A New Tool to Identify Pediatric Patients with Atypical Diabetes Associated with Gene Polymorphisms.

Background: Recent diabetes subclassifications have improved the differentiation between patients with type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus despite several overlapping features, yet without considering genetic forms of diabetes. We sought to facilitate the identification of monogenic diabetes by creating a new tool that we validated in a pediatric maturity-onset diabetes of the young (MODY) cohort. Methods: We first created the DIAgnose MOnogenic DIAbetes (DIAMODIA) criteria based on the pre-existing, but incomplete, MODY calculator. This new score is composed of four strong and five weak criteria, with patients having to display at least one weak and one strong criterion. Results: The effectiveness of the DIAMODIA criteria was evaluated in two patient cohorts, the first consisting of patients with confirmed MODY diabetes (n=34) and the second of patients with T1DM (n=390). These DIAMODIA criteria successfully detected 100% of MODY patients. Multiple correspondence analysis performed on the MODY and T1DM cohorts enabled us to differentiate MODY patients from T1DM. The three most relevant variables to distinguish a MODY from T1DM profile were: lower insulin-dose adjusted A1c score ≤9, glycemic target-adjusted A1c score ≤4.5, and absence of three anti-islet cell autoantibodies. Conclusion: We validated the DIAMODIA criteria, as it effectively identified all monogenic diabetes patients (MODY cohort) and succeeded to differentiate T1DM from MODY patients. The creation of this new and effective tool is likely to facilitate the characterization and therapeutic management of patients with atypical diabetes, and promptly referring them for genetic testing which would markedly improve clinical care and counseling, as well.

Efficacy and safety of a 4-year combination therapy of growth hormone and gonadotropin-releasing hormone analogue in pubertal girls with short predicted adult height.

Objectives: To improve adult height in pubertal girls with a poor height prediction, treatment with growth hormone (GH) can be used in combination with a gonadotropin releasing hormone agonist (GnRHa), to delay closure of the growth plates. However, there are few studies to support this practice, and they show conflicting results. The objective of this trial is to assess the safety and efficacy of this combination treatment in early pubertal girls with a short predicted height, in comparison with matched controls. Design patients and methods: We designed an open-label, multicenter, interventional case-control study. Early pubertal girls with predicted adult height (PAH) below -2.5 SDS, were recruited in tertiary care centers in Belgium. They were treated for four years with GH and GnRHa. The girls were followed until adult height (AH) was reached. AH vs PAH, AH vs Height at start, and AH vs Target Height (TH) were evaluated, as well as safety parameters. Control data were assembled from historical patient files or from patients who preferred not to participate in the study. Results: Sixteen girls with mean age ( ± SD) at start of 11.0 years (± 1.3) completed the study protocol and follow-up. Their mean height ( ± SD) increased from 131.3 ± 4.1 cm (-2.3 ± 0.7 SDS) at start of treatment to 159.8 ± 4.7 cm (-1.1 ± 0.7 SDS) at AH. In matched controls, height increased from 132.3 ± 4.2 cm (-2.4 ± 0.5 SDS) to 153.2 ± 3.4 cm (-2.1 ± 0.6 SDS) (p<0.001). AH surpassed initial PAH by 12.0 ± 2.6 cm in treated girls; and by 4.2 ± 3.6 cm in the controls (p<0.001). Most treated girls reached normal adult height (>-2SD) (87.5%) and 68.7% reached or superseded the target height (TH), which was the case in only a minority of the controls (37.5% and 6.2%, respectively) (p= 0.003 and 0.001). A serious adverse event possibly related to the treatment, was a fracture of the metatarsals. Conclusion: A four-year GH/GnRHa treatment in early pubertal girls with a poor PAH seems safe and results in a clinically relevant and statistically significant increase in AH compared with matched historical controls. Clinical trial registration: ClinicalTrials.gov, identifier NCT00840944.

Associations between endocrine disruptor contamination and thyroid hormone homeostasis in Belgian type 1 diabetic children.

PURPOSE: Humans are daily exposed to many environmental pollutants, some of which being suspected to be thyroid disruptors. Some populations could be particularly susceptible to thyroid disruption, such like diabetics due to the well-known relation between the thyroid function and the control of carbohydrate homeostasis by pancreas. Therefore, the aim of this study was to investigate the associations between the exposure to several persistent and non-persistent chemicals and thyroid hormones levels in children with type 1 diabetes. METHODS: Blood and urine sample were collected from 54 children diagnosed for type 1 diabetes mellitus. The concentrations of 7 phthalate metabolites, 4 parabens, 7 bisphenols, benzophenone 3 and triclosan were measured in urine, while 15 organochlorine pesticides, 4 polychlorinated biphenyls (PCBs) and 7 perfluoroalkyl substances were analyzed in serum samples. In the same time, the blood levels of free thyroxine (fT4), thyroid stimulating hormone (TSH) and glycated hemoglobin (Hb1Ac) were determined. RESULTS: We highlighted positive associations between serum perfluorohexane sulfonate and urinary monoethylphthalate levels, and TSH level in blood. We also found that PCB 138 was positively associated to fT4 while urinary levels of bisphenol F were negatively correlated to this hormone. Finally, we observed positive associations between Hb1Ac levels and the contamination by PCB 153 and two urinary phthalate metabolites: mono-2-ethyl-5-hydroxyhexyl phthalate and mono-2-ethyl-5-oxoxyhexyl phthalate. CONCLUSION: Our results showed that our small cohort of children with type 1 diabetes mellitus is potentially susceptible to thyroid disruptions by some pollutants. Moreover, for these children, both di-(2-ethylhexyl) phthalate metabolites would potentially hamper the glucose homeostasis. Nevertheless, additional studies are mandatory to further explore these findings.

Effect of nationwide reimbursement of real-time continuous glucose monitoring on HbA1c, hypoglycemia and quality of life in a pediatric type 1 diabetes population: The RESCUE-pediatrics study.

Objective: Real-time continuous glucose monitoring (RT-CGM) can improve metabolic control and quality of life (QoL), but long-term real-world data in children with type 1 diabetes (T1D) are scarce. Over a period of 24 months, we assessed the impact of RT-CGM reimbursement on glycemic control and QoL in children/adolescents with T1D treated with insulin pumps. Research design and methods: We conducted a multicenter prospective observational study. Primary endpoint was the change in HbA1c. Secondary endpoints included change in time in hypoglycemia, QoL, hospitalizations for hypoglycemia and/or ketoacidosis and absenteeism (school for children, work for parents). Results: Between December 2014 and February 2019, 75 children/adolescents were followed for 12 (n = 62) and 24 months (n = 50). Baseline HbA1c was 7.2 ± 0.7% (55 ± 8mmol/mol) compared to 7.1 ± 0.8% (54 ± 9mmol/mol) at 24 months (p = 1.0). Participants with a baseline HbA1c ≥ 7.5% (n = 27, mean 8.0 ± 0.3%; 64 ± 3mmol/mol) showed an improvement at 4 months (7.6 ± 0.7%; 60 ± 8mmol/mol; p = 0.009) and at 8 months (7.5 ± 0.6%; 58 ± 7mmol/mol; p = 0.006), but not anymore thereafter (endpoint 24 months: 7.7 ± 0.9%; 61 ± 10mmol/mol; p = 0.2). Time in hypoglycemia did not change over time. QoL for parents and children remained stable. Need for assistance by ambulance due to hypoglycemia reduced from 8 to zero times per 100 patient-years (p = 0.02) and work absenteeism for parents decreased from 411 to 214 days per 100 patient-years (p = 0.03), after 24 months. Conclusion: RT-CGM in pump-treated children/adolescents with T1D showed a temporary improvement in HbA1c in participants with a baseline HbA1c ≥ 7.5%, without increasing time in hypoglycemia. QoL was not affected. Importantly, RT-CGM reduced the need for assistance by ambulance due to hypoglycemia and reduced work absenteeism for parents after 24 months. Clinical trial registration: [ClinicalTrials.gov], identifier [NCT02601729].

Glycemic Variability Patterns Strongly Correlate With Partial Remission Status in Children With Newly Diagnosed Type 1 Diabetes.

OBJECTIVE: To evaluate whether indexes of glycemic variability may overcome residual ß-cell secretion estimates in the longitudinal evaluation of partial remission in a cohort of pediatric patients with new-onset type 1 diabetes. RESEARCH DESIGN AND METHODS: Values of residual ß-cell secretion estimates, clinical parameters (e.g., HbA1c or insulin daily dose), and continuous glucose monitoring (CGM) from 78 pediatric patients with new-onset type 1 diabetes were longitudinally collected during 1 year and cross-sectionally compared. Circadian patterns of CGM metrics were characterized and correlated to remission status using an adjusted mixed-effects model. Patients were clustered based on 46 CGM metrics and clinical parameters and compared using nonparametric ANOVA. RESULTS: Study participants had a mean (± SD) age of 10.4 (± 3.6) years at diabetes onset, and 65% underwent partial remission at 3 months. ß-Cell residual secretion estimates demonstrated weak-to-moderate correlations with clinical parameters and CGM metrics (r2 = 0.05-0.25; P < 0.05). However, CGM metrics strongly correlated with clinical parameters (r2 >0.52; P < 0.05) and were sufficient to distinguish remitters from nonremitters. Also, CGM metrics from remitters displayed specific early morning circadian patterns characterized by increased glycemic stability across days (within 63-140 mg/dL range) and decreased rate of grade II hypoglycemia (P < 0.0001) compared with nonremitters. Thorough CGM analysis allowed the identification of four novel glucotypes (P < 0.001) that segregate patients into subgroups and mirror the evolution of remission after diabetes onset. CONCLUSIONS: In our pediatric cohort, combination of CGM metrics and clinical parameters unraveled key clinical milestones of glucose homeostasis and remission status during the first year of type 1 diabetes.

ROHHAD(NET) Syndrome: Systematic Review of the Clinical Timeline and Recommendations for Diagnosis and Prognosis.

CONTEXT: Rapid-onset obesity with hypothalamic dysfunction, hypoventilation, autonomic dysregulation and neural crest tumor (ROHHHAD[NET]) is a rare and potentially fatal disease. No specific diagnostic biomarker is currently available, making prompt diagnosis challenging. Since its first definition in 2007, a complete clinical analysis leading to specific diagnosis and follow-up recommendations is still missing. OBJECTIVE: The purpose of this work is to describe the clinical timeline of symptoms of ROHHAD(NET) and propose recommendations for diagnosis and follow-up. DESIGN: We conducted a systematic review of all ROHHAD(NET) case studies and report a new ROHHAD patient with early diagnosis and multidisciplinary care. METHODS: All the articles that meet the definition of ROHHAD(NET) and provide chronological clinical data were reviewed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis individual patient data guidelines. The data were grouped into 7 categories: hypothalamic dysfunction, autonomic dysregulation, hypoventilation, NET, psychiatric symptoms, other clinical manifestations, and outcome. RESULTS: Forty-three individual patient data descriptions were analyzed. The timeline of the disease shows rapid-onset obesity followed shortly by hypothalamic dysfunction. Dysautonomia was reported at a median age of 4.95 years and hypoventilation at 5.33 years, or 2.2 years after the initial obesity. A NET was reported in 56% of the patients, and 70% of these tumors were diagnosed within 2 years after initial weight gain. CONCLUSION: Because early diagnosis improves the clinical management and the prognosis in ROHHAD(NET), this diagnosis should be considered for any child with rapid and early obesity. We propose guidance for systematic follow-up and advise multidisciplinary management with the aim of improving prognosis and life expectancy.

Endocrine consequences of neuroblastoma treatment in children: 20 years' experience of a single center.

Background Neuroblastoma (NBL) is a child neoplasia affecting extracranial tissue of neuroectodermal origin. It accounts for 10% of solid malignancies in children and is characterized by a survival rate approaching 70%, confronting physicians with the emergence of an adult survivor population who have been previously exposed to surgery, cytotoxic drugs, radiation therapy or metaiodobenzylguanidine (MIBG) therapy. All these treatments potentially affect the endocrine system. Our study consists in a retrospective review of late endocrine effects arising in survivors treated for NBL during childhood. Methods The medical files of 47 patients (M/F = 26/21) treated for NBL were reviewed. Collected data consisted of age, height, weight and biological hormonal values at diagnosis and at the last follow-up consultation. The incidence of late effects in our sample was compared to the data from the literature. Results Patients were between 0 and 15.8 years of age at diagnosis (median: 1.16 years) and between 1 and 25 years of age at last follow-up (median: 16 years). Twenty-six patients were treated with chemotherapy (CT), 11 underwent CT and radiation therapy and five were treated with CT and MIBG therapy. Ten percent of the patients died before reaching the end of therapy. Late effects occurred in 54% of the patients. Thirty-six percent of patients had non-endocrine complications (musculoskeletal, neurological, hematological or hepatic chronic conditions). Endocrine complications (28%) affected mainly patients treated with CT and consisted of gonadal dysfunction (up to 42% patients of over 12 years of age at follow-up) and hypothyroidism (21%). Our analysis revealed that CT had a significant impact on final height (p < 0.05). Conclusions Treatment for childhood malignancies exposes children to late effects affecting the endocrine system. In children treated for NBL, hypothyroidism, gonadal failure and impaired growth appear to be the main endocrine complications. Close follow-up of survivors is thus appropriate.

Insulin sensitivity modulates the growth response during the first year of high-dose growth hormone treatment in short prepubertal children born small for gestational age.

AIM: To study the relationship between insulin sensitivity and growth response in short children born small for gestational age (SGA) treated with growth hormone (GH). METHODS: Randomized, open-label, 24-month intervention study in 40 short prepubertal SGA children [age (mean ± SD) 5.3 ± 1.5 years], who either remained untreated (n = 20) or were treated with GH (66 µg/kg/day; n = 20). Changes in fasting glucose, insulin, quantitative insulin sensitivity check index (QUICKI), IGF-1 and leptin after 1 and 2 years were studied. RESULTS: Mean height SDS increased from -3.3 ± 0.7 to -2.3 ± 0.7 after 1 year, and to -1.9 ± 0.7 after 2 years of treatment. QUICKI decreased significantly (p = 0.008) in the first year of GH treatment and stabilized in the second year. Baseline QUICKI was positively associated (r = 0.40; p < 0.05) with the change in height SDS in the first year. CONCLUSION: Higher insulin sensitivity at the start of GH therapy is associated with greater first-year growth response to GH, and could be a promising parameter in selecting prepubertal short SGA children for GH treatment. However, this finding needs to be confirmed in larger studies.

Neuroendocrine disruption of pubertal timing and interactions between homeostasis of reproduction and energy balance.

The involvement of environmental factors such as endocrine disrupting chemicals (EDCs) in the timing of onset of puberty is suggested by recent changes in age at onset of puberty and pattern of distribution that are variable among countries, as well as new forms of sexual precocity after migration. However, the evidence of association between early or late pubertal timing and exposure to EDCs is weak in humans, possibly due to heterogeneity of effects likely involving mixtures and incapacity to assess fetal or neonatal exposure retrospectively. The neuroendocrine system which is crucial for physiological onset of puberty is targeted by EDCs. These compounds also act directly in the gonads and peripheral sex-steroid sensitive tissues. Feedbacks add to the complexity of regulation so that changes in pubertal timing caused by EDCs can involve both central and peripheral mechanisms. In experimental conditions, several neuroendocrine endpoints are affected by EDCs though only few studies including from our laboratory aimed at EDC involvement in the pathophysiology of early sexual maturation. Recent observations support the concept that EDC cause disturbed energy balance and account for the obesity epidemic. Several aspects are linking this system and the reproductive axis: coexisting neuroendocrine and peripheral effects, dependency on fetal/neonatal programming and the many factors cross-linking the two systems, for instance leptin, adiponectin, Agouti Related Peptide (AgRP). This opens perspectives for future research and, hopefully, measures preventing the disturbances of homeostasis caused by EDCs.

Oxytocin facilitates female sexual maturation through a glia-to-neuron signaling pathway.

It has been earlier proposed that oxytocin could play a facilitatory role in the preovulatory LH surge in both rats and humans. We here provide evidence that oxytocin also facilitates sexual maturation in female rats. The administration of an oxytocin antagonist for 6 d to immature female rats decreased GnRH pulse frequency ex vivo and delayed the age at vaginal opening and first estrus. The in vitro reduction in GnRH pulse frequency required chronic blockade of oxytocin receptors, because it was not acutely observed after a single injection of the antagonist. Hypothalamic explants exposed to the antagonist in vitro showed a reduced GnRH pulse frequency and failed to respond to oxytocin with GnRH release. Prostaglandin E(2) (PGE(2)) mimicked the stimulatory effect of oxytocin on GnRH pulse frequency, and inhibition of PG synthesis blocked the effect of oxytocin, suggesting that oxytocin accelerates pulsatile GnRH release via PGE(2). The source of PGE(2) appears to be astrocytes, because oxytocin stimulates PGE(2) release from cultured hypothalamic astrocytes. Moreover, astrocytes express oxytocin receptors, whereas GnRH neurons do not. These results suggest that oxytocin facilitates female sexual development and that this effect is mediated by a mechanism involving glial production of PGE(2).

Mechanisms of interaction of endocrine-disrupting chemicals with glutamate-evoked secretion of gonadotropin-releasing hormone.

In previous studies, we detected a dichlorodiphenyltrichloroethane (DDT) derivative in the serum of children with sexual precocity after migration from developing countries. Recently, we reported that DDT stimulated pulsatile gonadotropin-releasing hormone (GnRH) secretion and sexual maturation in the female rat. The aim of this study was to delineate the mechanisms of interaction of endocrine-disrupting chemicals including DDT with GnRH secretion evoked by glutamate in vitro. Using hypothalamic explants obtained from 15-day-old female rats, estradiol (E2) and DDT caused a concentration-related increase in glutamate-evoked GnRH release while p,p'-dichlorodiphenyldichloroethene and methoxychlor had no effect. The effective DDT concentrations in vitro were consistent with the serum concentrations measured in vivo 5 days after exposure of immature rats to 10 mg/kg/day of o,p'-DDT. Bisphenol A induced some stimulatory effect, whereas no change was observed with 4-nonylphenol. The o,p'-DDT effects in vitro were prevented partially by a selective antagonist of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of glutamate receptors. A complete prevention of o,p'-DDT effects was caused by an estrogen receptor (ER) antagonist as well as an aryl hydrocarbon receptor (AHR) antagonist and inhibitors of protein kinases A and C and mitogen-activated kinases. While an intermittent incubation with E2 caused no change in amplification of the glutamate-evoked GnRH release for 4 h, continuous incubation with E2 or o,p'-DDT caused an increase of this amplification after 3.5 h of incubation. In summary, DDT amplifies the glutamate-evoked GnRH secretion in vitro through rapid and slow effects involving ER, AHR, and AMPA receptor mediation.

Early maturation of gonadotropin-releasing hormone secretion and sexual precocity after exposure of infant female rats to estradiol or dichlorodiphenyltrichloroethane.

An increase in the frequency of pulsatile gonadotropin-releasing hormone (GnRH) secretion in vitro and a reduction in LH response to GnRH in vivo characterize hypothalamic-pituitary maturation before puberty in the female rat. In girls migrating for international adoption, sexual precocity is frequent and could implicate former exposure to the insecticide dichlorodiphenyltrichloroethane (DDT), since a long-lasting DDT derivative has been detected in the serum of such children. We aimed at studying the effects of early transient exposure to estradiol (E(2)) or DDT in vitro and in vivo in the infantile female rat. Using a static incubation system of hypothalamic explants from 15-day-old female rats, a concentration- and time-dependent reduction in GnRH interpulse interval (IPI) was seen during incubation with E(2) and DDT isomers. These effects were prevented by antagonists of alpha-amino-3-hydroxy-5-methylisoxazole-4 propionic acid (AMPA)/kainate receptors and estrogen receptor. Also, o,p'-DDT effects were prevented by an antagonist of the aryl hydrocarbon orphan dioxin receptor (AHR). After subcutaneous injections of E(2) or o,p'-DDT between Postnatal Days (PNDs) 6 and 10, a decreased GnRH IPI was observed on PND 15 as an ex vivo effect. After DDT administration, serum LH levels in response to GnRH were not different from controls on PND 15, whereas they tended to be lower on PND 22. Subsequently, early vaginal opening (VO) and first estrus were observed together with a premature age-related decrease in LH response to GnRH. After prolonged exposure to E(2) between PNDs 6 and 40, VO occurred at an earlier age, but first estrus was delayed. We conclude that a transient exposure to E(2) or o,p'-DDT in early postnatal life is followed by early maturation of pulsatile GnRH secretion and, subsequently, early developmental reduction of LH response to GnRH that are possible mechanisms of the subsequent sexual precocity. The early maturation of pulsatile GnRH secretion could involve effects mediated through estrogen receptor and/or AHR as well as AMPA/kainate subtype of glutamate receptors.

A de novo mutation in an already mutant nucleotide of the thyroid hormone receptor beta gene perpetuates resistance to thyroid hormone.

Resistance to thyroid hormone (RTH) is a syndrome of reduced sensitivity to thyroid hormone, most commonly caused by mutations in the thyroid hormone receptor (TR) beta gene. Mutations are mostly located in the ligand-binding domain of the TRbeta, decreasing T(3) binding to the mutant TRbeta molecule, which in turn interferes with the function of the wild-type (WT) TR. A total of 122 different TRbeta gene mutations have been identified so far, with 46 occurring in more than one family. We now report a family with two novel TRbeta mutations occurring in the same nucleotide. The proposita had two children from each of her two marriages. One daughter and one son from each marriage had severe RTH with free T(4) and T(3) levels 3- to 4-fold the mean normal values and unsuppressed TSH, mental retardation, and deafness. The proposita had a missense mutation (GTG to GGG) in codon 458 of the TRbeta gene, resulting in the replacement of the normal valine with glycine (V458G). Although this mutation was transmitted to her affected son, the mutated codon in her affected daughter was GAG, encoding glutamic acid (V458E). Haplotype analysis showed that this de novo mutation occurred on the already mutant allele of the proposita. Cotransfection of each of these mutant TRbetas with the wild-type TRbeta showed a potent dominant negative effect. Large amounts of T(3) were required to dissociate homodimers of the mutant TRbeta bound to DNA. In addition, and in contrast to other mutant TRbetas with severe T(3)-binding defects, homodimer release failed to recruit the steroid receptor coactivator. No defects in heterodimerization with retinoid X receptor-alpha or association with a nuclear receptor corepressor, were identified. These in vitro data are in agreement with the in vivo phenotype of severe RTH. Unique and previously unreported in human inherited diseases is the occurrence of a de novo mutation at an already mutant nucleotide. Because the occurrence by chance is extremely unlikely, it is postulated that the presence of three guanines in the sequence created by the mutant nucleotide of the proposita results in a mutagenic site prone to de novo mutation.

Factors accounting for perinatal occurrence of pulsatile gonadotropin-releasing hormone secretion in vitro in rats.

Our aim was to study the inhibitory and facilitatory factors possibly accounting for the undetectable activity of the GnRH pulse generator in late fetal life in vitro and its awakening in early postnatal life. Gamma aminobutyric acid (GABA(A)) receptor antagonism using SR 95 531 did not cause any secretory pulse in fetal explants, whereas a significant stimulation of GnRH pulse frequency was obtained at 5 and 15 days. GnRH secretory response to repeated N-methyl-D-aspartate (NMDA) stimulation showed progressive disappearance, indicating that the inhibitory autofeedback was operating. GnRH release caused by glutamine was respectively 9% and 20% of that evoked by glutamate in fetal and 5-day-old rats whereas both amino acids were equally active at 15 days. Explants obtained after cesarean section performed at onset of labor did not show any secretory pulse, while pulses could be observed with explants obtained 2 h after vaginal delivery. Incubation of fetal explants with oxytocin (10(-8) M) or prostaglandin E2 (PGE2) (10(-6) M) resulted in occurrence of GnRH secretory pulses. A facilitatory effect of the oxytocin was shown to persist on Days 1, 5, and 15 and inhibitory effects of an oxytocin receptor antagonist provided some evidence of endogenous oxytocin involvement. We conclude that, in the fetal rat hypothalamus, GnRH inhibitory autofeedback and GABAergic inputs do not account for the absence of pulsatile GnRH secretion in vitro. A low rate of glutamate biosynthesis from glutamine is a possibly limiting factor. Oxytocin and PGE2 can play a facilitatory role in the postpartal occurrence of pulsatile GnRH secretion.

In vitro paradigms for the study of GnRH neuron function and estrogen effects.

The elaboration of in vitro paradigms has enabled direct study of GnRH secretion and the regulation of this process. Common findings using different models are the pulsatile nature and calcium-dependency of GnRH secretion, the excitatory effect of glutamate, and the inhibitory or excitatory effect of GABA. Among the different paradigms, the fetal olfactory placode cultures exhibit the unique property of migration in vitro and may retain the capacity to undergo maturational changes in vitro. The short-term incubation of hypothalamic explants obtained at different ages enables one to study developmental changes as well. Estrogens may have important roles in the regulation of GnRH function and can act indirectly via the neighboring neuronal/glial apparatus and directly on GnRH neurons at the cell body and terminal levels. A direct effect is supported by the recent localization of ERalpha and ERbeta transcripts in GnRH neurons using most paradigms. Discrepant effects of estrogens on GnRH neurons were observed since GnRH biosynthesis is inhibited while GnRH secretion can be either stimulated, unaffected, or reduced. It is likely that the regulatory role of sex steroids including estradiol is very complex since it could involve direct and indirect effects on GnRH neurons through genomic and/or non-genomic mechanisms.