We identified a shift in the size distribution of tumor‐derived cfDNA fragments in these body fluids. We further analyzed cfDNA fragment sizes using whole‐genome sequencing, in urine samples from 35 glioma patients, 27 individuals with non‐malignant brain disorders, and 26 healthy individuals. cfDNA in urine of glioma patients was significantly more fragmented compared to urine from patients with non‐malignant brain disorders (P = 1.7 × 10−2) and healthy individuals (P = 5.2 × 10−9). Machine learning models integrating fragment length could differentiate urine samples from glioma patients (AUC = 0.80–0.91) suggesting possibilities for truly non‐invasive cancer detection.Glioma‐derived cell‐free DNA (cfDNA) is challenging to detect using liquid biopsy because quantities in body fluids are low. We determined the glioma‐derived DNA fraction in cerebrospinal fluid (CSF), plasma, and urine samples from patients using sequencing of personalized capture panels guided by analysis of matched tumor biopsies. By sequencing cfDNA across thousands of mutations, identified individually in each patient’s tumor, we detected tumor‐derived DNA in the majority of CSF (7/8), plasma (10/12), and urine samples (10/16), with a median tumor fraction of 6.4 × 10 Gliomas are challenging to detect using tumor derived cell‐free DNA (cfDNA) in body fluids. In this study, two novel analysis methods (tumor‐guided sequencing and sWGS) were developed to explore the potential of using plasma and urine cfDNA as a liquid biopsy for this malignancy. Compared to other disease types, detection of circulating cell‐free tumor DNA (cftDNA) in patients with brain tumors, in particular gliomas (GBM), is challenging. While analysis of cftDNA in cerebrospinal fluid (CSF) has improved detection frequencies, this bio‐fluid is both difficult to collect and associated with significant discomfort for the patient. As such, it is unlikely that analysis of cftDNA in CSF will be considered as a viable approach for longitudinal sampling going forward. On the other hand, minimally invasive liquid biopsy, in the form of plasma or urine, which do not face these same challenges, could be highly beneficial. However, their use is hampered by the presence of only minute levels of glioma‐derived cfDNA signal.First, using tumor‐guided sequencing in matched tissue and liquid biopsy, we compared the mutational burden and detection rate of cftDNA in CSF, plasma, and urine from GBM patients. We developed a whole exome sequencing approach that calls mutations that are private to or shared between multiple regions of the same tumor, in doing so affording greater confidence in the mutations calls. These mutations were then used to generate targeted panels for high depth sequencing of CSF, plasma, and urine. By integrating mutation signal across hundreds of mutations, we observed tumor‐derived signal in the majority of CSF, plasma, and urine samples.Then, a second more rapid and cost‐effective approach was developed using low coverage WGS. This revealed a possible difference in the fragment sizes of urine cftDNA in cancer patients as compared to healthy individuals. Subsequent application of a machine learning approach to this sequencing data led to the creation of classifiers that demonstrated an ‘area under the curve’ of between 0.8 and 0.91 for differentiating samples from patients and healthy controls.The non‐invasive nature of plasma and urine may permit more regular and less restrictive monitoring for GBM patients than CSF sampling. While the role of liquid biopsy for diagnosis has been the focus of much attention, both of the methods presented may provide utility in the follow‐up setting in combination with imaging.et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, Primary brain tumors, which are diagnosed in over 260,000 patients worldwide annually  fragmentation patterns . Among the five LGG, three were diffuse astrocytoma, one was an oligodendroglioma and one a pilocytic astrocytoma. Among the 30 HGG, 29 were glioblastomas (GBM) and one was an anaplastic oligodendroglioma , buffy coat, CSF, plasma, and urine Fig . Initialet al, et al, et al, et al, Sequencing data from the body fluids were analyzed using INVAR (INtegration of VAriants Reads), a recently developed pipeline  ctDNA was detected using INVAR in 7/8 CSF samples, 10/12 plasma samples, and 10/16 urine samples Fig . For thr CSF Fig . In CSF, CSF Fig . In the  CSF Fig . IMAFs w CSF Fig . Contrasence Fig .Of the 52 genes frequently mutated in glioma, mutations were detected in 41/52 genes in tumor tissue DNA, 14/52 genes in CSF, 6/52 genes in plasma, and 1/52 genes in urine samples Fig . In the et al, Detection of ctDNA in bio‐fluids is thought to be affected by intratumoral genomic heterogeneity . Such a shift was described previously for plasma samples of other cancer types  of mutant and non‐mutant cfDNA, i.e. reads carrying mutations previously identified in matched tissue and those not carrying mutations, in the CSF Fig , plasma We analyzed the cfDNA fragmentation patterns in 40 urine samples from 35 patients with gliomas (30 HGG and five LGG) collected pre‐treatment with paired‐end sWGS Fig . We alsoP = 5.2 × 10−9) and in urine of patients with other brain pathologies . We calculated the median empirical cumulative distribution function for each type of sample included in the study  and of other CNS pathologies . We analyzed the proportion of fragments in different size ranges and observed that the proportion of fragments between 30 and 60 bp was significantly increased in HGG and LGG cases as compared to healthy controls  and was also increased when compared to patients with other brain or CNS pathologies , and a second cluster with 45 data points and a mixture of non‐cancer and cancer samples . Analysis of cfDNA fragments using 10 bp bin sizes showed less pronounced clustering , which might affect the reproducibility of the models with an independent dataset, these results suggest that the cfDNA fragmentation patterns in urine samples may be a useful tool to provide information that can aid in the diagnosis of gliomas.Variable selection and the classification of samples as “non‐cancer” or “cancer” were performed using logistic regression (LR) and other machine learning models trained and validated on 40 cancer samples and 53 controls . The size of this cohort (n = 8 patients) will need to be increased to demonstrate clinical impact. However, this study reaffirms the high sensitivity and specificity of INVAR demonstrated with other cancer types  and early‐stage disease  and plasma (7/8) of patients with glioma. The median ctDNA fractions in plasma and urine were very low , patients with other CNS diseases (121 bp), and patients with gliomas (101 bp) was different from previous reports on other cancer types  of cfDNA fragments in urine samples showed larger differences between cancer cases and controls. These size ranges were similar to the size range enriched in mutant cfDNA in urine as observed using tumor‐guided capture panels. With four machine learning analyses, we identified and tested 10 size features that can be informative for classifying urine samples as being derived either from healthy individuals or from patients with glioma. The LR, RF, SVM, and GLMEN models correctly classified samples derived from patients with glioma in most of the cases . The GLMEN model correctly identified samples from cancer patients vs samples from controls with a sensitivity of 65% and specificity of 95% in a cohort of 93 urine samples (40 cancer samples and 53 control samples). These results from urine samples from glioma echoed our previous work, which identified 63% of plasma samples from glioma patients with 94% specificity using another RF model based on integration of fragmentation features in plasma cfDNA  study . Tumor tissue DNA was extracted and isolated as described previously  of all tumor tissue and germline buffy coat DNA samples. Fifty nanograms of DNA were fragmented to ˜ 120 bp by acoustic shearing (Covaris) according to the manufacturer’s instructions. Libraries were prepared using the Thruplex DNA‐Seq protocol (Rubicon Genomics) with 5× cycles of PCR. Libraries were quantified using quantitative PCR  and pooled for exome capture . Exome capture was performed with the addition of i5‐ and i7‐specific blockers (IDT) during the hybridization steps to prevent adaptor “daisy chaining.” Pools were concentrated using a SpeedVac vacuum concentrator . After capture, 8× cycles of PCR were performed. Enriched libraries were quantified using quantitative PCR , DNA fragment sizes were assessed by Bioanalyzer , and captures were pooled in equimolar ratio for paired‐end next‐generation sequencing on a HiSeq4000 (Illumina).http://broadinstitute.github.io/picard). Somatic SNV and indel mutations were called using GATK Mutect2 , (https://www.broadinstitute.org/gatk) in tumor‐normal pair mode using buffy coat as the normal.Sequencing reads were de‐multiplexed, allowing zero mismatches in barcodes. The reference genome was the GRCh37/b37/hg19 human reference genome—1000 Genomes GRCh37‐derived reference genome, which includes chromosomal plus unlocalized and unplaced contigs, the rCRS mitochondrial sequence (AC:NC_012920), Human herpesvirus 4 type 1 (AC:NC_007605) and decoy sequence derived from HuRef, Human Bac and Fosmid clones and NA12878. The sequence data of the patient samples were aligned to the reference genome using BWA‐MEM v0.7.15. The duplicate reads were marked using Picard v1.122 . Variants were annotated using Ensembl Variant Effect Predictor with details about consequence on protein coding, accession numbers for known variants and associated allele frequencies from the 1000 Genomes project.Hybrid‐based capture for the different body fluids  analysis was designed to cover the variants identified above for each patient using the SureDesign software (Agilent). In addition, 52 genes of interest for glioma were included in the tumor‐guided sequencing panel based on the TCGA databases. Patients were separated into 2 panels covering all the mutations included for those patients (4 patients per panel). Patients GB1, GB2, GB9, and GB16 were grouped in panel 1, and patients GB7, GB11, GB12, and GB19 were grouped in panel 2. Panel 1 covered in total 526 kbp  and panel 2 covered 526 kbp . Panels ranged in size between 1.430 Mb (panel 1) and 1.404 Mb (panel 2) with 120 bp RNA baits. Baits were designed with 5× tiling density, moderately stringent masking and balanced boosting. 99.7% of the targets had baits designed successfully.Indexed sequencing libraries were prepared using the Thruplex tag‐seq kits (Takara). Libraries were captured either in 1‐plex for plasma and urine samples or 3‐plex for CSF samples  using the Agilent SureSelectXTHS protocol, with the addition of i5 and i7 blocking oligos (IDT), as recommended by the manufacturer for compatibility with ThruPLEX libraries. Custom Agilent SureSelectXTHS baits were used. 13 cycles were used for amplification of the captured libraries. Post‐capture libraries were purified with AMPure XT beads, then quantified using quantitative PCR , and DNA fragment sizes controlled by Bioanalyzer . Capture libraries were then pooled in equimolar ratios for paired‐end next‐generation sequencing on a HiSeq4000 (Illumina).et al, et al, Sequencing reads were de‐multiplexed, allowing zero mismatches in barcodes. Cutadapt v1.9.1 was used to remove known 5′ and 3′ adaptor sequences specified in a separate FASTA of adaptor sequences. Trimmed FASTQ files were aligned to the UCSC hg19 genome using BWA‐mem v0.7.13 with a seed length of 19. Error suppression was carried out on ThruPLEX Tag‐seq library BAM files using CONNOR. The consensus frequency threshold (‐f) was set as 0.9 (90%), and the minimum family size threshold (‐s) was varied between 2 and 5 for characterization of error rates  were excluded in that sample, thereby suppressing outliers. The detailed calculation of IMAF was previously detailed  was determined across all loci passing pre‐INVAR data processing filters with mutant allele fraction at that locus of < 0.25; loci with signal > 0.25 mutant allele fraction were not included in the calculation because (i) loci would not be expected to have such high mutant allele fractions in body fluids of glioma patients (unless they are mis‐genotyped SNPs), and (ii) if the true IMAF of a sample is > 0.25, when a large number of loci are tested, they will show a distribution of allele fractions such that detection is still supported by having many low allele fraction loci with signal. Based on the ctDNA level of the sample, the binomial probability of observing each individual locus given the IMAF of that sample was calculated. Loci with a Bonferroni corrected Indexed sequencing libraries were prepared using the ThruPLEX‐Plasma Seq kit (Rubicon Genomics). Libraries were pooled in equimolar amounts and sequenced to < 0.4× depth of coverage on a HiSeq 4000 (Illumina) generating 150‐bp paired‐end reads. Sequence data were analyzed using an in‐house pipeline that consists of the following; paired end sequence reads were aligned to the human reference genome (GRCh37) using BWA‐mem following the removal of contaminating adapter sequences. PCR and optical duplicates were marked using MarkDuplicates (Picard Tools) feature, and these were excluded from downstream analysis along with reads of low mapping quality and supplementary alignments. When necessary, reads were down‐sampled to 10 million in all samples for comparison purposes.x bp peak is defined as the number of fragments with length x divided by the total number of fragments. To define local maxima, we selected the positions y such that y was the largest value in the interval . The same rationale was used to pick minima. PCA was calculated and visualized in R using the package ggbiplot. The tSNE analysis was performed in R with the Rtsne package using 1,000 iterations, Spearman correlations and a perplexity score of 8. Plots were generated in R using ggplot2. ROC curves were plotted in R with the plotROC package.The preliminary analysis was carried out on 93 samples (40 cancers and 53 non‐cancer controls). For each sample, the following features were calculated from sWGS data: P(30–60), P(61–90), P(91–120), P(121–150), P(151–180), P(181–210), P(211–240), P(241–270), and P(271–300). The data were arranged in a matrix where the rows represent each sample and the columns held the aforementioned features with an extra “class” column with the binary labels of “cancer” or “controls.” The amplitude of the 10 bp periodic peaks (OSC_10bp) was calculated from the sWGS data as follows: from the samples with clear peaks, the local maxima (“peak”) and minima  in the range 50–140 bp were calculated. The average of their positions across the samples was calculated: . We also used the ggplot2 (v3.2.0) and ggpubr (v0.2) packages.All statistics were performed using R (v3.4.3) programming language (Concept and design of the study: FM, CGS, NR, and RM; Methodology: FM, CGS, KH, JCMW, and RM; Investigation: FM, CGS, KH, MT, DG, IH, WC, JH, MG, JCMW, IH, WC, and RM; Sample collection: RM and CW; Data analysis: FM and RM; Fragmentation feature design and machine learning analysis: YvdP and FM; Computational analysis: FM, JS, YvdP, HZ, D‐LC, CGS, JM, ME, DC, and KH; Writing—original draft: FM and RM; Writing—review and editing: FM, RM, KH, IH, CGS, CW, KB, and NR; Funding acquisition: FM, CW, KB, NR, and RM; Supervision: FM, KB, NR, and RM.N.R. and D.G. are co‐founders, present/former officers or consultants and/or shareholders of Inivata Ltd, a cancer genomics company that commercializes circulating DNA analysis. C.G.S. has consulted for Inivata Ltd. Inivata had no role in the conception, design, data collection, and analysis of the study. Patent applications may be filed describing additional methods described in the manuscript. Other co‐authors have no conflict of interests.AppendixClick here for additional data file.Table EV1Click here for additional data file.Table EV2Click here for additional data file.Dataset EV1Click here for additional data file.Dataset EV2Click here for additional data file.Dataset EV3Click here for additional data file.Dataset EV4Click here for additional data file.Dataset EV5Click here for additional data file.Dataset EV6Click here for additional data file.Review Process FileClick here for additional data file.


Ber, a Chinese herbal monomer has been reported to exhibit an array of pharmacological activities related to the lowering of blood glucose and the treatment of polycystic ovarian syndrome (PCOS). In the present study, we aimed to elucidate the effect of berberine (Ber) on a rat model of PCOS mediated via the PI3K/AKT signaling pathway.A PCOS animal model was induced with the administration of letrozole, and animals were then randomized into untreated or Ber and metformin hydrochloride treated groups. After administration, fasting blood glucose, HOMA‐IR, fasting insulin (FINS) values, and the serum hormone levels were measured in PCOS rats. The ovarian tissues were stained with hematoxylin and eosin and terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling (TUNEL) for pathological and apoptosis examination. Moreover, the effect of Ber on the proliferation and apoptosis of granulosa cells was detected by CCK‐8 assays and flow cytometry. The influence of Ber on granulosa cells was confirmed by blockade of the PI3K/AKT pathway. In addition, the modulatory effect of the blockade of the PI3K/AKT pathway on the expression of related proteins was demonstrated via western blotting.We found that Ber was able to restore the serum hormone levels and improve IR in a PCOS rat model. The morphological lesions and apoptosis of the ovary were also restored by the Ber treatment. Blockade of the PI3K/AKT pathway attenuated the influences of Ber on the proliferation and apoptosis of granulosa cells.The beneficial effects of Ber on PCOS included alterations of the serum hormone levels, recovery of morphological lesions in the ovary, improvement of insulin resistance, and cell viability and inhibition of apoptosis, which were all mediated through the PI3K/AKT pathway. Polycystic ovarian syndrome (PCOS) is an endocrine syndrome characterized by persistent anovulation, hyperandrogenemia, or insulin resistance (IR).Coptis chinensis Franch and Phellodendron chinensis var. glabriusculum. It has been used in traditional Chinese medicine for many years and has been shown to be effective against IR and obesity, particularly against diabetes type 2 and hypercholesterolemia.Ber is an isoquinoline compound derived from many different plants like The PI3K/AKT signaling pathway is one of the most important insulin signaling pathways, which is not only closely related to IR, but also plays an important role in cell growth, proliferation, migration, invasion, inhibition of apoptosis, and angiogenesis.In the present study, a rat model of PCOS was constructed by daily gavage with a letrozole solution coupled with a high‐fat diet. Then, the ovarian granulosa cells of the model rats were extracted and cultured. The effects of Ber on IR, serum hormone levels, and ovary morphological lesions in the PCOS rats were explored, and the direct effects of Ber on the proliferation and apoptosis of granulosa cells were also investigated. In addition, LY294002 (a specific inhibitor of PI3K) and MK‐2206 (a specific inhibitor of AKT) were utilized to further verify the role of the PI3K/AKT signaling pathway on granulosa cell proliferation and apoptosis, along with an investigation of Ber's influence on the expression of related proteins.Female Sprague–Dawley (SD) rats (21–23‐day‐old), were purchased from the Center of Experimental Animals at the Shanghai Sippr‐BK Laboratory Animal Co. Ltd. . All of the rats were housed at a standard room temperature of 23 ± 2°C and humidity of 55%–70% under a 12 h light/dark cycle with access to food and water. All animal procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animal experiments were performed according to the guidelines of laboratory animal care and were authorized by the Ethic Committee of Zhejiang Traditional Chinese Medicine University . After the experiment, the rats were euthanized with carbon dioxide.After a week of adaptive feeding, 50 female SD rats were randomly assigned into five groups of 10 rats each: the control, PCOS, PCOS+ L‐Ber (95 mg/kg), PCOS+ H‐Ber (190 mg/kg), and metformin hydrochloride treated groups (PCOS+MH) (50 mg/kg).Twenty‐four hours after the last administration, blood from the rat aorta ventralis was centrifuged at 3000 rpm for 10 min, and following supernatant collection, the serum was stored at −80°C for subsequent use. A vaginal smear was conducted on 6‐week‐old rats, and the ovarian tissue was collected after fasting for a day. Pentobarbital sodium  was used for anesthesia, and then the bilateral ovaries were taken out. One ovary was rapidly fixed in 4% paraformaldehyde and the other was stored at −80°C until use.To assess IR in the study animals, the rats were fasted for 12 h after the last administration, then FBG and FINS were assessed using venous blood samples by enzyme‐linked immunosorbent assay (ELISA). We followed the homeostasis model assessment of insulin resistance (HOMA‐IR) approach. HOMA‐IR was calculated using the following formula:We used ELISA kits  to measure the serum concentrations of gonadotropins, including 17β‐estradiol (E2), follicle‐stimulating hormone (FSH), and luteinizing hormone (LH), and the steroid hormones, progesterone (P) and testosterone (T).At the study endpoint, the ovaries were quickly removed and dissected on dry ice, followed by formalin fixation/paraffin embedding; it was then cut into 4 μm sections and stained using hematoxylin and eosin (H&E). The morphological structure was observed under a light microscope and recognized by a morphological scoring standardPart of ovarian tissue was fixed with 4% paraformaldehyde, routinely dehydrated, paraffin‐embedded in paraffin, cut into 4‐μm sections, dewaxed, cleared, and then subjected to TUNEL staining. Microscopically, the nuclei of the normal ovaries were blue, and the nuclei of the apoptotic cells were brown and yellow. The apoptotic index (AI) was expressed as the percentage of apoptotic cells in the total number of ovaries.Twenty female SD rats were randomly assigned into 2 groups of 10 rats each: L‐Ber (95 mg/kg) and H‐Ber (190 mg/kg) group. These groups were administered gavage of 10 mL/kg of Ber once daily for seven consecutive days. One hour following the last intragastric administration, the abdominal artery blood was drawn from the rats in each group under aseptic conditions, placed at 4°C for 1 h, and centrifuged at 900 g and 4°C for 15 min. The drug‐containing serum samples from all of the rats in the same group were mixed, and then serum inactivation was conducted by placing it in a water bath which is 56°C for 30 min. Finally, the samples were sterilized by microporous membrane filtration, and then packed in tubes and stored at −70°C for the follow‐up experiments.6 cells/bottle) using DMEM/F12 medium supplemented with 10% fetal bovine serum protein (FBS) at 37°C with 5% CO2. After 24 h of preculture, when the cells reached approximately 70% confluence, the medium was replaced with fresh media every 2 days. When the cells reached approximately 80% confluence, they were digested with 0.25% trypsin. The cells were fixed using 4% paraformaldehyde for 10 min; then, they were stained using H&E for the identification of the rat ovarian granulosa cells.The in vitro experiments were conducted according to the protocol described previously.Experiment − A Blank)/(A Control − A Blank) × 100%.A cell counting kit‐8  was utilized to determine the granulosa cells viability and proliferation rates. Briefly, following exposure to various concentrations of Ber for various treatment times , 100 μL of cell suspension (approximately 5000 cells/well) was removed and 10 μL of CCK‐8 Solution was added to each well of a 96‐well plate. The plate was then incubated in a cell culture incubator. Finally, the optical density  was calculated with a microplate reader (CMaxPlus), and the absorbance of each experimental group was measured at 450 nm. Each treatment had three replicates. Cell viability = . Briefly, granulosa cells treated as above were collected and washed with ice‐cold PBS two times, and resuspended in 500 μL of binding buffer, then the mixture was centrifuged and the supernatant was discarded. The granulosa cells were resuspended in 100 μL Annexin V‐FITC binding solution, and stained for 15 min with 5 μL Annexin V‐FITC solution and 10 μL propidium iodide (PI) solution in the dark at room temperature. Finally, the apoptotic rate of the granulosa cells was measured by flow cytometry .g and 4°C for 5 min. The supernatant was collected, and the protein concentration in each group was detected using a bicinchoninic acid (BCA) protein quantification kit (pc0020). An appropriate amount of the protein sample was taken and mixed with 2 × sodium dodecyl sulfate (SDS) loading buffer (loading buffer: protein samples [v/v] = 4:1) to prepare the loading buffer system at an equal concentration, and the protein was denatured at 95°C for 5 min. Then, 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels were applied for electrophoresis under the constant voltage of 80 V. The protein was transferred onto a polyvinylidene difluoride (PVDF) membrane, which was blocked with freshly prepared 5% skim milk powder for 2 h and then washed. The membranes were then incubated with primary antibodies diluted according to the manufacturer's protocol overnight at 4°C, followed by incubation with a secondary antibody at room temperature for 1–2 h. The primary antibodies were directed against: GAPDH Antibody , Anti‐IGF1 Receptor Antibody , Anti‐AKT (phospho T308) Antibody , Anti‐pan‐AKT Antibody , Anti‐Bad Antibody , Anti‐Bcl‐2 Antibody , Bax Antibody , PI3 Kinase p85 Antibody  and PI3 Kinase p110α Antibody . The reactive bands were visualized with the ECL‐Plus reagent (Solarbio). The density of the band was quantified by Quantity One analytic software.The granulosa cells were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime), and centrifuged at 12 000χ¯ ± s. For the in vivo study, group comparisons were processed by using the LSD one‐way‐ANOVA test. For group comparisons in the in vitro study, we used Student's t‐test or the Kruskal–Wallis H test. In all cases, p < 0.05 was considered to indicate statistical significance.The statistical analyses were performed using SPSS 16.0 (IBM). Values are expressed as As shown in Figure p < 0.01), whereas the serum P level in the rats was significantly lower in the PCOS group than the control group  while the levels of LH, FSH, and P had no significant change (p > 0.05) in the PCOS+L‐Ber group compared to the PCOS group. In addition, the level of P was increased and the levels of LH, E2, and T were suppressed in the PCOS+H‐Ber and PCOS+MH groups compared to the PCOS group. Furthermore, the level of FSH had no significant change across all groups (p > 0.05).Results of the ELISA assays indicated that the serum E2, T, and LH levels in the rats were significantly higher in the PCOS group than in the control group . The 0.2 mg/mL dose of Ber was selected for subsequent experiments.To investigate the effects of Ber exposure on ovarian granulosa cells in vitro, primary ovarian granulosa cells were exposed for various lengths of time at various concentrations. The effects of letrozole exposure on cell viability and growth were determined using a CCK‐8 assay at four exposure time points  with concentrations of 0.1 and 0.2 mg/mL. The results suggested that the cell viability was significantly reduced in the letrozole treated groups when compared with that in the control groups after culturing for the four exposure times. We found 0.2 mg/mL Ber treatment for 48 h significantly increased cell viability (p < 0.05) compared with the PCOS group. The inhibitory effects of both 20 mM LY294002 and 20 mM MK‐2206 on cell growth were also confirmed. However, simultaneous addition of either LY294002 or MK‐2206 in combination with 0.2 mg/mL Ber blocked the letrozole‐induced cell growth but not significantly (p > 0.05). CCK‐8 analysis further revealed that 0.2 mg/mL Ber treatment significantly stimulated granulosa cell proliferation (p < 0.01), while this stimulatory effect was blocked in the presence of either LY294002 or MK‐2206 (p < 0.05) , but was elevated in the presence of either LY294002 or MK‐2206 (p < 0.05). Moreover, the anti‐apoptotic effects of Ber were blocked in combination with either LY294002 or MK‐2206  and an AKT inhibitor , were used. Cell proliferation and cell apoptosis were measured by the CCK‐8 assay and flow cytometry, respectively. In granulosa cells treated with 0.2 mg/mL Ber, a significant stimulatory effect of Ber on cell growth was observed  Figure . In addip < 0.01), while pharmacological inhibition of the PI3K/AKT pathway by LY294002 significantly decreased IGF‐1, Bcl‐2, p‐AKT, and p‐PI3K protein expression (p < 0.05 and p < 0.01) and MK‐2206 significantly decreased p‐PI3K, p‐AKT, and BAD protein expression (p < 0.05). In addition, the western blot assay also showed that LY294002 promoted the expression of BAD and Bax, while MK‐2206 promoted the expression of Bax in ovarian granulosa cells (Figure As shown in the western blotting analysis, the expression level of AKT and PI3K was no significantly different in all groups, but the protein expression levels of Bcl‐2, p‐AKT, and p‐PI3K were significantly enhanced by 0.2 mg/mL Ber treatment (PCOS is defined as a metabolic syndrome.IR and the dysregulation of glucose metabolism are common in PCOS patients.PI3K signaling cascades are important regulators of a host of cellular activities such as proliferation, differentiation, survival, apoptosis, and glucose homeostasis. Previous studies have shown that activation of the PI3K/AKT signaling pathway has important effects on insulin resistance.The PI3K/AKT pathway may also be involved in the regulation of ovarian granulosa cell apoptosis and proliferation. Previous works suggested that granulosa cell apoptosis is involved in many ovarian physiological processes including the female germ cell cyst breakdown and primordial follicle assembly, follicular atresia, and luteolysis.In summary, we reported on the beneficial effects of Ber in a PCOS rat model, including improvements in serum hormone levels, HOMA‐IR and the apoptosis and proliferation of ovarian granulosa cells. In addition, one of the important findings was the involvement of the PI3K/AKT pathway in the Ber‐induced beneficial effects on PCOS. This work provides experimental evidence to support the clinical application of Ber as a potential PCOS treatment.Yu Jia designed the experiment. Wang Chenye and Hua Zhoujuan performed the experiment and acquisition the data. Yu Jia, Ding Caifei, Jiang Xuejuan and Hua Zhoujuan analyzed the data. Yu Jia and Wang Chenye obtained the funding and wrote the manuscript. Wang Chenye revised the manuscript. All authors discussed the results, and approved the final manuscript.The authors declare that they have no conflict of interest.


Hypothyroxinemia is defined by low levels of thyroxine (T4) despite low or normal levels of thyroid-stimulating hormone (TSH). This study aimed to evaluate the factors associated with transient hypothyroxinemia of prematurity (THOP) in newborns admitted to the neonatal intensive care unit (NICU).n = 71) and the control group with euthyroid prematures (n = 73). The factors for THOP were identified by univariate analysis, followed by multivariate analysis.This is a single center, retrospective, case-control study. Premature newborns, between 24 and 34 weeks of gestation, hospitalised between January 2014–December 2019 in Istanbul University-Cerrahpasa Faculty of Medicine NICU were analyzed through their medical records. Thyroid function tests were routinely performed between the 10th and 20th days of postnatal life and were evaluated according to the gestational age references. Thirty six possible associated factors  were searched in the patient group with THOP (p = 0.606). The birth weight, small for gestational age (SGA), intraventricular hemorrhage (IVH), congenital heart disease (CHD) were found to be the possible associated factors for THOP in the univariate analysis and CHD , BW  and SGA  were found to be factors associated with THOP determined by univariate logistic regression analysis.Mean gestational ages of the study and the control groups were 29.7 ± 2.48 and 30.5 ± 2.30 weeks, respectively (Although some treatment practices might have had direct effects on pituitary–thyroid axis, related with the severity of the newborn clinical conditions, non of them was found to be a associated factor for THOP. However, CHD and SGA may be considered as associated factors with THOP detected in preterm infants. Transient hypothyroxinemia of prematurity (THOP) is defined by low levels of thyroxine T4) despite low or normal levels of thyroid-stimulating hormone (TSH) . Hypothy despite TSH screening is the most sensitive test for primary CH detection. However, a unique form of CH characterized by a delayed TSH elevation has been described in preterm infants . The scrPostpartum TSH and thyroid hormone peaks observed in term newborns are not so evident in preterms. Especially below 30 weeks’ gestation, TSH increase is late and weak, while T4 and FT4 levels remain low . In premThis is a single center, retrospective, case-control study. The THOP and control groups (euthyroid) of premature newborns with similar gestational age and closest date of birth time were compared. File records of 538 newborns who had been admitted to Istanbul University-Cerrahpasa Faculty of Medicine Neonatal Intensive Care Unit (NICU) between January 2014–December 2019 were retrospectively analyzed. Recruitment criteria: premature newborns between 24 and 34 GA, without multiple major congenital anomaly and whose serum thyroid function test were done between the 10th and 20th days of postnatal life. Cases with major multiple organ anomalies excluded from the study .n = 83), 47.5% (n = 86), 4.9% (n = 9) and 1.6% (n = 3), respectively. Infants of mothers with maternal hypothyrodism, pregnancy without follow-up was excluded from both THOP and euthyroid groups. Two groups were formed as the patient group  and the control group . The study design was planned as shown in Fig. During this 5-year retrospective study: THOP, euthyroid, primary hypothyroidism and subclinical hypothyroidism were diagnosed in 45.8% (n = 71) and the control group with euthyroid infants (n = 73). Data including applied medical treatments and clinical applications were collected from the date of birth to the measurement of TSH and FT4.Thirty six possible associated factors detected between birth to the time of blood samples collection  were compared between the patient group with THOP .Serum free thyroxine (FT4) and TSH levels were measured using commercially available kits  by electrochemiluminescence (ECLIA) method. The thyroid hormone levels were assessed based on the weeks’ gestational and postnatal age references. In the second postnatal week following intervals were accepted to be the normal range for FT4: 1.45 ± 0.5 ng/dl for 23–27 week, 1.65 ± 0.4 ng/dl for 28–30 week, 1.98 ± 0.4 ng/dl for 31–34 week and the normal range for serum TSH: 3.9 ± 2.7 mU/L for 23–27 week, 4.9 ± 11.2 mU/L for 28–30 week, 3.8 ± 9.3 mU/L for 31–34 week were taken [Bronchopulmonary dysplasia (BPD) was defined as the development of concomitant parenchymal lung injury requiring treatment with oxygen > 21% and/or positive pressure at postmenstrual gestational age at 36 weeks or at postnatal 56 days or at discharge (which one is earlier) . Echocarp < 0.2 were included in univariate logistic regression analysis. The results were given as the Odds Ratio (OR) and 95% confidence intervals (95% CI). The level of significance was taken as p < 0.05.The SPSS v.21  software was used for the statistical analyzes. The compatibility of the data with normal distribution was evaluated by the descriptive statistics  and Kolmogorov Smirnov Test. Continuous variables were shown with mean ± standart deviation and median (25th - 75th quarters), and categorical variables with frequency and percentage (%). The Mann-Whitney U test was used to compare the groups in terms of continuous variables. The Chi-squared test and Fisher’s exact test were used, where appropriate, in the analysis of the categorical variables. Possible associated factors with THOP which have p < 0.001), while there was no significant difference between the TSH levels  (p = 0.556). Mean birth weight was lower in the THOP group  compared to the control group  (p < 0.001) , 3 cases of ventricular septal defect (VSD), 1 case of transposition of the great arteries (TGA), 1 case of coarctation of the aorta (CoA), 1 case of atrial septal aneurysm, 1 case of mitral valve prolapse were detected. In the control group, 4 patients had ASD and 1 VSD. In THOP group, CHD was statistically significantly higher than control group  (p < 0.200) were analyzed. The CHD , BW  and SGA  were found to be factors associated with THOP , maternal smoking, gestational hypertension and diabetes, prenatal betamethasone treatments, central catheterisations, sepsis, pneumothorax, PDA, drug history , phototherapy, development of hypoglycemia, RDS, BPD and ROP, erythrocyte suspension (ES), thrombocyte suspension (TS) and fresh frozen plasma transfusions, mechanical ventilator and noninvasive ventilation support were not statistically significant Table . BW was In this study, The THOP and control groups (euthyroid) of premature newborns born at the same time and with the most similar gestational age were compared. Low birth weight, SGA and CHD were found to be as associated factors with THOP. These three associated factors are not affected by the gestational age, the severity of the illness and the medical and clinical treatment practices applied. It is already shown in the literature that the frequency of hypothyroxinemia changes in proportion to gestational age . EspeciaIn our study, interestingly although THOP group had similiar gestational age with the control group, had a low birth weight in THOP group. Consequently in this study, incidence of SGA was higher in the THOP group and increased the risk of the THOP 4.6 times. Each one hundred gram increase in BW reduced the THOP risk by 10%. We speculate that being SGA may result in further deficit in storage and adaptive mechanisms. A recent study by Chunhua et al., supported our results that SGA in preterms may be associated with thyroid dysfunction . A comprIt is already shown that thyroid and cardiac disorders can be associated . CongeniSome studies have shown that in preterms thyroid functions are affected by postpartum drugs and some perinatal conditions. Drugs frequently used in premature infants , respiratory distress syndrome, infections, disorders such as necrotizing enterocolitis, patent ductus arteriosus, malnutrition, chorioamnionitis, iodine deficiency or overload may suppress thyroid functions , 36–39. The medications are used widely as gestational age decrease and severity of the disorder increases. In a study with very low birth weight newborns, it was shown that the negative effect of dopamine and dobutamine use on thyroid function rapidly resolved after treatment was discontinued . There aIn this study, serum TFT results between ten and twenty days of life were evaluated according to the postnatal and gestational age reference values (1). However, there is no TFT level defined as “low or normal” and there is no consensus on the timing of measurements. The strength of this study is that it is one of the few studies , 40–44 eIn conclusion, although some treatment practices could have direct effects on the pituitary-thyroid axis in relation to the severity of neonatal clinical conditions, none of these were found to be an associated factor with THOP in our study. CHD and SGA were found to be as associated factors with THOP.


C. depressa peel for healthy elderly subjects.Nobiletin exerts beneficial effects on cognitive function in various animal models of Alzheimer's disease. The present study aimed to investigate the benefits and safety of a combination food of nobiletin‐rich extract from ®) comprised high‐purity nobiletin powder combined with dried root powder of K. parviflora and dried lead powder of P. japonicum and was administered to elderly Japanese subjects once a day for 16 weeks. The Japanese version of the Wechsler Memory Scaled‐Revised (WMS‐R) was used as a primary evaluation item for the assessment of global memory. Data from a protocol‐matched population (Per Protocol Set: PPS) (n = 108) were analyzed.The nobiletin‐containing test food  than in the placebo group (5.9 ± 7.70). An age‐stratified analysis of the WMS‐R test showed similar changes in subjects aged ≦74 years to those in the overall subject population. In the stratified analysis involving subjects with an MMSE‐J score of between 24 and 28, the “figural memory” subscale score in WMS‐R was significantly higher in the test food group than in the placebo group.The present results indicate that the nobiletin‐containing test food is beneficial for improving memory dysfunction in healthy elderly subjects. Nobiletin exerts beneficial effects on cognitive function in various animal models of Alzheimer's disease. The present results indicate that the nobiletin‐containing test food is beneficial for improving memory dysfunction in healthy elderly subjects. A large portion is processed into juice, leaving behind tons of citrus peels as industrial waste. The Okinawa Research Center Organization established a method to manufacture high‐purity nobiletin powder  from the residual by‐product, through isolation using ethanol extraction/concentration and an alkaline treatment  to improve the quality of life (QOL) of the elderly. One capsule contains the dried powder extract (containing 10.0 mg nobiletin and 5.8 mg tangeretin) of C. depressa peel, the dried root powder (126.7 mg) of K. parviflora and the dried leaf powder of P. japonicum (33.3 mg). These plant extracts were not included in the placebo capsules. Three capsules of the nobiletin‐containing test food or placebo food were administered to each subject once a day for 16 weeks.Sikwasa  (jRCTs041190073) on October 7, 2019. The present study was conducted in accordance with the Helsinki Declaration and “Clinical Research methods and regulations for Clinical Research methods”, considering subjects’ human rights and safety/welfare. Study participants were recruited by medical institutions and community organizations. The study contents were sufficiently explained to individuals wishing to participate in this study, and written informed consent was obtained. The study period was between October 7, 2019, and March 31, 2020. There were no changes in the protocol.2.3The number of subjects was 122, consisting of healthy subjects who consulted the Seishukai Takido Medical Clinic, Shujikai Hagiwara Medical Clinic, or Izu Health & Medical Center as well as elderly residents of the area who were able to participate/cooperate. Primary selection criteria included healthy males and females, aged ≥65 years, with memory lapses and an MMSE‐J score of ≥24. After obtaining written informed consent on recruitment, subjects who met the selection criteria, but not the exclusion criteria, were selected.2.4Written informed consent was obtained from the individual before participating in this study.Individuals aged ≥65 years, with memory lapses.Individuals who were able to consume the test food during the study period.Individuals who were able to write a diary during the consumption period.Individuals with an MMSE‐J .Individuals participating or planning to participate in other clinical studies at the start of this study.Individuals considered to be ineligible by the chief investigator.2.6The subjects of this study comprised 122 healthy elderly persons enrolled, and they were randomly assigned to a placebo or nobiletin‐containing test food group using the permuted block method. The assignment table was sealed, stored until the completion of this study, and opened after its completion. We confirmed that there were no significant differences in age or sex between the two groups.2.7Testing was conducted at the start of consumption and after 16 weeks. Compliance was confirmed by telephone in the midterm of the present study. When the subject withdrew from this study or when test‐food consumption was discontinued, the study was completed. Furthermore, complications or adverse events were recorded based on reports from subjects. In subjects who had taken drugs for other diseases before the start of the present study, neither the dose nor dosing method was changed during the study period.A primary evaluation criteria, the Japanese version of the Wechsler Memory Scale‐Revised (WMS‐R) , blood pressure, and pulse rate. Hematological parameters included the leukocyte count, erythrocyte count, hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, uric acid, urea nitrogen, aspartate aminotransferase (AST)(GOT), alanine aminotransferase (ALT)(GPT), γ‐glutamyl transpeptidase (GTP), alkaline phosphatase (ALP), lactic acid dehydrogenase (LDH), total bilirubin, total protein, albumin, creatinine (Cre), estimated glomerular filtration rate (e‐GFR), creatine phosphokinase (CPK), serum amylase, total cholesterol, high‐density lipoprotein (HDL)‐cholesterol, low‐density lipoprotein (LDL)‐cholesterol, neutral fat, blood glucose, HbA1c, Na, Cl, K, Mg, Ca, Fe, testosterone, and adiponectin (LA). In the urinalysis, qualitative protein/glucose/urobilinogen/bilirubin/occult blood reaction/ketone body tests, as well as specific gravity and pH tests were conducted. Blood, urine, and cognitive function tests were generally conducted twice at the start of food consumption and after 16 weeks. Safety assessment items included the number of patients with adverse events, adverse reactions, severe adverse events, or severe adverse reactions/their contents, and the number of patients in whom the oral administration of drugs for dementia was started/their contents. These items were evaluated based on the results of general and blood examinations.2.8t‐test. When comparing baseline values between the two groups, sex/age was compared using the chi‐squared test and the results of cognitive function tests using the unpaired t‐test. We used SPSS Statistics, ver. 25 software. In all tests, two‐sided tests were adopted. A p‐value of .05 was considered to indicate significant difference.To compare results between the test‐food and placebo groups, differences in scores after the completion of test food consumption from the baseline scores of the respective examination items were investigated using the unpaired Since the WMS‐R test is recommended for individuals aged ≤75 years, a stratified analysis was performed by dividing subjects into two age groups consisting of those aged 65 to 74 years and those aged ≥75 years, based on the number of subjects. Furthermore, a stratified analysis was conducted by dividing subjects into two groups based on the baseline MMSE‐J score (between 24 and 28 and ≥29) for comparisons.33.1n = 108), excluding 4 subjects meeting the exclusion criteria , no significant differences were noted between the test food and placebo groups Tables .3.3No significant differences were observed in any parameter in general/blood/urine examinations before and after food consumption between the test food and placebo groups. There were also no adverse events.4Natural medicines have gained popularity in many Western countries in recent years due to their therapeutic effects and affordability for the general public  was shown to significantly prevent the progression of cognitive impairments in these patients with no adverse events ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing‐original draft ; Writing‐review & editing . Michiyo Shirai: Data curation ; Formal analysis ; Methodology ; Project administration ; Writing‐original draft . Ken Ono: Conceptualization ; Data curation ; Investigation ; Project administration ; Supervision ; Validation ; Visualization . Toshiaki Teruya: Conceptualization ; Project administration ; Software ; Supervision ; Visualization . Aki Tamano: Data curation ; Formal analysis ; Methodology ; Software ; Visualization . Woo Je‐Tae: Conceptualization ; Funding acquisition ; Investigation ; Project administration ; Resources ; Software ; Supervision ; Validation ; Visualization ; Writing‐original draft ; Writing‐review & editing .This study was approved by the Clinical Research Examination Ethical Committee of Hamamatsu Medical University on October 4, 2019. The study conforms to the Declaration of Helsinki, US, and/or European Medicines Agency Guidelines for human subjects.


Diabetes insipidus (DI) can be a common cause of polydipsia and polyuria. Here, we present a case of congenital nephrogenic diabetes insipidus (CNDI) accompanied with central diabetes insipidus (CDI) secondary to pituitary surgery.A 24-year-old Chinese woman came to our hospital with the complaints of polydipsia and polyuria for 6 months. Six months ago, she was detected with pituitary apoplexy, and thereby getting pituitary surgery. However, the water deprivation test demonstrated no significant changes in urine volume and urine gravity in response to fluid depression or AVP administration. In addition, the genetic results confirmed a heterozygous mutation in arginine vasopressin receptor type 2 (AVPR2) genes.She was considered with CNDI as well as acquired CDI secondary to pituitary surgery. She was given with hydrochlorothiazide (HCTZ) 25 mg twice a day as well as desmopressin  0.1 mg three times a day. There is no recurrence of polyuria or polydipsia observed for more than 6 months. It can be hard to consider AVPR2 mutation in female carriers, especially in those with subtle clinical presentation. Hence, direct detection of DNA sequencing with AVPR2 is a convenient and accurate method in CNDI diagnosis. Diabetes insipidus (DI) can be a common cause of polyuria . Here, w. Later, she was treated with transnasal transsphenoidal resection for the pituitary mass, and the pathology findings suggested pituitary adenoma . She was then prescribed with methylprednisolone 40 mg once after pituitary surgery, and her cortisol level (8 am-4 pm-12 pm): 143.13μg/L-47.12μg/L-5.93μg/L, adrenocorticotropic hormone (ACTH) level (8 am-4 pm-12 pm): 33 pg/mL-18.8 pg/mL- < 5 pg/mL, 24-h urine cortisol level was 388μg, as well as gonadotrophins and thyroid hormones were all in the normal range at that period. Later, she developed severe polydipsia as well as polyuria, generally urinating every 2 h with 10 L per day in total. There was no backache, hand tremors, blurred vision, lower limb puffiness or weight loss. The laboratory examination revealed that the plasma osmolality was 301.00 mOsm/L, urine gravity was 1.002, potassium was 4.17 mmol/L, and serum sodium was 138.5 mmol/L. Then, she was diagnosed with pituitary surgery-derived central diabetes insipidus (CDI). However, therapeutic strategies with oral desmopressin , pituitrin and potassium chloride were of little efficacy, making the diagnosis undetermined. Later, she was speculated to have NDI and was started with extra HCTZ, along with DDAVP gradually reduced to stop. Surprisingly, her polyuria symptom was alleviated and was then given HCTZ twice a day out of the hospital. However, she stopped the treatment on her own and was admitted for polyuria a second time  determined pituitary apoplexy, with weakened signal in the posterior pituitary Fig. . Later, c.914dupC (p.Pro306fs). Further investigation revealed that she had slight polydipsia as well as polyuria for over 20 years. She primarily drank 5 L of water daily along with urine output every 3 h, totaling 4 L per day; however, she did not attach importance to it at that time. As a result of unfavorable therapeutic efficiency with HCTZ during previous discharge, the history of pituitary surgery as well as the slight remission after DDAVP taken during the water depression test, the diagnosis of CNDI and acquired CDI secondary to pituitary surgery should not be excluded, thereby, DDAVP was primarily utilized for 3 days alone for diagnosis therapy, and the treatment was subsequently changed to 25 mg HCTZ twice a day and 0.1 mg DDAVP three times a day. Currently, no recurrence of polyuria or polydipsia has been detected for more than 6 months, indicating the effectiveness of our therapeutic strategies.The physical examination this time revealed that her blood pressure was 108/71 mmHg, with a pulse rate of 121/min. Her skin was dry, and the rest of the physical examination was unremarkable. The laboratory examination revealed the following results: urine specific gravity, 1.002; serum sodium, 134.3 mmol/L; plasma osmolality, and 338.00 mOsm/(kg·H2O). Urinary ultrasonography scans showed normal results. A water deprivation test was performed later with an initial urine gravity of 1.006, plasma osmolality of 281 mOsm/L, and blood pressure of 124/72 mmHg. Two hours after AVP injection, the plasma osmolality was 299.00 mOsm/(kg·H2O), with urine gravity unchanged (more details are shown in Table _ex3 c.914dupC (p.Pro306fs), which makes the diagnosis of CNDI reliable. It is noteworthy that her mother as well as her grandmother also suffered from polydipsia and polyuria with a low specific urine gravity, despite two brothers and a sister of her mother as well as her grandfather not admitting to polyuria. In addition, this patient had a brother and two sisters who had no polyuria symptoms .The polyuria and polydipsia symptoms of this patient can be mainly attributed to NDI. Although CDI is likely with damage to vasopressin neurons by pituitary apoplexy or surgery, those patients normally develop polyuria 1–4 days after pituitary surgery and resolve spontaneously; less often, DI is permanent. Therefore, temporary treatment with desmopressin in those patients would be valid . FurtherDI is a kind of polyuria-polydipsia syndrome that is characterized by the excretion of large amounts of hypotonic urine. After exclusion of osmotic diuresis, such as diabetes mellitus, DI is normally classified into CDI, NDI and psychogenic polydipsia (PPD) . As a reRecently, more than 250 AVPR2 gene mutations have been identified in the Human Gene Mutation Database , among wAlthough the diagnosis of CNDI accompanied by CDI is conclusive according to her clinical history, the water depression test and genetic results, there still exist several anomalies in this patient. Since she had CDI, the use of Minirin or pituitrin should partially improve polyuria symptoms; nevertheless, there was no significant decrease in urine output after pituitary surgery with large doses of oral vasopressin. Further detailed review of this patient showed that she experienced obvious anxiety during the preoperative period and was given 60 mg of the antianxiety medicine duloxetine as well as 7.5 mg of mirtazapine once a day beginning at the point close to the HCTZ, which also suggested PPD. Unfortunately, as a result of the lack of strict water restriction during that time, the diagnosis of PPD was unreliable. In addition, after elimination of unfavorable emotion as well as withdrawal of the antidepressants, in which there is no spiritual factor interference, the familial NDI along with the CDI should be taken into primary consideration based on the results.


Tgfb1 mRNA levels. Kidney damage caused by the exposure to MHFD was associated with greater oxidative stress, renal inflammation, higher endoplasmic reticulum (ER)‐stress, and disruption of mitochondrial dynamics. In summary, our data demonstrate that obesity induced by a milder fat content diet is enough to establish renal injury, where oxidative stress, inflammation, ER‐stress, and mitochondrial damage take relevance, pointing out the importance of opportune interventions to avoid the long‐term consequences associated with obesity and metabolic syndrome.Most of the obesity murine models inducing renal injury use calorie‐enriched foods, where fat represents 60% of the total caloric supply, however, this strategy doubles the standard proportion of fat ingestion in obese patients. Therefore, it is crucial to study the impact of a high‐fat intake on kidney physiology that resembles common obesity in humans to understand the trigger mechanisms of the long‐term consequences of overweight and obesity. In this study, we analyzed whether chronic feeding with a moderately high fat diet (MHFD) representing 45% of total calories, may induce kidney function and structural injury compared to C57BL/6 mice fed a control diet. After 14 weeks, MHFD induced significant mice obesity. At the functional level, obese mice showed signs of kidney injury characterized by increased albuminuria/creatinine ratio and higher excretion of urinary biomarkers of kidney damage. While, at the structural level, glomerular hypertrophy was observed. Although, we did not detect renal fibrosis, the obese mice exhibited a significant elevation of  Here, we demonstrated that obesity induced by a MHFD is enough to establish kidney damage, where oxidative stress, renal inflammation, ER‐stress, and mitochondrial damage take relevance, pointing out the importance of opportune interventions to avoid the long‐term consequences associated with obesity. The Animal Care Committee at Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán approved our experimental study.All experiments involving animals were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals  aged 5–6 weeks and weighing 17–22 g were maintained in controlled conditions of temperature and humidity in our animal housing facility with 12:12 h day/night cycle, with free access to water and food. We did not use a method to generate the randomization sequence, the mice were only randomly assigned into two groups of at least 10 mice per group, as follows: (1) control mice fed with a control diet (C) and (2) obese mice (OB) fed with a moderately high fat diet (MHFD). The sample size was calculated according to the percentage of success and error test  to obtain blood samples by cardiac puncture. A slide of the kidney was fixed in paraformaldehyde 4% for histopathological analysis and both kidneys were weighed and preserved in liquid nitrogen (−80ºC) until analysis.Urinary creatinine concentration was measured with the Quantichrom creatinine assay kit (DICT‐500).2.2Analysis of body composition was performed in 6 animals per group using a 4‐ in‐1 small animal MRI . Serum analysis of glucose, triglycerides, and cholesterol were determined enzymatically with a SynchronCX auto analyzer (Beckman Coulter).2.3The determination of urinary hydrogen peroxide as an oxidative stress marker was carried out, using a commercial kit  following the manufacturer's instructions.2.4As a marker of kidney damage, urinary albumin concentration was analyzed by using a commercial kit Albuwell M  following the manufacturer's instructions. This assay is a competitive ELISA completed in a direct mode, consisting of mouse‐specific albumin recognition by the provided horseradish peroxidase‐conjugated antibody, absorbance spectrophotometry, and concentration estimation using linear regression of a standard curve, and were normalized by urinary creatinine (UCr).2.5Using Western blot technique, urinary HSP72 . The proteins concentration was assessed by Lowry protein assay . Renal protein levels were detected by Western blot, tissue proteins (20 μg) were electrophoresed in a denaturing 8.5% acrylamide gel with SDS. The membranes were incubated with the primary antibody IL‐6 , BiP‐1 , CHOP , FOXO3 , Mitofusin‐1+Mitofusin‐2 , Drp1  and HRP β‐Actin antibody [AC‐15]  overnight at 4℃. Three of 10‐min washes were performed with TBS‐1x Tween, and then incubated with a secondary antibody coupled to HRP, anti‐rabbit or anti‐mouse IgG . Tissue proteins assessed by Western blot were normalized by β‐actin detection.2.6.1Il‐6), tumor necrosis factor alpha (Tnfa), interleukin 10 (Il10), transforming growth factor (Tgfb1) vascular endothelial growth factor (Vegfa), catalase (Cat), superoxide dismutase (Sod2) and glutathione peroxidase 1 (Gpx1) were quantified by real‐time PCR on an ABI Prism 7300 Sequence Detection System . Probes were ordered as follows: Il6 (Mm00446190_m1), Tnfa (Mm0443258_m1), Il10 (Mm01288386_m1), Tgfb1 (Mm03024053_m1), Vegfa (Rn01511602_m1), Cat (Mm00437992_m1), Sod2 (Mm01313000_m1) and Gpx1 (Mm00656767_g1). As an endogenous control, eukaryotic 18S rRNA  was used. The relative quantification of each gene expression was performed using the comparative threshold cycle (Ct) method.Total RNA was isolated from whole kidneys using the TRIzol method  and RNA integrity was evaluated using 1% agarose gel electrophoresis, to analyze rRNA. To avoid DNA contamination, total RNA samples were treated with DNase . Reverse transcription (RT) was carried out with 1 μg of total RNA and 200 U of Moloney murine leukemia virus reverse transcriptase . The mRNA levels of Interleukin 6 . Twelve high‐power fields (Magnification 200×) were captured from the renal cortex of each kidney to ensure the presence of at least 12 glomeruli per kidney using a camera incorporated onto the microscope. The glomerulus size was evaluated by measuring glomerular area in each captured glomerulus per mouse using the NIS‐Elements software (Nikon Instruments Inc.). Because glomeruli are spheroidal in shape and in each section of the kidney, each glomerulus can be cut at the lower, upper, or middle pole, the glomerular area was organized by ranks and then the differences between the C and MHDF groups were determined by a contingency analysis, as we have previously reported .The results are presented as the mean ± SE. The significance of the differences between groups was assessed by unpaired 33.1p = NS). However, the ratio kidney/body weight (KW/BW) was reduced  by 48% compared to the control group Figure , withoutd Figure . We alsod Figure .https://figshare.com/s/753eb0b268daba362bf6) (https://doi.org/10.6084/m9.figshare.14224199) appears the corporal composition and metabolic parameters for the studied groups. Serum glucose and cholesterol levels were significantly increased by 35.6% and 45.1%, respectively in the MHDF group. In the MHFD group there was an increase in fat tissue percentage compared to the control group . Similarly, the visceral adipose tissue was greater in the MHFD than in the C group . These alterations resulted in a reduction in lean tissue percentage in the MHDF compared to the control group . At the end of the study, the MHFD group exhibited mild metabolic dysfunction characterized by a significant increase in serum glucose and in cholesterol, without changes in triglycerides levels.In Table Renal dysfunction induced by MHFD was evaluated by albuminuria/UCr Figure  and urin2) in the kidney microphotographs from obese mice, whereas the C group exhibited a normal distribution in the glomerular areas, where most of the glomeruli were between 2001 and 2400 μm2  mRNA   mRNA levels were significantly increased in the kidney of obese mice  mRNA levels was observed in the HFD group compared to the C group  Figure . Interese Figure . In addip Figure .2O2) and by antioxidant enzymes mRNA levels. Urinary H2O2 was clearly enhanced in the MHFD group compared to the C group (10.5 ± 2.3 vs. 4.2 ± 0.5 pMol/24 h)  mRNA levels between the studied groups  mRNA levels were significantly reduced in the MHFD group compared to the C group  or glutathione peroxidase (Gpx) mRNA levels were found  Figure . Althougs Figure , catalasp Figure , whereasd Figure . We alsod Figure . We found Figure .Additionally, we studied the protein levels of BiP‐1 and CHOP involved in ER‐stress in response to energetic or cellular stress. A significant increase by almost 2‐fold in BiP‐1 was observed in the MHFD group compared to the C group Figure . Similar3.3Finally, we evaluated the mitochondrial dynamics by analyzing the protein levels of Mitofusin‐1+Mitofusin‐2 (Mfn1/2) and Dynamin‐related Protein‐1 (Drp1) by western blot, as subrogates of mitochondrial damage. We found a significant increase in Drp1 protein levels Figure , whereas4In this study, we demonstrated that feeding male mice with a lesser content of fat (45%) for a relatively short period induced relevant renal alterations characterized by a significant increase in albuminuria, glomerular hypertrophy, and increased excretion of urinary kidney injury biomarkers , in spite of a discretional metabolic impairment. All these abnormalities were associated with altered balance of mitochondrial fission/fusion, renal inflammation, and increased oxidative and ER‐stress.2O2 excretion and a significant reduction in catalase mRNA levels. In addition, FOXO3 protein levels were reduced in the kidney from the MHFD group, which could contribute to more ROS generation. In this regard, it has been shown that the conditional deletion of FOXO3 exacerbates acute kidney injury (AKI) to CKD transition by reducing epithelial autophagy and lowering SOD2 expression. On the contrary, FOXO3 activation in hypoxic tubules prevents CKD  can lead to ER‐stress in several tissues. ER is essential for the biosynthesis of proteins, lipid metabolism and regulation of calcium metabolism  pathways, including nuclear factor κB (NF‐κB), mitogen‐activated protein kinase (MAPK), and glycogen synthase kinase‐3β (GSK‐3β)  generation. The abnormal crosstalk between VEGF‐A and NO pathways results in an increased oxidative stress (Tufro & Veron, In this study, we also found a significant increase in Emerging evidence suggest that dysfunctional mitochondria have a primary role in the development of CKD. There are some hallmark features of mitochondrial dysfunction, such as, changes in morphology, remodeling, and decrease in both mitochondrial biogenesis and ATP production (Galvan et al., In summary, this model of MHFD‐induced kidney damage has given us a great opportunity to detect key initiators, suggesting relevant interactions that can lead to the development of CKD. At the same time, it has taught us the possible kidney disorders that can occur in obese patients. We also believe that this model could be a useful tool to study the role of antioxidant molecules and drugs to protect ER and mitochondrial dynamics as new therapeutic targets for obesity‐induced kidney damage.The authors declare no conflict of interest.Conception and design: EZ and NAB. Inclusion and following of experimental mice, and metabolic analysis: DCCR and EZ. Performed renal functional and structural analysis, as well as biochemical and molecular assessment: ASN, MAMR, RICB, and RPV. Acquisition of data: ASN, and MAMR. Analysis and interpretation of data: ASN, MAMR, and NAB. Drafting the article and revising it critically for important intellectual content: N.A.B, A.S.N. and MAMR.Supplementary MaterialClick here for additional data file.Table S1Click here for additional data file.


Solitary sexual activity is a free, safe, and accessible way to experience sexual pleasure. Despite these advantages, research on masturbation in later life is highly understudied. Using data from a cross-sectional probability-based survey of 3816 European adults , we explored several sociodemographic, health, attitudinal, and sexual behavioral factors associated with reported masturbation frequency. Across all countries, between 41% and 65% of men and 27% and 40% of women reported any masturbation in the preceding month. Satisfaction with sexual activity and attitudes related to disapproval of sex without love were significant predictors of reported masturbation in almost all countries and in both genders. Age, education, self-perceived health, and depression were for the most part predictive of men’s reported masturbation, but not women’s. Generally, those believing sex is beneficial to older people were more likely to masturbate, while less permissive attitudes decreased the likelihood of reporting masturbation. To improve healthy sexual aging, misinformation about masturbation and sexual attitudes in older people need to be addressed. Although there has been an increasing interest in older adults’ sexual behavior, most research to date has focused on partnered sexual activity and not on solitary sexual behavior  could be reached again and among those, another 561 declined participation after they had received the mail survey. The Ipsos provided response rates were calculated by dividing the net sample by those who agreed to participate and received a questionnaire, and this resulted in a response rate of 68% in Norway, 57% in Belgium, 52% in Denmark, and 26% in Portugal.Second, for those who agreed to participate Ipsos registered the personal information (names and addresses) and sent them an anonymous questionnaire for self-completion with a freepost envelope. Prospective participants in Norway, Denmark, and Belgium received postal reminders about a week after they got the questionnaire. The 2000 previously recruited participants in Portugal were reminded by phone. However, only three quarters of these participants . Relationship status was measured by asking: “Do you currently have a steady/ committed relationship with anybody? A steady/ committed relationship also includes married/ cohabiting persons.” Response options were 1 = yes, 2 = no, and 3 = unsure. Twenty-one participants checked the category “unsure” and were subsequently dummy coded into the “no” category (2 + 3 = 0).Education was assessed by asking: “What is your highest level of formal education?” To reflect the educational system in each country, response options differed somewhat across countries. To allow for comparisons across countries, the response categories were recoded into three matching levels of education: 1 = primary (6–8 years at school), 2 = secondary (9–10 years or 12–13 years at school), and 3 = tertiary  to higher university level .Religiosity was measured by the following question: “Apart from special occasions such as weddings, funerals and baptisms, how often do you attend services or meetings connected with your religion?” Responses were 1 = never, 2 = less than once a year, 3 = once a year, 4 = twice a year, 5 = once a month, 6 = once every two weeks, and 7 = once a week or more.αby country = 0.78–0.82).Self-estimated health was indexed via the following item: “In general, would you say your health is:” Scores were 1 = excellent, 2 = very good, 3 = good, 4 = fair, and 5 = poor. Depression during the past four weeks was measured with a brief psychometrically validated depression scale . Responses were made on a 5-point Likert scale ranging from 1 = strongly agree to 5 = strongly disagree. Attitudes reflecting the idea that sex is legitimized by love were assessed with two items : “Having one-night stands is wrong,” and “A married person having sexual relations with someone other than their spouse is wrong.” The items were averaged into a composite indicator and reverse-recoded, so higher scores indicated negative feelings about having sex without love. The following two items were used to tap into feelings that contemporary society is too sexualized : “There’s too much sex in the media nowadays,” and “Young people today start having sex too early.” The two items were averaged and scale scores reverse-coded, so that higher scores reflected less liberal views about sexuality in modern society. Attitudes toward sexuality and aging were indexed by two separate items. One item assessed participants’ evaluation of older adults’ sexual ability, compared with younger ages (“The ability to have sex decreases as a person grows older”). The scale scores were reverse-coded, with higher scores reflecting the belief that sexuality decreases with increasing age. The other item asked participants to evaluate the beneficial aspects of sexual activity in later life . The responses were reversed-recoded, with higher scores indicating more positive attitudes toward sexuality at older age.Sexual attitudes were assessed using 6 items retrieved from the ELSA Sexual Relationship and Activities Questionnaire  and standardized coefficient (β), and multiple correlations squared (R2).All statistical analyses were performed using IBM SPSS version 27.0 statistical software package. In order to adjust for recruitment-based distortion in sex, age, and region, census-based post hoc weighting was applied for each country. Various group differences were estimated by using the Chi-squared test Table  and 4. RTable Table Table bs = − 0.06). For Portuguese men, there was no relationship between age and masturbation activity. Education was significantly associated with reported masturbation, with higher levels of education associated with more masturbation in Danish (b = 0.29) and Norwegian men (b = 0.21). In contrast, among Portuguese men, higher education was related to less masturbation frequency (b = − 0.35). For all men, except in Norway, poorer physical health was significantly associated with less masturbation . Higher levels of depression significantly predicted more frequent masturbation in Belgian (b = 0.43) and Norwegian men (b = 0.36).Age was significantly and negativity related to masturbation frequency in Norwegian, Danish, and Belgian men . Compared to Danish men, where intercourse activity was associated with less masturbation (b = − 0.16), the opposite relationship was found among men in Portugal (b = 0.25). With respect to sexual attitudes, several patterns of findings emerged. Believing that sex should happen within a loving relationship was significantly and negatively related to the level of reported masturbation for all men , except those in Portugal. Less permissive attitudes toward sexuality in modern society were negatively associated with masturbation activity in Norwegian men (b = − 0.22). Danish men who had less positive attitudes toward later life sexuality were less likely to report frequent masturbation than those who did not endorse these attitudes (b = − 0.21). Finally, believing that sexual activity is beneficial for older people predicted higher levels of reported masturbation in Danish (b = 0.41) and Belgian men (b = 0.24).Regarding sexual satisfaction, we found that greater satisfaction with one’s level of sexual activity significantly predicted lower levels of masturbation in men across all four countries (b = − 0.03). Second, relationship status was significantly related to masturbation activity; being in a relationship predicted less masturbation frequency in Portuguese (b = − 0.39) and Norwegian (b = − 0.36) women. In Belgium and Norwegian women, greater intercourse activity predicted more masturbation . Similar to men, being more satisfied with one’s level of sexual activity was negatively related to masturbation for Danish (b = − 0.20), Belgian (b = − 0.18), and Norwegian (b = − 0.11) women. Women in Norway and Portugal who believed that sex should only happen within loving relationships were less likely to report frequent masturbation than women with more permissive sexual attitudes . Finally, believing that sexual activity is beneficial for older people predicted higher levels of reported masturbation in Danish (b = 0.16) and Belgian women (b = 0.13).Results from the multiple linear regression analysis on masturbation activity for women are presented in Table In this European four-country study, we assessed several sociodemographic, health, attitudinal, and sexual behavioral factors associated with reported masturbation frequency among men and women aged 60–75 years. Despite marked cross-cultural and gender differences in masturbation frequency, predictors of masturbation were in most instances more similar than different across the four countries. Satisfaction with the level of sexual activity was a significant negative predictor of masturbation in Norwegian, Danish, and Belgian men and women, and Portuguese men. Another important predictor of frequent masturbation was sexual attitudes. Specifically, attitudes reflecting the idea that sex is legitimized by love were associated with masturbation in Norwegian, Danish, and Belgian men, and Norwegian and Portuguese women. While age, education, self-perceived health, and depression were significantly related to men’s reported masturbation, few sociodemographic and health factors were associated with masturbation activity among women.Satisfaction with sexual activity was significantly related to masturbation in almost all countries. Men and women in Norway, Denmark, and Belgium, and men in Portugal were less likely to report frequent masturbation if they were satisfied with their level of sexual activity. The central role of sexual contentment in predicting solitary sexual activity may reflect older adults’ tendency to view masturbation as a second-best alternative that is only needed if one desires more sex or partnered sex is not satisfying. Although this finding supports the idea that masturbation functions as a substitute among contemporary older populations, it is possible that this will not be the case for the coming generations. Findings from Finland and Germany indicate cultural changes in the meaning of masturbation, with younger generations increasingly considering it as an independent source of experiencing sexual pleasure (Dekker & Schmidt, Similar to previous findings, the association between intercourse and masturbation frequency was not unequivocal (Regnerus et al., Another key finding points to the important role of sexual attitudes in predicting sexual behavior in aging men and women. Sexual attitudes mirror prevailing sociocultural norms and the attached meaning of sexual behavior within a cultural context (Masters et al., Overall, more sociodemographic factors were predictive of men’s masturbation than women’s. As found by others (Corona et al., In terms of health, we observed a negative association between self-estimated health and reported masturbation among older men in Denmark, Belgium, and Portugal. Previous evidence on the relationship between general health and masturbation has been mixed Das, . One posIn addition to self-rated general health, we assessed the relationship between negative mood and solitary sexual activity. Interestingly, our findings demonstrated a significant association between depression and masturbation among Norwegian and Belgian men; the higher the level of depression, the more likely the reported masturbation. Although this finding seems counterintuitive, it is consistent with previous research (Cyranowski et al., Finally, regarding relationship status, we found that women in Norway and Portugal were less likely to report frequent masturbation if they were in a current relationship. This finding seems to corroborate results from previous studies among varied age groups (DeLamater & Moorman, Our survey had several strengths, including the large probability-based samples and the use of similar sampling methods, identical measures, and age cohorts across four European countries. Several limitations, however, should also be acknowledged. The sample size in Portugal, as well as the response rate, was much lower than in the other countries. Due to a less reliable sampling method, an overrepresentation of urban individuals, and a high refusal rate, the selection bias was possibly most substantial among Portuguese participants (Boughner, Previous research on solitary sexual activity highlighted the links between cultural-dependent sexual socialization processes and masturbation and how these change across different generations (Kontula & Haavio-Mannila,


Kaxial) and radial kurtosis  and the DTI metrics including diffusivity and fractional anisotropy (FA) were measured within the whole thalamus and segregated thalamic subregions. Strategic correlations by group, thalamo‐frontal connectivity, and canonical discriminant analysis (CDA) were used to demonstrate the discriminative ability of DKI for SIVD, AD, and NC. Whole and segregated thalamus analysis suggested that DKI metrics are less affected by white matter hyperintensities compared to DTI metrics. Segregated thalamic analysis showed that MK and Kradial were notably different between SIVD and AD/NC. The correlation analysis between Kaxial and MK showed a nonsignificant relationship in SIVD group, a trend of negative relationship in AD group, and a significant positive relationship in NC group. A wider spatial distribution of thalamo‐frontal connectivity differences across groups was shown by MK compared to FA. CDA showed a discriminant power of 97.4% correct classification using all DKI metrics. Our findings support that DKI metrics could be more sensitive than DTI metrics to reflect microstructural changes within the gray matter, hence providing complementary information for currently outlined pathogenesis of SIVD and AD.Differentiating between subcortical ischemic vascular disease (SIVD), Alzheimer's disease (AD), and normal cognition (NC) remains a challenge, and reliable neuroimaging biomarkers are needed. The current study, therefore, investigated the discriminative ability of diffusion kurtosis imaging (DKI) metrics in segregated thalamic regions and compare with diffusion tensor imaging (DTI) metrics. Twenty‐three SIVD patients, 30 AD patients, and 24 NC participants underwent brain magnetic resonance imaging. The DKI metrics including mean kurtosis (MK), axial kurtosis (  The patients with SIVD and AD shared common inclusion criteria including (a) cognitive complaints with interference in daily activities, (b) Clinical Dementia Rating (CDR) score ≥0.5 Morris, , and (c)2.2The MMSE is a 30‐point questionnaire  of 220 mm, MTX of 384 × 224, slice thickness (SL) of 5 mm, and 21 slices. For DKI, spin‐echo diffusion echo‐planar images were acquired with 30 diffusion gradient directions and two b‐values  along each direction. The other parameters were TR of 6 s, TE of 68 ms, FOV of 240 mm, MTX of 96 × 96, SL of 2.5 mm, and 60 slices. For each slice, a total of 65 images were acquired for DKI reconstruction, including five un‐weighted images  and 60 diffusion‐weighted images . WMHs were rated according to the Fazekas scale from T2‐FLAIR images, which semiquantifies periventricular and deep WMHs depending on the size and confluence of the lesions  with an eight‐channel phased‐array head coil. The MRI protocols included three‐dimensional T1‐weighted imaging (3D‐T1), T2 fluid‐attenuated inversion recovery imaging (T2‐FLAIR), and DKI. For 3D‐T1, a spoiled gradient echo with RF‐spoiling scheme was utilized with repetition time (TR) of 7.904 ms, echo time (TE) of 3.06 ms, inversion time (TI) of 450 ms, flip angle of 12°, matrix size (MTX) of 240 × 240 × 160 with isotropic voxel size of 1 mm2.4https://afni.nimh.nih.gov) . The quantitative metrics of DTI  and DKI  were then calculated from the DKI model.All image processing and registration steps were performed using AFNI software (ov) Cox, . The proov) Cox,  before Dov) Cox, . Least‐sAll DKI and DTI metrics were calculated first in each subject's space. The metrics for the whole thalamus were calculated by averaging the metrics derived from the bilateral thalami . For alignment of DWIs, the b0 images were first registered to each subject's own 3D‐T1 images via 12‐parameter affine alignment. The abovementioned T1‐related transformation matrix and warp parameters were applied onto the T1‐aligned b0 images to generate aligned b0 images in the MNI space. Finally, the DKI parametric maps were registered to MNI space by applying the transformations previously determined on b0 images. To reduce potential registration bias surrounding the edge of ventricular space and thalamus, a ventricle mask was generated from averaged b0 images of the SIVD group by AFNI software to avoid including unwanted ventricular voxels. A segregated thalamus atlas in MNI space . Three thalamic subregions, including ANT, MedioD, and VA, and ipsilateral superior frontal gyrus were selected as seeding regions to investigate the thalamo‐frontal connectivity using a deterministic fiber tracking algorithm based on DTI orientations  univariate data with Z score >2 or Z score <−2 were regarded as outliers then removed before entering variables into further statistical analysis, and (c) quantile–quantile plot for each set of data were visually examined for identifying other outlier and confirming their numeric distribution to be approximately normal. Analysis of covariance was used to examine between‐group differences by controlling for the Fazekas scale, age , 0.01 ≤ ηp2 < 0.06 denotes a small effect, 0.06 ≤ ηp2 < 0.14 denotes a median effect, while 0.14 ≤ ηp2 denotes a large effect  the data of all participants were explored according to their transformed  Lakens, . Linear 33.1p < .001). The NC group was younger than the SIVD and AD groups (both p < .001), and had a higher education level than the SIVD group (p = .023). Of 23 cases with SIVD, 12 cases (52%) were identified as having thalamic lesions such as lacunes and dilated Virchow Robin space within the thalamus by their conventional MRI scans.Table 3.2Kradial and MK of the bilateral thalami , and Daxial, Dradial, and MD of the right thalamus . Significant differences between SIVD and NC existed in MK of the bilateral thalami and Daxial of the right thalamus . After also controlling for the Fazekas scale, the SIVD group had lower MK values within the right thalamus than the AD group .Table p = .001–.006), compared to a nonsignificant correlation in SIVD (p = .100). With regards to MD‐MK relationships, significant negative correlations were found in all three groups (p = <.001–.021). The relationships between the mean value  and its axial and radial components were also examined . For DKI (d–f), in contrast to the significant positive correlations between Kradial and MK in all three groups (p < .001), the correlations between Kaxial and MK varied widely among the three groups. There was a nonsignificant correlation in SIVD (p = .982), a trend of negative correlation in AD (p = .059), and a significant positive correlation in NC (p = .019).Figure d Figure . For DTI3.3Kradial showed more significant between‐group differences. For MK, the significant between‐group differences after controlling for age, education, and effect of multiple comparisons were found for (a) SIVD < AD in the PUL, ANT, VLD, VLV of the left side and the ANT, MedioD, VLD, VA, VLV of the right side (p = <.001–.006) and (b) SIVD < NC in the PUL, ANT, and VLV of the left side and the PUL, C, and VLV of the right side . For Kradial, the significant between‐group differences after controlling for age, education, and effect of multiple comparisons were found for (a) SIVD < AD in the PUL and ANT of the left side and the ANT, MedioD, and VLV of the right side (p = <.001–.004) and (b) SIVD < NC in the left ANT . There were only two regions  of Kaxial showing significant differences on plain controlling for age and education (p = .016–.036); none has passed the FDR. On considering the Fazekas scale in addition to age and education, significant between‐group differences of MK existed in the bilateral PUL  and right VLV , Kradial in the right VLV , and Kaxial in the right VLV . None has passed the FDR. For comparison, the assessment of diffusivity metrics is shown in Figure Daxial, the significant between‐group differences after controlling for age, education, and effect of multiple comparisons were found for SIVD > NC in the bilateral VLV . For Dradial, the significant between‐group differences after controlling for age, education, and effect of multiple comparisons were found for SIVD > AD in the bilateral VLV and right VLD . For MD, the significant between‐group differences after controlling for age, education, and effect of multiple comparisons were found for (a) SIVD > AD in the bilateral VLV and right VLD (p = <.001–.002) and (b) SIVD > NC in the bilateral VLV . After also controlling for the Fazekas scale, none of the diffusivity metrics showed significant between‐group differences in any segregated thalamic regions.Figure 3.4p = .005, uncorrected) and right MedioD , while MK connectivity showed more significant between‐group differences, including SIVD < AD in all subregions , and SIVD < NC in all thalamic subregions  except for the right VA. Most aforementioned MK results passed the FDR examination, including SIVD < AD in all subregions (p ≤ .001) and SIVD < NC in all the left thalamic regions and the right MedioD . None of the FA results passed the FDR. No significant results were noted after also controlling for the Fazekas scale.Figure 3.5Kradial, and Kaxial) as independent variables. Two discriminant dimensions were derived, and suggested the group means appeared to differ as Wilks' Lambda values  of Function 1 to 2 and 2 were 0.033 and 0.246, respectively (both p ≤ .001). The canonical correlations for the first and second dimensions were 0.93 and 0.86, respectively. The Function 1/2 at group centroids are 0.048/2.626 for SIVD, 2.624/−1.142 for AD, and −3.325/−1.090 for NC. For discriminating these three groups, this method showed a discriminant power with 97.4% correct classification (p = .001). The discriminant power for all of the other combinations of diffusion MRI metrics is provided in Table Z value >2. The results by 1,000 times of simple sampling from the biased‐corrected and accelerated percentile bootstrap supported the original report, in which using all DKI metrics and composite metrics (MK + MD + FA) from segregated thalamic data equally achieved the best discriminant results. It was also noteworthy to point out a trend of compromised classification accuracy for those trials using single or two parameters derived from the overall thalamus data .Figure 3.6Kaxial within the bilateral PUL, and right C, (b) Kradial within the bilateral ANT, left VLV, and right MedioD, and (c) MK within the left PUL, bilateral ANT, and left VLV. MMSE showed significantly positive correlations with (a) Kradial within the left PUL and right ANT and (b) MK within the left PUL and ANT. The aforementioned variables were first entered into preliminary stepwise linear regression models, in which the Mahalanobis distance of each independent variable was derived. The following linear regression was conducted after removal of outliers, showing that the Kaxial in the right PUL  and MK in the right ANT  were the best metrics to predict dementia severity. No best predictor to predict global cognitive score was identified.Given that the best discriminant power was achieved using DKI metrics, correlations between DKI metrics and cognitive measures were examined Table . After c4In this study, we demonstrated that DKI metrics could more sensitively reflect the microstructural changes within the thalamus than DTI metrics. DKI can discriminate between SIVD, AD, and NC through multiple approaches, including intrinsic DKI metrics correlation, segregated thalamic assessment, thalamo‐frontal connectivity, and discriminant analysis. The utilization of DKI for delineating the thalamic microstructural changes could be a potentially useful diagnostic approach for dementia, which is consistent with the currently outlined pathogenesis of SIVD and AD  in segregated thalamic regions indicates that SIVD patients may have greater cellular damage within thalamic regions than AD patients even with comparable global cognition and consideration of demographic variables. Several reports addressing DKI changes within the thalamus among dementia patients provided interesting parallels to our current work. In one study recruiting patients with AD, volume reduction of the subcortical nuclei was reported to predate DKI modifications, while kurtosis metric within thalamus was unremarkable and of no correlations with MMSE score , and the contributive portion within the thalamus could be relatively small (~3%) .