N‐nonyl‐Acridine Orange. Inhibition of PDK with dichloroacetate resulted in increased mitochondrial permeability and cell death in tozasertib‐resistant glioma cell lines. Based on these results, we believe that PDK is a selective target for the tozasertib resistance phenotype and should be considered for further preclinical evaluations.Acquired resistance to conventional chemotherapeutic agents limits their effectiveness and can cause cancer treatment to fail. Because enzymes in the aurora kinase family are vital regulators of several mitotic events, we reasoned that targeting these kinases with tozasertib, a pan‐aurora kinase inhibitor, would not only cause cytokinesis defects, but also induce cell death in high‐grade pediatric and adult glioma cell lines. We found that tozasertib induced cell cycle arrest, increased mitochondrial permeability and reactive oxygen species generation, inhibited cell growth and migration, and promoted cellular senescence and pro‐apoptotic activity. However, sustained exposure to tozasertib at clinically relevant concentrations conferred resistance, which led us to examine the mechanistic basis for the emergence of drug resistance. RNA‐sequence analysis revealed a significant upregulation of the gene encoding pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), a pyruvate dehydrogenase (PDH) inhibitory kinase that plays a crucial role in the control of metabolic flexibility under various physiological conditions. Upregulation of PDK1, PDK2, PDK3, or PDK4 protein levels was positively correlated with tozasertib‐induced resistance through inhibition of PDH activity. Tozasertib‐resistant cells exhibited increased mitochondrial mass as measured by 10‐ Acquired resistance to conventional chemotherapeutic agents limits their effectiveness and can cause cancer treatment to fail. Sustained exposure to tozasertib at clinically relevant concentrations conferred resistance in glioma. Tozasertib‐resistant cells exhibited increased mitochondrial mass and upregulation of pyruvate dehydrogenase kinases (PDK). Addition of PDK inhibitor resulted in increased mitochondrial permeability and cell death in tozasertib‐resistant glioma cell lines. CCCPcarbonyl cyanide m‐chlorophenyl hydrazoneDCAdichloroacetateDEGdifferentially expressed genesFACSfluorescence activated cell sortingGBMglioblastomaHBSSHank's Balanced Salt SolutionKEGGKyoto Encyclopedia of Genes and GenomesNACN‐acetyl‐l‐cysteine)acetylcysteine  comprises a heterogeneous group of tumors with median survival ranges from 12 to 18 months. Despite maximum treatment efforts, survival improvements have been modest over several decades of technological advances in surgery, radiation therapy, and clinical trials of conventional and novel therapeutics. Though there is a wealth of information on basic underpinnings of glioma pathogenesis that has propelled meaningful progress in tumor subclassification, there is no satisfactory regimen available for adjuvant or salvage chemotherapy for these neoplasms  significantly reduced cell death  led to a substantial change in the cell cycle profile compared to untreated controls , cell death occurred to a significantly greater extent than in resistant cells (sustained exposure to tozasertib in 100 nmol·L−1), as shown by the appearance of a sub‐G1 peak in the DNA content analysis by flow cytometry . Genes identified using DESeq2 that featured an adjusted P value of < 0.05, and log2 fold change of +1.5 were regarded as differentially expressed genes (DEGs). DEGs are displayed in Table In order to identify gene expression signatures, and the molecules associated with acquired resistance, we isolated RNA from cells exposed to short‐term treatment with tozasertib and from a population of resistant cells (cultured in tozasertib for at least 8–10 weeks). RNA from drug‐naïve cells served as control Fig. . On averThe clusterProfiler package in R was utilized for the identification and visualization of enriched pathways among DEGs. Similar enrichment analysis was also conducted using GSEA. The dot plot depicts the significantly enriched KEGG pathways in tozasertib‐resistant cells when compared to the control cells. We then identified the emergence of a significant upregulation in pathways belonging to mitochondrial respiratory chain complex I, NADH dehydrogenase complex, NADH dehydrogenase activity, NADH dehydrogenase complex assembly, ATP synthesis coupled electron transport, oxidative phosphorylation, and mitochondrial gene expression in resistant cells Fig. , which i3.7P < 0.0005 in U87, SJG2, and SF8628), VER‐246608 , and DCA . Together, these results demonstrated and validated the potency of PDK inhibitors to inhibit cell growth and survival of tozasertib‐resistant glioma cells. Although multiple studies reported the use of DCA (but not PDK4‐IN‐1 hydrochloride or VER‐246608) in humans, particularly in the treatment of cancer [Multiple studies have highlighted the role of the PDH pathway and of its control by PDKs in cancer cell metabolism , 51, 52.f cancer , 54, witf cancer , we wantTo examine whether exogenous expression of PDK4 would reflect the resistant cell phenotype (on PDH activity and cell viability), logarithmically growing U87, SJG2, and SF8628 cells were transfected with PDK4 expression plasmids. Cells transfected with vector backbone served as control. Western blot analysis showed higher PDK4 expression than the vector‐transfected counterparts Fig. . In cont3.8N‐nonyl acridine orange (NAO), a metachromatic dye, which binds to the mitochondrial‐specific phospholipid, cardiolipin, where increased fluorescence intensity signal correlates well with increased mitochondrial content [−1 tozasertib for 72 h. However, we noticed a significant increase in the relative intensity of the fluorescent dye . We employed several strategies to elucidate the molecular basis for acquired resistance in these glioma models. Based on western blotting analysis, we observed aurora kinase A, B, and C protein expression was significantly upregulated in all resistant cell lines. In addition, cell cycle profile in resistant cells was similar to their drug‐naïve counterparts, and resistant cells proliferated at a similar rate as parental cells, suggesting that these cells had become refractory to clinically achievable concentrations of tozasertib. Importantly, the emergence of resistance to tozasertib was not associated with the p53 status of the tumor cell lines  and cell cycle analysis showed a paucity of cells with 4N or greater DNA content, a sharp contrast to short‐term treated cells. Because studies of drug‐induced resistance in cell line models can suggest strategies to circumvent drug resistance in the clinical setting, we examined the effect of alisertib , barasertib , and ZM447439 (AstraZeneca). Under or types , 75, 76,et al. [In this context, we noticed an extensive change in gene expression that accompanied tozasertib treatment. Between U87 and LNZ308 tozasertib‐resistant cell lines, 151 genes overlapped. Tozasertib‐resistant glioma cells significantly upregulated the expression of PDH kinase 4, a negative regulator of glucose oxidation, as assessed using RNA‐seq analysis, in comparison with drug‐naïve controls. Western blot analysis revealed that all four PDK isoenzymes  were upregulated in resistant cell lines. Moreover, the TCGA database showed that PDK4 mRNA levels were upregulated and are correlated with poor survival of GBM  and otheet al.  identifiet al. , 85, 86.et al. [In this report, we found the levels of PDK1, PDK2, PDK3, and PDK4 protein expression increased in the tozasertib‐resistant cells, with resultant inactivation of PDH activity. In accordance with Woolbright et al.  in bladdet al. , 78, 79.Although numerous mechanisms are responsible for cancer resistance to chemotherapy, an emerging concept suggests that metabolic change in cancer cells may be a critical determinant of resistance that is due, in part, to an attenuated mitochondrial function, which results from the inhibition of PDHC via increased expression of PDK1, PDK2, PDK3, and PDK4 , 88. Sev5The results suggest that clinically relevant concentrations of tozasertib, a pan‐aurora kinase inhibitor, has significant pro‐apoptotic activity in adult and pediatric glioma cell lines . However, the challenge presented is neoplastic cells acquire resistance at clinically achievable dosages, limiting the effectiveness of this therapeutic agent. We demonstrated that, over time, the emergence of this resistance to tozasertib is generated due to the neoplastic cells' highly integrated metabolic PDK/PDH network, driving mitochondrial biogenesis, and promoting this resistant cell growth. This convergent mechanism of resistance offers potential therapeutic opportunities to re‐sensitize the cells to tozasertib.The authors declare no conflict of interest.EPJ and DRP contributed equally to this work. IFP and DRP conceived and designed the study. EPJ, DRP, SA, and DR involved in development of methodology. EPJ, DRP, DR, MCR, and ST acquired data . EPJ, DRP, and DR analyzed and interpreted the data . Writing, review, and/or revision of the manuscript: DPR, DR and IFP. All the authors edited and approved the manuscript.https://publons.com/publon/10.1002/1878‐0261.13025.The peer review history for this article is available at Fig. S1. Cell cycle profiles in response to the inhibition of aurora kinases in malignant human glioma cells.  Logarithmically growing adult  and pediatric  glioma cells were seeded on 6‐well plates (1 × 105). On the following day cells were either transfected with non‐target siRNA (NT siRNA) or aurora kinase A or B or C siRNA. Seventy‐two hours post‐transfection, the cells were trypsinized, lifted, washed, and stained with PI. Fluorescence of the PI‐stained cells was measured using a Becton Dickinson flow cytometer. The color‐coded bar graphs show the percentage of glioma cells in various phase of cell cycle such as sub‐G1, G1, S, G2M and > 4N DNA content (A). B represents the average percentage of cells accumulated from three different experiments. (C) Logarithmically growing adult  and pediatric  cells were seeded on 6‐well plates (1 × 105). After overnight attachment period, cells were treated with tozasertib for 24 h or 72 h. After treatment, the cells were washed, trypsinized, and lifted. Then the cells were stained with PI and the fluorescence of the PI‐stained cells was measured using a flow cytometer. Quantitative measurements of cell cycle profile were performed, and the percentage of cells accumulated in different phases of the cell cycle from three different experiments are shown.Click here for additional data file.Fig. S2. Schematic of the approach taken to achieve drug resistance. Malignant human glioma cells were exposed to increasing concentrations of tozasertib. Starting with an initially low dose (10 nmol·L−1), the inhibitor concentration was progressively increased to 100 nmol·L−1 over a period of several weeks.Click here for additional data file.Fig. S3. Cross‐resistance to other aurora kinase inhibitors. (A) Drug‐naïve or tozasertib‐resistant (resistant) cells were allowed to attach overnight in a 6‐well plate. Drug‐naïve cells were either treated with tozasertib for 72 h (short‐term) or an equal amount of vehicle (DMSO). After treatment, the cells were trypsinized, lifted, washed, and stained with PI. Fluorescence of the PI‐stained cells was measured using a Becton Dickinson flow cytometer and the percentage of cells accumulated in different phases of cell cycle from three different experiments are shown. (B) Logarithmically growing drug‐naïve or tozasertib‐resistant cells (3 × 103) were seeded on 96‐well plates. After overnight attachment period, cells were exposed to indicated concentrations of inhibitors  for 72 h. DMSO (0) served as vehicle control. Cell growth was assessed spectrophotometric measurement of MTS bioreduction. Absorbance was recorded at 490 nm using a Synergy HTX multimode microplate reader. The values are represented as mean ± standard deviation of three separate experiments. (C) Whole cell extracts from drug‐naïve or cells treated with tozasertib for 72 h (short‐term) or tozasertib‐resistant cells were prepared. Twenty micrograms of protein were separated on a SDS/PAGE. Western blot analysis was performed with the polo‐like kinase 1 (PLK1) antibody. Expression of β‐actin served as loading control. These experiments were performed at least three times, and a representative blot is presented here.Click here for additional data file.Fig. S4. The effect of DCA on mitochondrial membrane potential of normal human astrocytes. Non‐neoplastic human astrocytes were treated with the indicated concentrations of DCA for 72 h. Then, the cells were subjected to mitochondrial membrane potential analysis by DiOC6 staining and flow cytometry. No significant change in the intrinsic cellular mitochondrial membrane potential was evident in the cells cultured in the presence of 2.5 mmol·L−1 DCA for 72 h. The values are represented as mean ± standard deviation of three independent experiments .Click here for additional data file.Table S1. List of up‐ and down‐ regulated DEG for the LNZ308 and U87 samples at a 5% threshold, and log‐fold change of 1.5.Click here for additional data file.Table S2. List of common genes between LNZ308 and U87 samples.Click here for additional data file.


This study aimed to analyze the clinicopathological characteristics of patients with paraproteinemia and renal damage.Ninety-six patients from 2014 to 2018 with paraproteinemia and renal damage were enrolled and the clinical data, renal pathology, treatment and prognosis data were collected.n  =  14). In the MGRS group, the main diseases were amyloidosis (n  =  25) and cryoglobulinemic glomerulonephritis (n  =  7), while in the MM group, the main diseases were cast nephropathy (n  =  9) and light chain deposit disease (n  =  3). In the MGUS group, it was mainly IgA nephropathy  and idiopathic membranous nephropathy (n  =  5); while in the renal monoIg group, most of the cases were IgAN (n  =  19). Chemotherapy was mainly administered to patients in the MM group, while immunosuppression therapy was mostly administered to patients in the renal monoIg group. Most patients with renal monoIg exhibited a major response, followed by the patients with MGUS and MGRS, while most of the patients with MM had a partial response but none had a major response. Approximately more than half (57.1%) of the patients with MM progressed to end-stage renal disease (ESRD), followed by MGRS (33.3%); however, the mortality rate was low in both the MGRS and MM groups. The survival analysis reviewed that serum creatinine, hemoglobin levels, and the serum κ/λ ratio were independent risk factors for ESRD in patients with MGRS.A total of 96 patients , accounting for 2.7% of all renal biopsies, were enrolled in this study. Among them, 42 were monoclonal gammopathy of renal significance (MGRS), 21 were renal monotypic immunoglobulin alone , and 19 were monoclonal gammopathy of undetermined significance (MGUS). Individuals with multiple myeloma (MM) accounted for the fewest number of patients (The clinicopathological changes in patients with MGRS were between those in patients with MM and MGUS. The treatment for MGRS and MM was more intensive, and the overall mortality rate was low. Both MGUS and renal monoIg alone exhibited slighter clinicopathological features than MGRS and MM, and the treatment was focused mostly on primary renal diseases. The detection rate of monoclonal immunoglobulin (MIg) has increased due to technological advances and the widespread use of serum and renal paraprotein analysis. Accordingly, renal damage with paraproteinemia is commonly diagnosed among older patients. A paraprotein is an MIg or immunoglobulin fragment, which is detected as abnormal serum or urine light and/or heavy chains, or an abnormal light chain ratio. Paraproteins that originate from disorders of the plasma and/or B cells are known as monoclonal gammopathy of undetermined significance (MGUS). MGUS is defined as the presence of a serum monoclonal protein at a concentration of 3.0 g per deciliter or less, no monoclonal protein or only modest amounts of monoclonal light chains in the urine, the absence of CRAB features  that are related to the monoclonal protein, and 10% or fewer monoclonal plasma cells in the bone marrow , 3. Thesn  =  42); a clonal plasma cell or B lymphocyte proliferation causing a renal lesion in the absence of hematologic malignancy or other myeloma-defining events . Major response was defined as a reduction in proteinuria to protein excretion  <  0.3 g/d with stable or improvement of eGFR to  ≥  60 mL/min/1.73 m.5 mg/dL , 15. ESR6 months . Patientt test or analysis of variance (ANOVA) for continuous variables, and by nonparametric tests for discontinuous variables. Categorical variables were expressed as percentages and between-group comparisons were assessed by Chi-square test or Fisher’s exact test. Death and renal endpoints were analyzed by univariate Cox regression followed by multivariate Cox regression. Predictive models were created using clinical data and pathological data. Univariate variables included the following variables: age, gender, lesion duration, hematuria, proteinuria, albumin, Hb, Scr, eGFR, low serum C3 level, abnormal serum and urine κ/λ ratio, abnormal sIFE and uIFE, presence of renal monotypic κ, glomerular global and segmental sclerosis, crescents, IFTA, interstitial inflammation, and podocyte effacement. Multivariate variables included Hb, Scr, eGFR, abnormal serum and urine κ/λ ratio, and glomerular global and segmental sclerosis. The Kaplan–Meier analysis was used to compare the survival rates. Results were expressed as hazard ratio with 95% confidence intervals. All P values were two-sided, and statistical significance was set at P  <  0.05.Statistical analysis was performed with SPSS 17.0. Normal distribution data were expressed as mean  ±  standard deviation (SD). The clinical and pathological characteristics among the groups were compared by n  =  42) of all cases, followed by the renal monoIg and MGUS groups . MM accounted for the fewest number of cases (14.6%). In the MGRS group, amyloidosis was the main disease, affecting 59.5% (n  =  25) of cases. In the MM group, cast nephropathy was the main disease (n  =  9), and three of these cases had light chain deposit disease (LCDD) combined with MM , followed by IMN (n  =  5), mesangial proliferation nephritis (n  =  2), acute tubular injury (n  =  1), and malignant hypertension (n  =  1). In the renal monoIg group, most of the cases had IgAN (n  =  19). The remaining two cases were lupus nephritis (LN) and hepatitis B-related nephritis. A comparison of the clinicopathological characteristics is shown in Table P  <  0.001). Additionally, the sIFE-positive detection rate was lower in the MGRS group than in the MM and MGUS groups (P  <  0.001). Patients with MM exhibited a much lower levels of hemoglobin, yet less hematuria , the highest SCr levels and the lowest eGFR than some of the other groups . Additionally, patients with MM had a higher abnormal κ/λ ratio in both the serum and urine compared to the other groups (P  =  0.024), and their interstitial fibrosis and inflammation were worse (P  =  0.029). The MGUS group exhibited the highest levels of hemoglobin and the lowest levels of serum creatinine . No renal monoclonal light chain deposits were detected. Renal monoIg patients were the youngest, and had more hematuria and less proteinuria than some of the other groups . No hematological paraproteins were detected according to the abnormal light chain ratio or IFE.A total of 96 patients , were enrolled in this study, which accounted for 2.7% of all renal biopsies, performed in our hospital during the study period. The mean age of the patients was 53.3  ±  12.8 years, and the participants were divided into four groups. MGRS accounted for 43.7% (n  =  7), proliferative glomerulonephritis with monoclonal IgG deposits , and monoclonal immunoglobulin deposition disease . The clinicopathological data of these patients are summarized in Table P  =  0.001), and their Hb level was the highest (P  <  0.001). Patients with Cryo-GN had the highest level of proteinuria while those with MIDD had the highest albumin levels . Patients with MIDD had the highest SCr levels and the lowest eGFR , followed by those with Cryo-GN. More patients with PGNMID had low serum C3 levels than those with amyloidosis. The abnormal κ/λ ratio and IFE were similar among the groups. Patients with amyloidosis exhibited a lower proportion of serum and renal monoclonal κ chains than those with Cryo-GN and PGNMID . Patients with MIDD exhibited worse chronic pathological features, including interstitial fibrosis and inflammation (P  <  0.001).Most patients with MGRS had amyloidosis n  =  25), followed by cryoglobulinemic glomerulonephritis . Immunosuppression therapy was administered mostly to patients in the renal monoIg group, followed by those in the MGUS and MGRS groups, each of which were significantly different (P  <  0.001). Long-term dialysis or renal transplantation was only applied in patients with MGRS and MM who had ESRD. The average follow-up period was 44.8  ±  16.9 months. The renal monoIg group had the highest proportion of patients with major response, followed by the MGUS and MGRS groups, each of which were significantly different (P  <  0.001). The MM group had a higher proportion of patients with partial response, but none of the patients showed a major response. More than half of the patients with MM (57.1%) reached ESRD, followed by those with MGRS (33.3%), although both the MGRS and MM groups had a low mortality rate without significant difference . The difference in survival time between these patients was also not significant (16.7% vs. 21.4%). Patients with MGUS demonstrated a very low rate of ESRD progression, and none of the patients with MGUS or renal monoIg died during the observational time.Most of the patients in the four groups underwent special treatment, including chemotherapy and immunosuppression Table . ChemothP  <  0.05), although it had no significant difference between the MGRS and MM groups , and it was also better in the MGUS group than the MM group , although the overall survival was not significantly different between them  and high affinity of mesangial region, causing selective deposition. However, Vignon et al. [λ restriction in renal tissue. We hypothesized that this was due to the selectivity difference of two light chains, according to the similar treatment and prognosis of LN or HBV-GN, and no MIg was detected during follow-up.There is a higher prevalence of both MGUS and chronic kidney disease (CKD) in older patients. The same patient could therefore have unrelated hematological conditions and CKD. As such, distinguishing the conditions is essential because of their different clinical characteristics and associated therapies . MGRS cae et al.  comparedn et al.  reportedκ chains, and milder IFTA and inflammation due to its fibril features without activation of the complement pathway [MGRS is complex and heterogenous, and manifests in diverse patterns of renal injury , 23, 24. pathway . Patient pathway . Patient pathway . Patient pathway , althoug pathway .κ/λ ratio were independent risk factors for ESRD in the MGRS group, no independent risk factors were found in the MM group. The overall mortality rate of MM and MGRS was low due to active chemotherapy and the clone-directed approach [Most of our patients underwent special treatment including chemotherapy, and immunosuppressive therapy. Chemotherapy was more widely administered in the MM group than in the MGRS group, and patients with MM exhibited more partial renal response, but no major response. While SCr, Hb, serum approach , 33. In approach –37. Patiapproach  reportedThe limitations of this study were its retrospective design and the lack of long-term follow-up. Additionally, we were unable to analyze abnormal free light chain due to limited detection in a few patients, and complete hematological data could not be analyzed during follow-up.The clinicopathological changes in patients with MGRS were between those in patients with MM and MGUS. The treatment of both MGRS and MM was more intensive, and the overall mortality rate was low. Both MGUS and renal monoIg alone exhibited fewer clinicopathological characteristics than MGRS and MM, and the treatment was mostly focused on primary renal diseases.


Optimal protein level in hypoallergenic infant formulas is an area of ongoing investigation. The aim was to evaluate growth of healthy term infants who received extensively hydrolyzed (EH) or amino acid (AA)-based formulas with reduced protein.7 CFU Lactobacillus rhamnosus GG/100 kcal. Anthropometrics were measured and recall of formula intake, tolerance, and stool characteristics was collected at 14, 30, 60, 90, 120 days of age. Primary outcome was weight growth rate (g/day) between 14 and 120 days of age . Medically confirmed adverse events were recorded throughout the study.In this prospective, multi-center, double-blind, controlled, parallel group study, infants were randomized to receive a marketed EH casein infant formula at 2.8 g protein/100 kcal (Control) or one of two investigational formulas: EH casein formula at 2.4 g protein/100 kcal (EHF) or AA-based formula at 2.4 g total protein equivalents/100 kcal (AAF). Control and EHF each had 2 × 10No group differences in weight or length growth rate from 14 to 120 days were detected. With the exception of significant differences at several study time points for males, no group differences were detected in mean head circumference growth rates. However, mean achieved weight, length, and head circumference demonstrated normal growth throughout the study period. No group differences in achieved weight or length  and head circumference  were detected and means were within the WHO growth 25th and 75th percentiles from 14 to 120 days of age. With the exception of Day 90, there were no statistically significant group differences in achieved head circumference for males; means remained between the WHO 50th and 75th percentiles for growth at Days 14, 30, and 60 and continued along the 75th percentile through Day 120. No differences in study discontinuation due to formula were detected. The number of participants for whom at least one adverse event was reported was similar among groups.This study demonstrated hypoallergenic infant formulas at 2.4 g protein/100 kcal were safe, well-tolerated, and associated with appropriate growth in healthy term infants from 14 to 120 days of age.NCT01354366. Registered 13 May 2011.ClinicalTrials.gov, ClinicalTrials.gov Identifier:  Cow’s milk allergy (CMA) is one of the most common allergies in infancy, with a clinically diagnosed prevalence estimated up to 3% (as reviewed –5). HypoIncreasing evidence supports lowering total protein content in both hypoallergenic formulas and routine infant formulas to be more in line with the amount of protein found in human milk. Human milk is dynamic in composition and declines in protein content as lactation progresses  whereas Though management of CMA is the primary goal of EH or AA formula usage, a reduction of protein in infant formula requires consideration with respect to growth and tolerance. The overall importance of adequate protein for infant growth and development is well understood. Both an EH and an AA-based formula that had protein at 2.8 g/100 kcal have been demonstrated to adequately support typical growth and safety . In the Good Manufacturing Practice guidelines are provided by the US Food and Drug Administration to insure that an infant formula meets the quality factor of normal physical growth , 16. ConHealthy 12- to 16-day old infants were recruited at 27 clinical sites in the United States. Eligible infants were singleton births at 37–42 weeks’ gestational age with birth weight ≥ 2500 g and solely receiving infant formula at least 24 h prior to randomization. Exclusion criteria included history of underlying disease or congenital malformation likely to interfere with normal growth and development or participant evaluation; feeding difficulties or history of formula intolerance; weight at randomization < 98% of birth weight; large for gestational age from mother who was diabetic at childbirth; and immunodeficiency.A computer-generated, randomization schedule stratified by sex was created by the study sponsor and provided in sealed consecutively numbered envelopes for each study site. At each study site the next sequential envelope was opened from the appropriate set to assign study formula. Two unique codes (known only to the sponsor) were used to designate each study formula. Study formulas were dispensed to parents at each study visit prior to completion or withdrawal. Product labels and the sealed envelopes did not allow direct unblinding by the study site. Study monitoring personnel were also blinded to study product identification. In the event of a medical emergency  blinding for a participant could be broken by study sponsor personnel. In this study, it was not necessary to break the study code prematurely.7 CFU Lactobacillus rhamnosus GG (LGG)/100 kcal  from 14 to 120 days of age: 1) an EH casein infant formula at 2.8 g protein/100 kcal ; 2) an investigational EH casein formula at 2.4 g protein/100 kcal (EHF); or 3) an investigational AA-based formula at 2.4 g total protein equivalents/100 kcal (AAF). Control and EHF each had 2 × 10Evaluation of growth and tolerance in healthy, term infants was the study objective. Body weight, length, and head circumference (anthropometric measures) were recorded at study visits corresponding to 14 , 30 (±3), 60 (±3), 90 (±3), 120 (±4) days of age.At all study visits parents completed a 24-h recall of tolerance (fussiness and gassiness) and stool characteristics (frequency and consistency); study formula intake (fluid oz./day) was reported beginning at the 30 days of age visit. We have previously characterized tolerance using the same reporting scales , 20, 21AWeight growth rate from 14 to 120 days of age was the primary outcome. Detection of a clinically relevant difference of 3 g/day in weight gain from 14 to 120 days of age (80% power) was used to determine the sample size. Enrollment of approximately 91 males and 66 females was needed in each group with expected completion of 59 male and 43 female participants per study group . Linear regression of weight on age was calculated for each participant and analysis of variance (ANOVA) was used to assess growth rates from 14 to 30, 60, 90, or 120 days of age. A one-tailed test, as outlined in guidance provided by the American Academy of Pediatrics (AAP) Task Force on Clinical Testing of Infant Formulas  was usedp-values reported from the analysis of weight gain based on one-tailed tests, all other p-values reported were based on two-tailed tests. When the overall comparison of the three groups was significant, unadjusted pairwise group comparisons were performed. All testing was conducted at α = 0.05. All analyses were conducted using SAS version 9.2 .Secondary outcomes included other anthropometric and tolerance measures and medically confirmed adverse events. Participant characteristics  and adverse events were analyzed by Fisher’s exact test. Achieved weight, length, and head circumference; length and head circumference growth rates; formula intake; and stool frequency were analyzed by ANOVA with two exceptions. For females, because a difference in head circumference was detected at enrollment, analysis of covariance (ANCOVA) with covariate head circumference at enrollment was used to analyze head circumference growth rates and achieved head circumference. Stool consistency, fussiness, and gas were analyzed using the Cochran-Mantel-Haenszel (CMH) row mean score test. With the exception of P = 0.004), no differences in body weight or length, or head circumference by sex were observed among groups at study enrollment . Participants who were randomized but consumed no study formula  were not included in subsequent analyses  or EHF ; however, the means for all groups plotted within the 50th and 75th percentiles of WHO growth charts at Days 14, 30, and 60 and continued along the 75th percentile through Day 120 ; lower in EHF or AAF versus Control at Day 60 (P ≤ 0.025); and lower in AAF versus Control or EHF at Day 90 (P ≤ 0.008) (Table P < 0.001). At each measured time point, more infants with “unformed or seedy” and fewer infants with a “hard” or “formed” stool consistency in the Control and EHF groups compared to the AAF group were the primary differences observed in stool consistency categories.fvNo group differences in parent-reported gassiness and fussiness were detected at any time point assessed, with the exception of fussiness at Day 30 Table . Mean st P ≤ 0.02; and lowP ≤ 0.036), there were no significant group differences by sex detected at Days 30, 60, 90, or 120 (Table P = 0.476). The incidence of adverse events categorized within Body as a Whole; Cardiovascular; Eyes, Ears, Nose, and Throat; Musculoskeletal; Respiratory; or Urogenital systems had no statistically significant group differences for specific events. In the Gastrointestinal (GI) System, there were no significant group differences in the most commonly reported specific adverse event, gastroesophageal reflux . The incidence of constipation was lower in the Control  or EHF  versus the AAF group ; the incidence of diarrhea was lower in the AAF  versus the Control  or EHF  groups. Within the Metabolic and Nutrition System, an overall significant difference was detected for poor weight gain  but no specific pairwise group differences were detected. Within the Skin System, the incidence of diaper rash was significantly higher in the Control  versus the AAF ; no significant differences were detected compared to the EHF  group. Any medically confirmed adverse event was considered serious if it met one or more of the following criteria: resulted in death, was life-threatening, required inpatient hospitalization or prolongation of existing hospitalization, resulted in persistent or significant disability/incapacity, or was a congenital anomaly/birth defect. A total of 16 participants experienced serious adverse events . All serious adverse events were individually evaluated by study site physicians and each was determined unrelated to study formula.With the exception of lower formula intake  at Day 60 for male infants in the Control (30.2 ± 1.0) versus the EHF (33.4 ± 1.1) or AAF (34.2 ± 1.1) groups  and an AA-based formula , were safe and well-tolerated when fed to healthy term infants from 14 to 120 days of age. Rate of weight gain (g/day), per guidance provided by the AAP Task Force on Clinical Testing of Infant Formulas , was useThe randomized, double-blind, controlled design is a key strength of this study. One limitation could be that an AA-based formula at 2.8 g protein/100 kcal was not included as a study formula. However, we have previously reported that an AA-based formula (vs a control casein EH formula) and a casein EH formula with added LGG (versus a control casein EH formula with no added LGG) were safe and supported growth in healthy, term infants in studies of comparable design , 25. TheIt is well known that infants fed formula grow on a different trajectory than infants exclusively receiving breast milk , 11, 26.Overall, acceptance and tolerance of study formulas were good. No differences in study discontinuation due to study formula were detected. Aside from fussiness at day 30, no significant group differences were detected in fussiness or gassiness. Mean stool frequency was not significantly different among groups by Day 120 but differed at other time points, with EHF and Control groups generally reporting more frequent stooling. Similarly, more infants in the EHF and Control groups reported “unformed or seedy” stool consistency and fewer “hard” or “formed” stools compared to infants in the AAF group.In summary, the results of the current study suggest that an EH casein formula and an AA-based formula, both with reduced protein content , were well tolerated and associated with normal growth in healthy, term infants from 14 to 120 days of age.


Failed induction of labor affects maternal and neonatal outcomes as well as the cost of healthcare, especially in low-resource setting regions in which the prevalence of failed induction is higher despite the incidence of labor induction is low. This study aimed to assess the prevalence of failed induction of labor in southwest Ethiopia.A hospital-based cross-sectional study was conducted among 441 induced women from March 1 to August 30, 2018. A systematic random sampling technique was used to select study participants. Data were collected using a pretested and structured questionnaire. Bivariable and multivariable logistic regression models were done and fitted to identify predictors of failed induction. An adjusted odds ratio with 95% confidence interval (CI) was calculated to determine the level of significance.Premature rupture of membrane was the most common cause of labor induction and the commonly used method of labor induction were oxytocin infusion. Cesarean section was done for 28.1% of induced women. Failed induction of labor was found to be 21%. Primiparous [AOR = 2.35 (1.35–4.09)], analgesia/anesthesia [AOR = 4.37 (1.31–14.59)], poor Bishop Score [AOR = 2.37 (1.16–4.84)], Birth weight ≥ 4 k grams [AOR = 2.12 (1.05–4.28)] and body mass index [AOR = 5.71 (3.26–10.01)] were found to be significantly associated with failed induction of labor.The prevalence of failed induction of labour was found to be high. Preparation of the cervix before induction in primi-parity women is suggested to improve the success of induction. To achieve the normal weight of women and newborns, proper nutritional interventions should be given for women of reproductive age. It is better to use analgesia/anesthesia for labor induction when it becomes mandatory and there are no other optional methods of no- pharmacologic pain management.The online version contains supplementary material available at 10.1186/s12884-021-03862-x. Induction of labor (IOL) is an interventional designed to artificially initiate uterine contractions . IOL shoInduction of labor is increased risk of poor maternal and perinatal outcomes  –8. CesarStudies were done in American, African, and Asian countries showed that low Apgar scores at 5 min, low birth weight, neonatal intensive care unit admission, stillbirth, and delayed breastfeeding increased when labor induced , 22, 23.The global prevalence of obesity among women has increased from 6% in 1975 to 15% in 2016 . Obese wAccording to research; maternal age, gestational age, parity, bishop score, PROM, postterm, previous obstetric complications, and birth weight are the most common contributing factors for failed induction , 20. HowThe study was conducted at Mizan Tepi University Teaching Hospital, Gebretsadik-shaw General Hospital and Tepi General Hospital from March 1 to August 30, 2018. Those 3 hospitals are the only hospitals which provide induction of labor service in the catchment area. Based on the 2017 Population forecast, a total of 2,218,689 population, of whom 1,123,834 females lived in Bench Maji Zone, Kaffa Zone, and Sheka zones . Women whose newborn birth weight ≥ 4000 g were 2 times as likely to have failed induction as compared with women whose newborn birth weight was 2500–3900 g [AOR = 2.12 (1.05–4.28)]. The odds of failed induction were 2.3 times more likely in Primiparous [AOR = 2.35 (1.35–4.09)] than in multiparous women. The odds of failed induction were 4.3 times more likely among women who were given analgesia/anesthesia [AOR = 4.37 (1.31–14.59)] than mothers who were not given. Women whose body mass index > 24 kg/m2 were 5.7 times more likely to have failed induction as compared to their counterparts [AOR = 5.71 (3.26–10.01)] . This is in line with studies done in Nigeria (24.1%), WHO survey in African and Asian countries (20%), southwest Ethiopia (21.4%), and Israel (21.6%) , 37, 38.This finding is higher than studies done in America (15.7%)  and soutPrimiparous women were 2.3 times more likely to had failed induction than multipara women. This study is similar to the previous studies; nulliparous women are at greater risk for both failed inductions and cesarean sections as compared to multiparous women , 40, 41.2 were 5.7 times more likely to had failed induction as compared with women whose body mass index ≤24 kg/m2. This finding is supported by previous findings [Women whose body mass index > 24 kg/mfindings , 41–43. findings , 45. Thefindings .Women who were given analgesia/anesthesia were 4.3 times more likely to had failed induction as compared to women who were not given analgesia/anesthesia. This finding is supported by studies in which the earlier epidural analgesia was given during labor, the higher the probability of cesarean delivery among induced women , 13, 18.Mothers whose pre-induction bishops score ≤ 5 were 2.3 times more likely to have failed induction as compare to mothers whose pre-induction bishop score > 5. This finding is comparable with the studies , 38, 40.Women whose newborn birth weight ≥ 4000 g were 2 times more likely to have failed induction as compared with women whose newborn birth weight was 2500–3900 g. This study is supported by the previous studies , 39. ThiThe economic situation of participants might be one of the causes that could affect the failed induction of labor. However, majority of the women who were participated in this study were house wives and did not remember the economic status the family. Some of the study variables had wider CI and this might not happened if the sample size was larger.2, bishop score ≤ 5, Primiparous, birth weight ≥ 4000 g, and using analgesia/anesthesia. Preparation of the cervix before starting induction in Primiparous women is recommended to improve the success of induction. To achieve the normal weight of women and newborns, proper nutritional interventions should be given for women of reproductive age. Consider the risk of failed induction in case of the provision of analgesia/anesthesia.The prevalence of failed induction of labor was high in the study area. Variables that increased the likelihood of failed induction were body mass index > 24 kg/mAdditional file 1.


Takotsubo cardiomyopathy (TCM), characterized by transient left ventricular dysfunction, was first reported in Japan in 1990. Current research suggests that TCM can be affected by conventional cardiovascular factors such as hypertension (HTN), diabetes, hyperlipidemia (HLD), and obesity. Despite the increasing interest in this disease, research on TCM remains limited. Conventional cardiovascular factors are clinically related to the outcome of TCM. We reviewed the publications published in PubMed database between January 01 2010 and January 15 2021, and summarized the most current available evidence on the correlation between TCM and the conventional cardiovascular factors. TCM patients are predominantly postmenopausal women. Men and young patients are less commonly seen, but are prone to acute adverse complications and poor prognosis. HTN is common in patients with recurrent TCM. Existing evidence suggests that obesity and chronic kidney disease are related to poor prognosis in TCM. HLD is reported to be associated with fewer complications, though current evidence is limited. Finally, the relationship between diabetes and TCM prognosis is ambivalent. Current evidence suggests conventional cardiovascular risk factors are associated with the outcome of TCM, especially with mortality and complications. More prospective studies are needed to clarify the relationship between each risk factor and the prognosis of TCM. ACSacute coronary syndromeAKIacute kidney injuryCIconfidence intervalCKDchronic kidney diseaseDKAdiabetic ketoacidosisDMdiabetes mellituseGFResti glomerular filtration rateESCEuropean society of cardiologyHLDhyperlipidemiaHRhazard ratioHTNhypertensionInter TAKInternational TakotsuboMeSHMedical Subject HeadingsORodds ratioPp valueSOsympathetic overactivitySTEMIST‐segment elevation myocardial infarctionTCMTakotsubo Cardiomyopathy1Takotsubo cardiomyopathy (TCM) is a reversible left ventricular dysfunction that is commonly known as “broken heart” syndrome. The first case of TCM was in Japan in 1990, and has since become increasingly reported worldwide.Studies demonstrated that TCM has distinct features in gender and age distribution, and its patient population is prominently postmenopausal women. In addition, conventional cardiovascular factors are relatively common in TCM patients, such as hypertension (HTN), hyperlipidemia (HLD), obesity, diabetes mellitus (DM), and chronic kidney disease (CKD).2We searched publications in the PubMed database published between January 01 2010 and January 15 2021. The following search terms were used, including Medical Subject Headings (MeSH): Takotsubo cardiomyopathy, Takotsubo syndrome, broken heart syndrome, stress cardiomyopathy, cardiovascular risk factors, hypertension, high blood pressure, diabetes mellitus, hyperlipidemia, obesity, CKD, age, gender and sex. Two investigators (J. L. and Y. X.) independently performed the literature research. Large prospective and retrospective studies that reporting mortality rate were included, and select case reports were also considered supplement the limited evidence available within the research topic.3Selective risk factors, including HTN, DM, HLD, obesity, CKD, gender, and age, and the current evidence of their association with TCM outcome are discussed below in detail and summarized in Table 3.1p = .03).HTN is common in TCM. In a systematic review of 1109 TCM patients, 54% of them carry a diagnosis of HTN.HTN is more prevalent in recurrent TCM patients compared to non‐recurrent cases. A prospective study of 749 TCM patients showed that the incidence of HTN in the TCM recurrence group was significantly higher than those in the non‐recurrence group .HTN, as a typical manifestation of pheochromocytoma,3.2DM is common in TCM patients, with its prevalence ranging from 12.6% to 22.8%.Though cases have been reported with complications of DM triggering TCM, the clear association between the two diseases are not well established. Diabetic ketoacidosis (DKA),Research on DM's effect on TCM outcome has generated conflicting results Table . A 3.8 yCounterintuitively, some retrospective studies argued that DM had a positive association with TCM outcome. A single‐center retrospective observational study of 114 TCM patients reported that the rate of cumulative event within 1 year, including all‐cause death, life‐threatening arrhythmias, thromboembolic events, rehospitalization for heart failure, TCM recurrence rate and stroke, were significantly lower in DM‐TCM group compared to non‐DM‐TCM group (p = .04).3.3The prevalence of HLD in TCM is thought to be as high as 43%.3.42.Nearly 9.7% of TCM patients are obese, defined as body mass index over 30 kg/mIn a retrospective cohort study of 1140 TCM patients, obese TCM patients were more likely to have major complications, such as acute myocardial infarction , cardiac arrest , and cardiogenic shock , although there was no significant difference in all‐cause hospital mortality, length of stay, and hospitalization expenses (p > .05).3.5CKD is also common in TCM patients, with 6.7% TCM patients carrying this diagnosis, according to a large retrospective study of 24 595 patients.2) and those without advanced CKD (eGFR>60 ml/min/1.73 m2) (p = .269).Current retrospective studies suggest that pre‐existing CKD is not associated with in‐patient mortality difference in TCM patients, though the long‐term mortality might be higher in CKD‐TCM patients compared to TCM patients without CKD. A retrospective study of 24 595 TCM patients found no statistical significance in the inpatient mortality between TCM patients with advanced CKD .3.7In TCM, men in general (50‐72 years) are younger than women (70–76 years).Limited studies have conducted to look at age's influence on the TCM mortality, and the current evidence available is yet to generate a consensus on this matter Table . A prospYounger patients are prone to have more complications, as compared to older age group. A retrospective study of 40 326 TCM patients showed the younger group was more likely to suffer from cardiogenic shock , cardiac arrest , and ventricular arrhythmia , compared with the middle‐aged group and the elderly group. The young, middle‐age, and elderly group were defined by age no more than 50 year‐old, age 51–74 year‐old, and age no less than 75 year‐old respectively. Young age was independently associated with risk of cardiac arrest , and ventricular arrhythmias .4Primarily manifested as ACS, TCM shares the conventional cardiovascular risk factors, similar to the other major cardiovascular events. The risk factors include but are not limited to HTN, DM, HLD, obesity, CKD, gender, and age. The impact of conventional cardiovascular risk factors on the mortality and complications of TCM is largely undefined, owing to the limited large sample sized studies available. Through our literature review, we found that conventional cardiovascular factors as stated above were relatively common in TCM. There are obvious gender differences in TCM patients as the majority of patients are post‐menopausal women. Male patients and younger patients are less common, but they are more prone to acute adverse complications and have a poor prognosis. HTN is more prevalent in recurrent TCM patients. The existing research indicated that obesity and CKD are related with a poor prognosis of TCM. On contrary, HLD was noted to be associated with less complications in TCM patients, though current evidence is limited. Lastly, DM poses unclear associations with TCM prognosis. More high quality, large sample size studies are required to further clarify each risk factor's association with TCM short‐term and long‐term outcomes.The authors report no conflicts of interest.


To explore the effect of nonthrombotic internal jugular venous stenosis (IJVS) exerted on cerebral venous thrombosis (CVT).Patients with imaging confirmed CVT were enrolled into this real‐world case–control study consecutively from January 2018 through April 2021, and were divided into CVT and IJVS‐CVT groups, according to whether or not with non‐thrombotic IJVS. Chi‐square and logistic regression models were utilized for between‐group comparison of thrombotic factors.p < 0.001). Multivariate logistic regression indicated that most identified thrombophilia were negatively related to IJVS‐CVT , including oral contraceptive use (β = −1.38), hyperhomocysteinemia (β = −1.58), hematology (β = −2.05), protein C/S deficiency (β = −2.28), connective tissue disease (β = −1.18) and infection (β = −2.77). All recruited patients underwent standard anticoagulation, 10 cases in IJVS‐CVT group also received jugular angioplasty for IJVS correction. Most participants obtained alleviations during 1‐year follow‐up. However, both clinical and imaging outcomes in IJVS‐CVT group were not as good as those in CVT group (both p < 0.05). Moreover, 8 cases with CVT and 7 cases with IJVS‐CVT were rehospitalized for CVT recurrences and underwent customized treatment.A total of 199 eligible patients entered into final analysis, including 92 cases of CVT and 107 cases of IJVS‐CVT. Chi‐square revealed that thrombophilic conditions were found in majority of CVT, while only minority in the IJVS‐CVT group  may predispose to cerebral venous thrombosis (CVT) formation through the obstructed venous outflow and hemodynamic changes, which could be supported by our results: firstly, a vast majority of simple CVT patients had thrombosis‐prone conditions, while such a situation only accounted for a minority in patients with CVT combined nonthrombotic IJVS; secondly, CVT relapse occurred in 8 cases with inherited thrombophilia in simple CVT group, and in 7 cases without any thrombophilic conditions in the IJVS‐CVT group, indicating that nonthrombotic IJVS could possibly promote the CVT recurrence like inherited thrombophilia did. Besides, nonthrombotic IJVS may be the candidate for anticoagulation and stenting to prevent CVT formation, although clinical trials with large simple size are still needed. Considering that IJV thrombosis (IJVT) may develop as a result of CVT extension,Demographics, clinical and neuroimaging data of all patients were obtained from our inpatient database. The conditions of bilateral IJVs in all patients enrolled were assessed into normal or stenotic conditions, according to the maps of head‐and‐neck contrast‐enhanced magnetic resonance venography (CE‐MRV) and/or computerized tomography venography (CTV). Premised on the results mentioned above, patients were divided into CVT group (isolated CVT without nonthrombotic IJVS) and IJVS‐CVT group . Worthy of note is that IJVS is defined as the inner diameter at the stenosis segment less than 50% of the one at the adjacent normal segment, accompanied with abnormal collaterals.Inclusion criteria: (a) imaging confirmed isolated CVT, or CVT coexisted with nonthrombotic IJVS; (b) no age and gender restrictions; (c) complete clinical and imaging data.Exclusion criteria: (a) MRBTI‐confirmed IJVT; (b) any other conditions that may influence intracranial pressure (ICP), such as arteriovenous fistula, cerebral space‐occupying lesions, idiopathic intracranial hypertension (ICH) and so on; (c) psychiatric disorders or other severe systematic illnesses; (d) incomplete clinical or radiological information.All patients were strictly selected with the following inclusion and exclusion criteria:2.22O or more was identified as ICH.2O. All CVT patients who fulfilled the screening criteria were included in the final analysis, whether or not with clear etiologies. Referring to literatures about the CVT etiologies,2), obstetric causes (pregnancy or postpartum), oral contraceptive use, hemopathy , protein C/S deficiency, nephrotic syndrome, connective tissue disease , hyperhomocysteinemia (>20.0 mmol/L), and infection  as well as other causes . Similarly, such thrombophilic assessments were achieved according to baseline check‐ups and electronic medical records at admission.Both diplopia and visual field defects were inducted into the category of visual disorders in this study. Ophthalmologists with extensive experience performed color fundus photography for all patients enrolled. The intensity of papilledema was graded using the Frisén scale. The Frisén value ranges from a minimum score of 0 to a maximum of 5, and the severer papilledema is awarded with the correspondingly higher Frisén score.2.3Imaging spectrums of all patients enrolled were retrospectively reviewed from medical imaging system to validate the nonthrombotic IJVS and preclude the MRBTI‐diagnosed IJVT or any conditions that might affect ICP. In our institution, magnetic resonance imaging (MRI) scanning was routinely performed in all patients with CVT except for those with claustrophobia and MR contraindications, based on which, the parenchymal lesions, such as venous ischemia or hemorrhagic transformation, were disclosed. A part of patients, especially the candidates for endovascular procedure, were also imaged with other diagnostic modalities, including digital subtract angiography (DSA). It was worth noting that all patients were positioned supine and their head were in neutral positions when being imaged.Locations  and laterals  of the thrombi in cerebral venous system were confirmed by MRBTI. In cases with CVT plus nonthrombotic IJVS, the stenotic types of IJVS  and the patterns of extrinsic compression  were also marked according to the results of CE‐MRV and/or CTV. Furthermore, to determine the relationship between ICH and accessory venous circulation, the intra‐ and extracranial collaterals were evaluated as well, and the extents of which were allocated as mild or severe degrees based on the venograms. In detail, the scoring scheme for dilated scalp veins, namely additional routes for intracranial venous drainage in regions with venous blockage, were assigned as no presence, mild dilation or severe dilatation through visual assessment.2.4All patients informed consent to this study were followed‐up by outpatient clinic or detailed telephone‐based interviews. Also, some patients with recurrent CVT were rehospitalized for optimized etiological therapy, which enabled us to access their clinical and neuroimaging data. The follow‐up investigation was undertaken to assess their posttherapeutic conditions through clinical and imaging analysis. Clinical follow‐up duration equaled to the time interval from the first hospitalization to outpatient clinic or rehospitalization. The period of imaging follow‐up was calculated based on the time span between the baseline and repeated imaging. Clinical outcomes were graded as improvement, no change or recurrence. Correspondingly, neuroimaging outcomes of CVT were assigned based on the recanalization degree as being improved, no change or worsen.2.5This study was approved by the Institutional Ethic Committee of Xuanwu Hospital, Capital Medical University, Beijing, China. The consent forms of all patients were signed prior to information collection.2.6T‐test; otherwise, they were presented as median  and analyzed by the Mann–Whitney U test. Categorical values were described as numbers (percentages) and examined by Pearson Chi‐square test or Fisher's exact tests. For further analysis, binary logistic regression model was built up to examine the confounding effects of thrombophilia conditions imposed on IJVS‐CVT. And the results were displayed as estimated β and odd ratio (OR) along with 95% confidence interval (95%CI). Furthermore, Stata 12.0 software was implemented to analyze the correlation and to construct the forest plots. Statistical significance was established at two‐tailed value of p < 0.05.Statistical Package for Social Sciences (SPSS) software version 25.0 was applied for our data processing. Numerical variables following the normal distribution were expressed as mean ± standard deviation (M ± SD) and calculated by two independent samples 33.1n = 92) and IJVS‐CVT (n = 107) groups, depending on the absence or presence of nonthrombotic IJVS. Details were displayed in Table 2) and IJVS‐CVT  group, both p > 0.05. However, the age difference between the two groups reached remarkable statistical significance .A total of 199 patients with MRBTI confirmed CVT were enrolled consecutively from January 2018 through April 2021, and were grouped into CVT . And further, 83 cases (90.2%) in the CVT group and 72 cases (67.3%) in IJVS‐CVT group underwent lumbar puncture, and the ratio of ICH in the former was obviously larger than that in the later . Meanwhile, the average CSF opening pressure in the CVT group was higher than that in the IJVS‐CVT group (288.6 ± 65.4 vs. 221.0 ± 76.4 mmH2O), and the ICH‐related symptoms and signs showed prominent preponderance in the CVT group over the IJVS‐CVT group , including headache (88.0% vs. 59.8%), nausea and vomiting (55.4% vs. 29.0%), visual disorders (30.4% vs. 13.1%), eyesight decline (43.5% vs. 29.0%), papilledema ratios (63.0% vs. 21.5%), and Frisén scores (median scores: 2.2 vs. 0.6). Additionally, the incidence of cerebral venous infarctions, irrespective of ischemia or hemorrhage, was mildly higher in the IJVS‐CVT group than that in the CVT group, even though not reaching statistical difference (p > 0.05). The history of hypertension, diabetes mellitus, hyperlipidemia, smoking, and alcohol use were balanced between the two groups .As in Table p < 0.001); 85 out of 107 patients (79.4%) in the IJVS‐CVT group failed to identified with any routine risk factors of CVT, which indicated that IJVS might be closely associated with CVT formation. Specifically, statistical differences reached significant levels in almost all of the CVT risks as aforementioned between the two groups , aside from obstetric causes (p = 0.556). An illustrated diagram was displayed in Figure β = −1.38, adjusted OR (95%CI) of 0.25 , p = 0.045], hyperhomocysteinemia , hematologic disorder , protein C/S deficiency , connective tissue disease , and infection . Considering the selection bias of this study, such results should be explained, as these thrombophilia profiles infrequently existed in CVT patients that were considered caused by non‐thrombotic IJVS, which was mainly secondary to external compression in this study. A corresponding forest plot is provided as Figure A vast majority of patients in the CVT group were implicated with clear thrombophilia, while such evidence was only found in minority cases in the IJVS‐CVT group , J2 (61 sides) or J1 (12 sides) segments. Among which, 41.1% accounted for bilateral, 36.4% involved the left side and 22.4% was at the right side. As regards the stenotic IJVs, 60.9% were observed with external bony compression, 14.6% were squeezed by carotid arteries, 19.9% were considered caused by venous wall contracture, and only 4.6% were pressed by the surrounding tissues .As in Table p < 0.001). In addition, the abnormal extended vertebral venous collaterals surrounded the stenotic IJVs were found in all IJVS‐CVT patients (100%) with 47.7% being mildly dilated and 52.3% severely. In contrast, only 47.8% of CVT patients were detected with enlarged vertebral veins with 84.1% in a mild degree and 15.9% in a severe degree (p < 0.001). More radiological details were obtainable in Table In terms of collateral venous drainage, expanded scalp veins were discovered in up to 77.2% (71/92) patients in the CVT group, among which, 45.1% 32/71) were mildly and 54.9% (39/71) were severely expanded; yet, in the cohort of IJVS‐CVT, only 43.9% (47/107) individuals harbored scalp venous expansion with 70.2% (33/47) in mild and 29.8% (14/47) in severe level  with CVT and 91 cases (85.0%) with IJVS‐CVT were conservative with isolated anticoagulation without other adjunctive interventions, while 15 cases (16.3%) with CVT and 16 cases (15.0%) with IJVS‐CVT underwent anticoagulation plus endovascular interventional treatment. A case example with CVT resolution after isolated anticoagulation was presented as Figure As for treatment patterns Table , due to 3.5p = 0.042), while 6.5% of the patients in the CVT group and 20.6% in the IJVS‐CVT group reported residual symptoms (p = 0.005). Yet, as showed in Table The average duration of clinical follow‐up in this study was 13.0 ± 11.7 months in the CVT group and 12.5 ± 10.4 months in the IJVS‐CVT group . All the results mentioned above revealed that IJVS might not only promote CVT formation but also impede CVT recanalization, despite the short conclusion reliability for the small number of CVT relapse.In the imaging follow‐up, only 42 cases (45.7%) in the CVT group and 39 cases (36.4%) in the IJVT‐CVT group underwent repetitive MRBTI scanning with an average interval of 4.6 months. Compared with their baseline MRBTI maps, 30 patients (71.4%) with CVT and 18 patients (46.2%) with IJVS‐CVT obtained recanalization, 1 Figure ; 8 cases9 Figure . The thr4Our findings have a directive significance for clinical practice, as coming up with a point of view that IJVS may be a promoting factor for CVT occurrence, which is independent of the currently well‐recognized risk factors. Specifically, both CVT and IJVS may manifest with headache as a corollary of elevated ICP or venous insufficiency, resulting in misinterpretation as either mere CVT or nonthrombotic IJVS in IJVS‐CVT patients. The findings in this study revealed that nonthrombotic IJVS might be a noteworthy danger for CVT, whereby patients with nonthrombotic IJVS should be screened for CVT and vice versa, which is not yet a clinical routine workflow. More importantly, this study indicated that patients with nonthrombotic IJVS might derive benefits from anticoagulation; and IJVS correction might inhibit IJVS‐mediated CVT occurrence and recurrence. Yet, only 10 cases in this study involved with IJVS correction and hence lacked conclusion reliability.4.1In this study, thrombophilic conditions were found in the majority of CVT cases (83.7%), whereas confined to a minority in those with IJVS‐CVT (20.6%). For which, we suspected that nonthrombotic IJVS might serve as a primary menace to CVT in patients without recognized thrombotic factors. Owing to the nature of the real‐world case study, we failed to recruit sufficient cases of healthy controls and perform the comparison as to the occurrence of CVT between the large cohorts with and without IJVS. Despite which, a cross‐sectional study with a large sample reported that jugular luminal narrowing could be noted in approximately 36.6% individuals in the general population.4.2According to the Virchow triad, entities that are responsible for endothelial dysfunction, blood hypercoagulability, and hemodynamic aberrations are the three elements for venous thrombus formation.4.3In this study, MRBTI was employed to display the direct visualization of thrombi.4.4According to our results, the dilated scalp veins and the relevant clinical symptoms, such as headache were more prevalent in CVT patients, whereas older ages, head noise, dizziness, insomnia, in addition to emotion changes and vertebral collaterals were more popular in IJVS‐CVT patients. Correspondingly, the IJVS in this study were mainly caused by the compression of bones or stiffened arteries, both of which were also age‐dependent. The above consisted with a notion that nonthrombotic IJVS tended to be acquired and develop with higher frequency during the aging process as revealed previously.4.5p = 0.005). Similarly, compared to baseline MRBTI, the thrombi loaded in 18 cases in IJVS‐CVT remained no remarkable changes in the follow‐up MRBTI maps, while this status was only seen in 8 cases with CVT (p = 0.009). For which, we suggested that CVT was triggered by long‐standing IJVS may presumably show more resistance to anticoagulation therapy because of the chronic process of thrombus formation. Of note, our previous work ever highlighted the adjunctive contribution of batroxobin to the dissolution of CVT, which enabled to accelerate thrombolysis and result in rapid recanalization.During more than 1‐year follow‐up, most patients in the two groups obtained relieves. However, treatment effects on IJVS‐CVT were not as sensitive as those on CVT (20.6% vs. 6.5%, Aside from the above, CVT relapse occurred in 8 cases with CVT and 7 cases with IJVS‐CVT. The leading causes in the CVT group were the poorly controlled risk factors of CVT plus discontinuation of standard anticoagulants, while in the IJVS‐CVT group, there was nothing except for the blame IJVS plus stopping anticoagulants. For this reason, it is, to some extent, rational to assume that preventive anticoagulation ought to be suggested for IJVS patients, despite not convincing enough. Given that not all participants finished the follow‐up imaging, conclusion regarding the critical role of nonthrombotic IJVS played in CVT prognosis could not be drawn in this study, and the mechanism of which need further clarification.This is a real‐world case–control study focusing on the role of nonthrombotic IJVS in CVT formation with a relatively large sample size. Previous studies insisted that nonthrombotic IJVS was a normal anatomic variant, as it was highly prevalent without considerable clinical impacts.Current guidelines neither take the nonthrombotic IJVS into account as a risk factor of CVT, nor provide insights into the relevant therapy. In our clinical practice, nonthrombotic IJVS patients were generally screened for CVT and vice versa. Additionally, they underwent long‐term anticoagulation if uncorrected by IJVS operation. Referring to our follow‐up results, the symptoms in most IJVS‐CVT patients were remised and even disappeared after anticoagulation. Besides, IJV correction may be a feasible therapy of choice to prevent CVT recurrence, in view that 10 cases with IJVS‐CVT derived great benefits from the IJVS reconstruction and ultimately obtained good clinical outcomes. However, it should be stressed that the efficacy of IJVS venoplasty in prohibiting CVT recurrence in patients with IJVS‐CVT is somewhat uncertain due to the small sample size of operated cases in this study. Additional studies with larger case numbers and long‐term follow‐up are still warranted.5Shortcomings are worthy noticing: Firstly, this study is only a mono‐center real‐world case–control study with a relatively short follow‐up period. Secondly, it remains undetermined whether or not nonthrombotic IJVS‐CVT should be granted with long‐term anticoagulation. Thirdly, the cut‐off value of the degree of IJVS on promoting CVT is still not clear. Last but not least, the accurate mechanisms of IJVS‐induced CVT were not elaborated very clearly due to the shortage of auxiliary modalities for a specific hemodynamic study. We hope findings from future studies can shed more light on the questions raised above.6To sum up, nonthrombotic IJVS may be a noteworthy risk factor for CVT occurrence, possibly due to the obstructed venous outflow induced by the IJVS. Besides, patients with nonthrombotic IJVS may possibly benefit from continuous anticoagulation and IJVS correction, although further well‐designed clinical trials with a large simple size are still needed to clarify these findings.XW, JY, DZ, YD, XJ, and RM report no conflicts of interest.XW and JY completed the study and drafted this manuscript. RM, DZ, XJ, and YD revised the manuscript and edited English. RM contributed to the conception and design of this study and proposed the amendments.The authors agree to publish.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Table S1Click here for additional data file.


Eyelid position and contour abnormality could lead to various diseases, such as blepharoptosis, which is a common eyelid disease. Accurate assessment of eyelid morphology is important in the management of blepharoptosis. We aimed to proposed a novel deep learning-based image analysis to automatically measure eyelid morphological properties before and after blepharoptosis surgery.This study included 135 ptotic eyes of 103 patients who underwent blepharoptosis surgery. Facial photographs were taken preoperatively and postoperatively. Margin reflex distance (MRD) 1 and 2 of the operated eyes were manually measured by a senior surgeon. Multiple eyelid morphological parameters, such as MRD1, MRD2, upper eyelid length and corneal area, were automatically measured by our deep learning-based image analysis. Agreement between manual and automated measurements, as well as two repeated automated measurements of MRDs were analysed. Preoperative and postoperative eyelid morphological parameters were compared. Postoperative eyelid contour symmetry was evaluated using multiple mid-pupil lid distances (MPLDs).p < .001), and the bias ranged from 0.09 mm to 0.15 mm. The ICCs between two repeated automated measurements were up to 0.999 (p < .001), and the bias was no more than 0.002 mm. After surgery, MRD1 increased significantly from 0.31 ± 1.17 mm to 2.89 ± 1.06 mm, upper eyelid length from 19.94 ± 3.61 mm to 21.40 ± 2.40 mm, and corneal area from 52.72 ± 15.97 mm2 to 76.31 ± 11.31mm2 . Postoperative binocular MPLDs at different angles (from 0° to 180°) showed no significant differences in the patients.The intraclass correlation coefficients (ICCs) between manual and automated measurements of MRDs ranged from 0.934 to 0.971 (This technique had high accuracy and repeatability for automatically measuring eyelid morphology, which allows objective assessment of blepharoptosis surgical outcomes. Using only patients’ photographs, this technique has great potential in diagnosis and management of other eyelid-related diseases. The eyelid acts as a protective layer of the eyeball and is vital to maintain a healthy ocular surface through regular blinking . Eyelid As is known to all, MRDs alone are not enough to define morphological properties of eyelids . Recent This study was approved by the Institutional Review Board of the Second Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou, China . Informed consent was obtained from patients aged ≥ 18 years and guardians of those aged < 18 years, in accordance with the Declaration of Helsinki.Patients who underwent blepharoptosis surgery in our oculoplastic clinic between January 2016 and September 2019 were invited to participate in this study. The surgeries were performed by a senior surgeon (J.Y.) who had more than 15 years of experience in oculoplastic surgery. Exclusion criteria were: variable ptosis (e.g. myasthenia gravis), coexisting eyelid diseases , strabismus, and abnormalities of pupil.In total, 103 patients (135 ptotic eyes) including 71 patients with unilateral blepharoptosis and 32 patients with bilateral blepharoptosis were included in this study. seventy-three patients were male and 30 patients were female. The mean age was 6.3 years old, ranging from 1 to 50 years old. 117 eyes underwent frontalis suspension, 17 eyes underwent levator resection, and one eye underwent levator aponeurosis repair. The mean follow-up duration was 21 weeks, ranging from 2 to 109 weeks.Preoperative and postoperative MRD1 and MRD2 of the operated eyes were manually measured by a senior surgeon (J.Y.). When the patients were gazing in the primary position, a penlight was used to produce the corneal light reflex and a ruler was used to judge the vertical distance from the corneal light reflection to the upper eyelid margin (MRD1) and to the lower eyelid margin (MRD2). If the upper eyelid covered the corneal light reflection, then the examiner raised the eyelid until the reflection was seen. The number of millimetres the eyelid must be raised was recorded as the MRD1 in negative numbers.Facial photographs were taken preoperatively and at the last follow up. A circular marker with a diameter of 10 mm was placed on the forehead of the patients. The patients were asked to gaze in the primary position and a digital camera Canon 500 D  was positioned at eye level at a distance of 1 m.The automated image analysis for comparisons of preoperative and postoperative eyelid morphology included the following four steps.Step 1: Regions of eyes were localized by an open-source project named Face Alignment . Facial Step 2: Preoperative and postoperative facial images of 103 patients (135 operated eyes) who underwent blepharoptosis surgery were used as the test set. The Attention R2U-Net based model predicted eyelid and cornea segmentation mask. Three points were randomly chosen from the cornea margin to fit the cornea circle and the circle centre was set as the pupil centre. Since the cornea is not perfectly round, the process was repeated 2000 times and the final pupil centre was localized by the mean shift algorithm with Gaussian kernel .Step 3: The pixel numbers of palpebral fissure length (including MRD1 and MRD2), lid length (including upper lid length and lower lid length), and palpebral fissure area  of the pre-operative and post-operative eyes were calculated . After l2.Step 4: Hough transform was used to detect the circular marker (10 mm in diameter) on the patient’s forehead . Then, tDice coefficient, as a statistical tool to measure the similarity of two samples, was calculated for the validation of eye segmentation algorithms . Intraclp < .001), indicating excellent agreement between the two methods (p < .001), which illustrated high repeatability of the automated method. The Bland–Altman plots also confirmed excellent agreement and acceptable limits of agreement between any two measurements for MRD1  in 135 ptotic eyes are shown in  methods . The ICCfor MRD1  and MRD2p < .001), upper eyelid length from 19.94 ± 3.61 mm to 21.40 ± 2.40 mm (p < .001), and corneal area from 52.72 ± 15.97 mm2 to 76.31 ± 11.31mm2 (p < .001), which indicated great improvement of eyelid morphology after blepharoptosis surgery.Preoperative and postoperative eyelid morphological parameters in 135 ptotic eyes are shown in N = 71) and patients with bilateral blepharoptosis (N = 32), respectively, are shown in Table S1. There were no significant differences in eyelid morphological parameters either between ptotic eyes and fellow eyes in patients with unilateral blepharoptosis, or between right eyes and left eyes in patients with bilateral blepharoptosis. Furthermore, postoperative binocular MPLDs at different angles (from 0° to 180°) were compared. Similarly, binocular MPLDs showed no significant differences in either patients with unilateral blepharoptosis or patients with bilateral blepharoptosis (Table S2), indicating ideal eyelid contour symmetry after surgery.Post-operative binocular eyelid morphological parameters in patients with unilateral blepharoptosis  ,6,7,20 aSome efforts had been made for measurement of eyelid contour ,11,23–25Several limitations should be noted in this study. Firstly, our algorithm assumed that pupil and iris boundaries are perfect circles sharing the same centre, and hence, pupil centre was located only using circle fitting methods. Secondly, this system was only developed for 2 D photographs and had not been tested in 3 D photographs. Despite a typical flat forehead in Asian population, the eyelid and the circular marker on the forehead were not always at the same coronal position. In addition, the measurements of medial and lateral area of palpebral fissure would be smaller than the true value since 2 D photographs were used. Thirdly, this was a single-centre study only involving evaluation of surgical outcomes in blepharoptosis patients. Anthropometry in other eyelid-related disorders, such as Graves’ ophthalmopathy, is worthy for future exploration.In conclusion, we proposed a novel image analysis technique to automatically measure eyelid morphological properties in blepharoptosis patients and to objectively evaluate surgical outcomes. Using only patients’ photographs, this technique could assist ophthalmologists with diagnosis, surgical planning and post-operative follow-up. The ease of accessing and transferring digital image also offers the possibility of telemedicine, benefiting people living in remote areas. Moreover, this new system would have wide potential application in other eyelid-related diseases, not limited to blepharoptosis.This article includes photographs of individual participants and the authors have fully anonymized them. The authors confirm that consent to publish these details has been obtained from legal guardians of these individuals.Click here for additional data file.


Laryngoscopy and tracheal intubation are strong stimuli that cause a reflex increase in blood pressure (BP), heart rate (HR), and serum catecholamine level. These can lead to myocardial infarction or cerebrovascular accidents. The purpose of this study is to compare the efficacy of dexmedetomidine and lidocaine combined with propofol in attenuating the hemodynamic response following laryngoscopy and endotracheal intubation.− 1) before induction. Group LP received lidocaine (1.5 mg kg− 1) before induction with additional propofol (0.5 mg kg− 1) before laryngoscopy. The primary endpoint was hemodynamic including systolic (S) BP, diastolic (D) BP, mean arterial blood pressure (MAP) and HR measured before and after induction and ≤ 10 min after intubation. Secondary outcome was complications/adverse effects.This study was a randomized controlled study and adhered to the CONSORT guidelines. One-hundred and six patients undergoing elective general anesthesia with endotracheal intubation were divided randomly into two groups. Group D received dexmedetomidine (1 μg kgP <  0.05). Group LP had a non-inferior effect in blunting BP at all time points except 1 and 2 min after induction, and 2 min after intubation. The mean difference in HR in group D was significantly lower than that in group LP at all time points (P <  0.001). Group D had significantly more episodes of bradycardia  and hypotension  than did group LP.After induction, the mean SBP, DBP, MAP and HR decreased significantly from baseline in both groups except for mean HR in group LP at 1 min. Differences in mean values of SBP, DBP, and MAP were significantly lower in group D after intubation at 4–10 min (− 1) with additional propofol (0.5 mg kg− 1) had a non-inferior effect compared with dexmedetomidine (1 μg kg− 1) in attenuating the hemodynamic response following laryngoscopy and endotracheal intubation, and had fewer adverse effects.Lidocaine (1.5 mg kgTRTC20190206002). Retrospectively registered 4 February 2019.Thai Clinical Trial Registry, ( General anesthesia requires laryngoscopy and intubation, which can result in increased plasma concentrations of catecholamines –3. An inSeveral agents have been employed to attenuate hemodynamic stress responses: dexmedetomidine, lidocaine, fentanyl, esmolol, propofol, or volatile anesthetic agents. Dexmedetomidine is a highly selective agonist of α-2 adrenergic receptors. It has a sympatholytic effect through a reduction in the norepinephrine concentration. Dexmedetomidine use can result in a decrease in blood pressure (BP) and heart rate (HR), so suppression of airway and circulatory reflexes during laryngoscopy and intubation is appropriate, , 7 but b− 1 bodyweight for 3 min before intubation to suppress the hemodynamic response. However, such suppression is not complete and a spike in systolic blood pressure (SBP) at 1-min and 3-min intervals post-intubation has been reported  was 25.65 mmHg)  in group LP (compared with group D) was used in our study. Group LP had a non-inferior effect in blunting BP at all time points except 1 min and 2 min after induction, and 2 min after intubation.A forest plot showing the difference in SBP between groups is shown as Fig. P < 0.001)  and increased significantly from baseline to the intubation phase  and 1 min after intubation  (Table The mean difference in HR in group D was significantly lower than that in group LP in each period from preinduction until 10 min after intubation (01) Fig. , Table 2Bradycardia and hypotension occurred in group D significantly more often than in group LP. The prevalence of bradycardia in group D was 18.87%, whereas bradycardia did not occur in group LP. The prevalence of hypotension in group D was 52.83%, whereas it was 15.09% in group LP  before intubation could improve intubation conditions significantly. In our study, lidocaine  given before induction combined with an additional dose of propofol (0.5 mg kg− 1) given 30 s before intubation led to all parameters having similar trends to those elicited using dexmedetomidine. SBP, DBP, and MAP in group LP decreased markedly after induction, and decreased after intubation. Furthermore, we showed that SBP at 2 min after induction decreased > 20% from that recorded at baseline. Hendrickx and colleagues [From the previous study, Dashti and colleagues  comparedlleagues  and Woodlleagues  reported− 1) for tracheal intubation, and the prevalence of hypotension (16%) and bradycardia (4%) was lower than that in our study. However, a study using lidocaine with an additional low dose of propofol before induction to blunt the cardiovascular reflex has not been caried out. The prevalence of hypotension and bradycardia in group LP was 15.09 and 0%, respectively, which were lower than those in group D and statistically significant. Our study had some limitations. First, our study included only healthy, non-obese, and young patients of ASA physical status I–II. Second, we did not measure the depth of anesthesia during the procedure in either group owing to a lack of facilities. An inadequate depth of anesthesia can lead to an increase in the hemodynamic response to intubation. Third, our study cohort was healthy, so we did not use invasive monitoring. The latter might be more sensitive and provide a more accurate measurement of hemodynamic change, including intravascular postoperative blood glucose was not analyzed Finally, anesthesiologists were not blinded to the group of study subjects. However, they would follow to the study protocol.Dexmedetomidine use has been reported to increase the risk of bradycardia and hypotension , 9. The Further study should include obese cases patients with ASA physical status >II or history of atrial fibrillation or other arrhythmias, implanted pacemakers, used of the antiarrhythmic drugs or beta blockers. In addition, a study in cost-effectiveness should be conducted.Our data suggest that lidocaine with propofol could be used as an alternative to a direct sympathomimetic response from laryngoscopy and laryngeal intubation. Moreover, administration of lidocaine and propofol is less expensive than dexmedetomidine administration and associated with fewer complications.− 1) with additional propofol (0.5 mg kg− 1) had a non-inferior effect compared with that of dexmedetomidine (1 μg kg− 1) for attenuating the hemodynamic response following laryngoscopy and endotracheal intubation only in healthy patients, and was also associated with fewer complications.Lidocaine (1.5 mg kg


Mechanisms underlying the adiposity–cancer relationship are incompletely understood. We quantified the mediating roles of C‐reactive protein (CRP), leptin, fasting insulin, and estradiol in the effect of adiposity on estrogen receptor (ER)‐positive breast, endometrial, and colorectal cancer risk in postmenopausal women.We used a case–cohort study within the Women's Health Initiative Observational Study, analyzed as a cumulative sampling case–control study. The study included 188 breast cancer cases, 98 endometrial cancer cases, 193 colorectal cancer cases, and 285 controls. Interventional indirect and direct effects on the risk ratio (RR) scale were estimated using causal mediation analysis.2 was 1.87 . The indirect effect RRs were 1.38 (0.79–2.33) through leptin and CRP, 1.58 (1.17–2.43) through insulin, and 1.11 (0.98–1.30) through estradiol. The direct effect RR was 0.82 (0.39–1.68). For endometrial cancer, the total effect RR was 2.12 (1.12–4.00). The indirect effect RRs were 1.72 (0.85–3.98) through leptin and CRP, 1.42 (0.96–2.26) through insulin, and 1.24 (1.03–1.65) through estradiol. The direct effect RR was 0.70 (0.23–2.04). For colorectal cancer, the total effect RR was 1.70 (1.03–2.79). The indirect effect RRs were 1.04 (0.61–1.72) through leptin and CRP, 1.36 (1.00–1.88) through insulin, and 1.02 (0.88–1.17) through estradiol. The direct effect RR was 1.16 (0.58–2.43).For breast cancer, the total effect RR for BMI ≥30 versus ≥18.5–<25 kg/mLeptin, CRP, fasting insulin, and estradiol appear to mediate the effect of high BMI on cancer risk to different extents, with likely varying degrees of importance between cancers. These insights might be important in developing interventions to modify obesity‐associated cancer risk in postmenopausal women. Mechanisms underlying the effect of adiposity on increased cancer risk are incompletely understood. Here, we quantified the roles of leptin and C‐reactive protein, fasting insulin, and estradiol in explaining the effect of high BMI on estrogen receptor‐positive breast, endometrial, and colorectal cancers in postmenopausal women. We used a novel causal mediation analysis approach and data from a case–cohort study within the Women's Health Initiative Observational Study. The assessed biomarkers explained the increased risk of the three cancers to different extents, and their importance varied between cancers. These insights might be important in developing cancer prevention interventions for postmenopausal women with obesity.1Excess body fat is a risk factor for several cancers, including estrogen receptor (ER)‐positive postmenopausal breast, endometrial, and colorectal cancers.Chronic low‐grade inflammation is a known consequence of excess adiposity. In the obese state, the production of adipocytokines and cytokines is disturbed, characterized by increased secretion of leptin, one of the most abundant adipocytokines with pro‐inflammatory properties.difference method. This method would entail estimating the adiposity–cancer association first without, then with the biomarker (mediator) in the regression model, and taking the difference between the coefficients from the two models as an estimate of the mediating (indirect) effect.A better understanding of the role these biomarker pathways play in mediating the effect of adiposity on cancer risk and their potentially differing importance for different cancers might help to identify risk‐reducing targets for individuals with obesity. In the present study, we made use of an existing case–cohort study of obesity‐related biomarkers and breast, endometrial, and colorectal cancers among postmenopausal women within the Women's Health Initiative Observational Study (WHI‐OS).2The Women's Health Initiative Observational Study (WHI‐OS) included 93,676 postmenopausal women aged 50–79 years old, recruited from 40 centers across the United States from 1993 to 1998.2.1We made use of an existing case–cohort study derived from the baseline WHI participants, which investigated inflammation, fasting insulin, and estradiol and the risk of endometrial, breast, and colorectal cancers.For the present analysis, we treated these data as if the design followed an unmatched case–control study with cumulative sampling because methods for mediation analysis for estimation of interventional indirect effects have not been adapted to the case–cohort design. However, since outcome was rare in the sub‐cohort and a small proportion of participants died or were lost to follow‐up, we expected that analyzing the study as cumulative sampling case–control study to not materially influence the results.Our analytic dataset included women diagnosed with ER‐positive breast, endometrial, or colorectal cancer in the original case–cohort study as cases, and women in the sub‐cohort who had not been diagnosed with those cancers, and had not died or were not lost to follow‐up by the end of follow‐up period as controls, if they met the following additional eligibility criteria Table .2. Women with diabetes or unknown diabetes status were excluded because both diabetes and diabetes medication use might change the adiposity–insulin signaling relationship.Confounder Selection), or biomarkers (see Biomarkers). Only for endometrial cancer, controls with history of hysterectomy at baseline were also excluded , or had BMI <18.5 kg/mThe analytic dataset included 188 ER‐positive breast cancer cases (median follow‐up 3.6 years [interquartile range 2.4–4.8]), 98 endometrial cancer cases (median follow‐up 3.3 years [interquartile range 2.6–4.8]), 193 colorectal cancer cases (median follow‐up 3.8 years [interquartile range 2.7–5.3]), and 285 controls  (median follow‐up 7.0 years [interquartile range 5.9–7.9])  it was included as a categorical variable in all analyses. Women were categorized based on the WHO cutoff values .Body mass index (BMI) was the measure of adiposity (exposure) and to assist with interpretation of the interventional indirect effects  preliminary analyses to investigate BMI–biomarker associations within the controls ; (ii) preliminary analyses to investigate the BMI–cancer and biomarker–cancer associations ; and iii) causal mediation analyses to decompose the total BMI–cancer association into the interventional indirect (mediated) and direct effects.2.3.1The causal diagram in Figure For the controls, for each biomarker, the geometric mean ratio (GMR) and 95% confidence intervals (CIs) were estimated in relation to BMI using a linear regression model fitted to the log‐transformed biomarker variable and adjusted for the pre‐exposure confounders.risk=1(1+exp‐logodds).log(1samplingfractionofcontrols) was subtracted from the constant term to take the cumulative case–control design into account.Risk ratios (RRs) and 95% CIs were estimated from the logistic regression models for the BMI–cancer and biomarker–cancer associations from the logistic regression models based on the formula The linearity of the mediator–cancer associations was investigated by fitting the biomarker variables as restricted cubic splines (with two degrees of freedom).The mediation analysis decomposes the total effect of BMI on cancer risk into indirect effects through the biomarkers, and a direct effect, which is the part of the effect that remains unexplained by the biomarkers. We followed the decomposition approach proposed by Moreno‐Betancur et al. A simulation‐based, regression‐standardization approach was used to estimate the indirect and direct effects, where the expected outcomes under different scenarios were averaged over mediator distributions based on a large number of Monte Carlo draws . There were positive associations with fasting insulin and estradiol, with the larger RR observed for fasting insulin . There was also evidence for positive associations with leptin and CRP, and the associations were somewhat attenuated after adjusting for insulin . There were positive associations with leptin and CRP, which were slightly attenuated after adjusting for insulin, and with fasting insulin, and estradiol. The strongest association was observed for estradiol    for BMI ≥30 versus ≥18.5–<25 kg/m2) Table .2, as there was no evidence for association comparing BMI ≥25–<30 kg/m2 versus ≥18.5–<25 kg/m2.For all cancer sites, there was no strong evidence for departure from linearity for any of the biomarkers Table . For eac3.32, we estimated a positive indirect effect through leptin and CRP (RR 1.38) but with high uncertainty , and a stronger indirect effect through fasting insulin . The indirect effect through estradiol was weaker . The RR for direct effect was 0.82  , and a stronger indirect effect through fasting insulin . The point estimate was indicative of even a stronger indirect effect through leptin and CRP (RR 1.72), but there was high uncertainty around the estimated effect . The RR for direct effect was 0.70   and estradiol , with high uncertainty around the estimated effects, but there was a positive indirect effect through fasting insulin  The RR for direct effect was 1.16   were ≤9%, estradiol and insulin tests were performed in duplicates and tests with, respectively, >20% and >10% CV were repeatedWe excluded cases diagnosed within the first year after baseline to reduce the possibility of cancer having influenced measures of adiposity or biomarkers. However, we cannot rule out reverse causation for the adiposity–biomarker associations. They were assessed at baseline, and we did not adjust for prior biomarker measurements. Our analyses relied on no unmeasured exposure–mediator, exposure–outcome, and mediator–outcome confounding.2 increment in BMI 50.0; 95%CI, 23.2–76.6), 24% was mediated through estradiol , with a larger mediating effect (49%) observed for ER‐positive breast cancers. In women without diabetes, of the total effect , 66% was mediated through fasting insulin .2 1.75; 95%CI, 1.05–2.91), conducted as a case–control study within the Melbourne Collaborative Cohort Study (MCCS), 72% of the effect was explained by free estradiol  and 28% by fasting insulin .Our results generally agree with a previous study that used data from two case–cohort studies within the WHI‐OS, one of which was the same dataset as that used here, and performed a causal mediation analysis to estimate the natural indirect effect of estradiol and fasting insulin in mediating the association of BMI with postmenopausal breast cancer in women not using hormone therapy.2 1.50; 95% CI, 1.43–1.57), the indirect effect through triglyceride glucose product  was essentially null , suggesting that the biomarker did not explain any of the total BMI–endometrial cancer risk association. The study also observed no evidence of an association between TyG and endometrial cancer risk (HR per 1 standard deviation (SD) increase in TyG 1.04; 95% CI, 0.98–1.11). In our study, we observed an association between fasting insulin and endometrial cancer risk, which was in line with results from a Mendelian randomization study 2 2.51; 95%CI, 1.26–5.02), the largest indirect effect was through a range of biomarkers of inflammatory status . A negligible indirect effect was estimated through C‐peptide (used as a proxy measure for insulin resistance) , and an indirect effect comparable to what we observed for estradiol, through free estradiol and estrone .In a study of six European cohorts that used causal mediation analysis to explain the effect of adiposity on endometrial cancer risk in pre‐ and postmenopausal women,In the UK Biobank cohort, a sequential causal mediation analysis approach was used to explain the effect of adiposity on colorectal cancer risk in postmenopausal women through CRP, glycated hemoglobin , and sex hormone‐binding globulin (SHBG) and testosterone.In summary, among postmenopausal women, we observed that leptin and CRP, fasting insulin, and estradiol might mediate associations of general obesity with ER‐positive breast, endometrial, and colorectal cancers, with suggestion of varying degrees of importance for the biomarkers between the three cancers. Studies with larger sample sizes are needed to further investigate and produce results with more certainty. Ideally, future studies will take advantage of novel causal mediation analysis approaches, similar to the method used in the present study, to appropriately account for dependence between biomarkers and to establish relevant biological targets for future prevention‐based interventions.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the institutional review boards of all participating institutions. Participants provided written informed consent at enrollment.Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy, or views of the International Agency for Research on Cancer/World Health Organization.The authors declare no conflict of interest.Table S1Click here for additional data file.