Although some patients with diffuse large B‐cell lymphoma (DLBCL) show a response to immunotherapy, there are still many who do not respond. This suggests that various immune checkpoints are complicatedly intertwined in the composition of the tumor microenvironment of DLBCL.To comprehensively understand the expression of various immune checkpoint genes in DLBCL, we performed NanoString assay in 98 patients to investigate 579 genes. In addition, we performed immunohistochemistry for LAG‐3 and PD‐L1 to compare the results with expression in NanoString assay.LAG3 was the highest in cluster C and the lowest in cluster A, showing an expression pattern opposite to that of other immune checkpoint genes. In Cluster A, the expression of genes related to T‐cell activity such as CD8A and GZMB was increased. In Cluster C, the expression of genes related to major histocompatibility complex molecules was the highest. Immunohistochemical stains showed modest agreement with the NanoString results but did not help clustering.As a result of hierarchical clustering of NanoString assay, 98 DLBCLs were classified into three tumor immune microenvironment clusters. Most immune checkpoint genes showed the highest expression in cluster A and the lowest in cluster C. However, the expression of LAG3 in DLBCL contrasts with that of other immune checkpoints. We suggest that the combination of anti‐PD‐1/PD‐L1 and anti‐LAG‐3 blockades in the immunotherapy of DLBCL patients can have a synergistic effect, improving the immunotherapy efficacy and outcome in DLBCL patients.Our results show that the unique expression pattern of  CAR‐T cell therapy allows us to control the T‐cells attacking tumor cells via expressing specific receptors through immunoediting.LAG3 gene is increased in a pattern opposite to that of most other immune checkpoint genes and is associated with immune depletion.To understand the TIME landscape, it is essential to examine the expression patterns of various immune checkpoints and inflammatory activity markers. Therefore, in this study, we attempted to comprehensively identify the activity of inflammation and various immune checkpoint genes in DLBCL using the NanoString assay consisting of a panel of genes related to human immunity. We found that the expression of the 22.1Patients diagnosed with DLBCL at Samsung Medical Center, Seoul, Korea, between January 2011 and December 2015 were enrolled in the study. From these, we selected 98 cases from various organs to compare TIME, and for whom formalin‐fixed paraffin‐embedded (FFPE) blocks were available. Based on the WHO classification2.210 transformed expression was considered the final gene expression matrix.https://software.broadinstitute.org/morpheus/).We analyzed RNA expression using nCounter® technology from NanoString in all 98 cases. The nCounter® Human Immunology V2 Panel with 579 human immune signature genes and 15 housekeeping genes (NanoString Technologies) was used for the NanoString nCounter assay. Total RNA was extracted from 3 to 4 μm thick FFPE tissue sections from representative blocks using the High Pure RNA Paraffin kit (Roche Diagnostics). RNAs (200 ng) were hybridized to the target sequence‐specific capture probes and fluorescence‐labeled reporter probes. The mRNA‐probe complexes were washed, immobilized, and quantified using fluorescence imaging. Two‐step normalization was performed for the gene expression matrix to remove the batch effect of nCounter gene expression. First, we performed within‐normalization using the NanoStringNorm R package with options CodeCount+Sum, Background=mean, and SampleContent=total.sum and adjusted the outliers to the median value with the outlier R package.2.32 were captured for two representative areas to measure the degree of infiltration of LAG‐3‐positive cells in tumors. The number of positive cells and the ratio (%) of positive cells to total cells in the captured images were investigated using QuPath software.Based on the results of the NanoString assay, we performed immunohistochemistry (IHC) for LAG‐3  and PD‐L1 . All slides were scanned using a Pannoramic 1000 digital slide scanner (3DHistech). An INFINITT DPS  was used as the image viewing system. Images with an area of 0.37 mm2.4p < 0.05 was considered statistically significant.The SPSS 27.0 statistical software program (IBM Corporation) was used for the statistical analyses. Pearson's chi‐square test was used for crossover analysis of clinicopathological features. A Kaplan–Meier curve (log‐rank test) was used for survival analysis. Pearson's correlation test was used to analyze the correlation between two continuous variables. Kruskal‐Wallis test was performed to compare two or more groups of continuous variables. 33.1The male‐to‐female ratio in the 98 patients was 63:35 (1.8:1), and the median age was 58 years . Ann Arbor stages I–II and III–IV were present in 67 (68.4%) and 31 (31.6%) patients, respectively. In DLBCL NOS, germinal center B‐cell (GCB) type were 26 (44.1%) and non‐GCB type were 33 (55.9%) cases. Based on the primary organ of tumor, 18, 24, and 4 patients had tumors in the lymph nodes, immune‐privileged organs (brain and testis), and mediastinum, respectively, and the remaining 52 had tumors in other extranodal sites.3.2As a result of unsupervised hierarchical clustering, 98 cases were classified into three clusters . Clusters A, B, and C had 25, 26, and 45 patients, respectively; the remaining 2 cases were unclassifiable. Figure  In the KCD19, BCL2, TNFRSF13B, TNFRSF13C, CD22, MS4A1, etc.), antigen processing and presentation, and major histocompatibility complex (MHC) class I protein binding . Genes related to T‐cell proliferation  or innate immune response  were highly expressed in clusters A and C but showed low expression in cluster B. Numerous immune‐related genes were highly expressed in cluster A and showed low expression in clusters B and C. Some genes were highly expressed in cluster C and showed low expression in cluster A, and these genes were associated with the regulation of innate immune response , regulation of cell adhesion, and MHC class II protein complex binding .Gene expression profile analysis Figure  revealed3.3BTLA, CD27, CD28, CD40, CD274, CD276, CTLA4, CYBB, HAVCR2, ICOS, IDO1, IL2RB, LAG3, PDCD1, and TNFRSF9) were selected from those included in the NanoString gene panel to compare the degree of expression by clusters. . PDCD1, CD4, CD80, CTLA4, PDCD1LG2, PTPN11, CD247, HOXD13, CD3E, HLA‐DRA, HLA‐DQA1, CD3D, HLA‐DRB1, CD28, HLA‐DQB1, CD3G, HLA‐DQA2, HLA‐DQB2, HLA‐DPA1, HLA‐DPB1, and HLA‐DRB5 genes were searched as predicted functional partners of CD274 . . . A number of immune checkpoint genes, including CD274, showed the highest expression in cluster A and the lowest expression in cluster C. By contrast, HLA‐A, HLA‐B, HLA‐C, HLA‐DRB1, HLA‐DPA1, HLA‐DPB1, and HLA‐DRA associated with MHC class I and II molecules showed the highest expression in cluster C along with LAG3. Representative T‐lymphocyte‐related genes  showed the highest expression in cluster A and the lowest expression in cluster C, whereas human leukocyte antigen (HLA)‐related genes showed the highest expression in cluster C. . . , and LAG‐3 positive ratio with LAG3 expression values in the NanoString assay  are shown in Figure The average CPS of PD‐L1 was 22.41 . The median values of PD‐L1 CPS were 13.50, 8.50, and 3.00 in clusters A, B, and C, respectively  cells, and some subsets of plasma cells.In a study conducted on TIL of DLBCL, LAG‐3 showed higher expression in CD4‐positive regulatory T‐cells (Treg) and CD8‐positive T cells than in CD4‐positive non‐Tregs.HLA genes associated with MHC class I and class II molecules was relatively higher in cluster C than in clusters A and B, although the overall inflammatory activity was low. This observation suggests that when LAG‐3 is blocked with an anti‐LAG‐3 antibody in DLBCL with cluster C, CD4 can bind to abundant MHC class II molecules on APC without competition with LAG‐3 and receive sufficient information about the antigen. This might hinder the immune evasion by tumor cells by increasing the activity of tumor‐specific host immunity. The anti‐human LAG‐3 antibody relatlimab was first introduced in 2019.In our NanoString assay results, the activity of the In a clinical trial in patients with previously untreated metastatic or unresectable melanoma (RELATIVITY‐047), inhibition of two immune checkpoints, LAG‐3 and PD‐1, provided a greater benefit with regard to progression‐free survival than inhibition of PD‐1 alone.CD274 and LAG3 genes, respectively, and did not help classify clusters A, B, and C. Therefore, we suggest trying a treatment strategy using a combination of anti‐PD‐1 and anti‐LAG‐3 blockade for all DLBCL patients requiring immunotherapy, rather than using them individually for the patients in each TIME cluster.In our study, immunohistochemical staining of PD‐L1 and LAG‐3 did not correlate well with the expression levels of In 2021, Kotlov et al. defined the TIME of DLBCL into four major categories, germinal center‐like, mesenchymal, inflammatory, and depleted, with distinct biological properties and clinical behavior through a transcriptomic analysis of 4655 DLBCLs.In summary, DLBCL can have various TIME. In a subgroup with TIME with active host immune reaction, most immune checkpoints, including PD‐L1, are highly expressed. In contrast, in another subgroup with TIME with depleted host immunity, the activity of most immune checkpoints is low, while the expression of LAG‐3 is increased. Our results show that the unique expression pattern of LAG‐3 in DLBCL contrasts with that of other immune checkpoints. The unique expression pattern of LAG‐3 in DLBCL shown by our analysis suggests that the combination of anti‐PD‐1/PD‐L1 and anti‐LAG‐3 inhibitors in the immunotherapy of DLBCL patients can have a synergistic effect and broaden the range of the TIMEs covered by immunotherapy, improving the immunotherapy efficacy and outcome in DLBCL patients. In addition, not only the administration of immunotherapeutic agents, but also immune editing that allows CAR‐T cells to ignore the inhibitory signals from PD‐L1 or LAG‐3 can also open new avenues for DLBCL immunotherapy. We need further studies on applying the synergistic dual checkpoint blockade of PD‐1/PD‐L1 and LAG‐3 to treat DLBCL.Hyunjee Lee: Formal analysis ; writing – original draft . Sang Eun Yoon: Resources . Seok Jin Kim: Resources . Won Seog Kim: Resources . Junhun Cho: Conceptualization (lead); formal analysis ; supervision (lead); writing – original draft ; writing – review and editing (lead).All authors declare no competing interests.


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 . Lrp5V667M 3‐month‐old mice, compared with control (CTL) mice, had decreased BMD at the femur (p < 0.01) and lumbar spine (p < 0.01) with normal microarchitecture and bone biomarkers. However, Lrp5V667M mice revealed a trend toward a lower femoral and vertebral stiffness (p = 0.14) and had a lower hydroxyproline/proline ratio compared with CTL, (p = 0.01), showing altered composition and quality of the bone matrix. Finally, higher tortuosity of retinal vessels was found in the Lrp5V667M mice and unspecific vascular tortuosity in two patients only. In conclusion, Lrp5V667M variant is associated with low BMD and impaired bone matrix quality. Retinal vascularization abnormalities were observed in mice. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.Early‐onset osteoporosis (EOOP) has been associated with several genes, including  667M mutant mice show low bone mineral density, reduced osteoblast function and hydroxylation of collagen matrix. Matrix failure explained bone fragility in early onset osteoporosis.Lrp5 But age, sex, and medical history explain only 20% to 40% of bone mineral density (BMD) variations, whereas heritability has been estimated responsible for 60% to 80% of these variations. The impact of genetic component is even higher in primary osteoporosis of men and women younger than 50 years, called early‐onset osteoporosis (EOOP). Genomewide‐association studies (GWAS) have identified a large number of genes as associated with low bone mass in the general population, some also responsible for monogenic bone disorders. The genes involved in the Wnt signaling pathway are particularly represented and, more specifically, the LRP5 (low‐density lipoprotein receptor‐related protein 5) gene. The encoded LRP5 protein acts as a coreceptor for Wnt ligands to activate the canonical Wnt pathway, known to regulate bone formation. Loss‐of‐function mutations of LRP5 are responsible for osteoporosis pseudoglioma syndrome , a rare autosomal recessive disorder characterized by severe osteoporosis in childhood associated with congenital blindness, whereas gain‐of‐function mutations are responsible for high‐bone‐mass disorders . Animal models based on Lrp5 total and conditional knockouts (KO) have consistently shown decreased BMD, and several LRP5 rare heterozygous variants or homozygous low‐frequency variants have been repeatedly detected in patients with EOOP. Hence, the p.Val667Met (V667M) low‐frequency variant, resulting from the missense single nucleotide mutation c.1999G > A in exon 9 of LRP5, has been reported as a genetic risk factor for fractures and low BMD in the general population and is overrepresented in patients with EOOP. Obligate carriers of LRP5 variant had reduced bone mass when compared with age‐ and sex‐matched controls. This homozygous variant was linked to reduced activation of the canonical Wnt signaling pathway in site‐directed mutagenesis experiments. Indeed, heterozygous harboring of this variant is associated with low BMD and EOOP but with a large heterogeneity in the severity of osteoporosis. Its contribution in bone fragility remains unclear as a causal relationship between the presence of the variant and clinical outcomes.The prevalence of osteoporosis raises with age, postmenopausal status,1 and LRP5 is also essential for the development of the retinal vasculature through a variant of Norrin/β‐catenin pathway. Mutations of LRP5 are responsible for loss of vision in the OPPG syndrome and other familial exudative vitreoretinopathy (FEVR). Previous studies reported ocular features in young adults with heterozygous LRP5 variants. However, whether osteoporotic patients have vitreoretinopathy or any vessel abnormalities remains unclear. Here, we aimed to characterize the bone and eye phenotype in patients with EOOP and in a mice model harboring the p.Val667Met variant.Z‐score <−2 standard deviation (SD) at the spine or total hip associated with osteoporotic fractures. For each patient, the following data were collected: age at diagnosis, number and site of fractures, family history of fracture, and bone marker levels before any treatment. None had signs of skeletal dysplasia or known eye abnormality. In this study, we included patients with LRP5 suspected pathogenic variants, who were offered a bone and eye evaluation. Clinical and genetic evaluation was part of the assessment of rare bone diseases. Written informed consent was obtained from all participants.We analyzed patients younger than 55 years who were referred to the clinic for osteoporosis and/or history of fracture. We excluded patients taking medications that interfered with bone metabolism. Extensive radiological and biochemical investigations allowed excluding secondary causes of osteoporosis. Tests included serum levels of total calcium, phosphorus, 25OH vitamin D and parathyroid hormone, electrophoresis of proteins, C‐reactive protein, thyroid‐stimulating hormone, and serum creatinine. Patients were <55 years old at diagnosis and had a BMD total hip30 ass Briefly, DNA samples were screened by targeted gene sequencing panel for variants in the following genes: LRP5, Plastin 3 (PLS3), Collagen type I α1 (COL1A1), Collagen type Ι α2 (COL1A2), Wnt family member 1 (WNT1), LDL receptor related protein 6 (LRP6) and dickkopf Wnt signaling pathway inhibitor 1 (DKK1), WNT3A, TNF superfamily member 11 (TNFSF11), TNF receptor superfamily member 11a (TNFRSF11A), TNF superfamily member 11b (TNFRSF11B), and vitamin D receptor (VDR). All variants were confirmed by Sanger technology. Variant pathogenicity was evaluated by using the prediction software Polyphen (http://genetics.bwh.harvard.edu/pph2), Mutationtaster (http://www.mutationtaster.org), and SIFT (http://sift.bii.a-star.edu.sg). Only the genes in the panels were investigated without other copy number variation (CNV) analysis.DNA was analyzed as described elsewhere.18 BriBlood samples were collected to assess bone‐remodeling biomarkers. We used the following methods to measure biomarkers: C‐terminal telopeptide of type I collagen (CTX), N‐terminal propeptide of type I collagen (P1NP), Osteocalcin , bone alkaline phosphatase , and TRAP5B levels by ELISA kit (IDS).Microarchitectural parameters were assessed at the right distal radius and tibia by HR‐pQCT , as previously described. The resolution (voxel size) was fixed at 80 μm. Quality control was performed by daily scans of the manufacturer's phantom. The volume of interest (VOI) was automatically separated into trabecular and cortical regions using a threshold‐based algorithm  to provide all parameters of interest.31Lrp5, generated by PCR using C57Bl/6N BAC DNA as template, which was linearized and electroporated into embryonic stem cells where it underwent homologous recombination. We used an FRT‐flanked neomycin resistance cassette, inserted in an unsuspicious region in intron 9 that was later excised by Flp recombination, upon breeding with germline Flp‐expressing mice (“Flp‐deletors”). The FLp‐mice used were gray flp‐deleter mice in C57/Bl6 background (C57BL/6‐Tg(CAG‐Flpe)2Arte). Mice obtained with the homozygous targeted mutation were thereafter called Lrp5V667M mice and were analyzed.The mutation in humans is caused by a c.1999G > A transition in the codon GTG, coding for valine, resulting in an exchange for methionine (ATG) in position 667 of the protein. We introduced the equivalent mutation in mice within exon 9 in position 1996, resulting in the p.Val666Met mutation. We used a targeting vector, containing two homology regions of 5.1 and 2.8 kb of All animal experiments were performed according to procedures approved by the local animal ethics committee and were approved by the French Ministry of Higher Education and Research (APAFIS#15223–2018052211589557).Lrp5V667M pups. Briefly, calvariae were digested for 10 × 2 minutes with 0.2% type IV collagenase  in phosphate‐buffered saline (PBS) with 4 mM EDTA, and then for 45 minutes with 0.2% collagenase in PBS for osteoblast release. The cells were expanded for 5 to 6 days in minimum essential medium‐alpha containing 10% fetal calf serum and plated at a density of 50,000 cells per well in 12‐well culture plates. Two separate experiments were performed in triplicate for each group. The culture medium was supplemented with 50 μm ascorbic acid and 10 mM beta‐glycerophosphate, which was replaced every 2 to 3 days. RNA was extracted for osteoblast gene expression. Determination of alkaline phosphatase activity was performed through alkaline phosphatase staining after 14 days of culture in osteogenic medium, using SigmaFast BCIP/NBT tablets (Sigma). Mineralization was assessed by alizarin red staining after 21 days of culture in osteogenic medium. Mean intensity was measured with the ImageJ software in arbitrary units and normalized to the mean value obtained for CTL.Primary osteoblasts were isolated from calvariae of 2‐ to 4‐day‐old control (CTL) and Sdha (Succinate Dehydrogenase Complex Flavoprotein Subunit A).mRNA was extracted using the Isolate II RNA Mini Kit , in accordance with the manufacturer's instructions. For each sample, 1 μg of total mRNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit  following manufacturer's instructions. Samples were subjected to quantitative PCR with SensiFAST SYBR (Bioline) on a Light Cycler 480 thermocycler (Roche). Primers will be available upon request. Results were analyzed with the ΔΔCt method, and reference gene was CTX and P1NP were measured in mice serum with ELISA method with the RatLaps (CTX‐I) EIA (IDS) and Rat/Mouse PINP EIA (IDS) kits, following the manufacturer's instructions.The right femur from each animal was collected after microCT measurement, dehydrated in xylene, and then embedded without demineralization in methylmethacrylate. Five micrometer‐thick coronal sections were cut parallel to the long axis of the femur, using an SM2500S microtome . Sections of 5 μm were stained with toluidine blue to quantify the osteoblastic, osteoid, and osteoclastic surfaces . Two 11‐μm‐thick unstained sections were taken for measurement of the dynamic parameters under UV light. The matrix apposition rate (MAR) was measured using the Microvision image analyzer  by a semiautomatic method using tetracycline and calcein double‐labeled bone surfaces. The mineralizing surfaces (MS/BS) were measured in the same areas using the objective eyepiece Leitz integrate plate II, and the bone formation rate was calculated using the two measured parameters. All the histomorphometric parameters were recorded in compliance with the recommendation of the American Society for Bone and Mineral Research Histomorphometry Nomenclature Committee.32Dual‐energy X‐ray absorptiometry (DXA) analysis of all animals was performed to determine the bone mineral density at the total body (excluding head), lumbar spine, and femurs. The measurement was performed on the animals under anesthesia with a Faxitron device .4 vertebrae were collected for bone microarchitecture analysis after fixation in 70% ethanol, without decalcification and after inclusion in methylmethacrylate for L4 vertebrae. They were analyzed with high‐resolution microCT using a Skyscan 1272 microCT . For femurs, measurements were made on the distal metaphysis for the trabecular bone and on the diaphysis for the cortical bone. Images were obtained using the following acquisition parameters: voltage 75 kV, intensity 125 μA, pixel size 6 μm, exposition time 1350 ms, and filter aluminum 0.25 mm. Images for L4 vertebrae were obtained for measurements on the trabecular bone with the following acquisition parameters: voltage 90 kV, intensity 111 μA, pixel size 6 μm, exposition time 1200 ms, filter aluminum 0.5 mm. NRecon, DataViewer, CTAn, and CTVox softwares were used for 3‐dimensional (3D) image reconstruction, analyzed, and expressed according to international guidelines. The following morphometric parameters were computed: bone volume/tissue volume , trabecular thickness , trabecular number , trabecular separation , cortical thickness , and cortical area .Right femurs and Lidelines.33 The2 vertebrae were collected after euthanization and immediately frozen at −20°C, in a compress soaked in saline solution. Samples were thawed in physiological saline solution 24 hours before mechanical tests at 4°C. The 3‐point bending test was used for femurs, with the Instron 5942 device . The load of the actuator was applied in the center of the shaft at a speed of 2 mm/min until fracture. The span of the lower support was 10 mm. The load–displacement curve was recorded with the Bluehill 3 software (Instron), providing the stiffness, which was defined as the slope of the linear elastic deformation, the yield load at the boundary between the elastic deformation and plastic deformation, and the ultimate load, which is the maximum sustained force at the point of fracture. Ultimate displacement and post‐yield displacement were also computerized, as well as work to fracture. The yield load was calculated with the 0.2% offset method. Elastic modulus was estimated through the beam theory. Vertebral strength was assessed by a compression test in the cranio‐caudal axis of L2 vertebrae, with a loading speed of 1 mm/min until fracture.Left femurs and Lfracture.34 UltQuantitative Backscattered Electron Imaging (qBEI) experiments were performed on right midshaft femurs embedded in methylmethacrylate. The blocks were carbon‐coated and observed with a scanning electron microscope  equipped with a five‐quadrant semiconductor backscattered electron detector. Method details are provided in Appendix All patients underwent a detailed ocular exam including best‐corrected visual acuity using a Snellen chart, slit‐lamp biomicroscopy, measurement of intraocular pressure (IOP) and autorefractometry , and retinal imaging. All eyes underwent ultrawide‐field color fundus photographs images obtained using Optos  and optical coherence tomography (OCT)‐angiography (OCTA). Capillary density (CD) was quantified in OCTA image and was converted into binary images using ImageJ . CD was calculated as the ratio of the area occupied by vessels divided by the total image area. Details can be found in the Appendix Eyes were collected immediately after death, fixed in 4% paraformaldehyde overnight, washed, and then stored in PBS at 4°C.Eyes were dissected to obtain flat‐mounted retinas, immersed in permeabilization and saturation buffer during 1 hour at room temperature, and then incubated overnight with 1/400 anti‐COLIV goat antibody solution  and revealed using Donkey anti‐goat 488 antibodies . They were mounted with spacers and a Permafluor mounting medium. Images were obtained using confocal microscopy  at ×20 magnification . Three regions of interest (ROI) were picked in peripheral retina. A 3‐dimensional modeling software  was used to model a total volume of COLIV‐labeled retinal vessels. Vascular tortuosities were marked manually, allowing for volume quantification. Because atrophies were difficult to model, atrophic segments were counted in each ROI, regardless of their volume.Eyes were dissected to obtain eyecups that were cryoprotected and frozen. Serial transversal slices (12 μm thick) were performed with cryostat , then immersed in permeabilization and saturation buffer during 1 hour at room temperature, incubated overnight with 1/400 Iba1 goat  and 1/500 GFAP rabbit  antibody solutions and revealed using Donkey anti‐goat 488 and anti‐rabbit 594 antibodies . Images were obtained using confocal microscopy  at ×40 magnification.p < 0.05.Data are expressed as median with interquartile range. Mann–Whitney test was used, except when stated otherwise . The GraphPad  software was used for analysis. A difference was considered as significant when LRP5 variants considered as pathogenic and loss‐of‐function. They were 6 patients with the V667M variant (5 heterozygous and 1 homozygous carriers) and 5 patients with other pathogenic variants  . Serum bone biomarkers were within the reference range: BAP (12.4 μg/L [12.0–14.5] and 12.6 μg/L [10.4–23.0]), P1NP (48.9 ng/mL [39.6–53.2] and 45.7 ng/mL [39.8–83.5]), CTX (339.0 pg/mL [255.0–416.5] and 338.0 pg/mL [255.0–410.0]), TRAP5b (2.6 U/L [2.15–4.0] and 2.8 U/L [2.2–3.8]).LRP5 variant, mice carrying the mutation were generated  were fertile, macroscopically normal, with no difference in size or weight compared with CTL mice. Analysis of long bones without bone marrow showed lower mRNA expression of Osx, Col1, and osteocalcin in Lrp5V667M bones than in CTL ones   and osteocalcin (p < 0.0001 for interaction time × genotype)  Fig. A, suggepe) Fig. B, confipe) Fig. C.Lrp5V667M mice, compared with CTL mice, had indeed lower BMD at all the measuring sites , femur , and lumbar spine . However, microarchitecture parameters at the femur and vertebrae were similar in Lrp5V667M and CTL mice ; BV/TV ; Tb.Th  were not different, as well as the indices at L4 lumbar vertebrae ; Tb.Th . In addition, serum bone biomarkers were comparable in the 2 groups   or either for bone resorption   Fig. .Lrp5V667M mice tended to have a lower femoral and vertebral stiffness . For femurs, Young's elastic modulus was estimated and appeared lower than for CTL mice , meaning the altered stiffness does not result from the bones' geometry but rather from modified intrinsic bone biomechanical properties. Work to fracture was similar in the two groups  but the yield load seemed again lower for Lrp5V667M femurs , which therefore could undergo plastic nonreversible deformation for lower applied forces and accumulate more microdamage through time, increasing the risk of fracture.To investigate whether the variant could contribute to bone fragility, we measured the bone biomechanical properties Fig. A, B. LrLrp5V667M femurs compared with CTL ones . It is possible that the mineral‐to‐matrix ratio was also impacted in Lrp5V667M mice. Indeed, when estimated through the phosphate‐to‐proline+hydroxyproline ratio, it trended to be lower than in CTL mice   in bone  or prolyl 4‐hydroxylase  and in the deep capillary complex (DCC) were not different from a population of reference  (Fig. In mice retinas, total retinal vascular volume was similar in the two groups Fig. A, B. VaLRP5 is associated with variations in BMD and risk of fracture. Although heterozygous harboring of V667M variant has been repeatedly described in early‐onset osteoporosis and this variant has been suggested as pathogenic, the contribution of LRP5 variants in the osteoporotic phenotype remains unclear. In addition, the mechanisms of bone fragility are not completely understood. Here, we generated a murine model carrying the LRP5 p.V667M variant, the most frequently reported as associated with low BMD, and showed that this variant is indeed responsible for a reduced BMD. Mechanisms involved include impairment of osteoblastic differentiation and bone fragility can be explained by modifications of the extracellular matrix.LRP5 variants had low lumbar spine BMD, similarly to previously reported data. BMD, measured by DXA and HR‐pQCT, revealed low BMD compared with the general population at the trabecular but also at the cortical compartments, with alterations in bone microarchitecture, as reported in young osteoporotic patients without mention of LRP5 variants. Bone remodeling markers are usually normal or low, as observed in our patients.Here, patients with EOOP carrying ted data.35 BMD and is therefore relevant to estimate the effect of the V667M in humans. However, we could not evidence any modification of bone microarchitecture in mice, in contrast to what was observed in our patients or previously reported in Lrp5 KO mice. Indeed, we cannot rule out a selection bias because patients were selected on low BMD and presence of fractures with no evidence of secondary osteoporosis. They could be considered as having a mild form of EOOP as the incidence of fracture was relatively low. Because the copy number variation or exome analyses were not performed, we cannot rule out that undetected variant or deletion in another gene could be responsible of low BMD in addition to the LRP5 variant. The bone profile observed in patients could be related to other mutations in unexplored sequences such as introns, alterations in modifier genes, differences in epigenetic factors, or undescribed mutations in other genes. It is also possible that unknown nongenetic factors could play a role and explain why the phenotype was more severe than that observed in mice.Our rodent model reproduced the variation of BMD observed in V667M patients, as shown by DXA measurements4 and Lrp5 KO mice, but the phenotype is expected to be stronger with a KO than with a single‐nucleotide mutation, as in this model. We cannot completely rule out an effect of the V667M variant on bone microarchitecture though, as modifications might be too small to be detected with our experimental plan, based on mice with a C57Bl/6 background, known to have one of the lowest bone mass among frequently used laboratory mice. In particular, the cortical thickness could be slightly reduced. As the low BMD and bone fragility could not be explained by impaired microarchitecture, we investigated the bone quality through estimation of its biomechanical properties. We observed that the stiffness and yield load appeared lower in Lrp5V667M mice. This could promote accumulation of microdamage in bone and therefore increase bone fragility. Lower stiffness is usually associated with a lower mineral content of the bone matrix. We could not evidence a clear reduction in the mineral‐to‐matrix ratio compared with CTL mice, but a slight reduction could exist as a trend was observed regardless of the method for organic matrix content estimation. A reduction in matrix mineralization could result from the reduced osteoblastic activity and be responsible for the low BMD observed. A more in‐depth analysis of the bone matrix showed a reduced hydroxyproline‐to‐proline ratio of collagen. A reduction in posttranslational proline hydroxylation was reported to favor overmodification of collagen chains and affect the stability of the collagen triple helix, the assembly of collagen fibrils, and could have an impact on mineralization of the bone matrix. Mutations in P3h1, one of the enzymes responsible for hydroxylation of proline residues in collagen chains, are responsible for some forms of osteogenesis imperfecta. However, expression of P3h1 that might alter the hydroxylation of collagen was not significantly different in our model. The exact mechanism through which the V667M variant of LRP5 impacts proline hydroxylation warrants further investigation. Our findings provide new insights into mechanisms of bone fragility induced by mutations of LRP5. It highlights that LRP5‐related EOOP involves modifications in the quality of the extracellular matrix and further emphasizes the role of collagen posttranslational modifications. Interestingly, LRP5‐related EOOP has recently been classified in the same group as osteogenesis imperfecta in the classification of genetic skeletal disorders. Our results strengthen the idea of a continuum between LRP5 EOOP and osteogenesis imperfecta, as LRP5 variants can be responsible for alterations of the collagen structure of bone extracellular matrix. The discrepancy between a marked osteoblastic phenotype and the mild bone phenotype was an unexpected finding and might involve compensation mechanisms and modification of signaling metabolic pathways.Altered bone microarchitecture was observed in  KO mice,43 but Whereas high blood pressure was present in one of these patients, the other one did not have any history of these conditions. Also, arteriolar tortuosity was observed in the retinopathy of prematurity, the presentation of which is similar to FEVR, and arterial and venous tortuosity was also described in FEVR, which can result from LRP5 mutations. Therefore, LRP5 variants could have contributed to the observed tortuosity. The phenotype appeared clearer in mice with a higher rate of retinal vascular tortuosity in Lrp5V667M than in CTL mice. Vessel tortuosity can be related to disturbed blood flow, endothelial cell dysfunction, or hypoxia. As collagen type 1 is impacted in Lrp5V667M mice, it is conceivable that the vessel tortuosity in our murine model could be related to anomalies in collagen type IV, as in familial retinal arteriolar tortuosity, a condition linked to mutations in COL4A1. However in this condition, increased tortuosity of the second‐ and third‐order arteries is observed, whereas in the two Lrp5V667M patients, the tortuosity involved first‐order arteries.Regarding the retinal vasculature, nonspecific vascular tortuosity was found in two patients, the significance of which remains uncertain at this point. Indeed, tortuosity can be observed in other diseases such as diabetes, high blood pressure, and obstructive sleep apnea.55 WheLRP5, and more generally loss‐of‐function mutations at heterozygous level of LRP5, can indeed have an impact on bone mineralization and alteration of the quality of the bone matrix. These results highlight the benefit of screening EOOP patients, investigating LRP5 variant as a susceptibility factor of bone fragility. LRP5 also plays a role in retinal vascularization development, and the V667M variant might increase retinal vascular tortuosity.In conclusion, the homozygous p.V667M low‐frequency variant of Stéphanie Fabre: Conceptualization; data curation; formal analysis; validation; writing – original draft; writing – review and editing. Morgane Bourmaud: Conceptualization; data curation; formal analysis; methodology; writing – original draft; writing – review and editing. guillaume mabilleau: Data curation; formal analysis; validation; writing – review and editing. Ruben Goulet: Data curation; formal analysis; writing – review and editing. Aude Couturier: Data curation; formal analysis; writing – original draft; writing – review and editing. Alexandre Dentel: Data curation; formal analysis; writing – original draft; writing – review and editing. Serge Picaud: Conceptualization; writing – review and editing. Thomas Funck‐Brentano: Data curation; formal analysis; writing – original draft; writing – review and editing. Corinne Collet: Conceptualization; formal analysis; writing – original draft; writing – review and editing. Martine Cohen‐Solal: Conceptualization; data curation; formal analysis; funding acquisition; methodology; project administration; resources; validation; writing – original draft; writing – review and editing.The authors report no conflicts of interest in relation to the work.https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/jbm4.10741.The peer review history for this article is available at Appendix S1. Supplementary InformationClick here for additional data file.


Dioscorea opposite Thunb.) has been used as food and medicine to treat diarrhea for thousands of years. This article aimed to elucidate the potential mechanism of Dioscoreae Rhizoma starch in alleviating chronic diarrhea induced by rhubarb based on gut microbiotas and fecal metabolome. The administration of the Dioscoreae Rhizoma aqueous extracts, crude polysaccharides, and starch could improve diarrhea and alleviate intestinal injury in chronic diarrhea rats. The Dioscoreae Rhizoma starch displayed the most apparent effect on regulating intestinal microbiotas by increasing the abundance and diversity of microbiotas. At the genus level, there were 17 changed intestinal microbiotas in model rats, and the treatment with Dioscoreae Rhizoma starch regulated 11 microbiotas. Metabolomics analysis revealed that Dioscoreae Rhizoma starch could regulate abnormal fecal metabolites to alleviate diarrhea, and these metabolites are involved in phenylalanine, tyrosine, and tryptophan biosynthesis; tyrosine metabolism; vitamin B6 metabolism; and purine metabolism. This study will contribute to the further research and development of Dioscoreae Rhizoma starch.Chinese yam ( Yam starch alleviated the symptoms of chronic diarrhea in rats, and its effect is better than that of the water extract and crude polysaccharides of yam; it improved intestinal injury of rats with chronic diarrhea; it increased the abundance and diversity of intestinal microbiotas in rats with chronic diarrhea and regulated the abnormal intestinal microbiotas; and it displayed a beneficial role by regulating phenylalanine, tyrosine, and tryptophan biosynthesis; tyrosine metabolism; vitamin B6 metabolism; and purine metabolism. Dioscoreae Rhizoma (dried yam) possesses the effect of improving the functions of the spleen, stomach, lungs, and kidneys. It is always used for treating inappetence, long‐term diarrhea, asthma and cough, spermatorrhea, morbid leukorrhea, frequent urination, and diabetes in the clinic  were bought from Zhangzhongjing pharmacy , and these pieces were confirmed by Professor Suiqing Chen from the Henan University of Chinese Medicine to ensure their authenticity.High‐performance liquid chromatography‐grade methanol, acetonitrile, and formic acid were purchased from Thermo Fisher Technology Co., Ltd. . Other analytical pure reagents were purchased from Tianjin Fuyu Fine Chemical Co., Ltd. .2.22.2.1The preparation of the Dioscoreae Rhizoma aqueous extract was carried out according to the methods reported by Zeng et al.  with min2.2.2The Dioscoreae Rhizoma crude polysaccharides, nonstarch polysaccharides, were prepared according to the methods reported by Wang et al.  with min2.2.3Dioscoreae Rhizoma starch was prepared according to the method reported by Huang et al.  with min2.2.4Ten times the volume of distilled water was added to the flask containing slices of rhubarb and then soaked at 50°C for 1 h, the soaking was repeated once after filtration. The two filtrates were combined and concentrated to 1 g/mL by a rotary evaporator at 40°C.2.3a.m. and 4:00 p.m.  were purchased from Shandong Jinan Pengyue Experimental Animal Breeding Co., Ltd. Rats were raised in the Animal Experimental Center of the Henan University of Chinese Medicine. The rats were kept in the animal house with 12 h of light and 12 h darkness. Moreover, the condition temperature was 23 ± 2°C and the humidity was 60 ± 5%. Animal experiments were approved by the Animal Ethics Committee of the Henan University of Chinese Medicine (NO: DWLL202103192). All animal experiments followed the National Institutes of Health guide for the care and use of laboratory animals. After 1 week of adaptation, rats were randomly divided into five groups, including blank control (B) group, model (M) group, Dioscoreae Rhizoma aqueous extract (SY) group, Dioscoreae Rhizoma crude polysaccharide (SYP) group, and Dioscoreae Rhizoma starch (SYS) group, with seven rats in each group. A chronic diarrhea model was prepared by intragastric administration of rhubarb extract. During the modeling process, rats in the M, SY, SYP, and SYS groups were administered the water extract of rhubarb at a dose of 10 mL/kg for 7 days, twice a day, at 8:00 The rats were anesthetized with 10% pentobarbital sodium. The colon content and colon tissue of the animals were collected. The colon contents were stored in a sterile sampling tube, then quickly frozen in liquid nitrogen, and stored in a −80°C refrigerator to analyze intestinal microbiotas. Four centimeters from the anus, fresh colon tissues were collected and immersed in 4% paraformaldehyde for histopathological analysis. After adequately storing in paraformaldehyde, the colon tissues were dehydrated, embedded in paraffin, and sliced into 4 μm sections. After hydrated and deparaffinized, the paraffin sections were stained with hematoxylin and eosin. Changes in colon histological sections were observed under an optical microscope  with barcode sequences for multiplexing reads of each sample. Then, the sequencing was performed on an Illumina NovaSeq platform and 250 bp paired end reads were generated. Paired end reads were assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Paired end reads were merged using fast length adjustment of short reads (FLASH), a high‐speed and accurate analysis tool designed to merge paired end reads when at least some of the reads overlap the read generated from the opposite end of the same DNA fragment, and the splicing sequences were called raw tags. Quality filtering on the raw tags was performed under specific filtering conditions to obtain the high‐quality clean tag according to the QIIME quality‐controlled process . Beta diversity is the comparative analysis of microbial community composition in different samples. First, according to the species annotation results of all samples and the abundance information of OTUs, the same classification of OTUs information was combined to obtain the species abundance information table. At the same time, the unweighted Unifrac distance is further calculated by using the system occurrence relationship between OTUs. Finally, the differences between different samples (groups) are found through multivariate statistical methods, including principal coordinates analysis (PCoA) and nonmetric multi‐dimensional scaling (NMDS) analysis  were weighed accurately and added with 400 μL of 50% methanol  using an UHPLC system . The mobile phase consisted of solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile), and the chromatographic separation was carried out with gradient elution; the mobile phase gradient program was 0 → 15 min, 3% B → 15% B; 15 → 19 min, 35 %B → 40% B; 19 → 25 min, 40%B → 50% B; 25 → 33 min, 70%B → 90%B, 33 → 40 min, 90%B, and the post run time was 3 min. The column temperature was maintained at 35°C and the injection volume was 1 μL.Mass spectrometry data were acquired on an Agilent 6546 Q‐TOF/MS . The instrument was operated with an electrospray ion (ESI) source in positive and negative modes. The ionization source conditions were as follows: drying gas temperature was 320°C, drying gas flow rate was 9 L/min, nebulizer pressure was 38 psi, sheath gas temperature was 350°C, sheath gas flow rate was 11 L/min, and the fragmentor voltage was 110 V. Capillary voltage and nozzle voltage were different in positive and negative modes, as follows: 3500 V and 500 V in positive mode, 4000 V and 1000 V in negative mode, respectively. The mass scanning range was 50–1100 m/z, and the collision energy was 10 V, 20 V, and 30 V for the target MS/MS analysis.2.5.3t test. The fold change (FC) was analyzed by MPP. SIMCA‐P 14.1  was used for principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS‐DA), and permutation testing was used to evaluate the accuracy and reliability of the constructed OPLS‐DA model. The confirmation of the importance of the variables was represented by the variable importance in the projection (VIP). The biomarkers were confirmed based on the candidate compounds with VIP value >1, p < .05, and the absolute value of FC >2 , then processed by Profinder 10.0 , including peak extraction, peak alignment, and retention time correction. The exported data from Profinder 10.0 were normalized and filtered by Mass Profiler Professional 15.0 . One‐way analysis of variance (ANOVA) was used to analyze the differences between different groups, and the difference between every two groups was performed by Student's 2.6t test or nonparametric test was used for comparative analysis between the two different groups. The p < .05 was considered statistically significant.Statistical analysis was performed using SPSS 24.0 software . Comparison among multiple groups was analyzed using one‐way ANOVA. Data are presented as mean ± standard deviation. The 33.1p < .05, shown in Figure During the experiment, rats in the B group displayed a normal mental state, normal activity, spindle‐shaped stool, and shiny hair. The rats in the M group showed less activity, loose stool, filthy anus, withered hair, and skin laxity, with a 100% diarrhea rate. After administration, the diarrhea rates in the SY, SYP, and SYS groups were 42.7%, 28.6%, and 14.3%, respectively. Rats in the SY, SYP, and SYS groups had daily mental status improved, showing increased activity, improved hair appearance, and improved diarrhea. At the end of the experiment, the weight of rats in the SYS group was significantly higher than that in the M group . The PCoA results of intestinal microorganisms in rats of different groups are shown in Figure The beta‐diversity analysis manifests the differences in microbial composition among animals Clarke, . PCoA isNMDS analysis is a nonlinear sorting method that can overcome the shortcomings of linear model analysis, such as PCoA. The sample is reflected in the multidimensional space in the form of points according to the sample's information. The distance between points reflects the difference between different samples. Finally, the two‐dimensional spatial positioning point map of the sample is obtained. Figure 3.3.3Gut microbiotas at the phylum level mainly consisted of Bacteroidota, Firmicutes, Verrucomicrobiota, Proteobacteria, unidentified bacteria, Actinobacteriota, Cyanobacteria, Campylobacterota, Acidobacteriota, and Desulfobacterota, and these microbiotas represented more than 97% relative abundance Figure . The relFirmicutes and Bacteroidetes are the two bacteria with the highest relative abundance in the intestinal microbiotas of rats. Their ratios significantly increased in the M group. After treatment with Dioscoreae Rhizoma starch, the ratios are significantly improved Figure . HoweverAlloprevotella, Akkermansia, Prevotella, Bacteroides, Parasutterella, Escherichia‐Shigella, Lactobacillus, Christensenellaceae_R‐7 group, UCG‐005, Staphylococcus, Bifidobacterium, Parabacteroides, unidentified_Ruminococcaceae, Lachnoclostridium, Ruminococcus, Allobaculum, Roseburia, Phascolarctobacterium, Lachnospiraceae_NK4A136_group, Butyricimonas, Candidatus_Saccharimonas, Romboutsia, Rodentibacter, NK4A214_group, Colidextribacter, Izhakiella, Acinetobacter, Alistipes, Helicobacter, and Prevotellaceae_UCG‐001, as shown in Figure The relative abundance changes of microbiotas at genus levels were analyzed to further understand the detailed alternations of gut microbiotas. At the genus level, the top 30 relative abundance of rat gut microbiotas include Alloprevotella, Bacteroides, Parasutterella, Escherichia‐Shigella, UCG‐005, Bifidobacterium, Parabacteroides, unidentified_Ruminococcaceae, Lachnoclostridium, Ruminococcus, Allobaculum, Phascolarctobacterium, Butyricimonas, Candidatus_Saccharimonas, Colidextribacter, Alistipes, Prevotellaceae_UCG‐001.The abundance of 17 intestinal microbiotas in group M significantly changed, including p < .05). Compared with M group, 11 microbiotas showed significant change in group SYS  in positive and negative modes are shown in Figure 3.4.1PCA, a typical unsupervised multivariate statistical analysis method, was employed for metabolomics analysis. The sample points in each were clustered . The mass tolerance between the measured m/z values and the exact mass of the components was within 5 ppm. If the accurate mass of markers could not match the databases or correlative descriptions were not endogenous, they were excluded from the interested list. Finally, 46 potential biomarkers related to chronic diarrhea were identified, as shown in Table The potential biomarkers were selected based on VIP >1, the absolute value of fold change >2, and 3.6http://www.metaboanalyst.ca/) for the metabolic pathway analysis  and the results were shown in Figure Spearman's correlation analysis was performed to elucidate the interaction between changed metabolites and intestinal microbiotas in group SYS. The correlation analysis between metabolic markers and gut microbiota was analyzed by Omicstudio (Alloprevotella and Parasutterella are two bacteria closely related to metabolites. These related metabolites include 4‐(3‐pyridyl)‐3‐butenoic acid, 3‐methoxytyramine, uric acid, pyridoxamine, hippuric acid, l‐tyrosine, 6‐hydroxy‐1H‐indole‐3‐acetamide, sebacic acid, 1‐nitro‐5,6‐dihydroxy‐dihydronaphthalene, 5‐hexyltetrahydro‐2‐oxo‐3‐furancarboxylic acid, deoxyhypusine, dodecanedioic acid, 3‐hydroxydodecanedioic acid, ascorbalamic acid, imazamethabenz, 1beta, 3alpha, 7alpha, 12alpha‐Tetrahydroxy‐5beta‐cholanoic acid, PE(15:0/0:0), LysoPE(0:0/16:0), PE/0:0), tauroursocholic acid, and taurallocholic acid. Interestingly, the correlations of Alloprevotella and Parasutterella with these metabolites showed opposite trends.For example, 4Dioscoreae Rhizoma is a commonly used Chinese medicine with the effect of invigorating the spleen, which can be widely used to treat gastrointestinal diseases such as chronic diarrhea. At present, there are few experimental studies on Dioscoreae Rhizoma for diarrhea treatment. The aqueous extract , and nitrogen oxide ; data curation (lead); funding acquisition ; investigation (lead); validation ; writing – original draft (lead); writing – review and editing (lead). Xu Zhang: Investigation ; methodology . Qing Wang: Investigation ; methodology ; writing – original draft . Suiqing Chen: Funding acquisition (lead); project administration (lead); supervision ; writing – review and editing .The authors declared that they have no conflicts of interest.


In a patient with Parkinson's disease (PD) who underwent spine surgery 13 h after the last anti‐Parkinson medications, negative pressure pulmonary edema from upper airway obstruction developed immediately after extubation. Although oxygenation improved with high‐flow nasal cannula therapy, such complications might develop due to abrupt discontinuation of medication for PD. Preoperative examinations, including blood and urine tests, an electrocardiogram, and a chest X‐ray, showed no abnormal findings. The patient was taking oral medications for PD, including levodopa‐carbidopa (q.i.d). Symptoms of PD were well‐controlled so that he was able to live independently. He was admitted to the hospital the day before surgery. The patient took the last medications the night before surgery, 13 h before induction of anesthesia. Considering the well‐controlled PD status, anti‐Parkinson medications on the morning of surgery were neither administered orally nor replaced by those via alternative routes.2) was maintained between 97% and 99%, and arterial oxygen tension (PaO2) was 201 mmHg with inspired oxygen fraction (FIO2) of 0.5. Fentanyl (150 μg) and acetaminophen (1000 mg) were intravenously administered for immediate postoperative analgesia. Durations of surgery and anesthesia were 161 and 254 min, respectively. Volumes of fluid infusion, blood loss, and urine output were 1410 mL, 60 mL, and 400 mL, respectively.General anesthesia was induced and maintained with continuous infusions of propofol and remifentanil. Rocuronium (40 mg) was given to facilitate tracheal intubation. After the patient was placed in the prone position before surgery, neuromuscular relaxation was antagonized with sugammadex (150 mg) for monitoring of motor‐evoked potentials, which remained unsuppressed throughout surgery. Arterial oxygen saturation   was applied to the spontaneously breathing patient using an anesthesia circuit. PaO2 increased to 105 mmHg.After emergence from anesthesia, a sufficient tidal volume and respiratory rate were confirmed. The patient could take a deep breath and move extremities voluntarily, as requested by anesthesiologists. Immediately after tracheal extubation, however, the patient presented with labored breathing. Inspiratory stridor was heard. Systolic blood pressure exceeded 200 mmHg. SpOIO2 of 0.6 and a flow rate of 50 L/min. NPPE improved by the morning of postoperative day (POD) 1; PaO2 increased to 204 mmHg with FIO2 0.6. Oral anti‐Parkinson medications were resumed. On POD 2, the patient was weaned from HFNC, and returned to the ward without any oxygen supply.The patient was transferred to the intensive care unit. High‐flow nasal cannula therapy (HFNC) was initiated with F3Although UAO can develop as a manifestation of PD,The patient took the last anti‐Parkinson medications 13 h before anesthesia. The withdrawal of drugs might be long enough to diminish their effects, thereby causing UAO. Reportedly, duration of action of levodopa‐carbidopa to reduce motor disability lasts as short as 3–6 h.According to a recent review, patients with PD are advised to have their anti‐Parkinson medications at fixed intervals daily to prevent fluctuations in motor and non‐motor symptoms of PD.In our patient, UAO and NPPE improved with HFNC without a resumption of anti‐Parkinson medications. HFNC is able to deliver a consistent oxygen supply to the alveoli by constantly applying positive pressure, enabling the patient to maintain a high level of oxygen supply.4We experienced upper airway obstruction and negative pressure pulmonary edema that developed immediately after anesthesia and extubation in a patient with Parkinson's disease. Our experience suggests that in patients with Parkinson's disease, anti‐Parkinson medications should be continued perioperatively to minimize risks of such complications, and high‐flow nasal cannula therapy can improve upper airway obstruction and negative pressure pulmonary edema in such patients.Ayaka Hasegawa: Writing – original draft. Naoko Niimi: Writing – review and editing. Chieko Mitaka: Writing – review and editing. Masakazu Hayashida: Writing – review and editing.None.The authors declare that they have no competing interests.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


The success can further be extended to ampere-hour-scale pouch cells. This work uncovers the SEI dissolution and its correlation with Li corrosion, enabling the durable operation of Li metal batteries by reducing the Li loss.Reactive negative electrodes like lithium (Li) suffer serious chemical and electrochemical corrosion by electrolytes during battery storage and operation, resulting in rapidly deteriorated cyclability and short lifespans of batteries. Li corrosion supposedly relates to the features of solid-electrolyte-interphase (SEI). Herein, we quantitatively monitor the Li corrosion and SEI progression  in typical electrolytes through devised electrochemical tools and cryo-electron microscopy. The continuous Li corrosion is validated to be positively correlated with SEI dissolution. More importantly, an anti-corrosion and interface-stabilizing artificial passivation layer comprising low-solubility polymer and metal fluoride is designed. Prolonged operations of Li symmetric cells and Li | |LiFePO Lithium metal electrodes suffer from both chemical and electrochemical corrosion during battery storage and operation. Here, the authors show that lithium corrosion is due to dissolution of the solid-electrolyte interphase and suppress this by utilizing a multifunctional passivation layer. To satisfy the multiple concerns of energy, environment, and resource2, expeditions on novel and powerful battery systems boom7. Lithium (Li) metal battery emerges as the next-generation energy storage technique, which holds promise to break through the specific energy limit (>500 Wh kg−1) of conventional battery systems9. Substantial progress has been made in the development of Li metal batteries by tackling the typical issues of highly reactive Li metal, mainly including Li dendrite growth10, accumulation of inactive Li11, and unstable electrode/electrolyte interfaces12. However, the stability and lifespan of Li metal batteries still remain formidable challenges, which are highly related to the irreversible Li loss associated with incomplete Li stripping, interface evolutions, and corrosion behaviors.With the approaching of an electrified era, rechargeable batteries have been widely adopted, which greatly change the patterns of daily life and industries15, involving such diverse factors as material, coating, environment, microbiology, stresses, and/or electromagnetism  at the Li metal–liquid electrolyte interface19. Similar to the corrosion of structural metals21, Li corrosion highly relates to the features  and progression  of such a passivation layer . Very recently, Cui and Zhang et al. have validated the SEI dissolution in alkali metal battery systems23. In detail, the native SEI suffers a dynamic progression of repeated swelling, dissolution, breakage, and reformation, leaving Li with high redox power exposed to electrolytes. Consequently, the direct chemical depletion of both electrolyte and active Li sources is constantly undergone. Moreover, Li corrosion can be electrochemically aggravated by forming localized galvanic couples comprising dissimilar metals  or regions  in electrical contact and electrolyte25. The chemical and electrochemical corrosion patterns continuously undergo during battery storage and operation, together contributing to serious Li loss26. The Li corrosion and SEI dissolution emerge as additional failure mechanisms of reactive Li metal negative electrodes.Corrosion is an irreversible system phenomenon of materials 32. Though some progress has been made to stabilize SEI and conveniently inhibit Li corrosion, the improvement of battery lifespan is still far from ideal. Such a dilemma highly relates to the limited recognition of the Li corrosion science, and corrosion-induced Li loss cannot be eliminated35, appealing for more expeditions to uncover the correlation between dynamic Li corrosion and SEI progression  and introducing artificial SEI −2), and the optimized Li||LiFePO4 cell exhibits superior cycling stability (1500 cycles at 1.3 mA cm−2). Such a shielding strategy can be preliminarily translated into real ampere-hour-scale Li||LiFePO4 and Li||LiNi0.5Co0.2Mn0.3O2 (NCM523) pouch cells. The role of artificial SEI has been extendedly understood from the perspective of anti-corrosion, which distinguishes it from conventional designs. Most importantly, this work uncovers the dynamic Li corrosion and SEI dissolution, which promises Li metal batteries with improved lifespans by reducing the corrosion-induced Li loss.In this work, Li corrosion behaviors referring to both chemical and electrochemical corrosion have been quantitatively monitored and revealed by devised electrochemical tools along with cryo-electron microscopy. The underlying correlation among Li corrosion, SEI dissolution, and battery failure has been understood. The dissolution of native SEI is proved to be responsible for the continuous Li corrosion. From a corrosion science perspective, a shielding strategy by employing a functional passivation layer of low-solubility polymer and embedded metal fluoride is designed to reduce Li corrosion by 74%. Consequently, Li||Cu cells with superiorly stable interfaces are enabled to deliver high average Coulombic efficiency  can be found in typical ether and ester electrolytes  and extended open-circuit pauses were conducted in Li||Cu cell36. Note that no Li deposits formed during this test above 0 V vs. Li/Li+. The cell was firstly cycled to form a relatively stable SEI at the initial cycles, after which variable durations of rest were conducted  during battery storage and operation should be established to prevent Li corrosion.SEI as the passivation layer of Li corrosion by electrolyte cannot totally eliminate the side reactions at the Li metal–liquid electrolyte interface. Consequently, corroded Li deposits on the current collector with damaged morphologies after short durations of rest in the electrolytes can be frequently monitored Fig. 34. Such tes Fig.  and 2. Otes Fig. . Notablytes Fig. . Subsequyte Fig. . A reasoyte Fig.  and 834.x@PVDF, M = Li, Na, Mg, Al, Zn, La, and Ca, x = 1, 2, or 3)39. The PVDF matrix is supposed to inhibit the Li corrosion by isolating electrolytes and electron tunneling. The embedded metal fluorides promise the formation of LiF and lithiophilic alloy  at the interface, which promotes superior ion transport and uniform Li deposition40. By a facile coating methodology    and Li (PDF#15-0401) can be found after the introduction of an artificial passivation layer onto Li metal  , which can effectively withstand the swelling and solvation of electrolytes to eliminate Li corrosion. Besides, this passivation layer should exhibit superior mechanical and (electro)chemical stability and facilitate fast ion transport. Such concerns promote the application of an artificial passivation layer comprised of low-solubility poly(vinylidene fluoride) (PVDF) and metal fluoride  was quantified to be decreased by 74%. With the increase in resting time, the capacity loss of Li deposits shows a gradual increase (84 μAh for 500 h). Notably, the CE of cells with MgF2@PVDF layers remains above 92% as a function of time, also indicating the apparently suppressed Li corrosion. In addition, the passivation of the bare PVDF layer is weaker than that of MgF2@PVDF, delivering higher capacity loss and lower CE, which validates the synergistic effect of PVDF and MgF2 in Li protection. An ideal artificial passivation layer should not only passivate the negative electrode but also promise sufficient interfacial kinetics. Consequently, the ionic conductivity of swollen MgF2@PVDF and bare PVDF layer was measured. With the adoption of fluoride, the ionic conductivity of PVDF layer is largely improved  enabled the fast diffusion of Li+ at the interface, benefiting the homogenous Li deposition. The gradual increase of exchange current density may be ascribed to the increase in the intrinsic rate of Li+ exchange of the SEI or the electrochemically active area for Li/Li+ redox45.The direct correlation between the artificial passivation layer and the inhibited Li corrosion was first validated by the aforementioned electrochemical protocol Fig. . After a2@PVDF layer was directly coated onto the Cu grid for TEM, where Li was deposited  promoted rapid ion transport at the interface. In contrast, the SEI of Li deposits without the artificial passivation layer was thick and comprised of abundant crystals  due to the Li corrosion and net growth of SEI  was employed to uncover the role of the artificial passivation layers in Li protection from perspectives of microscopic morphology and structure. The MgF−2, 1.0 mAh cm−2), MFx@PVDF-Cu delivered much higher CE and better cyclic stability than that of bare Cu electrode (CE of 85% at the 194th cycle)  Fig. . Of note-Cu Fig. –26. Moredes Fig. , benefitans Fig. . Additioans Fig. . Of note4 positive electrodes was assembled and evaluated. Fig. 2@PVDF layer. The cell with MgF2@PVDF delivered superior rate performance with capacities of 166, 164, 156, 142, 127, and 99 mAh g−1 at 0.13, 0.26, 0.65, 1.3, 2.6, and 6.5 mA cm−2, respectively. Notably, the MgF2@PVDF layer enabled the cell to be stably operated even at an extremely fast charging/discharging rate of 13 mA cm−2. In great contrast, the cell without the protection of an artificial passivation layer exhibited deteriorated rate performances, which can hardly work at large rates like 6.5 and 13 mA cm−2. As seen in the voltage profiles at different current densities, the cell with MgF2@PVDF-Li obviously delivered lower overpotential and higher capacity than that of the cell with bare Li foil  and NCM523 (3.2 mAh cm−2). Expectedly, the Li||LiFePO4 pouch cell with MgF2@PVDF layer delivered twice better cycling stability than the control group, exhibiting a capacity retention of 77% after 70 cycles  can serve as a typically ideal artificial passivation layer to reduce the corrosion of reactive negative electrodes like Li metal by 74%. The PVDF matrix enables the isolation of Li metal and native SEI from electrolytes, while the fluorides react with Li to form corrosion-resistant Li alloy and LiF to suppress Li corrosion further. Additionally, the artificial passivation layer successfully facilitates ion transport and deposition, preventing dendrite growth and dead Li accumulation. Consequently, both coin-type and pouch cells exhibited superior cycling stability and remarkably extended lifespans. This work supplements the lack of fundamental recognition of Li corrosion and its correlation with SEI progression. The findings and strategy can further be extended to other battery systems using reactive metallic negative electrodes  and promote the development of corrosion science in battery research.In conclusion, Li corrosion behaviors associated with SEI dissolution have been quantitatively uncovered. Such dynamic evolutions at the Li metal–liquid electrolyte interface largely decide the stability and performances of Li metal batteries. A prototypical strategy is to introduce a protective layer to inhibit the SEI dissolution and Li corrosion by electrolyte. The matrix of such an artificial passivation layer should be stable against electrolyte, Li metal, and interfacial stress , while the filler inside the layer can promise uniform ion transport and Li deposition . Notably, the composite comprised of polymer and fluorides  were purchased and used after grinding. A slurry comprising fluorides, NMP , and PVDF (1:2:40 by weight) was prepared with a mixer. The slurry of artificial passivation layer was brushed onto the Cu or Li foil (~14 μL cm−2) and dried at 50 °C in a glove box or dry room. Notably, the mass loading of an artificial passivation layer on each metal foil was calculated to be ~1.1 mg cm−2, and the content of fluorides in each layer was set to be ~30 wt% in this work. Li foil (a thickness of 0.4 mm and a diameter of 12 mm) and Li belt (a thickness of 50 μm) with a high purity of 99.95% were purchased from China Energy Lithium Co., Ltd. Two kinds of battery-grade Cu were used, including a Cu foam  and a Cu foil , which were obtained from Jinghong New Energy Co., Ltd.PVDF  and micron-sized metal fluorides . The morphology and microstructure of samples in this work were observed by SEM  and TEM . Elemental analysis was performed on an EDX spectrometer attached to TEM. Cryo-TEM was characterized by using a cryo-transfer holder of Gatan 698. Notably, a dilute solution of PVDF and fluoride was applied to reduce the thickness of the artificial passivation layer with the purpose of better visualizing the interface during TEM characterization. EELS was conducted by a FEI Titan G2 transmission electron microscope with an aberration corrector for a condenser lens operated at an accelerating voltage of 300 kV. XPS measurements were performed using an Al Kα monochromatic X-ray source . Infrared absorption spectra were measured at room temperature on an FTIR spectrometer .XRD patterns of samples in this work were conducted on an X’Pert Pro diffractometer deploying Cu Kx@PVDF layers with an electrolyte amount of 50 μL. MFx@PVDF-Cu electrodes were paired with Li foil to prepare Li||Cu cells. The symmetric cells were obtained by pre-depositing Li onto Cu in Li||Cu cells with an area capacity of 10.0 mAh cm−2. The coin cells were tested in galvanostatic mode with Neware battery testing equipment . For CE of Li||Cu cells, 1.0 mAh cm−2 Li was plated into the MFx@PVDF-Cu electrodes at a specific current density. Then the cell was charged to 1.5 V at the same current to strip out the plated Li. The efficiency was decided by the ratio between the amount of deposited and stripped Li. The symmetric cells were charged and discharged at certain current densities with a Li cycling capacity of 1.0 mAh cm−2. The Li||LiFePO4 cells were composed of LiFePO4 positive electrodes and MgF2@PVDF-Li or bare Li foil. LiFePO4 positive electrode  was prepared by coating the slurry of 80% LiFePO4, 10% PVDF, and 10% Super P conducting carbon onto Al foil. The liquid electrolyte consisted of 1.0 M LiTFSI  and 1.0 wt% LiNO3  dissolved in a mixture of DOL and DME (v/v = 1:1) was selected as the electrolyte and Celgard 2400 as the separator. EIS was measured with a CHI660E electrochemical workstation with a frequency ranging from 100 kHz to 0.1 Hz. Tafel plots were obtained from a cyclic voltammetry test in Li||Li cells with a scanning rate of 1.0 mV s−1. The Tafel plot of the log current versus cell polarization was linearly fit over the voltage range from −25.0 to −50.0 mV. The value of exchange current density can be obtained from the intersection of the extrapolated linear part of the lg j with the equilibrium potential (η = 0) line. The pouch cells were assembled by pairing 4.0 cm × 5.0 cm double-sided LiFePO4  or NCM523 positive electrodes  with 4.0 cm × 5.0 cm × 50.0 μm double-sided Li foil copper inlay, employing the 5.0 cm × 6.0 cm separators. 1.0 M LiTFSI in DOL/DME (v/v = 1:1) with 1.0 wt% LiNO3 and 1.0 M LiPF6 in FEC/dimethyl carbonate (DMC) (v/v = 1:4) electrolytes were used in Li||LiFePO4 and Li | |NCM523 pouch cells, respectively. All the cells were tested in a battery testing chamber  with a setting temperature of 25 °C.Typical 2032 coin-type cells were assembled to evaluate the effects of various MF−2) was first plated onto the Cu foil. Next, the cell was stopped for a certain time , during which Li was corroded by the electrolyte. After that, Li was totally stripping. The amount of Li for SEI formation can be obtained from the cell without the rest treatment. Consequently, the capacity loss between Li plating and stripping can indicate the degree of Li corrosion. Li||Cu cell was also used to determine the SEI dissolution rates in different electrolytes. The cells were cycled from 0.05 to 2.0 V vs. Li/Li+ for 5 cycles to produce SEI on the electrode before pausing for 50 h. The same procedure was repeated with a pause time of 30, 15, and 5 h. The extra reductive capacity after the pauses indicates the degree of SEI dissolution. The electrolytes include 1.0 M LiTFSI in DOL/DME (v/v = 1:1), LiTFSI in DOL/DME with 1.0 wt% LiNO3 (v/v = 1:1), LiPF6 in EC/DMC/DEC (v/v/v = 1:1:1), LiPF6 in EC/EMC/DEC with 1.0 wt% FEC (v/v/v = 1:1:1), and LiPF6 in FEC/DMC (v/v = 1:4). The electrolyte amount used in cells for Li corrosion and SEI dissolution tests was 50 μL.Li||Cu in typical 2032 coin-type cells were assembled to quantify the Li corrosion. A certain amount of Li (1.0 mAh cmSupplementary informationPeer Review FileSource Data


Baseline characteristics were similar across treatment groups . At 12 months, BMD gains were greater with abaloparatide compared with placebo at the lumbar spine (least squares mean percentage change [standard error]: 8.48 [0.54] versus 1.17 [0.72]), total hip (2.14 [0.27] versus 0.01 [0.35]), and femoral neck (2.98 [0.34] versus 0.15 [0.45]) . The most common (≥5%) treatment‐emergent adverse events were injection site reaction, dizziness, nasopharyngitis, arthralgia, bronchitis, hypertension, and headache. During 12 months of abaloparatide treatment, men with osteoporosis exhibited rapid and significant improvements in BMD with a safety profile consistent with previous studies. These results suggest abaloparatide can be considered as an effective anabolic treatment option for men with osteoporosis. © 2022 Radius Health Inc and The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).Abaloparatide significantly increased bone mineral density (BMD) in women with postmenopausal osteoporosis and decreased risk of vertebral, nonvertebral, and clinical fractures compared with placebo. The Abaloparatide for the Treatment of Men with Osteoporosis  study evaluated the efficacy and safety of abaloparatide compared with placebo in men. Eligible men aged 40 to 85 years with osteoporosis were randomized 2:1 to daily subcutaneous injections of abaloparatide 80 μg or placebo for 12 months. The primary endpoint was change from baseline in lumbar spine BMD. Key secondary endpoints included BMD change from baseline at the total hip and femoral neck. A total of 228 men were randomized  from baseline in lumbar spine BMD at 12 months was 8.48% (0.54%) with abaloparatide compared with 1.17% (0.72%) with placebo (p < 0.0001).Change from baseline in bone mineral density. * Approximately one in four men over 50 years of age will incur a fragility fracture in their lifetime, with men accounting for up to 30% of the societal burden of osteoporosis and fractures. Although osteoporosis prevalence is lower in men than in women, men have greater fracture‐related morbidity and mortality. Further, lower proportions of men initiate an appropriate osteoporosis treatment after a fracture, which contributes to consequent morbidity and mortality. A retrospective review of Medicare claims data from 2012–2016 showed that the rates of testing and/or treatment of osteoporosis among patients ≥65 years of age with fractures were lower in men (5.7%) than in women (12.1%). Similarly, a study with a 1‐ to 5‐year follow‐up after hip fracture found fewer men (27%) than women (71%) treated for osteoporosis. A more recent study showed that only 24% of patients (men and women combined) were treated after hip fracture and also reported that the proportion of men treated within 1 year after hip fracture was lower than women. Thus, the widely appreciated care gap after an osteoporosis‐related fracture is even greater in men than in women.Osteoporosis in men is an important but underappreciated public health problem.1 Appr Because the prevalence of osteoporosis and fractures is higher in women than in men, effectiveness of available osteoporosis treatments has been more extensively studied in women. In part because of the underlying differences in testosterone and estrogen levels and their changes with aging, it is unclear whether treatments that are effective in women are equally so in men. Thus, the US Preventative Services Task Force has indicated that more trials for the treatment of osteoporosis that focus on or include men are needed.Currently, medications approved for the treatment of osteoporosis in men include bisphosphonates , denosumab, and teriparatide.9 BecaIn the Abaloparatide Comparator Trial in Vertebral Endpoints (ACTIVE), abaloparatide increased bone mineral density (BMD) in women with postmenopausal osteoporosis, decreased the risk of vertebral, nonvertebral, and clinical fractures compared with placebo, and decreased the risk of major osteoporotic fracture compared with teriparatide and placebo.12The primary objective of the Abaloparatide for the Treatment of Men with Osteoporosis (ATOM) study was to evaluate the efficacy and the safety of abaloparatide compared with placebo for osteoporosis treatment in men.ATOM NCT03512262), a randomized, double‐blind, placebo‐controlled, multicenter phase 3 study evaluating the efficacy and safety of abaloparatide compared with placebo for osteoporosis treatment in men, was conducted from May 3, 2018, to September 8, 2021. After a 1‐week pretreatment period, eligible volunteers were randomized 2:1 to receive daily subcutaneous (SC) injections of abaloparatide 80 μg or placebo SC for 12 months  or ≤ −1.5 and with radiologic vertebral fracture at screening or history of low‐trauma nonvertebral fracture in the past 5 years were required. Men >65 years of age who did not meet fracture criteria but who had BMD T‐scores ≤ −2.0 were also eligible. Hypogonadism at study entry was based on medical history or the laboratory reference range for serum testosterone at screening. Stable doses of androgen replacement therapy for at least 12 months were required. Good general health with a body mass index of 18.5 to 33 kg/m2 and a 25‐hydroxyvitamin D level of ≥20 ng/mL were also required.Men aged 40 to 85 years with primary osteoporosis or osteoporosis associated with hypogonadism were eligible to participate in the study. BMD Men with a history of fragility fracture within the past 12 months, >2 moderate vertebral fractures, or <2 radiologically evaluable lumbar vertebrae within L1 to L4 were excluded. Other exclusion criteria were prior treatment with parathyroid hormone (PTH)‐ or parathyroid hormone‐related peptide (PTHrP)‐derived drugs, intravenous bisphosphonates at any time, oral bisphosphonates within the past 3 years, denosumab within the past 18 months, or calcitonin or tibolone within the past 6 months. Men with serum levels of calcium , PTH, thyroid‐stimulating hormone (TSH), phosphorus, and alkaline phosphatase outside the normal range and men with clinical signs of hypogonadism or low testosterone at screening but who had not yet initiated treatment were not eligible. Men with bone disorders  other than osteoporosis, a history of prior external beam or implant radiotherapy to the bone, any cancer in the past 5 years , or osteosarcoma at any time were also ineligible. Incident vertebral fractures were recorded with a VSQ score increase of ≥1. For clinical fractures at other anatomic locations , radiographs were obtained at follow‐up visits as clinically warranted.The primary efficacy endpoint was percentage change from baseline in lumbar spine BMD by dual‐energy X‐ray absorptiometry (DXA) at 12 months. Key secondary endpoints were percentage change from baseline in total hip and femoral neck BMD at 12 months. Additional secondary endpoints included percentage change from baseline in BMD at 3 and 6 months for lumbar spine, total hip, and femoral neck; percentage change in BMD for the ultradistal radius and one‐third radius at 3, 6, and 12 months; and the incidence of new clinical fractures at 12 months. DXA scans were centrally adjudicated . Clinical assessments for fracture were obtained on day 1 and at months 1, 3, 6, 9, and 12. Radiographs were read locally, and reports were reviewed at a central location. Lateral spine radiographs obtained at screening were graded according to the Genant semiquantitative scoring method for vertebral fractures (VSQ).13 IncAdditional efficacy endpoints included the change from baseline in serum procollagen type I N‐terminal propeptide (s‐PINP) and serum carboxy‐terminal cross‐linking telopeptide of type I collagen (s‐CTX), for which samples were collected pre‐dose at 1, 3, 6, and 12 months. Samples were batched and analyzed by Centre Académique de Recherche et d'Expérimentation en Santé (CARES) .Safety data were collected throughout the study and included treatment‐emergent adverse events (TEAEs), changes in vital signs (including orthostatic blood pressure at each blood pressure assessment), injection site reactions, and antibodies for immunogenicity testing. Laboratory tests  were conducted at screening and months 1, 6, and 12. Electrocardiograms were performed pre‐dose and 1 hour post‐dose on day 1 and at month 12. All adverse events (AEs) were coded using MedDRA.α level of 0.01, assuming a standard deviation of 6.0% and drop‐out rate of 10%. For efficacy analyses, the window convention was used to select data . Primary and key secondary endpoints were analyzed by ANCOVA models using the intention‐to‐treat (ITT) population. Missing data were imputed using the wash‐out multiple imputation method.A sample size of 225 was determined to provide 99% power to detect a mean difference of 6.5% change from baseline in lumbar spine BMD at 12 months between treatment groups at an To claim statistical significance at the 2‐sided level of 1%, the following three fixed‐sequence tests were performed in sequential order: (i) percentage change from baseline in BMD at the lumbar spine at 12 months; (ii) percentage change from baseline in BMD at the total hip at 12 months; (iii) percentage change from baseline in BMD at the femoral neck at 12 months. If the treatment difference was not statistically significant at the 1% level at any of the steps, all the subsequent comparisons following the fixed sequence could not be claimed statistically significant.For the primary endpoint and secondary BMD endpoints, subgroup analyses were performed. Treatment groups were compared using ANCOVA models within subgroups defined by age, race, ethnicity, body mass index, geographic region, smoking status, BMD at baseline, fracture history, or s‐PINP levels at baseline.Bone turnover markers were analyzed by ANCOVA models based on the ratio of the post‐baseline value relative to baseline at each visit, using a natural log transformation. Missing data were imputed using the wash‐out multiple imputation method.A sensitivity analysis using the mixed model for repeated measures (MMRM) was performed to assess the impact of missing data. To investigate the effect of the COVID‐19 pandemic, several sensitivity analyses were performed, including subjects who completed or withdrew from the study before March 1, 2020, compared with subjects who completed or withdrew from the study on or after March 1, 2020. The nominal visit, excluding BMD measurements collected outside the allowable time period for data collection and excluding BMD measurements collected more than 30 days after the ideal visit days, were used.Safety data were summarized descriptively.n = 149; placebo, n = 79) with 178 (78.1%) completing the study: 114 (76.5%) in the abaloparatide group and 64 (81.0%) in the placebo group  and mean lumbar spine BMD p < 0.0001) . BMD gains at the lumbar spine, total hip, and femoral neck at 3 months were 3.76% (0.32%), 1.07% (0.19%), and 1.43% (0.24%), respectively, with abaloparatide compared with 1.06% (0.44%), 0.24% (0.25%), and 0.18% (0.32%) for placebo . At 6 months, BMD change at the lumbar spine, total hip, and femoral neck were 5.54% (0.41%), 1.39% (0.21%), and 1.48% (0.27%) with abaloparatide compared with 0.64% (0.55%), 0.03% (0.28%), and −0.19% (0.36%) for placebo .For the primary outcome, least squares mean percentage change (standard error [SE]) from baseline in lumbar spine BMD at 12 months was 8.48% (0.54%) with abaloparatide compared with 1.17% (0.72%) with placebo (01) Fig. A. Least01) Fig. B, C werp < 0.05). However, BMD change compared with placebo at the one‐third radius was not statistically significant at any time point  , 1.55% (0.36%), and 1.44% (0.42%), respectively, with abaloparatide, and −0.35% (0.45%), −0.32% (0.48%), −0.19% (0.57%) for placebo  and in the placebo group there were three patients with a new clinical fracture .p < 0.0001). Median s‐CTX level peaked at month 6 (0.476 ng/mL) and was 0.448 ng/mL at month 12 .In the abaloparatide group, median s‐PINP level peaked at month 1 (111.17 ng/mL) and was 85.7 ng/mL at month 12 Fig. A. Geomeonth 6 0.76 ng/mL The proportion of participants with any TEAE was similar between groups  (Table .2%) (Tab Orthostatic hypotension was reported in no patients in the placebo group and in 2 (1.3%) patients treated with abaloparatide. Neither of these events was severe or serious. Hypercalcemia was reported in 2 (1.3%) men treated with abaloparatide. No hypercalcemia events were reported in the placebo group. Hypercalciuria was reported in both treatment groups . The only patient to discontinue due to hypercalciuria was in the placebo group.Prior studies in women have suggested that orthostatic hypotension, hypercalcemia, and hypercalciuria may occur with abaloparatide treatment.14 OrtMean changes in heart rate at 1 hour post‐dose were 5.7 beats/min (bpm) on day 1 and 5.3 bpm on month 12 in the abaloparatide group compared with −1.3 bpm on day 1 and 0.6 bpm on month 12 in the placebo group. A consistent and low percentage of men had QT shifts  over the course of the study. No difference between treatment groups was observed in the incidence of clinically important QTcF values, the number of men with shifts in QTcF, or in the overall assessment of electrocardiograms. A subgroup analysis in the current study also showed that BMD increases at each anatomic location were consistent regardless of s‐PINP levels at baseline.In the current study in men with osteoporosis, the use of abaloparatide for 12 months resulted in significant and rapid increases in BMD at the lumbar spine, total hip, and femoral neck compared with placebo. Statistically significant changes in bone turnover markers were observed in the abaloparatide group, consistent with observed BMD changes and with previously reported changes in bone turnover markers in women treated with abaloparatide.12 A sNo new safety concerns with abaloparatide were observed and several of the most frequently reported AEs in men were also among the most frequent previously reported in the ACTIVE study in women .12 evidence for osteoporosis treatment efficacy in men is limited. Separate studies have demonstrated a decreased rate of new morphometric vertebral fractures with zoledronic acid and denosumab compared with placebo. Compared with alfacalcidol, alendronate and risedronate have also decreased vertebral fractures, and there are observational data to suggest that fracture risk is reduced with teriparatide. Additionally, men on androgen deprivation therapy for prostate cancer treated with denosumab sustained fewer vertebral fractures than men who received placebo. However, most studies that have been conducted in men have been too small to assess fracture outcomes. The majority of studies have relied on surrogate outcomes such as BMD and bone turnover markers to assess efficacy, an approach that is further supported by a more recent study showing the benefits of using surrogate threshold endpoints for BMD improvements by anatomic location for the prediction of fracture efficacy. Based on this evidence, alendronate, risedronate, zoledronic acid, denosumab, and the anabolic therapy teriparatide are approved for use in men in the United States.Although some recent guidelines have recommended age‐ and risk‐based osteoporosis screening for men,17 evi Additionally, increases in BMD with abaloparatide exceeded surrogate threshold estimates of BMD increases for predicting the clinical effectiveness for fracture reduction at all anatomic locations evaluated. Rapid rises in BMD and serum bone turnover markers in the current study are consistent with a recent study of romosozumab in men and previous studies of anabolic treatments in women, suggesting that antifracture efficacy for dual action and anabolic agents in women may be extrapolated to men.The current study provides further evidence for the use of anabolic therapy in men. Although comparisons between studies should be done with caution, BMD improvements in the current study appear similar to those observed in men treated with denosumab and zoledronic acid.24 Add The similar BMD and bone turnover marker outcomes between men in this study and those previously reported in women suggest that abaloparatide may provide an effective treatment option in men.Limitations include the short 12‐month duration of this study and the relatively small sample size. Because of the small number of clinical fractures in this study, no meaningful comparison could be made between groups in terms of fracture incidence outcomes. Although larger studies in men that include fracture endpoints and active comparators would be informative, the use of BMD changes at the total hip has been proposed as a surrogate for anti‐fracture efficacy.18 TheAbaloparatide rapidly improved lumbar spine and proximal femur BMD, suggesting that it may provide an effective treatment option for men with osteoporosis. Among men with osteoporosis, the self‐administration of abaloparatide was generally safe and well tolerated.EC reports travel support from Amgen and institutional funding from Radius Health, Inc., to conduct this study. JC and RP received institutional funding from Radius to conduct this study. CR has received research support and grants from Amgen, CytoDyn, Eli Lilly, Genentech, Integra, Novartis, Radius, Roche, and Senhwa and institutional funding from Radius to conduct this study. TV reports consulting fees and study funding from Takeda, study funding from Ascendis, and consulting and speaker fees from Radius outside of this work, and institutional funding from Radius to conduct this study. KGS has received research support from Horizon, Sobi, and Shanton and consulting fees from Arthrosi, Atom Bioscience, Horizon, Inflazome, L.G. Pharma, Mallinckrodt, Takeda, and Sobi pharmaceuticals and institutional funding from Radius to conduct this study. NCB reports consulting fees from Amgen and institutional funding from Radius to conduct this study. EML reports consulting fees and study funding from Amgen and Radius outside of this work and institutional funding from Radius to conduct this study. JA has provided consultancy to Amgen, Gilead, and Paladin Pharma; received research funding from Amgen and Radius; is an advisory board member of Amgen and Gilead; and participated in speaker bureaus for Amgen and Gilead. DKn received institutional funding from Radius to conduct this study. DKe reports speaker and consulting fees from Amgen, consulting fees from Paladin Pharma and Biosynt, and institutional funding from Radius to conduct this study. EO reports research support from Mereo, consulting fees from Amgen, Sanofi, Biogen, Bayer, NextCure, and Ultragenyx, and consulting fees from Radius in relation to this study. YC is a former employee of and holds equity in Radius. LP, YHL, and BM are employees of and hold equity in Radius.Edward Czerwinski: Investigation; resources; writing – review and editing. Jose Cardona: Investigation; resources; writing – review and editing. Rafal Plebanski: Investigation; resources; writing – review and editing. Chris Recknor: Investigation; resources; writing – review and editing. Tamara J. Vokes: Investigation; resources; writing – review and editing. Kenneth G Saag: Investigation; resources; writing – review and editing. Neil Binkley: Investigation; resources; writing – review and editing. E Michael Lewiecki: Investigation; resources; writing – review and editing. Jonathan Adachi: Investigation; resources; writing – review and editing. Dorota Knychas: Investigation; resources; writing – review and editing. David Kendler: Investigation; resources; writing – review and editing. Eric Orwoll: Conceptualization; visualization; writing – review and editing. Yinzhong Chen: Data curation; formal analysis; resources; visualization; writing – review and editing. Leny Pearman: Resources; writing – review and editing. Y Heather Li: Resources; writing – review and editing. Bruce Mitlak: Conceptualization; funding acquisition; methodology; resources; supervision; visualization; writing – review and editing.https://publons.com/publon/10.1002/jbmr.4719.The peer review history for this article is available at