Search

Objective: Klinefelter syndrome (KS) (47,XXY and variants, KS) is the most common sex chromosome disorder in humans. However, little is known about the onset and progression of puberty in patients with KS. In this study, we describe the onset and progression of puberty in a large series of boys with KS in a single tertiary centre. Design and Patients: Retrospective data (Tanner stages, testicular length, testosterone supplementation, levels of luteinizing hormone [LH] and testosterone) before possible testosterone treatment on 72 KS patients with 47,XXY karyotype were reviewed, and G (n = 59 patients) and P (n = 56 patients) stages were plotted on puberty nomograms. Measurements and Results: One boy had a delayed onset of puberty, as he was at the G1 stage at the age of 13.8 years (-2.2 SDs). No observations of delay were made of boys at Stage G2. The progression of G stages was within normal limits in the majority of patients; only few boys were late at G3 (4.1%; 1 out of 24) and G4 (7.4%; 2 out of 27). Testosterone supplementation was started at the average age of 15.5 years to 35 boys (47%), 2 of whom were over 18 years old. LH level was on average 18.2 IU/L (SD: 6.3 IU/L) and testosterone 9.1 nmol/L (SD: 3.1 nmol/L) when testosterone supplementation was started. Conclusions: Our results suggest that puberty starts within the normal age limits in boys with KS, and testosterone supplementation is not needed for the initial pubertal progression in the majority of patients.

Objective The hybrid close-loop system (HCL) is a rapidly emerging treatment method for type 1 diabetes (T1D), but the long-term effectiveness of the system remains unclear. This study investigates the influence of the HCL on glycemic control in children and adolescents with T1D in a real-life setting during the first year on HCL. Research design and methods This retrospective study included all the patients (n = 111) aged 3 to 16 years with T1D who initiated the HCL system between 1st of December 2018 and 1st of December 2019 in the Helsinki University Hospital. Time in range (TIR), HbA1c, mean sensor glucose (SG) value, time below range (TBR), and SG coefficient of variance (CV) were measured at 0, 1, 3, 6, and 12 month. The changes over time were analyzed with a repeated mixed model adjusted with baseline glycemic control. Results After the initiation of HCL, all measures of glycemic control, except HbA1c, improved and the effect lasted throughout the study period. Between 0 and 12 month, TIR increased (beta = -2.5 [95%CI: -3.6 - (-1.3)], p < 0.001), whereas mean SG values (beta = -0.7 [95%CI: -0.9 - (-0.4)]), TBR (beta = -2.5 [95%CI: -3.6 - (-1.3)]), and SG CV (beta = -4.5 [95%CI: -6.3 - [-2.8]) decreased significantly (p < 0.001). Importantly, the changes occurred regardless of the age of the patient. Conclusions Measurements of glycemic control, except HbA1c, improved significantly after the initiation of the HCL system and the favorable effect lasted throughout the follow-up. These results support the view that HCL is an efficacious treatment modality for children and adolescents with T1D of all ages.

Objective The role of miRNA as endocrine regulators is emerging, and microRNA mir-30b has been reported to repress Mkrn3. However, the expression of miR-30b during male puberty has not been studied. Design and methods Circulating relative miR-30b expression was assessed in sera of 26 boys with constitutional delay of growth and puberty (CDGP), treated with low-dose testosterone (T) (n =11) or aromatase inhibitor letrozole (Lz) (n =15) for 6 months and followed up to 12 months (NCT01797718). The associations between the relative expression of miR-30b and hormonal markers of puberty were evaluated. Results During the 12 months of the study, circulating miR-30b expression increased 2.4 ± 2.5 (s.d.) fold (P = 0.008) in all boys, but this change did not correlate with corresponding changes in LH, testosterone, inhibin B, FSH, or testicular volume (P = 0.25-0.96). Lz-induced activation of the hypothalamic–pituitary–gonadal (HPG) axis was associated with more variable miR-30b responses at 3 months (P < 0.05), whereas those treated with T exhibited significant changes in relative miR-30b levels in the course the study (P < 0.01–0.05). Conclusions Circulating miR-30b expression in boys with CDGP increases in the course of puberty, and appears to be related to the activity of the HPG axis.

Patients with deletions on chromosome 9q31.2 may exhibit delayed puberty, craniofacial phenotype including cleft lip/palate, and olfactory bulb hypoplasia. We report a patient with congenital HH with anosmia (Kallmann syndrome, KS) and a de novo 2.38 Mb heterozygous deletion in 9q31.2. The deletion breakpoints (determined with whole-genome linked-read sequencing) were in the FKTN gene (9:108,331,353) and in a non-coding area (9:110,707,332) (hg19). The deletion encompassed six protein-coding genes (FKTN, ZNF462, TAL2, TMEM38B, RAD23B, and KLF4). ZNF462 haploinsufficiency was consistent with the patient’s Weiss–Kruszka syndrome (craniofacial phenotype, developmental delay, and sensorineural hearing loss), but did not explain his KS. In further analyses, he did not carry rare sequence variants in 32 known KS genes in whole-exome sequencing and displayed no aberrant splicing of 15 KS genes that were expressed in peripheral blood leukocyte transcriptome. The deletion was 1.8 Mb upstream of a KS candidate gene locus (PALM2AKAP2) but did not suppress its expression. In conclusion, this is the first report of a patient with Weiss–Kruszka syndrome and KS. We suggest that patients carrying a microdeletion in 9q31.2 should be evaluated for the presence of KS and KS-related features.

Accumulating evidence indicates that gut microbiota may regulate sex-hormone levels in the host, with effects on reproductive health. Very little is known about the development of intestinal microbiota during puberty in humans. To assess the connection between pubertal timing and fecal microbiota, and to assess how fecal microbiota develop during puberty in comparison with adult microbiota, we utilized a Finnish allergy-prevention-trial cohort (Flora). Data collected at 13-year follow-up were compared with adult data from a different Finnish cohort. Among the 13-year-old participants we collected questionnaire information, growth data from school-health-system records and fecal samples from 148 participants. Reference adult fecal samples were received from the Health and Early Life Microbiota (HELMi) cohort (n = 840). Fecal microbiota were analyzed using 16S rRNA gene amplicon sequencing; the data were correlated with pubertal timing and compared with data on adult microbiota. Probiotic intervention in the allergy-prevention-trial cohort was considered as a confounding factor only. The main outcome was composition of the microbiota in relation to pubertal timing (time to/from peak growth velocity) in both sexes separately, and similarity to adult microbiota. In girls, fecal microbiota became more adult-like with pubertal progression (p = 0.009). No such development was observed in boys (p = 0.9). Both sexes showed a trend towards increasing relative abundance of estrogen-metabolizing Clostridia and decreasing Bacteroidia with pubertal development, but this was statistically significant in girls only (p = 0.03). In girls, pubertal timing was associated positively with exposure to cephalosporins prior to the age of 10. Our data support the hypothesis that gut microbiota, particularly members of Ruminococcaceae, may affect pubertal timing, possibly via regulating host sex-hormone levels.Trial registration The registration number for the allergy-prevention-trial cohort: ClinicalTrials.gov, NCT00298337, registered 1 March 2006—Retrospectively registered, https://clinicaltrials.gov/show/NCT00298337. The adult-comparison cohort (HELMi) is NCT03996304.

Publisher Copyright: © 2021 The Authors Background: Extremely tall children (defined as height SDS (HSDS) ≥+3) are frequently referred to specialized healthcare for diagnostic work-up. However, no systematic studies focusing on such children currently exist. We investigated the aetiology, clinical features, and auxological clues indicative of syndromic tall stature in extremely tall children subject to population-wide growth monitoring and screening rules. Methods: Subjects with HSDS ≥+3 after three years of age born between 1990 and 2010 were identified from the Helsinki University Hospital district growth database. We comprehensively reviewed their medical records up to December 2020 and recorded underlying diagnoses, auxological data, and clinical features. Findings: We identified 424 subjects (214 girls and 210 boys) who fulfilled the inclusion criteria. Underlying growth disorder was diagnosed in 61 (14%) patients, in 36 (17%) girls and 25 (12%) boys, respectively (P=0•15). Secondary causes were diagnosed in 42 (10%) patients and the two most frequent secondary diagnoses, premature adrenarche, and central precocious puberty were more frequent in girls. Primary disorder, mainly Marfan or Sotos syndrome, was diagnosed in 19 (4%) patients. Molecular genetic studies were used as a part of diagnostic work-up in 120 subjects. However, array CGH or next-generation sequencing studies were seldom used. Idiopathic tall stature (ITS) was diagnosed in 363 (86%) subjects, and it was considered familial in two-thirds. Dysmorphic features or a neurodevelopmental disorder were recorded in 104 (29%) children with ITS. The probability of a monogenic primary growth disorder increased with the degree of tall stature and deviation from target height. Interpretation: A considerable proportion of extremely tall children have an underlying primary or secondary growth disorder, and their risk is associated with auxological parameters. Clinical features related to syndromic tall stature were surprisingly frequent in subjects with ITS, supporting the view that syndromic growth disorders with mild phenotypes may be underdiagnosed in extremely tall children. Our results lend support to comprehensive diagnostic work-up of extremely tall children. Funding: Päivikki and Sakari Sohlberg Foundation, Foundation for Pediatric Research, and Helsinki University Hospital research grants.

In approximately half of congenital hypogonadotropic hypogonadism (cHH) patients, the genetic cause remains unidentified. Since the lack of certain miRNAs in animal models has led to cHH, we sequenced human miRNAs predicted to regulate cHH-related genes (MIR7-3,MIR141,MIR429andMIR200A-C) in 24 cHH patients with Sanger sequencing. A heterozygous variant inMIR200A(rs202051309; general population frequency of 0.02) was found in one patient. Our results suggest that mutations in the studied miRNAs are unlikely causes of cHH. However, the complex interplay between miRNAs and their target genes in these diseases requires further investigations.

Objective Congenital hypogonadotropic hypogonadism (CHH) is associated with impaired bone mineral density in adulthood, whereas the estimates on bone structure in adolescents with CHH has not been previously evaluated. Design This cross-sectional study describes bone structure in CHH patients and compares it to that in boys with constitutional delay of growth and puberty (CDGP). Methods Peripheral quantitative computed tomography (pQCT) of non-dominant arm and left leg were performed in five treatment-naive males diagnosed with CHH and in 24 males with CDGP. Volumetric bone mineral density (BMD), bone mineral content, and area in trabecular and cortical bone compartments were evaluated, and bone age-adjusted Z-scores for the bone parameters were determined. Results The participants with CHH had more advanced bone age and were older, taller, and heavier than the CDGP boys, yet they had lower trabecular BMD in distal radius (147.7 mg/mm3 [95%CI, 128-168 mg/mm3 ] vs. 181.2 mg/mm3 [172-192 mg/mm3 ], p=0.002) and distal tibia (167.6 mg/mm3 [145-190 mg/mm3 ] vs. 207.2 mg/mm3 [187-227 mg/mm3 ], p=0.012), respectively. CHH males had lower cortical thickness at diaphyseal tibia than the participants with CDGP (p=0.001). These between-group differences remained significant in corresponding Z-scores adjusted for bone age and height (p=0.001). In CDGP group, serum testosterone correlated positively with trabecular BMD (r=0.51, p=0.013) at distal radius, and estradiol levels correlated positively with trabecular BMD at the distal site of tibia (r=0.58, p=0.004). Conclusions Five treatment-naive male patients with CHH exhibited poorer trabecular BMD than untreated males with CDGP. We speculate that timely low-dose sex steroid replacement in CHH males may benefit skeletal health in adulthood.

Background Paternally inherited loss-of-function mutations in MKRN3 underlie central precocious puberty (CPP). We describe clinical and genetic features of CPP patients with paternally inherited MKRN3 mutations in two independent families. Methods The single coding exon of MKRN3 was analyzed in three patients with CPP and their family members, followed by segregation analyses. Additionally, we report the patients' responses to GnRH analog treatment. Results A paternally inherited novel heterozygous c.939C>G, p.(Ile313Met) missense mutation affecting the RING finger domain of MKRN3 was found in a Finnish girl with CPP (age at presentation 6 years). Two Polish siblings (a girl presenting with B2 at the age of 4 years and a boy with adult size testes at the age of 9 years) had inherited a novel heterozygous MKRN3 mutation c.1237_1252delGGAGACACATGCTTTT p.(Gly413Thrfs*63) from their father. The girls were treated with GnRH analogs, which exhibited suppression of the hypothalamic-pituitary-gonadal axis. In contrast, the male patient was not treated, yet he reached his target height. Conclusions We describe two novel MKRN3 mutations in three CPP patients. The first long-term data on a boy with CPP due to an MKRN3 mutation questions the role of GnRH analog treatment in augmenting adult height in males with this condition. Impact We describe the genetic cause for central precocious puberty (CPP) in two families. This report adds two novel mutations to the existing literature. One of the mutations, p.(Ile313Met) affects the RING finger domain of MKRN3, which has been shown to be important for repressing the promoter activity of and . MKRN3KISS1TAC3We describe the first long-term observation of a male patient with CPP due to a paternally inherited MKRN3 loss-of-function mutation. Without GnRH analog treatment, he achieved an adult height that was in accordance with his mid-parental target height.

Objective: To describe the etiology of severe short stature in the Helsinki University Hospital district covering a population of 1.2 million that is subject to frequent growth monitoring and screening rules during childhood. Design: Retrospective cohort study. Methods: We identified all subjects born 1990 or later with a height SD score Results: A pathological cause for short stature was diagnosed in 76% of the girls and 71% of the boys (P = NS). Syndromes were the most numerous pathological cause (n = 160; 20%), followed by organ disorders (n = 127; 16%), growth hormone deficiency (GHD, n = 94; 12%), SGA without catch-up growth (n = 73; 9%), and skeletal dysplasias (n = 57; 7%). Idiopathic short stature (ISS) was diagnosed in 210 (27%) subjects. The probability of growth-related pathology, particularly of a syndrome or skeletal dysplasia, increased with the shorter height SD score and the greater deviation from the target height. Sitting height to height SDS was increased in subjects with ISS, GHD, and SGA (all P < 0.01). Conclusions: Height

Purpose: To evaluate short-term testicular outcome after torsion in children. Methods: Fifty-four children and adolescents were evaluated after 6 months of the operation for testicular torsion. Testicular volume was measured and circulating Inhibin B, FSH, LH and testosterone levels were checked. Results: Delay from the onset of symptoms to surgery was shorter in the orchidopexy group (n = 47), than in the orchiectomy group (n = 7, p = 0.001). In the orchidopexy group, the median volume of the affected testis was 83% (IQR 43-104) of the contralateral testis (p = 0.002). The plasma hormone levels in orchidopexy and orchiectomy groups were: 148 ng/l (IQR 108-208) vs. 129 ng/l (IQR, 123-138, p = 0.269) for Inhibin B; 4.5 IU/L (IQR2.6-6.9) vs. 11.7 IU/L (IQR 4.3-12.8, p = 0.037) for FSH; 2.9 IU/L (IQR 1.3-3.7) vs. 4.8 (IQR 3.0-5.6, p = 0.066) for LH; and 13.6 nM(IQR 6.5-18.0) vs. 14.5 nM(IQR 6.7-15.9, p = 0.834) for testosterone. The association between FSH, LH as well as testosterone levels was most clear with the volume of the contralateral testis (Rho = 0.574, p

STUDY QUESTIONDoes treatment of constitutional delay of growth and puberty (CDGP) in boys with aromatase inhibitor letrozole (Lz) or conventional low-dose testosterone (T) have differing effects on developing seminiferous epithelium?SUMMARY ANSWERAnti-Müllerian hormone (AMH) declined similarly in both treatment groups, and the two Sertoli cell-derived markers (AMH and inhibin B (iB)) exhibited differing responses to changes in gonadotrophin milieu.WHAT IS KNOWN ALREADYBoys with CDGP may benefit from puberty-inducing medication. Peroral Lz activates gonadotrophin secretion, whereas intramuscular low-dose T may transiently suppress gonadotrophins and iB.STUDY DESIGN, SIZE, DURATIONSera of 28 boys with CDGP who participated in a randomised, controlled, open-label trial at four paediatric centres in Finland between August 2013 and January 2017 were analysed. The patients were randomly assigned to receive either Lz (2.5 mg/day) (n = 15) or T (1 mg/kg/month) (n = 13) for 6 months.PARTICIPANTS/MATERIALS, SETTING, METHODSThe 28 patients were at least 14 years of age, showed first signs of puberty, wanted medical attention for CDGP and were evaluated at 0, 3, 6 and 12 months of visits. AMH levels were measured with an electrochemiluminescence immunoassay and Lz levels with liquid chromatography coupled with tandem mass spectrometry.MAIN RESULTS AND THE ROLE OF CHANCEAMH levels decreased in both treatment groups during the 12-month follow-up (P LIMITATIONS, REASONS FOR CAUTIONThe original trial was not blinded for practical reasons and included a limited number of participants.WIDER IMPLICATIONS OF THE FINDINGSIn early puberty, treatment-induced gonadotrophin stimulus was unable to counteract the androgen-mediated decrease in AMH, while changes in iB levels were associated with changes in gonadotrophin levels. AMH decreased similarly in both groups during the treatment, reassuring safety of developing seminiferous epithelium in both treatment approaches. Since a fixed dose of Lz induced variable serum Lz levels with a desired puberty-promoting effect in all boys, more research is needed to aim at a minimal efficient dose per weight.STUDY FUNDING/COMPETING INTEREST(S)This study was supported by the Academy of Finland, the Foundation for Pediatric Research, the Emil Aaltonen Foundation, Sigrid Juselius Foundation and Helsinki University Hospital Research Funds. The authors have nothing to disclose.TRIAL REGISTRATION NUMBERNCT01797718

Context Congenital pituitary hormone deficiencies with syndromic phenotypes and/or familial occurrence suggest genetic hypopituitarism; however, in many such patients the underlying molecular basis of the disease remains unknown. Objective To describe patients with syndromic hypopituitarism due to biallelic loss-of-function variants in TBC1D32, a gene implicated in Sonic Hedgehog (Shh) signaling. Setting Referral center. Patients A Finnish family of 2 siblings with panhypopituitarism, absent anterior pituitary, and mild craniofacial dysmorphism, and a Pakistani family with a proband with growth hormone deficiency, anterior pituitary hypoplasia, and developmental delay. Interventions The patients were investigated by whole genome sequencing. Expression profiling of TBC1D32 in human fetal brain was performed through in situ hybridization. Stable and dynamic protein-protein interaction partners of TBC1D32 were investigated in HEK cells followed by mass spectrometry analyses. Main Outcome Measures Genetic and phenotypic features of patients with biallelic loss-of-function mutations in TBC1D32. Results The Finnish patients harboured compound heterozygous loss-of-function variants (c.1165_1166dup p.(Gln390Phefs*32) and c.2151del p.(Lys717Asnfs*29)) in TBC1D32; the Pakistani proband carried a known pathogenic homozygous TBC1D32 splice-site variant c.1372 + 1G > A p.(Arg411_Gly458del), as did a fetus with a cleft lip and partial intestinal malrotation from a terminated pregnancy within the same pedigree. TBC1D32 was expressed in the developing hypothalamus, Rathke’s pouch, and areas of the hindbrain. TBC1D32 interacted with proteins implicated in cilium assembly, Shh signaling, and brain development. Conclusions Biallelic TBC1D32 variants underlie syndromic hypopituitarism, and the underlying mechanism may be via disrupted Shh signaling.

Gonadotropin-releasing hormone (GnRH) neurons provide a fundamental signal for the onset of puberty and subsequent reproductive functions by secretion of gonadotropin-releasing hormone. Their disrupted development or function leads to congenital hypogonadotropic hypogonadism (CHH). To model the development of human GnRH neurons, we generated a stable GNRH1-TdTomato reporter cell line in human pluripotent stem cells (hPSCs) using CRISPR-Cas9 genome editing. RNA-sequencing of the reporter clone, differentiated into GnRH neurons by dual SMAD inhibition and FGF8 treatment, revealed 6461 differentially expressed genes between progenitors and GnRH neurons. Expression of the transcription factor ISL1, one of the top 50 most upregulated genes in the TdTomato-expressing GnRH neurons, was confirmed in 10.5 gestational week-old human fetal GnRH neurons. Among the differentially expressed genes, we detected 15 genes that are implicated in CHH and several genes that are implicated in human puberty timing. Finally, FGF8 treatment in the neuronal progenitor pool led to upregulation of 37 genes expressed both in progenitors and in TdTomato-expressing GnRH neurons, which suggests upstream regulation of these genes by FGF8 signaling during GnRH neuron differentiation. These results illustrate how hPSC-derived human GnRH neuron transcriptomic analysis can be utilized to dissect signaling pathways and gene regulatory networks involved in human GnRH neuron development. This article has an associated First Person interview with the first author of the paper., Summary: Here, we generated a GNRH1-reporter cell line in hPSCs and investigated transcriptomes of GNRH1-expressing neurons and their progenitors, potentially leading to validation of new genes related to GnRH neuron function.

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disorder characterized by infertility and the absence of puberty. Defects in GnRH neuron migration or altered GnRH secretion and/or action lead to a severe gonadotropin-releasing hormone (GnRH) deficiency. Given the close developmental association of GnRH neurons with the olfactory primary axons, CHH is often associated with anosmia or hyposmia, in which case it is defined as Kallmann syndrome (KS). The genetics of CHH are heterogeneous, and >40 genes are involved either alone or in combination. Several CHH-related genes controlling GnRH ontogeny encode proteins containing fibronectin-3 (FN3) domains, which are important for brain and neural development. Therefore, we hypothesized that defects in other FN3-superfamily genes would underlie CHH. Next-generation sequencing was performed for 240 CHH unrelated probands and filtered for rare, protein-truncating variants (PTVs) in FN3-superfamily genes. Compared to gnomAD controls the CHH cohort was statistically enriched for PTVs in neuron-derived neurotrophic factor (NDNF) (p = 1.40 x 10(-6)). Three heterozygous PTVs (p.Lys62*, p.Tyr128Thrfs*55, and p.Trp469*, all absent from the gnomAD database) and an additional heterozygous missense mutation (p.Thr201Ser) were found in four KS probands. Notably, NDNF is expressed along the GnRH neuron migratory route in both mouse embryos and human fetuses and enhances GnRH neuron migration. Further, knock down of the zebrafish ortholog of NDNF resulted in altered GnRH migration. Finally, mice lacking Ndnf showed delayed GnRH neuron migration and altered olfactory axonal projections to the olfactory bulb; both results are consistent with a role of NDNF in GnRH neuron development. Altogether, our results highlight NDNF as a gene involved in the GnRH neuron migration implicated in KS.

Background We describe the phenotypic spectrum and timing of diagnosis and management in a large series of patients with disorders of sexual development (DSD) treated in a single pediatric tertiary center. Methods DSD patients who had visited our tertiary center during the survey period (between 2004 and 2014) were identified based on an ICD-10 inquiry, and their phenotypic and molecular genetic findings were recorded from patient charts. Results Among the 550 DSD patients, 53.3% had 46,XY DSD; 37.1% had sex chromosome DSD and 9.6% had 46,XX DSD. The most common diagnoses were Turner syndrome (19.8%, diagnosed at the mean age of 4.7 ± 5.5 years), Klinefelter syndrome (14.5%, 6.8 ± 6.2 years) and bilateral cryptorchidism (23.1%). Very few patients with 46,XY DSD (7%) or 46,XX DSD (21%) had molecular genetic diagnosis. The yearly rate of DSD diagnoses remained stable over the survey period. After the release of the Nordic consensus on the management of undescended testes, the age at surgery for bilateral cryptorchidism declined significantly (P Conclusions Our results show that (i) Turner syndrome and Klinefelter syndrome, the most frequent single DSD diagnoses, are still diagnosed relatively late; (ii) a temporal shift was observed in the management of bilateral cryptorchidism, which may favorably influence patients’ adulthood semen quality and (iii) next-generation sequencing methods are not fully employed in the diagnostics of DSD patients.

Study question What is the peripubertal outcome of recombinant human FSH (r-hFSH) treatment during minipuberty in boys with congenital hypogonadotropic hypogonadism (CHH)? Summary answer Sertoli-cell response to r-hFSH, given during the minipuberty of infancy, appears insufficient to maintain Sertoli cell function throughout childhood, as evaluated by inhibin B measurements. What is known already Severe CHH in boys can be diagnosed during the minipuberty of infancy. Combined gonadotropin treatment at that age is suggested to improve testicular endocrine function and future fertility, yet long-term evidence is lacking. Study design, size, duration In this retrospective cohort study, we describe five CHH boys treated with r-hFSH in Helsinki University Hospital or Kuopio University Hospital between 2004 and 2018. Immediate follow-up data (0.1-1.4 months after cessation of the gonadotropin therapy) was available for four boys and long-term observations (at the age of 10.0-12.8 years) was available for three boys. As a retrospective control cohort, we provide inhibin B values of eight untreated CHH boys at the age of 12.7-17.8 years. Participants/materials, setting, methods Four patients had combined pituitary hormone deficiency, and one had CHARGE syndrome due to a CHD7 mutation. The patients were treated at the age of 0.7-4.2 months with r-hFSH (3.4 IU/kg-7.5 IU/kg per week in 2 or 3 s.c. doses for 3-4.5 months) combined with T (25 mg i.m. monthly for three months for the treatment of micropenis). Inhibin B was chosen as the primary outcome measure. Main results and the role of chance During the r-hFSH + T treatment, inhibin B increased from 76 ± 18 ng/l to 176 ± 80 ng/l (P = 0.04) and penile length increased by 81 ± 50% (P = 0.04). Unexpectedly, two boys with robust inhibin B responses in infancy demonstrated low inhibin B values in peripuberty: declining from 290 ng/l (4 months) to 16 ng/l (12.4 years), and from 207 ng/l (6 months) to 21 ng/l (12.8 years). All boys underwent orchiopexy at 2.0 ± 0.7 years of age. Inhibin B values in long-term follow-up, available for the three boys, did not significantly differ from the untreated CHH controls. Limitations, reasons for caution Limitations of this retrospective study are the small number and heterogeneity of the patients and their treatment schemes. Wider implications of the findings We describe the first long-term follow-up data on CHH boys treated with r-hFSH and T as infants. The results from this small patient series suggest that the effects of infant r-hFSH treatment may be transient, and further longitudinal studies are required to determine the efficacy of this treatment approach to optimise the fertility potential in this patient population. Study funding/competing interest(s) This work was supported by the Finnish foundation for Pediatric Research, the Academy of Finland and the Emil Aaltonen Foundation. The authors have no competing interests. Trial registration number Non-applicable.

Background: Aromatase inhibitors (Als) have been used in boys with idiopathic short stature (ISS) to promote growth despite the lack of actual data regarding treatment effect on adult height. In this study, we characterized adult heights and long-term follow-up in Al-treated boys with ISS. Methods: Adult heights and long-term follow-up data, including spine MRIs, of a randomized, double-blind, placebo-controlled trial of boys who were treated with letrozole (Lz) (2.5 mg/d) or placebo (PI) for 2 years during prepuberty and early puberty. The mean bone ages at treatment cessation were 10.2 and 10.8 years, respectively. Results: Adult heights were similar between the boys treated with Lz (n = 10) and those who received PI (n = 10) (164.8 +/- 4.0 vs. 163.7 +/- 3.7 cm, p = 0.49, respectively). In either group, the adult heights did not differ from predicted adult heights at start of the study [PI: 163.7 (3.7) cm vs. 166.9 (3.3), p = 0.06; Lz: 164.8 (4.0) cm vs. 167.6 (7.9), p = 0.20, respectively]. Long-term follow-up data showed that the frequency of subjects with a vertebral deformity was similar between the groups (Lz, 29% and PI, 22%, p = 0.20), and no single comorbidity was clearly enriched in either group. Conclusions: The Lz-treated boys had similar adult heights with the subjects who received PI for 2 years, which indicates that the treatment is not beneficial when given to pre- or early-pubertal boys. Previously observed vertebral deformities ameliorated during follow-up, which supports the skeletal safety of Lz therapy in children and adolescents.

Paternally-inherited loss-of-function mutations in makorin ring finger protein 3 gene (MKRN3) underlie central precocious puberty. To investigate the puberty-related mechanism(s) of MKRN3 in humans, we generated two distinct bi-allelic MKRN3 knock-out human pluripotent stem cell lines, Del 1 and Del 2, and differentiated them into GNRH1-expressing neurons. Both Del 1 and Del 2 clones could be differentiated into neuronal progenitors and GNRH1-expressing neurons, however, the relative expression of GNRH1 did not differ from wild type cells (P = NS). Subsequently, we investigated stable and dynamic protein-protein interaction (PPI) partners of MKRN3 by stably expressing it in HEK cells followed by mass spectrometry analyses. We found 81 high-confidence novel protein interaction partners, which are implicated in cellular processes such as insulin signaling, RNA metabolism and cell-cell adhesion. Of the identified interactors, 20 have been previously implicated in puberty timing. In conclusion, our stem cell model for generation of GNRH1 -expressing neurons did not offer mechanistic insight for the role of MKRN3 in puberty initiation. The PPI data, however, indicate that MKRN3 may regulate puberty by interacting with other puberty-related proteins. Further studies are required to elucidate the possible mechanisms and outcomes of these interactions.

Summary Gonadotropin-releasing hormone (GnRH) neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs) into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs) by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders., Graphical Abstract, Highlights • GnRH neurons regulate puberty and reproduction • We generated GnRH-expressing and secreting neurons from hPSCs • These neurons can be used to study diseases affecting the reproductive system, Raivio and colleagues have developed a protocol for differentiating GnRH-expressing neurons from hPSCs. The cells were first differentiated into NPCs and then with combined FGF8 stimulation and Notch inhibition to GnRH-secreting neural cells. This protocol will provide an important platform for investigating the development and function of the human hypothalamic-pituitary-gonadal axis.

IntroductionWe studied if motivational interviewing (MI) added to standard educational care (SEC) improves vascular health in adolescents with poorly controlled type 1 diabetes.Research design and methods47 adolescents with type 1 diabetes of at least 2 years duration and hemoglobin A1c >75 mmol/mol (>9.0%) on two visits were randomized to MI+SEC or SEC. We also compared vascular health parameters of patients with type 1 diabetes at trial baseline with a group of healthy historical controls matched for age and body size.Results39 adolescents (20 MI+SEC) completed the vascular health study. At 12 months, parameter changes were not statistically significantly different between MI+SEC and SEC (carotid-femoral pulse wave velocity (cfPWV): mean difference 0.052 m/s (95% CI −0.395 to 0.500, p=0.81); carotid-radial PWV (crPWV): 0.118 m/s (95% to 0.478 to 0.713, p=0.69), carotid intima-media thickness (IMT): 0.002 mm (95% CI −0.37 to 0.40, p=0.93), systolic blood pressure (BP) z-score: 0.495 (95% CI −0.099 to 1.09, p=0.10). At baseline, duration of type 1 diabetes was associated with radial IMT (r=0.430, p=0.007) and cfPWV (r=0.373, p=0.018), and carotid, femoral and brachial IMT were correlated with continuous glucose monitoring (CGM) SD (r=0.440, p=0.017; r=0.377, p=0.048; r=0.387, p=0.038). There was an inverse association between CGM time-in-range (3.9–10.0 mmol/L) and crPWV (r=−0.476, p=0.022) changes. Systolic BP change was associated with body mass index change (r=0.374, p=0.019) and IMT change (r=0.461, p=0.016 for carotid IMT; r=0.498, p=0.010 for femoral IMT). PWVs were higher and common carotid compliance lower among patients with type 1 diabetes at baseline compared with healthy controls, but no other differences were found.ConclusionThere was no effect of MI added to SEC on vascular health parameters. Although disease duration and glycemic control were associated with vascular health at baseline, there were only limited associations between glycemic control and vascular health parameter changes. Vascular health parameter changes were interrelated suggesting clustering of cardiovascular risk.Trial registration numberNCT02637154.

Context Recombinant human FSH (r-hFSH), given to prepubertal boys with hypogonadotropic hypogonadism (HH), may induce Sertoli cell proliferation and thereby increase sperm-producing capacity later in life. Objective To evaluate the effects of r-hFSH, human chorionic gonadotropin (hCG), and testosterone (T) in such patients. Design and Setting Retrospective review in three tertiary centers in Finland between 2006 and 2016. Patients Five boys: ANOS1 mutation in two, homozygous PROKR2 mutation in one, FGFR1 mutation in one, and homozygous GNRHR mutation in one. Prepubertal testicular volume (TV) varied between 0.3 and 2.3 mL; three boys had micropenis, three had undergone orchidopexy. Interventions Two boys received r-hFSH (6 to 7 months) followed by r-hFSH plus hCG (33 to 34 months); one received T (6 months), then r-hFSH plus T (29 months) followed by hCG (25 months); two received T (3 months) followed by r-hFSH (7 months) or r-hFSH plus T (8 months). Main Outcome Measures TV, inhibin B, anti-Müllerian hormone, T, puberty, sperm count. Results r-hFSH doubled TV (from a mean ± SD of 0.9 ± 0.9 mL to 1.9 ± 1.7 mL; P < 0.05) and increased serum inhibin B (from 15 ± 5 ng/L to 85 ± 40 ng/L; P < 0.05). hCG further increased TV (from 2.1 ± 2.3 mL to 8.6 ± 1.7 mL). Two boys with initially extremely small testis size (0.3 mL) developed sperm (maximal sperm count range, 2.8 to 13.8 million/mL), which was cryopreserved. Conclusions Spermatogenesis can be induced with gonadotropins even in boys with HH who have extremely small testes, and despite low-dose T treatment given in early puberty. Induction of puberty with gonadotropins allows preservation of fertility.

Biallelic, partial loss-of-function mutations in GNRHR cause a wide spectrum of reproductive phenotypes from constitutional delay of growth and puberty to complete congenital hypogonadotropic hypogonadism. We studied the frequency of GNRHR, FGFR1, TAC3, and TACR3 mutations in nine adolescent and young adult females with clinical cues consistent with partial gonadotropin deficiency (stalled puberty, unexplained secondary amenorrhea), and describe phenotypic features and molecular genetic findings of monozygotic twin brothers with stalled puberty. Two girls out of nine (22%, 95% CI 6-55%) carried biallelic mutations in GNRHR. The girl with compound heterozygous c. 317A>G p.(Gln106Arg) and c. 924_926delCTT p.(Phe309del) GNRHR mutations displayed incomplete puberty and clinical signs of hypoestrogenism. The patient carrying a homozygous c. 785G> A p.(Arg262Gln) mutation presented with signs of hypoestrogenism and unexplained secondary amenorrhea. None of the patients exhibited mutations in FGFR1, TAC3, or TACR3. The twin brothers, compound heterozygous for GNRHR mutations c. 317A> G p.(Gln106Arg) and c. 785G>A p.(Arg262Gln), presented with stalled puberty and were discordant for weight, and the heavier of them had lower testosterone levels. These results suggest that genetic testing of the GNRHR gene should be offered to adolescent females with low-normal gonadotropins and unexplained stalled puberty or menstrual dysfunction. In male patients with partial gonadotropin deficiency, excess adipose tissue may suppress hypothalamic-pituitary-gonadal axis.

WDR11 has been implicated in congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS), human developmental genetic disorders defined by delayed puberty and infertility. However, WDR11’s role in development is poorly understood. Here we report that WDR11 modulates the Hedgehog (Hh) signalling pathway and is essential for ciliogenesis. Disruption of WDR11 expression in mouse and zebrafish results in phenotypic characteristics associated with defective Hh signalling, accompanied by dysgenesis of ciliated tissues.Wdr11null mice also exhibit early onset obesity. We found that WDR11 shuttles from the cilium to the nucleus in response to Hh signalling. WDR11 was also observed to regulate the proteolytic processing of GLI3 and cooperate with EMX1 transcription factor to induce the expression of downstream Hh pathway genes and gonadotrophin releasing hormone production. The CHH/KS-associated human mutations result in loss-of-function of WDR11. Treatment with the Hh agonist purmorphamine partially rescued the WDR11-haploinsufficiency phenotypes. Our study reveals a novel class of ciliopathy caused by WDR11 mutations and suggests that CHH/KS may be a part of the human ciliopathy spectrum.

Radial glial cells play an essential role through their function as guides for neuronal migration during development. Disruption of metabotropic glutamate receptor 5 (mGluR5) function retards the growth of radial glial processes in vitro. Neuregulins (NRG) are activated by proteolytic cleavage and regulate (radial) glial maintenance via ErbB3/ErbB4 receptors. We show here that blocking ErbB4 disrupts radial process extension. Soluble NRG acting on ErbB4 receptors is able to promote radial process extension in particular where process elongation has been impeded by blockade of mGluR5, the nonselective cation channel canonical transient receptor potential 3 (TRPC3), or matrix metalloproteases (MMP). NRG does not restore retarded process growth caused by ErbB4 blockade. Stimulation of muscarinic receptors restores process elongation due to mGluR5 blockade but not that caused by TRPC3, MMP or ErbB4 blockade suggesting that muscarinic receptors can replace mGluR5 with respect to radial process extension. Additionally, NRG/ErbB4 causes Ca2+ mobilization in a population of cells through cooperation with ErbB1 receptors. Our results indicate that mGluR5 promotes radial process growth via NRG activation by a mechanism involving TRPC3 channels and MMPs. Thus neurotransmitters acting on G-protein coupled receptors could play a central role in the maintenance of the radial glial scaffold through activation of NRG/ErbB4 signaling.

Animal studies have suggested that angiopoietin-like 4 (Angptl4) regulates adiposity through central and peripheral mechanisms. The aim of this study was to investigate whether serum concentration and adipose tissue expression of Angptl4 are associated with obesity-related parameters in humans. Altogether, 75 dizygotic (DZ) and 46 monozygotic (MZ) twin pairs were studied, from the FinnTwin12 and FinnTwin16 cohorts. Among the MZ pairs, 21 were discordant for body mass index (BMI) (intra-pair BMI-difference >2.5 kg/m², age 23-33 years). Serum Angptl4 (s-Angptl4) levels were measured by ELISA, and adipose tissue gene expression was analyzed by genome-wide transcript profiling. In MZ twin pairs discordant for BMI, s-Angptl4 and adipose tissue ANGPTL4 mRNA (at-ANGPTL4) levels were significantly decreased (P = 0.04 and P = 0.03, respectively) in obese twins as compared with their nonobese cotwins. In all twins, intra-pair differences in s-Angptl4 levels were inversely correlated with intra-pair differences in BMI (r = -0.27, P = 0.003). In individual MZ twins, at-ANGPTL4 expression was inversely correlated with BMI (r = -0.44, P = 0.001) and positively correlated with at-LIPE (r = 0.24, P = 0.01) and at-ABHD5 (r = 0.41, P = 0.005) expression. Our results demonstrated that variation in Angptl4 concentration was only modestly accounted for by genetic factors and suggest a role for Angptl4 in acquired obesity in humans.

Human embryonic stem cells (hESCs) are able to proliferatein vitroindefinitely without losing their ability to differentiate into multiple cell types upon exposure to appropriate signals. Particularly, the ability of hESCs to differentiate into neuronal subtypes is fundamental to develop cell-based therapies for several neurodegenerative disorders, such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In this study, we differentiated hESCs to dopaminergic neurons via an intermediate stage, neural progenitor cells (NPCs). hESCs were induced to neural progenitor cells by Dorsomorphin, a small molecule that inhibits BMP signalling. The resulting neural progenitor cells exhibited neural bipolarity with high expression of neural progenitor genes and possessed multipotential differentiation ability. CBF1 and bFGF responsiveness of these hES-NP cells suggested their similarity to embryonic neural progenitor cells. A substantial number of dopaminergic neurons were derived from hES-NP cells upon supplementation of FGF8 and SHH, key dopaminergic neuron inducers. Importantly, multiple markers of midbrain neurons were detected, includingNURR1, PITX3, andEN1, suggesting that hESC-derived dopaminergic neurons attained the midbrain identity. Altogether, this work underscored the generation of neural progenitor cells that retain the properties of embryonic neural progenitor cells. These cells will serve as an unlimited source for the derivation of dopaminergic neurons, which might be applicable for treating patients with Parkinson’s disease.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSC) by the forced expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Pluripotent reprogramming appears as a slow and inefficient process because of genetic and epigenetic barriers of somatic cells. In this report, we have extended previous observations concerning donor age and passage number of human fibroblasts as critical determinants of the efficiency of iPSC induction. Human fibroblasts from 11 different donors of variable age were reprogrammed by ectopic expression of reprogramming factors. Although all fibroblasts gave rise to iPSC colonies, the reprogramming efficiency correlated negatively and declined rapidly with increasing donor age. In addition, the late passage fibroblasts gave less reprogrammed colonies than the early passage cell counterparts, a finding associated with the cellular senescence-induced upregulation of p21. Knockdown of p21 restored iPSC generation even in long-term passaged fibroblasts of an old donor, highlighting the central role of the p53/p21 pathway in cellular senescence induced by both donor age and culture time. (C) 2015 The Authors. Published by Elsevier B.V.

BACKGROUND: Congenital hypogonadotropic hypogonadism (HH) is a rare cause for delayed or absent puberty. These patients may recover from HH spontaneously in adulthood. To date, it is not possible to predict who will undergo HH reversal later in life. Herein we investigated whether Finnish patients with reversal of congenital hypogonadotropic hypogonadism (HH) have common phenotypic or genotypic features. METHODS AND FINDINGS: Thirty-two male HH patients with anosmia/hyposmia (Kallmann Syndrome, KS; n = 26) or normal sense of smell (nHH; n = 6) were enrolled (age range, 18-61 yrs). The patients were clinically examined, and reversal of HH was assessed after treatment withdrawal. KAL1, FGFR1, FGF8, PROK2, PROKR2, CHD7, WDR11, GNRHR, GNRH1, KISS1R, KISS1, TAC3, TACR3, and LHβ were screened for mutations. Six HH patients (2 KS, 4 nHH) were verified to have reversal of HH. In the majority of cases, reversal occurred early in adulthood (median age, 23 yrs; range, 21-39 yrs). All had spontaneous testicular growth while on testosterone replacement therapy (TRT). One nHH subject was restarted on TRT due to a decline in serum T. Two reversal variants had a same GNRHR mutation (R262Q), which was accompanied by another GNRHR mutation (R139H or del309F). In addition, both of the KS patients had a mutation in CHD7 (p.Q51X) or FGFR1 (c.91+2T>A). CONCLUSIONS: Considerable proportion of patients with HH (8% of KS probands) may recover in early adulthood. Spontaneous testicular enlargement during TRT was highly suggestive for reversal of HH. Those with the GNRHR mutation R262Q accompanied by another GNRHR mutation may be prone to reversal, although even patients with a truncating mutation in CHD7 or a splice-site mutation in FGFR1 can recover. We recommend that all adolescents and young adults with congenital HH should be informed on the possibility of reversal.

AIM: To study whether high-sensitivity C-reactive protein (hs-CRP) measurement can aid the assessment of disease activity and glucocorticoid treatment in paediatric inflammatory bowel disease (IBD). METHODS: CRP levels were measured in 39 children with IBD undergoing colonoscopy [median age 12.8 years, Crohn’s disease (CD) n = 20], in 22 other children with IBD followed for acute response to glucocorticoids, and in 33 paediatric non-IBD patients. When standard CRP level was below detection limit (< 5 mg/L), hs-CRP was analyzed. RESULTS: Sixty-four percent (25/39) of the children with IBD undergoing colonoscopy displayed undetectable (< 5 mg/L) standard CRP levels. Of these, the hs-CRP measurement could not differentiate between active (median, 0.2 mg/L, range, 0.007-1.37, n = 17) or quiescent (0.1 mg/L, 0.01-1.89, n = 8, P = NS) disease. Patients with ileocolonic CD had higher CRP levels (14 mg/L, 0.06-45, n = 13) than patients with no ileal involvement (0.18 mg/L, 0.01-9, n = 7, P < 0.01) or ulcerative colitis (UC) (0.13 mg/L, 0.007-23, P < 0.05). In children with active IBD treated with systemic glucocorticoids, the standard CRP was undetectable in 59% of the patients. The hs-CRP levels did not differ between patients that responded to steroid therapy and in non-responders. CONCLUSION: The measurement of hs-CRP did not prove useful in the assessment of disease activity or glucocorticoid treatment in paediatric IBD patients that had undetectable standard CRP.