93 children. METHODS: All the children underwent two follow-up examinations, the first one at a mean age of 1 year and the second at a mean age of 3 years. At the first follow-up, a skin-prick test against egg white was performed, and serum IgG antibodies against RSV were measured. At the second follow-up, serum IgE antibodies were measured using screening tests for common food and inhalant antibodies, and skin-prick tests against egg white, cat, birch, and mite allergen were performed. Hereditary and environmental factors (passive smoking, indoor furred animals) and duration of breast-feeding were recorded. RESULTS: At the first follow-up, 89% in the RSV group and 27% in the control group had IgG antibodies against RSV (P < .001). At the second follow-up, asthma, defined as three episodes of bronchial obstruction verified by a physician, was found in 11 of 47 children (23%) in the RSV group and in 1 of 93 children (1%) in the control group (P < .001). A positive test for IgE antibodies was noted in 14 of 44 (32%) RSV children and in 8 of 92 (9%) children in the control group (P = .002). An analysis of risk factors for the development of asthma and IgE antibodies on the whole group of 140 children showed that RSV bronchiolitis was the most important risk factor, and a family history of atopy or asthma further increased the risk. CONCLUSIONS: Respiratory syncytial virus bronchiolitis during the first year of life apparently is an important risk factor for the development of asthma and sensitization to common allergens during the subsequent 2 years, particularly in children with hereditary for atopy/asthma.

Changes in body composition and leptin levels during growth hormone (GH) treatment in short children with various GH secretory capacities.

OBJECTIVE: The aim of this study was to follow changes in body composition, estimated by dual-energy X-ray absorptiometry (DXA), in relation to changes in leptin during the first year of GH therapy in order to test the hypothesis that leptin is a metabolic signal involved in the regulation of GH secretion in children. DESIGN AND METHODS: In total, 33 prepubertal children were investigated. Their mean (S.D.) chronological age at the start of GH treatment was 11.5 (1.6) years, and their mean height was -2.33 (0.38) S.D. scores (SDS). GH was administered subcutaneously at a daily dose of 0.1 (n=26) or 0.2 (n=7) IU/kg body weight. Ten children were in the Swedish National Registry for children with GH deficiency, and twenty-three children were involved in trials of GH treatment for idiopathic short stature. Spontaneous 24-h GH secretion was studied in 32 of the children. In the 24-h GH profiles, the maximum level of GH was determined and the secretion rate estimated by deconvolution analysis (GHt). Serum leptin levels were measured at the start of GH treatment and after 10 and 30 days and 3, 6 and 12 months of treatment. Body composition measurements, by DXA, were performed at baseline and 12 months after the onset of GH treatment. RESULTS: After 12 months of GH treatment, mean height increased from -2.33 to -1.73 SDS and total body fat decreased significantly by 3.0 (3.3)%. Serum leptin levels were decreased significantly at all time points studied compared with baseline. There was a significant correlation between the change in total body fat and the change in serum leptin levels during the 12 months of GH treatment, whereas the leptin concentration per unit fat mass did not change. In a multiple stepwise linear regression analysis with 12 month change in leptin levels as the dependent variable, the percentage change in fat over 12 months, the baseline fat mass (%) of body mass and GHt accounted for 24.0%, 11.5% and 12.2% of the variability respectively. CONCLUSIONS: There are significant correlations between changes in leptin and fat and endogenous GH secretion in short children with various GH secretory capacities. Leptin may be the messenger by which the adipose tissue affects hypothalamic regulation of GH secretion.

The decrease of IGF-I, IGF-binding protein-3 and bone alkaline phosphatase isoforms during gluten challenge correlates with small intestinal inflammation in children with coeliac disease.

OBJECTIVE: In children with coeliac disease, the ingestion of gluten causes small intestinal inflammation and a clinical picture of malabsorption, weight reduction and short stature. Decreased alkaline phosphatase (ALP) during gluten challenge was found in a previous study. ALP is a marker of bone formation and ALP activities are correlated with growth velocity. The aim of this study was to characterise the previously observed decrease of total ALP by investigating three specific bone ALP isoforms (bone/intestinal, B1 and B2) and three specific liver ALP isoforms (L1, L2 and L3) and, moreover, to correlate these ALP isoforms with other growth factors and growth markers. In addition, we also studied the association with possible weight changes, small intestinal mucosa inflammation, sex, age and gluten dose during gluten challenge. MATERIALS AND METHODS: Bone and liver ALP isoforms, IGF-I, IGF-binding protein (IGFBP)-3 and serum cross-linked carboxy-terminal telopeptide of type I collagen (ICTP) were measured together with change in weight and small intestinal mucosa histopathology in 54 children with earlier enteropathy who participated in a 4-week gluten challenge. RESULTS: We observed a decreased total ALP activity after 4 weeks of gluten challenge, 7.8+/-1.8 to 6.5+/-1.7 microkat/l (means +/- s.d.), which was mainly due to a reduction of the bone ALP isoforms. The sum of all three bone ALP isoforms decreased from 6.3+/-1.7 to 5.1+/-1.6 microkat/l. The decreased activities of the bone ALP isoforms correlated with the observed reductions of IGF-I (r=0.74, P<0.001), IGFBP-3 (r=0.51, P<0.001) and ICTP (r=0.57, P<0.001). The decrease of the growth factors and growth markers correlated with weight reduction, but when influences from the change in weight were adjusted for, the partial correlation of the small intestinal mucosa inflammation was significant for IGF-I (r=-0.56, P<0.001) and IGFBP-3 (r=-0.55, P<0.001). CONCLUSION: The decrease of total ALP was due to a reduction of bone ALP. The decrease of IGF-I and IGFBP-3 was independently correlated with weight change and small intestinal inflammation.

Serum leptin in short children born small for gestational age: relationship with the growth response to growth hormone treatment. The Swedish Study Group for Growth Hormone Treatment.

The product of the obese (ob) gene, leptin, is an adipocyte-derived hormone that is involved in the regulation of appetite and body weight. This study was undertaken in order to describe the basal serum levels of leptin in prepubertal short children born small for gestational age (SGA) and their relationship with growth parameters, before and during growth hormone (GH) treatment. Eighty-nine prepubertal short children (66 boys, 23 girls; height standard deviation score (SDS), -5.4 to -2.0; age, 2.0 to 12.8 years) born SGA, 12 of whom (9 boys, 3 girls) had signs of Silver-Russell syndrome, were included in the study. Serum leptin concentrations were measured by radioimmunoassay. Leptin levels in the children born SGA were compared with those in a reference group of 109 prepubertal healthy children born at an appropriate size for gestational age (AGA). The mean (S.D.) change in height SDS was 0.11 (0.22) during the year before the start of GH therapy (0.1 IU/kg/day) and increased to 0.82 (0.44) during the first year (P < 0.001) and to 1.28 (0.59) during the 2-year period of GH therapy (P < 0.001). The children born SGA were significantly leaner than the reference group. An inverse correlation was found between leptin and chronological age in the SGA group (r = -0.31, P < 0.01). The mean serum level of leptin in the children born SGA who were older than 5.5 years of age was 2.8 micrograms/l which was significantly lower than the mean value of 3.7 micrograms/l found in the children born AGA of the same age range. The difference remained after adjustment of leptin levels for sex, age, body mass index (BMI) and weight-for-height SDS (WHSDSSDS). Leptin correlated with WHSDSSDS (r = 0.32, P < 0.001) and BMI (r = 0.36, P < 0.01) in the reference population, but not in the SGA group. No correlation was found between leptin and spontaneous 24-h GH secretion, insulin-like growth factor (IGF)-I or IGF-binding protein-3 levels, or with fasting insulin or cortisol levels. Leptin levels at the start of GH treatment were correlated with the growth response over both 1 year (r = 0.46, P < 0.001) and 2 years (r = 0.51, P < 0.001) of GH therapy. Using multiple regression analysis, models including leptin levels at the start of GH therapy could explain 51% of the variance in the growth response after 1 year and 44% after 2 years of GH treatment. In conclusion, serum leptin levels are reduced in short children born SGA and are inversely correlated with chronological age. Leptin concentrations correlate with the growth response to GH treatment and might be used as a marker for predicting the growth response to GH treatment.