Transcriptome profiling of kisspeptin neurons from the mouse arcuate nucleus reveals new mechanisms in estrogenic control of fertility.

Kisspeptin neurons in the mediobasal hypothalamus (MBH) are critical targets of ovarian estrogen feedback regulating mammalian fertility. To reveal molecular mechanisms underlying this signaling, we thoroughly characterized the estrogen-regulated transcriptome of kisspeptin cells from ovariectomized transgenic mice substituted with 17ß-estradiol or vehicle. MBH kisspeptin neurons were harvested using laser-capture microdissection, pooled, and subjected to RNA sequencing. Estrogen treatment significantly (p.adj. < 0.05) up-regulated 1,190 and down-regulated 1,139 transcripts, including transcription factors, neuropeptides, ribosomal and mitochondrial proteins, ion channels, transporters, receptors, and regulatory RNAs. Reduced expression of the excitatory serotonin receptor-4 transcript (Htr4) diminished kisspeptin neuron responsiveness to serotonergic stimulation. Many estrogen-regulated transcripts have been implicated in puberty/fertility disorders. Patients (n = 337) with congenital hypogonadotropic hypogonadism (CHH) showed enrichment of rare variants in putative CHH-candidate genes (e.g., LRP1B, CACNA1G, FNDC3A). Comprehensive characterization of the estrogen-dependent kisspeptin neuron transcriptome sheds light on the molecular mechanisms of ovary-brain communication and informs genetic research on human fertility disorders.

Standardising the biochemical confirmation of adult male hypogonadism: A joint position statement by the Society for Endocrinology and Association of Clinical Biochemistry and Laboratory Medicine.

BACKGROUND: Inter-assay variation between different immunoassays and different mass spectrometry methods hampers the biochemical confirmation of male hypogonadism. Furthermore, some laboratories utilise assay manufacturer reference ranges that do not necessarily mirror assay performance characteristics, with the lower limit of normality ranging from 4.9 nmol/L to 11 nmol/L. The quality of the normative data underlying commercial immunoassay reference ranges is uncertain. DESIGN: A working group reviewed published evidence and agreed upon standardised reporting guidance to augment total testosterone reports. RESULTS: Evidence-based guidance on appropriate blood sampling, clinical action limits, and other major factors likely to affect the interpretation of results are provided. CONCLUSIONS: This article aims to improve the quality of the interpretation of testosterone results by non-specialist clinicians. It also discusses approaches for assay harmonisation which have been successful in some but not all healthcare systems.

Vitamin D and COVID-19-Revisited.

Vitamin D, when activated to 1,25-dihydroxyvitamin D, is a steroid hormone that induces responses in several hundred genes, including many involved in immune responses to infection. Without supplementation, people living in temperate zones commonly become deficient in the precursor form of vitamin D, 25-hydroxyvitamin D, during winter, as do people who receive less sunlight exposure or those with darker skin pigmentation. Studies performed pre-COVID-19 have shown significant but modest reduction in upper respiratory infections in people receiving regular daily vitamin D supplementation. Vitamin D deficiency, like the risk of severe COVID-19, is linked with darker skin colour and also with obesity. Greater risk from COVID-19 has been associated with reduced ultraviolet exposure. Various studies have examined serum 25-hydroxyvitamin D levels, either historical or current, in patients with COVID-19. The results of these studies have varied but the majority have shown an association between vitamin D deficiency and increased risk of COVID-19 illness or severity. Interventional studies of vitamin D supplementation have so far been inconclusive. Trial protocols commonly allow control groups to receive low-dose supplementation that may be adequate for many. The effects of vitamin D supplementation on disease severity in patients with existing COVID-19 are further complicated by the frequent use of large bolus dose vitamin D to achieve rapid effects, even though this approach has been shown to be ineffective in other settings. As the pandemic passes into its third year, a substantial role of vitamin D deficiency in determining the risk from COVID-19 remains possible but unproven.

The p190 RhoGAPs, ARHGAP35, and ARHGAP5 are implicated in GnRH neuronal development: Evidence from patients with idiopathic hypogonadotropic hypogonadism, zebrafish, and in vitro GAP activity assay.

PURPOSE: The study aimed to identify novel genes for idiopathic hypogonadotropic hypogonadism (IHH). METHODS: A cohort of 1387 probands with IHH underwent exome sequencing and de novo, familial, and cohort-wide investigations. Functional studies were performed on 2 p190 Rho GTPase-activating proteins (p190 RhoGAP), ARHGAP35 and ARHGAP5, which involved in vivo modeling in larval zebrafish and an in vitro p190A-GAP activity assay. RESULTS: Rare protein-truncating variants (PTVs; n = 5) and missense variants in the RhoGAP domain (n = 7) in ARHGAP35 were identified in IHH cases (rare variant enrichment: PTV [unadjusted P = 3.1E-06] and missense [adjusted P = 4.9E-03] vs controls). Zebrafish modeling using gnrh3:egfp phenotype assessment showed that mutant larvae with deficient arhgap35a, the predominant ARHGAP35 paralog in the zebrafish brain, display decreased GnRH3-GFP+ neuronal area, a readout for IHH. In vitro GAP activity studies showed that 1 rare missense variant [ARHGAP35 p.(Arg1284Trp)] had decreased GAP activity. Rare PTVs (n = 2) also were discovered in ARHGAP5, a paralog of ARHGAP35; however, arhgap5 zebrafish mutants did not display significant GnRH3-GFP+ abnormalities. CONCLUSION: This study identified ARHGAP35 as a new autosomal dominant genetic driver for IHH and ARHGAP5 as a candidate gene for IHH. These observations suggest a novel role for the p190 RhoGAP proteins in GnRH neuronal development and integrity.

Society for Endocrinology guidelines for testosterone replacement therapy in male hypogonadism.

Male hypogonadism (MH) is a common endocrine disorder. However, uncertainties and variations in its diagnosis and management exist. There are several current guidelines on testosterone replacement therapy that have been driven predominantly by single disciplines. The Society for Endocrinology commissioned this new guideline to provide all care providers with a multidisciplinary approach to treating patients with MH. This guideline has been compiled using expertise from endocrine (medical and nursing), primary care, clinical biochemistry, urology and reproductive medicine practices. These guidelines also provide a patient perspective to help clinicians best manage MH.

Genetics of congenital hypogonadotropic hypogonadism: peculiarities and phenotype of an oligogenic disease.

A genetic basis of congenital isolated hypogonadotropic hypogonadism (CHH) can be defined in almost 50% of cases, albeit not necessarily the complete genetic basis. Next-generation sequencing (NGS) techniques have led to the discovery of a great number of loci, each of which has illuminated our understanding of human gonadotropin-releasing hormone (GnRH) neurons, either in respect of their embryonic development or their neuroendocrine regulation as the "pilot light" of human reproduction. However, because each new gene linked to CHH only seems to underpin another small percentage of total patient cases, we are still far from achieving a comprehensive understanding of the genetic basis of CHH. Patients have generally not benefited from advances in genetics in respect of novel therapies. In most cases, even genetic counselling is limited by issues of apparent variability in expressivity and penetrance that are likely underpinned by oligogenicity in respect of known and unknown genes. Robust genotype-phenotype relationships can generally only be established for individuals who are homozygous, hemizygous or compound heterozygotes for the same gene of variant alleles that are predicted to be deleterious. While certain genes are purely associated with normosmic CHH (nCHH) some purely with the anosmic form (Kallmann syndrome-KS), other genes can be associated with both nCHH and KS-sometimes even within the same kindred. Even though the anticipated genetic overlap between CHH and constitutional delay in growth and puberty (CDGP) has not materialised, previously unanticipated genetic relationships have emerged, comprising conditions of combined (or multiple) pituitary hormone deficiency (CPHD), hypothalamic amenorrhea (HA) and CHARGE syndrome. In this review, we report the current evidence in relation to phenotype and genetic peculiarities regarding 60 genes whose loss-of-function variants can disrupt the central regulation of reproduction at many levels: impairing GnRH neurons migration, differentiation or activation; disrupting neuroendocrine control of GnRH secretion; preventing GnRH neuron migration or function and/or gonadotropin secretion and action.

Phenotypic continuum between Waardenburg syndrome and idiopathic hypogonadotropic hypogonadism in humans with SOX10 variants.

PURPOSE: SOX10 variants previously implicated in Waardenburg syndrome (WS) have now been linked to Kallmann syndrome (KS), the anosmic form of idiopathic hypogonadotropic hypogonadism (IHH). We investigated whether SOX10-associated WS and IHH represent elements of a phenotypic continuum within a unifying disorder or if they represent phenotypically distinct allelic disorders. METHODS: Exome sequencing from 1,309 IHH subjects (KS: 632; normosmic idiopathic hypogonadotropic hypogonadism [nIIHH]: 677) were reviewed for SOX10 rare sequence variants (RSVs). The genotypic and phenotypic spectrum of SOX10-related IHH (this study and literature) and SOX10-related WS cases (literature) were reviewed and compared with SOX10-RSV spectrum in gnomAD population. RESULTS: Thirty-seven SOX10-associated IHH cases were identified as follows: current study: 16 KS; 4 nIHH; literature: 16 KS; 1 nIHH. Twenty-three IHH cases (62%; all KS), had ≥1 known WS-associated feature(s). Moreover, five previously reported SOX10-associated WS cases showed IHH-related features. Four SOX10 missense RSVs showed allelic overlap between IHH-ascertained and WS-ascertained cases. The SOX10-HMG domain showed an enrichment of RSVs in disease states versus gnomAD. CONCLUSION: SOX10 variants contribute to both anosmic (KS) and normosmic (nIHH) forms of IHH. IHH and WS represent SOX10-associated developmental defects that lie along a unifying phenotypic continuum. The SOX10-HMG domain is critical for the pathogenesis of SOX10-related human disorders.

Iatrogenic hypoglycaemia following glucose-insulin infusions for the treatment of hyperkalaemia.

OBJECTIVES: To study the incidence of, and risk factors for, iatrogenic hypoglycaemia following GwI infusion in our institution. CONTEXT: Hyperkalaemia is a life-threatening biochemical abnormality. Glucose-with-insulin (GwI) infusions form standard management, but risk iatrogenic hypoglycaemia (glucose ≤ 3.9 mmol/L). Recently updated UK guidelines include an additional glucose infusion in patients with pretreatment capillary blood glucose (CBG) < 7.0 mmol/L. DESIGN: Retrospective analysis of outcomes for GwI infusions prescribed for hyperkalaemia from 1 January to 28 February 2019, extracted from the Newcastle upon Tyne Hospitals NHS Foundation Trust electronic platform (eRecord). PARTICIPANTS: 132 patients received 228 GwI infusions for hyperkalaemia. MAIN OUTCOME MEASURES: Incidence, severity and time to onset of hypoglycaemia. RESULTS: Hypoglycaemia incidence was 11.8%. At least 1 hypoglycaemic episode occurred in 18.2% of patients with 6.8% having at least 1 episode of severe hypoglycaemia (< 3.0 mmol/L). Most episodes (77.8%) occurred within 3 h of treatment. Lower pretreatment CBG (5.9 mmol/L [4.1 mmol/L-11.2 mmol/L], versus 7.6 mmol/L [3.7 mmol/L-31.3 mmol/L], P = .000) was associated with hypoglycaemia risk. A diagnosis of type 2 diabetes and treatment for hyperkalaemia within the previous 24 h were negatively associated. CONCLUSIONS: Within our inpatient population, around 1 in 8 GwI infusions delivered as treatment for hyperkalaemia resulted in iatrogenic hypoglycaemia. Higher pretreatment CBG and a diagnosis of type 2 diabetes were protective, irrespective of renal function. Our findings support the immediate change to current management, either with additional glucose infusions or by using glucose-only infusions in patients without diabetes. These approaches should be compared via a prospective randomized study.

Kisspeptin-54 Accurately Identifies Hypothalamic Gonadotropin-Releasing Hormone Neuronal Dysfunction in Men with Congenital Hypogonadotropic Hypogonadism.

BACKGROUND: Hypogonadotropic hypogonadism (HH) is hypogonadism due to either hypothalamic or pituitary dysfunction. While gonadotropin-releasing hormone (GnRH) can directly test pituitary function, no specific test of hypothalamic function exists. Kisspeptin-54 (KP54) is a neuropeptide that directly stimulates hypothalamic GnRH release and thus could be used to specifically interrogate hypothalamic function. Congenital HH (CHH) is typically due to variants in genes that control hypothalamic GnRH neuronal migration or function. Thus, we investigated whether KP54 could accurately identify hypothalamic dysfunction in men with CHH. METHODS: Men with CHH (n = 21) and healthy eugonadal men (n = 21) received an intravenous bolus of either GnRH (100 µg) or KP54 (6.4 nmol/kg), on 2 occasions, and were monitored for 6 h after administration of each neuropeptide. RESULTS: Maximal luteinizing hormone (LH) rise after KP54 was significantly greater in healthy men (12.5 iU/L) than in men with CHH (0.4 iU/L; p < 0.0001). KP54 more accurately differentiated CHH men from healthy men than GnRH (area under receiver operating characteristic curve KP54: 1.0, 95% CI 1.0-1.0; GnRH: 0.88, 95% CI 0.76-0.99). Indeed, all CHH men had an LH rise <2.0 iU/L following KP54, whereas all healthy men had an LH rise >4.0 iU/L. Anosmic men with CHH (i.e., Kallmann syndrome) had even lower LH rises after KP54 than did normosmic men with CHH (p = 0.017). Likewise, men identified to have pathogenic/likely pathogenic variants in CHH genes had even lower LH rises after KP54 than other men with CHH (p = 0.035). CONCLUSION: KP54 fully discriminated men with CHH from healthy men. Thus, KP54 could be used to specifically interrogate hypothalamic GnRH neuronal function in patients with CHH.

DCC/NTN1 complex mutations in patients with congenital hypogonadotropic hypogonadism impair GnRH neuron development.

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disease characterized by absent puberty and infertility due to GnRH deficiency, and is often associated with anosmia [Kallmann syndrome (KS)]. The genetic etiology of CHH is heterogeneous, and more than 30 genes have been implicated in approximately 50% of patients with CHH. We hypothesized that genes encoding axon-guidance proteins containing fibronectin type-III (FN3) domains (similar to ANOS1, the first gene associated with KS), are mutated in CHH. We performed whole-exome sequencing in a cohort of 133 CHH probands to test this hypothesis, and identified rare sequence variants (RSVs) in genes encoding for the FN3-domain encoding protein deleted in colorectal cancer (DCC) and its ligand Netrin-1 (NTN1). In vitro studies of these RSVs revealed altered intracellular signaling associated with defects in cell morphology, and confirmed five heterozygous DCC mutations in 6 probands-5 of which presented as KS. Two KS probands carry heterozygous mutations in both DCC and NTN1 consistent with oligogenic inheritance. Further, we show that Netrin-1 promotes migration in immortalized GnRH neurons (GN11 cells). This study implicates DCC and NTN1 mutations in the pathophysiology of CHH consistent with the role of these two genes in the ontogeny of GnRH neurons in mice.

Pathogenic mosaic variants in congenital hypogonadotropic hypogonadism.

PURPOSE: Congenital hypogonadotropic hypogonadism (CHH) is a rare disorder resulting in absent puberty and infertility. The genetic architecture is complex with multiple loci involved, variable expressivity, and incomplete penetrance. The majority of cases are sporadic, consistent with a disease affecting fertility. The current study aims to investigate mosaicism as a genetic mechanism for CHH, focusing on de novo rare variants in CHH genes. METHODS: We evaluated 60 trios for de novo rare sequencing variants (RSV) in known CHH genes using exome sequencing. Potential mosaicism was suspected among RSVs with altered allelic ratios and confirmed using customized ultradeep sequencing (UDS) in multiple tissues. RESULTS: Among the 60 trios, 10 probands harbored de novo pathogenic variants in CHH genes. Custom UDS demonstrated that three of these de novo variants were in fact postzygotic mosaicism-two in FGFR1 (p.Leu630Pro and p.Gly348Arg), and one in CHD7 (p.Arg2428*). Statistically significant variation across multiple tissues (DNA from blood, buccal, hair follicle, urine) confirmed their mosaic nature. CONCLUSIONS: We identified a significant number of de novo pathogenic variants in CHH of which a notable number (3/10) exhibited mosaicism. This report of postzygotic mosaicism in CHH patients provides valuable information for accurate genetic counseling.

DLG2 variants in patients with pubertal disorders.

PURPOSE: Impaired function of gonadotropin-releasing hormone (GnRH) neurons can cause a phenotypic spectrum ranging from delayed puberty to isolated hypogonadotropic hypogonadism (IHH). We sought to identify a new genetic etiology for these conditions. METHODS: Exome sequencing was performed in an extended family with autosomal dominant, markedly delayed puberty. The effects of the variant were studied in a GnRH neuronal cell line. Variants in the same gene were sought in a large cohort of individuals with IHH. RESULTS: We identified a rare missense variant (F900V) in DLG2 (which encodes PSD-93) that cosegregated with the delayed puberty. The variant decreased GnRH expression in vitro. PSD-93 is an anchoring protein of NMDA receptors, a type of glutamate receptor that has been implicated in the control of puberty in laboratory animals. The F900V variant impaired the interaction between PSD-93 and a known binding partner, Fyn, which phosphorylates NMDA receptors. Variants in DLG2 that also decreased GnRH expression were identified in three unrelated families with IHH. CONCLUSION: The findings indicate that variants in DLG2/PSD-93 cause autosomal dominant delayed puberty and may also contribute to IHH. The findings also suggest that the pathogenesis involves impaired NMDA receptor signaling and consequently decreased GnRH secretion.

Is calcium supplementation always needed in patients with hypoparathyroidism?

Oral calcium salts are recommended for the treatment of chronic hypoparathyroidism (HypoPT), although dosimetry is variable between individual patients and clinicians. However, patient feedback on calcium salts can be negative, particularly due to gastrointestinal side effects and hypercalciuria-related complications. We begin with a clinical case of a HypoPT patient taking oral calcium salts following thyroid surgery, who requested support in reducing her dose of these with a view to stopping entirely. To evaluate her request, we first describe the usual treatment of HypoPT according to current guidance and then present data from (a) a case note review of a cohort of 24 HypoPT patients managed with a "no calcium" treatment regimen by single physician (b) a comprehensive online survey of HypoPT patients' treatment and experiences (n = 330). The case note review found that target range serum calcium levels were successfully achieved in all 24 patients since transitioning to a "no calcium" regimen, without any breakthrough hypocalcaemia-related symptoms, the development of new renal stones, the occurrence of calcium-related hospital admissions or the finding of significant hypercalciuria. The online survey identified 36% of HypoPT patients who continued to take activated vitamin D, but had discontinued calcium supplements. HypoPT patients not currently taking calcium reported a significantly lower prevalence of adverse effects and outcomes, both compared with their previous experiences whilst taking calcium and also compared with the 64% of patients who continued to take oral calcium. We conclude that, subject to methodological limitations, there are significant issues of tolerability arising from conventional calcium-based treatment regimens for patients with chronic HypoPT. For selected patients, it may be reasonable to facilitate a managed therapeutic transition to "no calcium" regimen, and we also propose that calcium-based regimes be prospectively evaluated against calcium-free (or calcium-low) alternatives.

Evaluating CHARGE syndrome in congenital hypogonadotropic hypogonadism patients harboring CHD7 variants.

PURPOSE: Congenital hypogonadotropic hypogonadism (CHH), a rare genetic disease caused by gonadotropin-releasing hormone deficiency, can also be part of complex syndromes (e.g., CHARGE syndrome). CHD7 mutations were reported in 60% of patients with CHARGE syndrome, and in 6% of CHH patients. However, the definition of CHD7 mutations was variable, and the associated CHARGE signs in CHH were not systematically examined. METHODS: Rare sequencing variants (RSVs) in CHD7 were identified through exome sequencing in 116 CHH probands, and were interpreted according to American College of Medical Genetics and Genomics guidelines. Detailed phenotyping was performed in CHH probands who were positive for CHD7 RSVs, and genotype-phenotype correlations were evaluated. RESULTS: Of the CHH probands, 16% (18/116) were found to harbor heterozygous CHD7 RSVs, and detailed phenotyping was performed in 17 of them. Of CHH patients with pathogenic or likely pathogenic CHD7 variants, 80% (4/5) were found to exhibit multiple CHARGE features, and 3 of these patients were reclassified as having CHARGE syndrome. In contrast, only 8% (1/12) of CHH patients with nonpathogenic CHD7 variants exhibited multiple CHARGE features (P = 0.01). CONCLUSION: Pathogenic or likely pathogenic CHD7 variants rarely cause isolated CHH. Therefore a detailed clinical investigation is indicated to clarify the diagnosis (CHH versus CHARGE) and to optimize clinical management.

Fertility induction in hypogonadotropic hypogonadal men.

Men with hypogonadotropic hypogonadism (HH) are typically azoospermic, and yet HH is one of the few treatable forms of male infertility. Sperm induction protocols using gonadotrophins aim to replicate the natural endocrine control of spermatogenesis. Previously virilised men with adult-onset HH and normal testicular volume respond well to monotherapy in which human chorionic gonadotrophin (hCG) acts as a long-acting LH-analogue stimulating spermatogenesis. However, this approach is rarely successful for men with congenital HH (CHH) (eg, Kallmann syndrome), for whom combined gonadotrophin therapy (hCG + follicle-stimulating hormone [FSH]) is an absolute requirement to maximise fertility potential. Key baseline predictors of successful spermatogenesis-induction include prior spontaneous testicular development (ie, testicular volume [TV] > 4 mL), serum inhibin B (IB ) concentration >60 pg/mL and no history of maldescended testes (cryptorchidism).