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Zebrafish are now widely used to study skeletal development and bone-related diseases. To that end, understanding osteoblast differentiation and function, the expression of essential transcription factors, signaling molecules, and extracellular matrix proteins is crucial. We isolated Sp7-expressing osteoblasts from 4-day-old larvae using a fluorescent reporter. We identified two distinct subpopulations and characterized their specific transcriptome as well as their structural, regulatory, and signaling profile. Based on their differential expression in these subpopulations, we generated mutants for the extracellular matrix protein genes col10a1a and fbln1 to study their functions. The col10a1a−/− mutant larvae display reduced chondrocranium size and decreased bone mineralization, while in adults a reduced vertebral thickness and tissue mineral density, and fusion of the caudal fin vertebrae were observed. In contrast, fbln1−/− mutants showed an increased mineralization of cranial elements and a reduced ceratohyal angle in larvae, while in adults a significantly increased vertebral centra thickness, length, volume, surface area, and tissue mineral density was observed. In addition, absence of the opercle specifically on the right side was observed. Transcriptomic analysis reveals up-regulation of genes involved in collagen biosynthesis and down-regulation of Fgf8 signaling in fbln1−/− mutants. Taken together, our results highlight the importance of bone extracellular matrix protein genes col10a1a and fbln1 in skeletal development and homeostasis.

ABSTRACT Early‐onset osteoporosis (EOOP) has been associated with several genes, including LRP5, coding for a coreceptor in the Wnt pathway. Variants in LRP5 were also described in osteoporosis pseudoglioma syndrome, combining severe osteoporosis and eye abnormalities. Genomewide‐association studies (GWAS) showed that LRP5 p.Val667Met (V667M) variant is associated with low bone mineral density (BMD) and increased fractures. However, despite association with a bone phenotype in humans and knockout mice, the impact of the variant in bone and eye remains to be investigated. Here, we aimed to evaluate the bone and ocular impact of the V667M variant. We recruited 11 patients carrying the V667M variant or other loss‐of‐function variants of LRP5 and generated an Lrp5V667M mutated mice. Patients had low lumbar and hip BMD Z‐score and altered bone microarchitecture evaluated by HR‐pQCT compared with an age‐matched reference population. Murine primary osteoblasts from Lrp5V667M mice showed lower differentiation capacity, alkaline phosphatase activity, and mineralization capacity in vitro. Ex vivo, mRNA expression of Osx, Col1, and osteocalcin was lower in Lrp5V667M bones than controls (all p

Osteoarthritis is a degenerative articular disease affecting mainly aging animals and people. The extracellular matrix protein Efemp1 was previously shown to have higher turn-over and increased secretion in the blood serum, urine, and subchondral bone of knee joints in osteoarthritic patients. Here, we use the zebrafish as a model system to investigate the function of Efemp1 in vertebrate skeletal development and homeostasis. Using in situ hybridization, we show that the efemp1 gene is expressed in the brain, the pharyngeal arches, and in the chordoblasts surrounding the notochord at 48 hours post-fertilization. We generated an efemp1 mutant line, using the CRISPR/Cas9 method, that produces a severely truncated Efemp1 protein. These mutant larvae presented a medially narrower chondrocranium at 5 days, which normalized later at day 10. At age 1.5 years, µCT analysis revealed an increased tissue mineral density and thickness of the vertebral bodies, as well as a decreased distance between individual vertebrae and ruffled borders of the vertebral centra. This novel defect, which has, to our knowledge, never been described before, suggests that the efemp1 mutant represents the first zebrafish model for spinal osteoarthritis.

Aim: To describe the presenting features, bone characteristics and molecular genetics in a large monocentric cohort of children and young adults with idiopathic primary osteoporosis. Methods: Sixty-six patients (19 children, 47 adults; 28 males, 38 females; age at referral: 3.8 to 65 years) diagnosed with primary osteoporosis were included in this study; patients with features of osteogenesis imperfecta or other known syndromes associated with osteoporosis were excluded. For each patient, the following data were collected by retrospective chart review: family and personal history of fracture and osteoporosis, mineral homeostasis parameters and markers of bone formation and resorption, bone mineral density (BMD) of the lumbar spine (LS-BMD), the total body less head (TB-BMD), and total hip levels (TH-BMD) measured by DXA. As part of the initial assessment process, a bone fragility gene panel sequencing was performed in all of these patients. Results: There was a higher predominance of males in the children (63%) and of females in the adults (66%) (p = 0.030). Compared to the adults, the children had a significantly lower frequency of vertebral fractures (26 vs 57%, p = 0.022) and a higher frequency of peripheral fractures (84 vs 53%; p = 0.019). Bone fragility gene panel sequencing allowed the identification of the heterozygous pathogenic variant in 27% of patients (most frequently in LRP5, WNT1 and COL1A1 or 2 genes) and the heterozygous p.(Val667Met) LRP5 variant in 11% of them. The frequency of pathogenic variants tended to be higher in the children compared to the adults without reaching statistical significance (42 vs 19%; p = 0.053). The frequency of the p.(Val667Met) LRP5 variant was similar in children and adults. No significant differences were found regarding the various clinical, biological and radiological characteristics of the patients according to genotype. Conclusion: In this study, we reported the presenting features and bone characteristics in a large cohort of children and young adults with idiopathic primary osteoporosis. Bone fragility gene panel sequencing allowed the identification of genetic variants in a significant proportion of these patients. Molecular diagnosis in these patients is important in order to be able to offer genetic counselling and organise patient management.

Objective: Faciocraniosynostoses (FCS) are malformations affecting the development of the bones of the skull and face, due to the premature closure of one or more craniofacial sutures, mostly secondary to activating Fibroblast Growth Factor Receptor (FGFR) 1–3 mutations. Gain-of-function FGFR3 mutations are also responsible for various conditions referred to as osteochondrodysplasia (OCD), characterized by structural and functional abnormalities of growth plate cartilages. We hypothesized that patients with FGFR-related faciocraniosynostoses may present extra-cranial growth anomalies. Study design: We retrospectively collected height and weight data from a cohort of 70 patients. Included patients were admitted for FGFR-related FCS between 2000 and 2021 at the Craniofacial Unit of Necker – Enfants Malades University Hospital in Paris, France. Results: We showed that FGFR-related faciocraniosynostoses had significantly reduced heights and weights relative to controls, and that two specific time periods (1–3 years and > 8 years of age) were associated with lower height and weight values. Four patients had received growth hormone treatment but remained below normal values for growth in height and weight. Conclusions: Patients with FGFR-related faciocraniosynostoses have clinically significant extra-cranial anomalies which are not currently investigated and managed in usual protocols; these patients could benefit from a systematic pre-pubertal endocrine assessment. More generally, our results extend the scope of extracranial anomalies in FGFR-related faciocraniosynostoses and support the hypothesis that all conditions with activating FGFR mutations affect both membranous ossification and long bones.

Abstract Background Cherubism is a rare autosomal dominant genetic condition caused by mutations in the SH3BP2 gene. This disease is characterized by osteolysis of the jaws, with the bone replaced by soft tissue rich in fibroblasts and multinuclear giant cells. SH3BP2 is a ubiquitous adaptor protein yet the consequences of SH3BP2 mutation have so far been described as impacting only face. Cherubism mouse models have been generated and unlike human patients, the knock-in mice exhibit systemic bone loss together with a systemic inflammation. Case presentation In light of these observations, we decided to search for a systemic cherubism phenotype in a 6-year-old girl with an aggressive cherubism. We report here the first case of cherubism with systemic manifestations. Bone densitometry showed low overall bone density (total body Z-score = − 4.6 SD). Several markers of bone remodelling (CTx, BALP, P1NP) as well as inflammation (TNFα and IL-1) were elevated. A causative second-site mutation in other genes known to influence bone density was ruled out by sequencing a panel of such genes. Conclusions If this systemic skeletal cherubism phenotype should be confirmed, it would simplify the treatment of severe cherubism patients and allay reservations about applying a systemic treatment such as those recently published (tacrolimus or imatinib) to a disease heretofore believed to be localised to the jaws.

Abstract Background Early‐onset osteoporosis (EOOP) is defined by low bone mineral density (BMD), which increases the risk of fracture. Although the prevalence of osteoporosis at a young age is unknown, low BMD is highly linked to genetic background. Heterozygous pathogenic variants in low‐density lipoprotein receptor‐related protein 5 (LRP5) are associated with EOOP. This study aimed to investigate the genetic profile in patients with EOOP to better understand the variation in phenotype severity by using a targeted gene sequencing panel associated with bone fragility. Method and Results We used a sequencing panel with 17 genes reported to be related to bone fragility for analysis of 68 patients with EOOP. We found a high positivity rate of EOOP with LRP5 variants (14 patients, 20.6%). The remaining 79.4% of patients with EOOP but without LRP5 variants showed variable disease severity, as observed in patients with at least one variant in this gene. One patient, with multiple fractures and spine L1‐L4 BMD Z‐score −2.9, carried a novel pathogenic homozygous variant, c.2918T>C, p.(Leu973Pro), without any pseudoglioma. In addition to carrying the LRP5 variant, 2 other patients carried a heterozygous variant in Wnt signaling pathway genes: dickkopf WNT signaling pathway inhibitor 1 (DKK1) [NM_012242.4: c.359G>T, p.(Arg120Leu)] and Wnt family member 3A (WNT3A) [NM_033131.3: c.377G>A, p. (Arg126His)]. As compared with single‐variant LRP5 carriers, double‐variant carriers had a significantly lower BMD Z‐score (−4.1 ± 0.8) and higher mean number of fractures (6.0 ± 2.8 vs. 2.2 ± 1.9). Analysis of the family segregation suggests the inheritance of BMD trait. Conclusion Severe forms of EOOP may occur with carriage of 2 pathogenic variants in genes encoding regulators of the Wnt signaling pathway. Two‐variant carriers of Wnt pathway genes had severe EOOP. Moreover, DKK1 and WNT3A genes should be included in next‐generation sequence analyses of bone fragility.

Abstract Background Wilson disease is an autosomal recessive metabolic disorder resulting from accumulation of excess copper especially in the liver and brain. This disease is mainly characterized by hepatic disorders and less frequently by neuro‐psychiatric disturbances. This recessive disease is due to mutation in ATP7B, which codes for an ATPase involved in copper‐transport across the plasma membrane. Molecular diagnosis of WD is positive in approximately 98% of cases. Also, in few cases, WD patients present a single deleterious mutation (heterozygous) or no mutation after sanger and NGS standard sequencing analysis of ATP7B. Therefore, in these problematic WD cases, we hypothesized that deleterious mutations reside in intronic regions of ATP7B. Methods Complete ATP7B gene was sequenced by Next Generation Sequencing including its promoter. Results Five unrelated families with Wilson disease shared the same novel, deep intronic NG_008806.1 (ATP7B_v001):c.2866‐1521G>A variant in ATP7B. Analysis of RNA transcripts from primary fibroblasts of one patient confirmed the deleterious impact of the intronic variant on splicing and its likely pathologic effect in this compound heterozygote. Conclusion This discovery of a novel intronic mutation in ATP7B has improved the molecular diagnosis of WD in the French patient cohort to greater than 98%. Thus, we recommend complete sequencing of ATP7B gene, including introns, as a molecular diagnostic approach in cases of clinically confirmed WD which lack pathogenic exon or promoter variants in one or both alleles.

Abstract Background Wilson’s disease (WD) is a rare autosomal recessive metabolic disease caused by ATP7B gene mutations tat cause excessively high copper levels, particularly in the liver and brain. The WD phenotype varies in terms of its clinical presentation and intensity. Diagnosing this metabolic disorder is important as a lifelong treatment, based on the use of copper chelating agents or zinc salts, is more effective if it’s started early. Worldwide prevalence of WD is variable, with an average of 1/30,000. In France, a recent study based on French health insurance data estimated the clinical prevalence of the disease to be around 3/200,000. Methods To estimate the genetic prevalence of WD in France, we analysed the ATP7B gene by Next Generation Sequencing from a large French cohort of indiscriminate subjects. Results We observed a high heterozygous carrier frequency of ATP7B in France. Among the 697 subjects studied, 18 variants classified as pathogenic or probably pathogenic were found at heterozygous level in 22 subjects (22 alleles/1394 alleles), yielding a prevalence of 0.032 or 1/31 subjects. Conclusions This considerable and unexplained discrepancy between the heterozygous carrier frequency and the clinical prevalence of WD may be explained by the clinical variability, the incomplete penetrance and the existence of modifiers genes. It suggests that the molecular analysis of ATP7B should be interpreted with caution, always alongside copper assays (ceruloplasmin, relative exchangeable copper, 24 h-urinary copper excretion) with particular respect to exome sequencing.

Mutations in Fibroblast Growth Factor Receptor II (FGFR2) have been identified in patients with Crouzon and Pfeiffer syndrome, among which rare mutations of the intracellular tyrosine kinase domain. Correlating subtle phenotypes with each rare mutation is still in progress. In Necker-Enfants Malades Hospital, we identified three patients harboring three different pathogenic variants of the same amino acid residue Asn-549 located in this domain: in addition to a very typical crouzonoid appearance, they all developed clinically relevant hydrocephalus, which is an inconstant feature of Crouzon and Pfeiffer syndrome. Overall, FGFR2 tyrosine kinase domain mutations account for 5/67 (7.4%) cases in our hospital. We describe a novel mutation, p.Asn549Ser, and new cases of p.Asn549His and p.Asn549Thr mutations, each reported once before. Our three cases of Asn-549 mutations, alongside with rare previously reported cases, show that these patients are at higher risk of hydrocephalus. Clinical and imaging follow-up, with possible early surgery, may help prevent secondary intellectual disability.

Background: Calcium pyrophosphate (CPP) microcrystal deposition is associated with wide clinical phenotypes, including acute and chronic arthritis, that are interleukin 1β (IL-1β)-driven. Two CPP microcrystals, namely monoclinic and triclinic CPP dihydrates (m- and t-CPPD), have been identified in human tissues in different proportions according to clinical features. m-CPP tetrahydrate beta (m-CPPTβ) and amorphous CPP (a-CPP) phases are considered as m- and t-CPPD crystal precursors in vitro.Objectives: We aimed to decipher the inflammatory properties of the three crystalline phases and one amorphous CPP phase and the intracellular pathways involved.Methods: The four synthesized CPP phases and monosodium urate crystals (MSU, as a control) were used in vitro to stimulate the human monocytic leukemia THP-1 cell line or bone marrow-derived macrophages (BMDM) isolated from WT or NLRP3 KO mice. The gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR; IL-1β, IL-6 and IL-8 production by ELISA; and mitogen-activated protein kinase (MAPK) activation by immunoblot analysis. NF-κB activation was determined in THP-1 cells containing a reporter plasmid. In vivo, the inflammatory potential of CPP phases was assessed with the murine air pouch model via cell analysis and production of IL-1β and CXCL1 in the exudate. The role of NF-κB was determined by a pharmacological approach, both in vivo and in vitro.Results:In vitro, IL-1β production induced by m- and t-CPPD and m-CPPTβ crystals was NLRP3 inflammasome dependent. m-CPPD crystals were the most inflammatory by inducing a faster and higher production and gene expression of IL-1β, IL-6, and IL-8 than t-CPPD, m-CPPTβ and MSU crystals. The a-CPP phase did not show an inflammatory property. Accordingly, m-CPPD crystals led to stronger activation of NF-κB, p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) MAPKs. Inhibition of NF-κB completely abrogated IL-1β and IL-8 synthesis and secretion induced by all CPP crystals. Also, inhibition of JNK and ERK1/2 MAPKs decreased both IL-1β secretion and NF-κB activation induced by CPP crystals. In vivo, IL-1β and CXCL1 production and neutrophil infiltration induced by m-CPPD crystals were greatly decreased by NF-κB inhibitor treatment.Conclusion: Our results suggest that the inflammatory potential of different CPP crystals relies on their ability to activate the MAPK-dependent NF-κB pathway. Studies are ongoing to investigate the underlying mechanisms.

Osteoporosis-pseudoglioma (OPPG) syndrome is a very rare autosomal recessive disorder, caused by mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene. It manifests by severe juvenile osteoporosis with congenital or infancy-onset visual loss. We describe a case of OPPG due to novel mutation in LRP5 gene, occurring in a female Mauritanian child. This 10-year-old female child was born blind, and after then multiple fragility fractures appeared. PCR amplification and sequencing revealed a novel homozygous nonsense mutation in exon 10 of the LRP5 gene (c.2270G>A; pTrP757⁎); this mutation leads to the production of a truncated protein containing 757 amino acids instead of 1615, located in the third β-propeller domain of the LRP5 protein. Both parents were heterozygous for the mutation. This is the first case of the OPPG described in black Africans, which broadens the spectrum of LRP5 gene mutations in OPPG.

Osteoporosis is due to an imbalance between decreased bone formation by osteoblasts and increased resorption by osteoclasts. Deciphering factors controlling bone formation is therefore of utmost importance for the understanding and the treatment of osteoporosis. Our previous in vivo results showed that bone formation is reduced in the absence of the serotonin receptor 5-HT2B, causing impaired osteoblast proliferation, recruitment, and matrix mineralization. In this study, we investigated the signaling pathways responsible for the osteoblast defect in 5-HT2BR(-/-) mice. Notably, we investigated the phospholipase A2 pathway and synthesis of eicosanoids in 5-HT2BR(-/-) compared to wild type (WT) osteoblasts. Compared to control osteoblasts, the lack of 5-HT2B receptors was only associated with a 10-fold over-production of prostacyclin (PGI2). Also, a specific prostacyclin synthase inhibitor (U51605) rescued totally osteoblast aggregation and matrix mineralization in the 5-HT2BR(-/-) osteoblasts without having any effect on WT osteoblasts. Prostacyclin is the endogenous ligand of the nuclear peroxisome proliferator activated receptor ß/δ (PPAR-ß/δ), and its inhibition in 5-HT2BR(-/-) cells rescued totally the alkaline phosphatase and osteopontin mRNA levels, cell-cell adhesion, and matrix mineralization. We conclude that the absence of 5-HT2B receptors leads to the overproduction of prostacyclin, inducing reduced osteoblast differentiation due to PPAR-ß/δ -dependent target regulation and defective cell-cell adhesion and matrix mineralization. This study thus reveals a previously unrecognized cell autonomous osteoblast defect in the absence of 5-HT2BR and highlights a new pathway linking 5-HT2B receptors and nuclear PPAR- ß/δ via prostacyclin.

Background There are over 2000 pregnancies annually in women with type 1 diabetes in the UK. Despite recent improvements in diabetes technology, most women cannot achieve and maintain the recommended pregnancy glucose targets. Thus, one in two babies experience complications requiring neonatal care unit admission. Recent studies demonstrate that hybrid closed-loop therapy, in which algorithms adjust insulin delivery according to continuous glucose measurements, is effective for managing type 1 diabetes outside of pregnancy, but efficacy during pregnancy is unclear. Objective To examine the clinical efficacy of hybrid closed-loop compared to standard insulin therapy in pregnant women with type 1 diabetes. Design A multicentre, parallel-group, open-label, randomised, controlled trial in pregnant women with type 1 diabetes. Setting Nine antenatal diabetes clinics in England, Scotland and Northern Ireland. Participants Pregnant women with type 1 diabetes and above-target glucose levels, defined as glycated haemoglobin A1c of ≥ 48 mmol/mol (6.5%) in early pregnancy. Interventions A hybrid closed-loop system compared to standard insulin delivery (via insulin pump or multiple daily injections) with continuous glucose monitoring. Outcome measures The primary outcome is the difference between the intervention and control groups in percentage time spent in the pregnancy glucose target range (3.5–7.8 mmol/l) as measured by continuous glucose monitoring from 16 weeks’ gestation until delivery. Secondary outcomes include overnight time in range, time above range (> 7.8 mmol/l), glycated haemoglobin A1c, safety outcomes (diabetic ketoacidosis, severe hypoglycaemia, adverse device events), psychosocial functioning obstetric and neonatal outcomes. Results The percentage of time that maternal glucose levels were within target range was higher with closed-loop than standard insulin therapy: 68.2 ± 10.5 in closed-loop and 55.6 ± 12.5 in the control group (mean‑adjusted difference 10.5 percentage points, 95% confidence interval 7.0 to 14.0; p < 0.001). Results were consistent in secondary outcomes, with less time above range (−10.2%, 95% confidence interval −13.8 to −6.6%; p < 0.001), higher overnight time in range (12.3%, 95% confidence interval 8.3 to 16.2%; p < 0.001) and lower glycated haemoglobin A1c (−0.31%, 95% confidence interval −0.50 to −0.12%; p < 0.002) all favouring closed-loop. The treatment effect was apparent from early pregnancy and consistent across clinical sites, maternal glycated haemoglobin A1c categories and previous insulin regimen. Maternal glucose improvements were achieved with 3.7 kg less gestational weight gain and without additional hypoglycaemia or total daily insulin dose. There were no unanticipated safety problems (six vs. five severe hypoglycaemia cases, one diabetic ketoacidosis per group) and seven device-related adverse events associated with closed-loop. There were no between-group differences in patient-reported outcomes. There was one shoulder dystocia in the closed-loop group and four serious birth injuries, including one neonatal death in the standard care group. Limitations Our results cannot be extrapolated to closed-loop systems with higher glucose targets, and our sample size did not provide definitive data on maternal and neonatal outcomes. Conclusions Hybrid closed-loop therapy significantly improved maternal glycaemia during type 1 diabetes pregnancy. Our results support National Institute for Health and Care Excellence guideline recommendations that hybrid closed-loop therapy should be offered to all pregnant women with type 1 diabetes. Future work Future trials should examine the effectiveness of hybrid closed-loop started before pregnancy, or as soon as possible after pregnancy confirmation. Trial registration This trial is registered as ISRCTN56898625. Funding This award was funded by the National Institute of Health and Care Research (NIHR) Efficacy and Mechanism Evaluation (EME) programme (NIHR award ref: 16/35/01) and is published in full in Efficacy and Mechanism Evaluation; Vol. 11, No. 7. See the NIHR Funding and Awards website for further award information. Dexcom supplied the continuous glucose monitoring systems used by AiDAPT intervention- and control-arm participants at reduced cost. Plain language summary Pregnancy in women with type 1 diabetes is associated with complications for both mother and baby. Recent improvements in diabetes technology have not been sufficient to help most women achieve and maintain recommended pregnancy glucose targets. Hybrid closed-loop technology (an insulin pump, smartphone app and continuous glucose monitor), which automatically adjusts insulin doses according to glucose measurements, is effective in managing type 1 diabetes outside of pregnancy, but its effectiveness during pregnancy was unclear. To examine the effectiveness of hybrid closed-loop therapy compared to standard insulin therapy in pregnant women with type 1 diabetes, we conducted a randomised controlled trial in nine maternity clinics in England, Scotland and Northern Ireland. We enrolled pregnant women with type 1 diabetes and above-target glucose levels, defined as glycated haemoglobin A1c of ≥ 48 mmol/mol, in early pregnancy. Women were randomly assigned to either a hybrid closed-loop system or standard insulin delivery (insulin pump or multiple daily injections) with continuous glucose monitoring. This study found that hybrid closed-loop therapy improved maternal glucose levels during type 1 diabetes pregnancy, resulting in higher time spent in the target glucose range and less time above range. Mothers in the closed-loop group had less weight gain during pregnancy, without additional hypoglycaemia or insulin dose. The benefits of closed-loop were consistent across maternity clinics, maternal glucose levels and previous insulin pump or injection therapy. There were no unanticipated safety problems associated with using closed-loop. However, the study was too small to provide definite information on pregnancy outcomes, and the results cannot be inferred to other closed-loop systems with higher glucose targets. Future trials should examine the effectiveness of closed-loop started before pregnancy or as soon as possible after pregnancy confirmation. Our results support National Institute for Health and Care Excellence guideline recommendations that hybrid closed-loop therapy should be offered to all pregnant women with type 1 diabetes. Scientific summary Background There are over 2000 pregnancies annually in women with type 1 diabetes. These pregnancies are complicated by high and increasing rates of preterm births, large for gestational age birthweight babies and neonatal care unit admissions. Obstetric and neonatal complications are lowest in mothers who achieve target glucose levels, which requires unrelenting attention to diabetes self-management and insulin dose adjustment throughout pregnancy. Despite improvements in diabetes technology, including continuous glucose monitoring (CGM) and insulin pumps, most pregnant women with type 1 diabetes cannot achieve or maintain the pregnancy glucose targets. National audit data confirm that only 15% of women enter pregnancy with the recommended glycated haemoglobin A1c (HbA1c) target of 10 mmol/l). The primary outcome was the percentage of time spent with CGM glucose levels between 3.5 and 7.8 mmol/l between 16 weeks’ gestation and delivery. Safety outcomes included the number and severity of diabetic ketoacidosis (DKA), severe hypoglycaemia (SH) and adverse device events. Patient-reported outcomes were reported at around 34–36 weeks’ gestation using the following validated questionnaires: Insulin Delivery Systems: Perspectives, Ideas, Reflections and Expectations (INSPIRE); EuroQol-5 Dimensions health-related quality-of-life questionnaire (EQ-5D), Diabetes Distress Scale (DDS), hypoglycaemia fear survey II (HFS – worry scale only) and Pittsburgh Sleep Quality Index (PSQI). Maternal and neonatal outcomes were documented at hospital discharge following delivery. Protocol amendments implemented during the COVID-19 pandemic allowed participants the option to continue using CGM with standard or closed-loop insulin delivery as per their initial randomisation for up to 6 months post partum. Outcomes for those who participated in the observational post-partum extension study will be reported separately. Details of the clinical study protocol are published. Primary outcome analysis was by intention-to-treat using a linear mixed-effects regression model adjusted for baseline CGM TIR, insulin delivery and clinical site. Missing primary end-point data were handled using multiple imputation (Rubins and direct likelihood methods) with all randomised participants included. For secondary outcomes, analyses were similar to the primary analysis, without imputation. False discovery rate (FDR)-adjusted p-values were calculated for selected secondary outcomes (overall, overnight, and by-trimester sensor glucose metrics, HbA1c, insulin doses, subgroup analyses, questionnaires) using Benjamini–Hochberg methods. For attainment of sensor glucose targets, a mixed-effects logistic regression model was fitted adjusting for baseline TIR, insulin delivery and clinical site as a random effect. All p-values are two-tailed. Analyses were performed using SAS® 9.4 (SAS Institute Inc., Cary, NC, USA; SAS and all other SAS Institute Inc. product or service names are registered trademarks or trademarks of SAS Institute Inc. in the USA and other countries.® indicates USA registration). Results Participants Between September 2019 and May 2022, 334 participants were assessed for eligibility, with 126 enrolled and 124 randomised: 61 to the closed-loop intervention group and 63 to the standard care control group. Participants were from nine NHS maternity clinics and spanned a range of maternal age, body weight and glycaemic categories. Almost all (98%) were using CGM and approximately half were using insulin pump therapy at enrolment. Participants in the closed-loop group had more previous pregnancies, while those in the standard care group reported more previous DKA events. Two participants switched from their randomised allocation group: one intervention participant for whom lockdown restrictions prevented closed-loop training and one standard care participant who procured closed-loop (CamAPS FX) outside of the trial. Seven participants in each group discontinued their allocated treatment. Despite the impact of the COVID-19 pandemic, the proportion of completed study visits was high (approximately 95%). Participants in the standard care group had more additional clinic visits (1.5 vs. 1.1) and more unscheduled contacts (9.6 vs. 6.1), mostly for pregnancy and diabetes-related reasons. The frequency of sensor use was consistently high: median 97% across both treatment groups. The frequency of closed-loop use was high (median 96%) and remained > 95% throughout pregnancy. Primary efficacy end point The mean (± standard deviation) percentage of time that maternal glucose levels were within the pregnancy target range increased from 47.8 ± 16.4% to 68.2 ± 10.5% in the closed-loop group and from 44.5 ± 14.4% to 55.6 ± 12.5% in the control group [mean‑adjusted difference 10.5 percentage points, 95% confidence interval (CI) 7.0 to 14.0 percentage points; p < 0.001]. Adjustment for potential confounding variables, including previous pregnancies and DKA episodes, did not change the treatment difference. There were no variations in the treatment effect between trial sites and no differential effects across maternal age, HbA1c or insulin delivery categories. The large treatment difference was consistent between intention-to-treat and per-protocol analyses and using multiple imputation methods. Secondary glycaemic outcomes Participants randomised to closed-loop spent less time with glucose levels above target range (mean difference −10.2%, 95% CI −13.8% to −6.6%; p < 0.001). This was accompanied by decreased hyperglycaemia across milder (> 6.7 mmol/l) and more pronounced (> 10.0 mmol/l) categories, as well as lower mean glucose and lower HbA1c (mean difference −0.31%, 95% CI −0.50% to −0.12%; p < 0.002). These changes are notable since participants in both groups spent approximately 70% of time in the near-optimal glucose range of 3.5–10.0 mmol/l at enrolment. Furthermore, in those who started closed-loop therapy during the first trimester, a 5% higher TIR was observed by the end of 12 weeks’ gestation. The effects of the intervention during the overnight period (23.00–07.00) closely followed the 24-hour results (12.3% higher TIR, 95% CI 8.3% to 16.2%; p < 0.001). This was accompanied by less nocturnal hypoglycaemia and fewer nocturnal hypoglycaemic events. Attainment of the sensor glucose target of > 70% time (16 hours 48 minutes) within the pregnancy-specific range was achieved by 28 (47%) closed-loop and 7 (11%) standard care participants. Attainment of the sensor glucose target of 95%) throughout pregnancy, and without apparent safety problems, including among those new to insulin pump therapy. Indeed, participants who continued standard care had more clinic visits and more unscheduled contacts, suggesting that beyond initial training, closed-loop use did not require additional healthcare professional input. Recent trials have demonstrated the benefits of CamAPS FX to those with newly diagnosed type 1 diabetes and young children, and these results further extend the evidence for closed-loop therapy to pregnant women. During pregnancy, women in the closed-loop group increased the percentage of time with near-target glucose levels (3.5–10.0 mmol/l) from 71 to 87%. This is, to the best of our knowledge, the tightest glycaemic control yet achieved through use of closed-loop therapy. Alongside women’s motivation to minimise pregnancy complications, closed-loop use facilitated attainment of 70% time in pregnancy-specific target range throughout gestation. This suggests that tighter glycaemic control could also be feasible outside of pregnancy, when clinically warranted. Given the rapid increases in TIR observed within 1 week of therapy initiation in this trial, and within 1 day in a recent trial, we speculate that further benefits may be obtained from starting closed-loop before pregnancy, or as soon as possible, after pregnancy is confirmed. The current trial participants gained an additional 10% TIR above and beyond the 10% increment achieved by CGM and standard insulin therapy across pregnancy. Previous studies demonstrated that every 5% increased TIR is associated with improved obstetric and neonatal outcomes. Our trial was not powered for pregnancy outcomes, but we infer that this additional 10% time in the pregnancy target range would be expected to have additional health benefits for mothers and their babies. The strengths of our trial include its parallel-group, randomised controlled design, generalisability of our patient population, including those naive to insulin pump therapy and a large proportion who initiated therapy during the first trimester, and a flexible pragmatic trial protocol that facilitated virtual or in-person visits. There was no evidence of increased clinical contacts, frequently observed in investigational device trials. This trial had certain limitations. We did not undertake a health economic evaluation and the current sample size did not provide definitive data on maternal and neonatal health outcomes. Furthermore, our data are applicable only to the CamAPS FX closed-loop system and cannot be extrapolated to systems with higher glucose targets. Closed-loop therapy was effective in type 1 diabetes pregnancy, safely accommodating the marked gestational changes in insulin doses across a range of maternal body weight and glycaemic categories. It gave additional clinical advantage above and beyond that which can be achieved by CGM and standard insulin therapy, supporting NICE guideline recommendations that hybrid closed-loop therapy should be offered to all pregnant women with type 1 diabetes. Trial registration This trial is registered as ISRCTN56898625. Funding This award was funded by the National Institute of Health and Care Research (NIHR) Efficacy and Mechanism Evaluation (EME) programme (NIHR award ref: 16/35/01) and is published in full in Efficacy and Mechanism Evaluation; Vol. 11, No. 7. See the NIHR Funding and Awards website for further award information. Dexcom supplied the continuous glucose monitoring systems used by AiDAPT intervention- and control-arm participants at reduced cost.