New liquid chromatography tandem mass spectrometry reference data for estradiol show mini-puberty in both sexes and typical pre-pubertal and pubertal patterns.

CONTEXT: Reliable estradiol (E2) reference intervals (RIs) are crucial in pediatric endocrinology. OBJECTIVES: This study aims to develop a sensitive ultra-performance liquid chromatographic tandem mass spectrometry (UPLC-MS/MS) method for E2 in serum, to establish graphically represented RI percentiles and annual RIs for both sexes, and to perform a systematic literature comparison. METHODS: First, a UPLC-MS/MS method for E2 was developed. Second, graphically represented RI percentiles and annual RIs covering 0-18 years were computed (cohort of healthy children [1181 girls and 543 boys]). Subsequently, RIs were compared with published data by systematic searches. RESULTS: Lower limit of quantification was 11 pmol/L, indicating high sensitivity. Estradiol first peaked during mini-puberty in both sexes (girls up to 192 pmol/L; boys up to 225 pmol/L). As could be expected, girls showed higher pubertal E2 (up to 638 pmol/L). However, boys' RIs (up to 259 pmol/L) overlapped considerably. We found 4 studies in the literature that also used LC-MS/MS to determine E2 and published RIs for the complete pediatric age range. Reference intervals varied considerably. Pre-pubertal and pubertal phases were present in all studies. Higher E2 during the time of mini-puberty in both sexes was documented in 3 studies including ours. CONCLUSIONS: Variability of RIs for E2 between studies illustrates the importance of laboratory-specific RIs despite using a LC-MS/MS reference method. In boys, the striking E2 peak during mini-puberty as well as high pubertal E2 without phenotypic estrogenization in regular male puberty indicates that the role of E2 in children and, especially in boys, requires better functional understanding.

An overview of the outreach of the 2019-2021 Endo-ERN knowledge generation webinars.

The current study aims to assess the development of the knowledge generation program of the European Reference Network on Rare Endocrine Conditions (Endo-ERN) from its start in 2019 until December 2021, with special reference to webinars. We analyzed the number of webinars and live/postevent participants and whether participation and engagement of the attendees changed over time. A total of 30 (86%) self-prepared webinars comprising 300 h of knowledge and competence sharing were broadcasted (2019 - 3; 2020 - 13; 2021 - 14). A total of six webinars were broadcasted live prior to the coronavirus disease 2019 pandemic (https://endo-ern.eu/events/webinars/). The most active main thematic group (MTG) was MTG3 Genetic Disorders of Glucose and Insulin Homeostasis with eight (27%) webinars. Two (25%) MTGs fulfilled the goal to prepare at least two to three webinars per year. Patients were actively involved in 20% of the accounted webinars as both creators and presenters. The total number of live and postevent participants was 3023. The availability of the webinars after the live broadcast increased their outreach with a larger number of postevent viewers (n = 1629, 54%). Within the formal structured evaluation of the webinars, 40-85% of the participants replied on separate occasions and helped improve content. The free webinar access is among the perceived reasons for the rapidly increasing number of total hits to the Endo-ERN website. In conclusion, for its short existence, the Endo-ERN rapidly developed educational outreach, and further efforts to attract creators and learners are warranted.

DHX37 and NR5A1 Variants Identified in Patients with 46,XY Partial Gonadal Dysgenesis.

The group of disorders known as 46,XY gonadal dysgenesis (GD) is characterized by anomalies in testis determination, including complete and partial GD (PGD) and testicular regression syndrome (TRS). Several genes are known to be involved in sex development pathways, however approximately 50% of all cases remain elusive. Recent studies have identified variants in DHX37, a gene encoding a putative RNA helicase essential in ribosome biogenesis and previously associated with neurodevelopmental disorders, as a cause of PGD and TRS. To investigate the potential role of DHX37 in disorders of sexual development (DSD), 25 individuals with 46,XY DSD were analyzed and putative pathogenic variants were found in four of them. WES analyses were performed on these patients. In DHX37, the variant p.(Arg308Gln), recurrent associated with DSD, was identified in one patient; the p.(Leu467Val), predicted to be deleterious, was found together with an NR5A1 loss-of-function variant in patient 2; and, the p.(Val999Met) was identified in two unrelated patients, one of whom (patient 3) also carried a pathogenic NR5A1 variant. For both patients carrying DHX37 and NR5A1 pathogenic variants, a digenic inheritance is suggested. Our findings support the importance of DHX37 variants as a cause of disorders of sex development, implying a role in testis development.

Effects of Burosumab Treatment on Mineral Metabolism in Children and Adolescents With X-linked Hypophosphatemia.

CONTEXT: Burosumab has been approved for the treatment of children and adults with X-linked hypophosphatemia (XLH). Real-world data and evidence for its efficacy in adolescents are lacking. OBJECTIVE: To assess the effects of 12 months of burosumab treatment on mineral metabolism in children (aged <12 years) and adolescents (aged 12-18 years) with XLH. DESIGN: Prospective national registry. SETTING: Hospital clinics. PATIENTS: A total of 93 patients with XLH (65 children, 28 adolescents). MAIN OUTCOME MEASURES: Z scores for serum phosphate, alkaline phosphatase (ALP), and renal tubular reabsorption of phosphate per glomerular filtration rate (TmP/GFR) at 12 months. RESULTS: At baseline, patients showed hypophosphatemia (-4.4 SD), reduced TmP/GFR (-6.5 SD), and elevated ALP (2.7 SD, each P < .001 vs healthy children) irrespective of age, suggesting active rickets despite prior therapy with oral phosphate and active vitamin D in 88% of patients. Burosumab treatment resulted in comparable increases in serum phosphate and TmP/GFR in children and adolescents with XLH and a steady decline in serum ALP (each P < .001 vs baseline). At 12 months, serum phosphate, TmP/GFR, and ALP levels were within the age-related normal range in approximately 42%, 27%, and 80% of patients in both groups, respectively, with a lower, weight-based final burosumab dose in adolescents compared with children (0.72 vs 1.06 mg/kg, P < .01). CONCLUSIONS: In this real-world setting, 12 months of burosumab treatment was equally effective in normalizing serum ALP in adolescents and children, despite persistent mild hypophosphatemia in one-half of patients, suggesting that complete normalization of serum phosphate is not mandatory for substantial improvement of rickets in these patients. Adolescents appear to require lower weight-based burosumab dosage than children.

Transition for adolescents with a rare disease: results of a nationwide German project.

PURPOSE: The transition process from paediatric/adolescent to adult medical care settings is of utmost importance for the future health of adolescents with chronic diseases and poses even more difficulties in the context of rare diseases (RDs). Paediatric care teams are challenged to deliver adolescent-appropriate information and structures. Here we present a structured transition pathway which is patient-focused and adoptable for different RDs. METHODS: The transition pathway for adolescents 16 years and older was developed and implemented as part of a multi-centre study in 10 university hospitals in Germany. Key elements of the pathway included: assessment of patients' disease-related knowledge and needs, training/educational and counselling sessions, a structured epicrisis and a transfer appointment jointly with the paediatric and adult specialist. Specific care coordinators from the participating university hospitals were in charge of organization and coordination of the transition process. RESULTS: Of a total of 292 patients, 286 completed the pathway. Deficits in disease-specific knowledge were present in more than 90% of participants. A need for genetic or socio-legal counselling was indicated by > 60%. A mean of 2.1 training sessions per patient were provided over a period of almost 1 year, followed by the transfer to adult care in 267 cases. Twelve patients remained in paediatric care as no adult health care specialist could be identified. Targeted training and counselling resulted in improved disease-specific knowledge and contributed to empowering of patients. CONCLUSION: The described transition pathway succeeds to improve health literacy in adolescents with RDs and can be implemented by paediatric care teams in any RD specialty. Patient empowerment was mainly achieved by individualized training and counselling.

Pubertal and Gonadal Outcomes in 46,XY Individuals with Partial Androgen Insensitivity Syndrome Raised as Girls.

INTRODUCTION: Although it was common in the 1970s-1990s to assign female gender of rearing to 46,XY infants with limited virilization of varying etiologies, including those with partial androgen insensitivity syndrome (PAIS), long-term data on outcomes for these individuals are sparse. Therefore, our goal was to use the power of an international registry to evaluate clinical features, surgical management, and pubertal data in patients with a molecularly confirmed diagnosis of PAIS who were born before 2008 and were raised as girls. METHODS: The current study interrogated the International Disorders of Sex Development Registry for available data on management and pubertal outcomes in individuals with genetically confirmed PAIS who were raised as girls. RESULTS: Among the 11 individuals who fulfilled the key criteria for inclusion, the external masculinization score (EMS) at presentation ranged from 2 to 6 (median 5); 7 girls underwent gonadectomy before the age of 9 years, whereas 4 underwent gonadectomy in the teenage years (≥ age 13). Clitoral enlargement at puberty was reported for 3 girls (27%) who presented initially at the time of puberty with intact gonads. In the 9 individuals (82%) for whom gonadal pathology data were provided, there was no evidence of germ cell tumor at median age of 8.1 years. All girls received estrogen replacement, and 8/11 had attained Tanner stage 4-5 breast development at the last assessment. CONCLUSION: In general, although it appears that female assignment in PAIS is becoming uncommon, our data provide no evidence to support the practice of prophylactic prepubertal gonadectomy with respect to the risk of a germ cell tumor.

Persistence of foetal testicular features in patients with defective androgen signalling.

OBJECTIVE: Congenital defects of androgen synthesis or action in 46,XY individuals can result in impaired virilisation, despite the apparent testicular development. In a recent case, report of a young adult with complete androgen insensitivity syndrome (CAIS), tumourous gonadal tissue was shown to express HSD17B3 in Sertoli cells (SCs) and not in Leydig cells (LCs). This expression pattern differs from the typical adult human testis and resembles a foetal mouse testis, suggesting an underlying testicular development and function defect. Here, we investigate the effect of altered androgen signalling in gonads from five 46,XY individuals with defects in androgen synthesis or action. METHODS: Gonadal tissue sections from four patients with CAIS, one with CYP17A1 deficiency, and one control were immunostained for LC developmental and steroidogenic markers. The expression of some of these markers during development was investigated by reanalysing previously published single-cell RNA sequencing (scRNA-seq) data from normal human testicular tissues. RESULTS: All gonadal tissues from the patients show an exclusive expression of HSD17B3 in SCs and an expression of the foetal/immature LC marker DLK1 in a subset of LCs, suggesting an androgen-dependent differentiation defect of adult SCs and LCs. Furthermore, reanalysis of scRNA-seq data reveals an expression of HSD17B3 in foetal and neonatal SCs that is downregulated in adult SCs. CONCLUSIONS: Androgen signalling may affect the differentiation of adults, but possibly not foetal SCs or LCs, and may induce a shift of testosterone production from the tubular compartment in the foetal phase to the interstitial compartment in the adult phase.

Disruption of the topologically associated domain at Xp21.2 is related to 46,XY gonadal dysgenesis.

BACKGROUND: Duplications at the Xp21.2 locus have previously been linked to 46,XY gonadal dysgenesis (GD), which is thought to result from gene dosage effects of NR0B1 (DAX1), but the exact disease mechanism remains unknown. METHODS: Patients with 46,XY GD were analysed by whole genome sequencing. Identified structural variants were confirmed by array CGH and analysed by high-throughput chromosome conformation capture (Hi-C). RESULTS: We identified two unrelated patients: one showing a complex rearrangement upstream of NR0B1 and a second harbouring a 1.2 Mb triplication, including NR0B1. Whole genome sequencing and Hi-C analysis revealed the rewiring of a topological-associated domain (TAD) boundary close to NR0B1 associated with neo-TAD formation and may cause enhancer hijacking and ectopic NR0B1 expression. Modelling of previous Xp21.2 structural variations associated with isolated GD support our hypothesis and predict similar neo-TAD formation as well as TAD fusion. CONCLUSION: Here we present a general mechanism how deletions, duplications or inversions at the NR0B1 locus can lead to partial or complete GD by disrupting the cognate TAD in the vicinity of NR0B1. This model not only allows better diagnosis of GD with copy number variations (CNVs) at Xp21.2, but also gives deeper insight on how spatiotemporal activation of developmental genes can be disrupted by reorganised TADs causing impairment of gonadal development.

Starting point for benchmarking outcomes and reporting of pituitary adenoma surgery within the European Reference Network on Rare Endocrine Conditions (Endo-ERN): results from a meta-analysis and survey study.

Objective: The European Reference Network on Rare Endocrine Conditions (Endo-ERN) aims to organize high-quality healthcare throughout Europe, including care for pituitary adenoma patients. As surgery is the mainstay of treatment, we aimed to describe the current surgical practice and published surgical outcomes of pituitary adenoma within Endo-ERN. Design and Methods: Systematic review and meta-analysis of studies reporting surgical outcomes of pituitary adenoma patients within Endo-ERN MTG6 pituitary reference centers between 2010 and 2019. A survey was completed by reference centers on their current surgical practice. Results: A total of 18 out of 43 (42%) reference centers located in 7 of the 20 (35%) MTG6-represented countries published 48 articles. Remission rates were 50% (95% CI: 42-59) for patients with acromegaly, 68% (95% CI: 60-75) for Cushing's disease, and 53% (95% CI: 39-66%) for prolactinoma. Gross total resection was achieved in 49% (95% CI: 37-61%) of patients and visual improvement in 78% (95% CI: 68-87). Mortality, hemorrhage, and carotid injury occurred in less than 1% of patients. New-onset hypopituitarism occurred in 16% (95% CI: 11-23), transient diabetes insipidus in 12% (95% CI: 6-21), permanent diabetes insipidus in 4% (95% CI: 3-6), syndrome of inappropriate secretion of antidiuretic hormone (SIADH) in 9% (95% CI: 5-14), severe epistaxis in 2% (95% CI: 0-4), and cerebrospinal fluid leak in 4% (95% CI: 2-6). Thirty-five (81%) centers completed the survey: 54% were operated endoscopically and 57% were together with an ENT surgeon. Conclusion: The results of this study could be used as a first benchmark for the outcomes of pituitary adenoma surgery within Endo-ERN. However, the heterogeneity between studies in the reporting of outcomes hampers comparability and warrants outcome collection through registries.

Contexts of care for people with differences of sex development: Diversity is still missing in the laboratory routine.

The 2006 Chicago consensus statement of management of disorders/difference of sex development (DSD) has achieved advantages in clinical care and diagnosis for patients and families affect by DSD. This article provides a brief overview of contexts of care for physicians, and points out specific challenges in clinical practice that have arisen from the transformations of the sex/gender system in recent years. We focus on the impact of diagnosis and laboratory measurements. Both laboratory measurements and hormonal therapies still depend on the binary system. One problem is the lack of reference intervals for the different forms of DSD, which means that diversity is often neglected. In the following, we will give a brief insight into this complex topic.

Is sex still binary?

In this perspective article we discuss the limitations of sex as a binary concept and how it is challenged by medical developments and a better understanding of gender diversity. Recent data indicate that sex is not a simple binary classification based solely on genitalia at birth or reproductive capacity but encompasses various biological characteristics such as chromosomes, hormones, and secondary sexual characteristics. The existence of individuals with differences in sex development (DSD) who do not fit typical male or female categories further demonstrates the complexity of sex. We argue that the belief that sex is strictly binary based on gametes is insufficient, as there are multiple levels of sex beyond reproductivity. We also explore the role of sex in sex determination, gene expression, brain development, and behavioural patterns and emphasize the importance of recognizing sex diversity in personalized medicine, as sex can influence disease presentation, drug response, and treatment effectiveness. Finally, we call for an inter- and transdisciplinary approach to study sex diversity and develop new categories and methodologies that go beyond a binary model.

Monthly video consultation for children and adolescents with type 1 diabetes mellitus during the COVID-19 pandemic.

AIMS: To explore the impact of primarily telemedical care for children and adolescents with type 1 diabetes by monthly video consultations on metabolic control and parents' treatment satisfaction and disease-specific burden during the COVID-19 pandemic. METHODS: In this 12-month multicenter observational follow-up VIDIKI 2.0 study, 100 participants (3-18 years) received monthly video consultations, which partially replaced quarterly outpatient clinic appointments during the pandemic. The children's metabolic parameters as well as the parents' treatment satisfaction and diabetes specific burden were assessed at study entry and 12 months later. RESULTS: During the study, 912 video consultations took place (mean 0.84 ±â€¯0.23 / patient/month). The children's HbA1c remained stable, while mean sensor glucose level and glucose management indicator decreased. Simultaneously, parents' treatment satisfaction significantly increased, and their diabetes-specific burden and distress decreased. CONCLUSIONS: Primarily telemedical care of children and adolescents with type 1 diabetes during the COVID-19 pandemic via monthly video consultations resulted in a significant improvement in parents' treatment satisfaction and their diabetes-specific burden and distress. It was associated with a slight improvement in mean sensor glucose and glucose management indicator, while HbA1c remained stable. Thus, video consultations offer great potential to enhance standard care for children and adolescents with diabetes.

The genetic diagnosis of rare endocrine disorders of sex development and maturation: a survey among Endo-ERN centres.

Differences of sex development and maturation (SDM) represent a heterogeneous puzzle of rare conditions with a large genetic component whose management and treatment could be improved by an accurate classification of underlying molecular conditions, and next-generation sequencing (NGS) should represent the most appropriate approach. Therefore, we conducted a survey dedicated to the use and potential outcomes of NGS for SDM disorders diagnosis among the 53 health care providers (HCP) of the European Reference Network for rare endocrine conditions. The response rate was 49% with a total of 26 HCPs from 13 countries. All HCPs, except 1, performed NGS investigations for SDM disorders on 6720 patients, 3764 (56%) with differences of sex development (DSD), including 811 unexplained primary ovarian insufficiency, and 2956 (44%) with congenital hypogonadotropic hypogonadism (CHH). The approaches varied from targeted analysis of custom gene panels (range: 11-490 genes) in 81.5% of cases or whole exome sequencing with the extraction of a virtual panel in the remaining cases. These analyses were performed for diagnostic purposes in 21 HCPs, supported by the National Health Systems in 16 cases. The likelihood of finding a variant ranged between 7 and 60%, mainly depending upon the number of analysed genes or criteria used for reporting, most HCPs also reporting variants of uncertain significance. These data illustrate the status of genetic diagnosis of DSD and CHH across Europe. In most countries, these analyses are performed for diagnostic purposes, yielding highly variable results, thus suggesting the need for harmonization and general improvements of NGS approaches.

MYRF: A New Regulator of Cardiac and Early Gonadal Development-Insights from Single Cell RNA Sequencing Analysis.

De novo variants in the myelin regulatory factor (MYRF), a transcription factor involved in the differentiation of oligodendrocytes, have been linked recently to the cardiac and urogenital syndrome, while familiar variants are associated with nanophthalmos. Here, we report for the first time on a patient with a de novo stop-gain variant in MYRF (p.Q838*) associated with Scimitar syndrome, 46,XY partial gonadal dysgenesis (GD) and severe hyperopia. Since variants in MYRF have been described in both 46,XX and 46,XY GD, we assumed a role of MYRF in the early development of the bipotential gonad. We used publicly available single cell sequencing data of human testis and ovary from different developmental stages and analysed them for MYRF expression. We identified MYRF expression in the subset of coelomic epithelial cells at stages of gonadal ridge development in 46,XX and 46,XY individuals. Differential gene expression analysis revealed significantly upregulated genes. Within these, we identified CITED2 as a gene containing a MYRF binding site. It has been shown that Cited2-/- mice have gonadal defects in both testis and ovary differentiation, as well as defects in heart development and establishment of the left-right axis. This makes MYRF a potential candidate as an early regulator of gonadal and heart development via upregulation of the transcriptional cofactor CITED2.

Development and evaluation of a patient education programme for children, adolescents, and young adults with differences of sex development (DSD) and their parents: study protocol of Empower-DSD.

BACKGROUND: Differences in sexual development (DSD) are rare diseases, which affect the chromosomal, anatomical or gonadal sex differentiation. Although patient education is recommended as essential in a holistic care approach, standardised programmes are still lacking. The present protocol describes the aims, study design and methods of the Empower-DSD project, which developed an age-adapted multidisciplinary education programme to improve the diagnosis-specific knowledge, skills and empowerment of patients and their parents. METHODS: The new patient education programme was developed for children, adolescents and young adults with congenital adrenal hyperplasia, Turner syndrome, Klinefelter syndrome or XX-/or XY-DSD and their parents. The quantitative and qualitative evaluation methods include standardised questionnaires, semi-structured interviews, and participatory observation. The main outcomes (assessed three and six months after the end of the programme) are health-related quality of life, disease burden, coping, and diagnosis-specific knowledge. The qualitative evaluation examines individual expectations and perceptions of the programme. The results of the quantitative and qualitative evaluation will be triangulated. DISCUSSION: The study Empower-DSD was designed to reduce knowledge gaps regarding the feasibility, acceptance and effects of standardised patient education programmes for children and youth with DSD and their parents. A modular structured patient education programme with four generic and three diagnosis-specific modules based on the ModuS concept previously established for other chronic diseases was developed. The topics, learning objectives and recommended teaching methods are summarised in the structured curricula, one for each diagnosis and age group. At five study centres, 56 trainers were qualified for the implementation of the training programmes. A total of 336 subjects have been already enrolled in the study. The recruitment will go on until August 2022, the last follow-up survey is scheduled for February 2023. The results will help improve multidisciplinary and integrated care for children and youth with DSD and their families. TRIAL REGISTRATION: German Clinical Trials Register, DRKS00023096 . Registered 8 October 2020 - Retrospectively registered.

Can Non-Coding NR5A1 Gene Variants Explain Phenotypes of Disorders of Sex Development?

INTRODUCTION: NR5A1 is an essential transcription factor that regulates several target genes involved in reproduction and endocrine function. Pathogenic variants in this gene are responsible for a wide spectrum of disorders/differences of sex development (DSD). METHODS: The molecular study involved Sanger sequencing, in vitro assays, and whole exome sequencing (WES). RESULTS: Four variants were identified within the NR5A1 non-coding region in 3 patients with 46,XY DSD. In vitro analyses showed that promoter activity was affected in all cases. WES revealed variants in SRA1, WWOX, and WDR11 genes. DISCUSSION/CONCLUSION: Evaluation of clinical and phenotypic significance of variants located in a non-coding region of a gene can be complex, and little is known regarding their association with DSD. Nevertheless, based on the important region for interaction with cofactors essential to promote appropriated sex development and on our in vitro results, it is feasible to say that an impact on gene expression can be expected and that this may be correlated with the DSD pathophysiology presented in our patients. Considering the number of cases that remain elusive after screening for the well-known DSD related genes, we emphasize the importance of a careful molecular analysis of NR5A1 non-coding region which is commonly neglected and might explain some idiopathic DSD cases.

Metabolic effects of estradiol versus testosterone in complete androgen insensitivity syndrome.

PURPOSE: To study differences in metabolic outcomes between testosterone and estradiol replacement in probands with complete androgen insensitivity syndrome (CAIS). METHODS: In this multicentre, double-blind, randomized crossover trial, 26 women with CAIS were included of whom 17 completed the study. After a two-months run in phase with estradiol, probands either received transdermal estradiol followed by crossover to transdermal testosterone or vice versa. After six months, differences in lipids, fasting glucose, insulin, hematocrit, liver parameters and blood pressure between the treatment phases were investigated. RESULTS: Linear mixed models adjusted for period and sequence did not reveal major group differences according to treatment for the investigated outcomes. In each treatment group, there were however significant uniform changes in BMI and cholesterol. BMI increased significantly, following six months of estradiol ( + 2.7%; p = 0.036) as well as testosterone treatment ( + 2.8%; p = 0.036). There was also a significant increase in total ( + 10.4%; p = 0.001) and LDL-cholesterol ( + 29.2%; p = 0.049) and a decrease in HDL-cholesterol (-15.8%; p < 0.001) following six months of estradiol as well as six months of testosterone treatment (total cholesterol: + 14.6%; p = 0.008; LDL-cholesterol: + 39.1%; p = 0.005, HDL-cholesterol: -15.8%; p = 0.004). Other parameters remained unchanged. CONCLUSION: Transdermal estradiol as well as testosterone treatment in women with CAIS results in worsening in lipid profiles. Given the relatively small sample size, subtle group differences in other metabolic parameters may have remained undetected.