Hypogonadism is a worldwide problem among men causing sexual, physical and mental problems. Testosterone therapy is the first‐choice treatment for male hypogonadism, with several side effects, that is, subfertility. Clomiphene citrate (CC) is an alternative off‐label therapy for a certain group of hypogonadal males, especially for those with an active or future child wish. There is scarce literature in usage of CC for men with hypogonadism. The aim of this retrospective study was to evaluate the effectiveness and safety of CC for hypogonadal males.In this single‐centre study, men treated with CC for hypogonadism were evaluated retrospectively. Primary outcome was hormonal evaluation including total testosterone (TT), free testosterone (FT), luteinizing hormone (LH) and follicle stimulating hormone (FSH). Secondary outcomes were hypogonadal symptoms, metabolic and lipid parameters, haemoglobin (Hb), haematocrit (Ht), prostate specific antigen (PSA), side effects, the effect of a trial without medication and potential predictors for biochemical and clinical response.In total, 153 hypogonadal men were treated with CC. Mean TT, FT, LH and FSH increased during treatment. TT increased from 9 to 16 nmol/L, with a biochemical increase in 89% of the patients. In patients who continued CC treatment, an increased level of TT persisted after 8 years of treatment. With CC treatment, 74% of the patients experienced hypogonadal symptom improvement. LH at the lower normal range before CC treatment was predictive for better TT response. During CC therapy, few side effects were reported and no clinical important changes in PSA, Hb and Ht were found.Clomiphene citrate is an effective therapy on short and long term, improving both clinical symptoms and biochemical markers of male hypogonadism with few side effects and good safety aspects. This retrospective study shows the good short and long‐term outcomes of clomiphene citrate on testosterone in hypogonadal males with good safety aspects. CC is a selective oestrogen receptor modulator, occupying oestrogen receptors in the hypothalamus and pituitary gland, leading to increased secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) and so, stimulating testicular endogenous testosterone production and preserving spermatogenesis.Since the 1960s, CC has been often used for ovulation induction in females. For males, the United States Food and Drug Administration (FDA) did not approve the medicine with reason of unclear clinical effect because of the lack of well‐controlled and well‐powered controlled trials.To support the outcomes in the scarce literature on the efficacy of CC therapy, the purpose of this retrospective study was to evaluate the effectiveness and safety of CC therapy for male hypogonadism.22.1A retrospective, single‐centre study was conducted in the University Medical Center (UMC) Utrecht. Hypogonadal men, who wanted to preserve their endogenous testicular function or men who had other reasons for not willing to use TTh and therefore treated with CC within the time frame from January 2012 to January 2021 were included. Ethical approval for this retrospective study was obtained from the local Research Ethics Committee, UMC Utrecht, the Netherlands (WAG/mb/20/500309). All patients were informed that CC is used as an off‐label therapy.2.2Men aged above 18 years with low Hypogonadal symptoms were scored by the experienced treating physicians, no validated questionnaires were used. Patients were excluded in case: hypogonadotropic hypogonadism, no assessment of TT/FT was done before or during treatment, CC was used in combination with TTh, human chorionic gonadotrophin (hCG) or aromatase inhibitors (AI) or if these therapies were used within 12 months of CC initiation.2, testicular volume, medical history  and additional medication for erectile dysfunction during treatment were obtained manually from patient files.With the clinical knowledge in 2012 based on expert opinion at that time, men received CC, with a starting dosage of 25 mg every other day, or 25 mg a day in case of bodyweight above 100 kg. Dosage was raised to 50 mg daily when there was little biochemical response. Data of duration of CC therapy, patient characteristics , obesity (BMI >25 kg/m))2.3Total testosterone (TT) from early morning blood draws (<11 am) (laboratory standard (lab. st.): 7.0–31 nmol/L)Free testosterone (FT) from early morning blood draws (<11 am) (lab. st.: 230–600 pmol/L)LH (lab. st.: 1.0–9.0 IU/L)FSH (lab. st.: 1.0–19.0 IU/L)Estradiol (lab. st.: 70–200 pmol/L)Sex‐hormone binding globulin (SHBG) (lab. st.: 10–40 nmol/L)Albumin (lab. st.: 35.0–50.0 g/L)Prolactin (lab. st.: 0.10–0.65 IU/L)Study primary outcome was hormonal assessment at baseline and at 1, 3, 6 and 12 months during CC therapy and annually thereafter measured with immunoassay  the immunoassay was replaced in 2014, however, hormonal values were comparable using both immunoassay tools:2.4Presence of hypogonadal symptoms measured at baseline. Subjective symptom improvement was evaluated during follow‐up (FU).BMI, HbA1c, serum total cholesterol, triglycerides, low‐density lipoprotein (LDL)‐, high‐density lipoprotein (HDL)‐ and non‐HDL cholesterol, before and during treatment.Self‐reported side effects were noted during treatment.Haematocrit (Ht), haemoglobin (Hb), thrombocytes, alanine aminotransferase (ALAT), aspartate aminotransaminase (ASAT), alkalic phosphatases (AF), gamma‐glutamyl transferase (gGT) and total prostate‐specific antigen (PSA) before and during treatment.Data on the presence of a trial without CC therapy, the reason and duration of trial without medication (TWM), TT, FT and symptom changes during TWM, and whether patients restart CC therapy after TWM, were collected.The classification of biochemical responder, non‐responders or a reversed effect on TT. Patients were classified as biochemical responder if there was a significant increase in TT and/or FT. Non‐responders were classified if there was no change in TT and/or FT. A reversed effect was defined as a significant decrease in TT and/or FT.2.5p‐value ≤.05 was considered statistically significant. Statistical analysis was performed by using SPSS .Data were recorded and tabulated with Microsoft Excel® software. Values were presented as number (%), mean (standard deviation [SD]) or median (25th–75th percentile interquartile range [IQR]) if data were not normally distributed. Normality of continuous variables was tested with the Shapiro–Wilk‐test, histograms and normality‐quantile plots. Paired sample t‐test was used to compare before‐ and during treatment hormonal levels, safety aspects, serum lipids, BMI. The Wilcoxon signed ranked test was used if the data did not meet the assumptions for normality. Assumptions for correlation analysis were normality, homoscedasticity and linearity. The Pearson test was used for bivariate correlation, if data did not meet the assumption for normality the Spearman test was used. A 33.1n = 22, 14%) in case of minimal TT response or decreased in case of side effects within the first year . In 145 patients (95%) pre‐treatment TT levels were <12.1 nmol/L. Two patients (1%) had TT level <15 nmol/L, but FT levels <243 pmol/L. Six patients (4%) had TT levels <15 nmol/L and FT levels <280 pmol/L. All of these eight patients with TT levels >12 nmol/L and < 15 nmol/L had clear hypogonadal symptoms.In total, 173 male patients treated with CC were screened for inclusion. Twenty patients were excluded with reason; four used CC combined with TTh or hCG, six had no wash‐out period longer than 12 months between TTh and before start of CC therapy, two had no before treatment TT and/or FT measurement and eight were lost to FU before any hormonal evaluation. Finally, 153 patients were included for analysis. Data on patient characteristics, comorbidities and dosage at start of treatment, are provided in Table 3.2p ≤ .05)  were classified before treatment as hypergonadotropic hypogonadism. In this hypergonadotropic hypogonadism group 6/16 were biochemical responders (38%), of which the patient with Klinefelter. The Klinefelter patient showed an increase in TT of 6 nmol/L tot 14 nmol/L and FT from 140 pmol/L to 280 pmol/L during CC treatment. In the orchiectomy patient group, 11/41 patients were hypergonadotropic, and 9/11 patients hypergonadotropic hypogonadal orchiectomy patients were biochemical non‐responders. Two hypergonadotropic patients (1%) had a reversed response of TT after start of CC therapy (see section 4.8).3.3n = 11) only the two biochemical responders described symptom improvement.Table 3.4p < .05). However, it is unclear if all the values were based on fasting blood tests. BMI and triglycerides did not change before and during treatment. Data are available in of the Appendix Total cholesterol, LDL‐, HDL‐ and non‐HDL cholesterol decreased during treatment (3.5n = 5), agitation (n = 4), visual changes (n = 2), nipple tenderness (n = 2), mood change (n = 1), weight gain (n = 1), blurred vision (n = 1), headache (n = 1), dizziness (n = 1).There were no changes in Ht, Hb, thrombocytes, liver enzymes and PSA , presence of side effects  , run out of medication , or extremely elevated estradiol . During TWM, TT decreased significant in 48 of these 52 patients (94%), with a mean TT during CC treatment of 19.1 nmol/L(5.6) and during TWM of 10.8 nmol/L (3.1) (p = .000). Of these patients in whom TT decreased during TWM, 22 patients were obese and 16 patients had an orchiectomy in the past of which two were hyperchonadotropic. The 4/52 patients in whom TT did not decreased, three of them had an orchiectomy in the past, two patients had TT levels between >12 nmol/L and 15 nmol/L before start treatment and LH levels before start of treatment were in all patients not available. Mean FT during CC therapy was 373.2 pmol/L (126.6) and during TWM 203.3 pmol/L (63.5) (p = .000). Clinically, 20/52 patients noticed a worsening of symptoms during TWM (38%), fatigue (n = 12), libido (n = 8), depressive thoughts (n = 3), erectile dysfunction (n = 2), agitation (n = 2). Eighteen /52 patients (35%) restarted CC therapy after the TWM, three of them were the patients with side effects. In 44% of the patients without noticing a difference in symptoms during TWM, TT levels were >12 nmol/L during TWM. Eight/35 patients (25%) who did not restart CC after TWM had TT levels before initiating CC treatment of >12 nmol/L. There was no correlation between discontinuation CC therapy and pre‐treatment TT levels, age or hypogonadism aetiology. Only 14/35 (32%) of the physician‐initiated TWM patients continued CC therapy after TWM.Fifty‐two patients had a trial without CC therapy (34%), with a median duration of 3 months . Reasons for TWM were; physician‐initiated testing of CC effect . A second correlation was found between a lower range of normal LH and FSH before treatment and a higher increase in TT at first measurement during treatment . A third correlation was found between a low‐normal LH before treatment and improvement of symptoms with CC therapy . Further, a correlation was found between a lower increase in TT at first measurement and an orchiectomy in the medical history . No correlations were found between first measurement during TT treatment and age, BMI, testis volume, and different subgroups of patients with a presence/history of overweight, cardiovascular disease, chemo‐ and/or radiation therapy, DM, Klinefelter, other malignancies.A correlation was found between symptom improvement during treatment and a higher increase in TT at first measurement during treatment .Levels of TT, FT, LH, FSH and SHBG increased after start treatment. Increased testosterone levels lasted with ongoing treatment until 8 years of FU. This improvement of hormone levels has been described in previous studies, showing TT at baseline ranging between 7 and 11 nmol/L and at first measurement during treatment ranging between 16 and 24 nmol/L  there was clinical improvement (increased libido). Considering of inclusion of these patients is supported by the study of Zitzmann (2006), showing that testosterone deficiency symptoms may also be seen with TT levels as high as 15 nmol/L.n = 29–120) indicated that after 24–52 months of CC use, hormone levels were stable.n = 27) demonstrating during trial without CC therapy that 78% of the patients had decreased TT levels of <10 nmol/ after 3 months, and all patients had decreased TT levels of <10 nmol/L after 6 months.It is unknown whether CC is effective on the long term. In our study, there was a sustained biochemical effect of CC, with the longest long‐term FU of 8 years. Three earlier studies (n = 178) who showed that younger age (<55 years) and absence of diabetes is a predictor for better TT response.Furthermore, this retrospective study is the first study showing that a better early response of TT is correlated with a better clinical response. The study also shows that low‐normal LH before treatment predicted symptom improvement and a higher increase in TT during CC treatment. This potential predictor was also seen in the study of Mazzola et al. (2014).n = 77).n = 76), who found only one patient with increased Ht (0.51) during CC therapy.n = 363).n = 1180)n = 13).Side effects were reported in 10% of our population and included agitation, hot flushes, nipple tenderness, mood changes, weight gain, visual changes, headache and dizziness. These side effects were also reported in the literature, with a prevalence between 4% and 11%.A reversed effect of CC on testosterone levels was found in two young hypergonadotropic patients (<1%). Although this phenomenon has been described before in the literature, the physiology of this paradoxical effect of CC is unclear.There are several limitations to address for this study. First, it has a retrospective character, with data obtained manually from patient files. There were no strict inclusion criteria with predefined baseline and outcome measurements. No validated instrument for reporting hypogonadal symptoms could be used because there does not exist a Dutch validated hypogonadism questionnaire, but self‐reported hypogonadal symptoms and subjective symptom improvement was evaluated during follow‐up. Second, because this was not a placebo controlled trial, it is difficult to conclude if clinical improvement is the result of CC therapy. Third, in 130 patients, the presence of side effects was not reported, and side effects were not systematically classified. This may have caused an underestimation of the actual prevalence of side effects. Fourth, dosage at start of therapy was chosen based on weight, based on expert opinion in 2012. For future research and for clinical practice it is recommended with this gap in knowledge to start with the minimal dosage not depending on weight and to titrate dosage up if there is no sufficient effect. As last to mention, the included patients had different origins of hypogonadism. This is causing heterogeneity and does not imply that the effect of the study do count for all different subgroups. Nevertheless, with the encouraging outcomes of this study, we believe it is very valuable to execute a prospective study, especially for young hypogonadal males with an active or potential future child wish who are not eligible for TTh, with clear inclusion criteria, adequate measurement of hormonal levels, symptomatology with the usage of a validated questionnaire and registration of side effects.5Clomiphene citrate therapy for hypogonadal males shows promising effect on both clinical symptoms and the biochemical testosterone insufficiency with few reported side effects and good safety aspects compared with TTh. Therefore, it is worth to be considered especially in males presenting with symptoms of hypogonadism and low testosterone who wish to preserve their testicular function and are not eligible for TTh. Low to normal LH at baseline seems to predict biochemical and clinical effectiveness. Further research with clear inclusion criteria, adequate measurement of hormonal levels and registration of symptoms and side effects is recommended.M.H., T.L., V.d.K., J.B., L.d.K., H.v.B., contributed to the design, to the analysis of the results and to the writing of the manuscript.The authors did not receive funding for this study.There is no conflict of interest.Appendix S1–S2.Click here for additional data file.


The cerebrospinal fluid (CSF) provides mechanical protection for the brain and serves as a brain dispersion route for nutrients, hormones, and metabolic waste. The CSF secretion rate is elevated in the dark phase in both humans and rats, which could support the CSF flow along the paravascular spaces that may be implicated in waste clearance. The similar diurnal CSF dynamics pattern observed in the day-active human and the nocturnal rat suggests a circadian regulation of this physiological variable, rather than sleep itself. To obtain a catalog of potential molecular drivers that could provide the day–night-associated modulation of the CSF secretion rate, we determined the diurnal fluctuation in the rat choroid plexus transcriptomic profile with RNA-seq and in the CSF metabolomics with ultraperformance liquid chromatography combined with mass spectrometry. We detected significant fluctuation of 19 CSF metabolites and differential expression of 2,778 choroid plexus genes between the light and the dark phase, the latter of which encompassed circadian rhythm–related genes and several choroid plexus transport mechanisms. The fluctuating components were organized with joint pathway analysis, of which several pathways demonstrated diurnal regulation. Our results illustrate substantial transcriptional and metabolic light–dark phase–mediated changes taking place in the rat choroid plexus and its encircling CSF. The combined data provide directions toward future identification of the molecular pathways governing the fluctuation of this physiological process and could potentially be harnessed to modulate the CSF dynamics in pathology. The cerebrospinal fluid (CSF) secretion rate and the associated intracranial pressure are elevated in the dark phase in both humans and rats, irrespective of sleep pattern. Potential molecular drivers may be found within the catalog of fluctuating choroid plexus transcripts and metabolites of the surrounding CSF. Both of these components demonstrate substantial diurnal regulation, which, on their own or jointly, could contribute to the light–dark phase–mediated modulation of the CSF dynamics.Cerebrospinal fluid (CSF) fills the ventricular spaces and encircles the mammalian brain and spinal cord, providing protection against mechanical insult, and serves as a dispersion route for nutrients, metabolic waste, and signaling molecules between various brain cells and structures. Aberrant accumulation of CSF is observed in connection with a number of cerebral pathologies, i.e. congenital or secondary hydrocephalus and stroke-induced cerebral edema, which may associate with life-threatening elevation of the intracranial pressure (ICP) if left untreated. Standard care of such conditions relies on invasive neurosurgical procedures including ventriculoperitoneal shunt insertion, which, although lifesaving, are hampered by frequent shunt failures, infections, and other complications , 2. EffiThe majority of the CSF is produced by the choroid plexus, which is a specialized epithelium localized in each of the four ventricles . The choHere, we determine the light–dark phase fluctuations in the rat choroid plexus transcriptome and in the rat CSF metabolome to create a catalog of potential regulatory properties of these compartments that could contribute to the circadian modulation of the CSF secretion rate.N-acetyl-tyrosine, and N-acetyl-glutamine . One hundred twenty-six different metabolites were identified in the CSF Table  and werene Table .To determine whether the detected metabolites were arranged into relevant biological pathways, these were grouped by pathway analysis . Pathway analysis assesses the biological relevance of pathways by identifying the collected impact of pathway-specific metabolites detected, against the probability of the metabolite composition observed being a function of that particular pathway. The biological pathways detected in the rat CSF were mainly related to amino acid metabolism 6/12), with the remaining pathways belonging to either carbohydrate metabolism, metabolism of other amino acids, genetic translation, or nucleotide metabolism  with CSF extracted from anesthetized rats in the peak dark phase . A principal component analysis (PCA) plot of the tested metabolites demonstrated that the CSF extracted at these two time points separated into two groups Fig. . Among t2-fold change) in the diurnally modulated transcript fluctuations  and from rats in the peak dark phase (zeitgeber time 20 h) Table . A PCA pons Fig.  and S7, ons Fig. . Figure ons Fig. A. The da+,HCO3− cotransporter 2 NBCe2 , the water channel aquaporin 1 , the sodium-coupled neutral amino acid transporter 3 SNAT3 , and the folate transporter 1 FOLT . Among the transporter genes that were most up-regulated in the dark phase, we detected the monocarboxylate transporter MCT6 , the organic anion transporter OATP1 , the Na+-dependent phosphate transporter 2 PIT2 , the potassium-dependent sodium/calcium exchanger NCX4 , and the transient receptor potential vanilloid 4 ion channel TRPV4 . These data could suggest that diurnal regulation of choroid plexus transporters could contribute to the fluctuating CSF secretion rate. However, with the relatively modest numerical changes, we speculated whether differences in choroid plexus metabolic activity could modulate the energy-dependent transporters supporting the CSF secretion and, thus, in addition, determined the diurnal fluctuation in transcripts encoding mitochondrial genes  and adenosylhomocysteinase like 2   enrichment analysis was performed on the differentially expressed genes with exclusion of the 902 genes  that did not have a protein class assigned. Eleven different protein classes each contained ≤2% of the transcripts and were combined in “Others” . The majority of the differentially expressed genes encode metabolite interconversion enzymes (15%) and protein-modifying enzymes (15%), followed by the category comprising transporters (9%) and various other smaller categories Fig.  and S10.s Tables  and S11.s Tables , inset. s Tables , and dius Tables , inset. se) Fig. .P-value change occurring with the inclusion , which is involved in regulation of CLOCK genes  at 2 cm, and 10% sigh for a ∼400-g rat. Body temperature during the experiment was maintained at 37°C by a homeothermic monitoring system (Harvard Apparatus). A cisterna magna puncture was performed with a glass capillary , and approximately 100 µl CSF was collected and centrifuged . The supernatant was transferred to a new microtube  and immediately placed on dry ice prior to placement at −80°C until analysis. Rat choroid plexus  was acutely isolated following sacrificing of the anesthetized rat and stored in RNAlater (Sigma) at −80°C until analysis. All animal experimental works conformed to the legislations for animal protection and care in the European Community Council Directive (2010/63/EU) and were approved by the Danish Animal Experiments Inspectorate (license no. 2016-15-0201-00944).Male Sprague–Dawley rats, 9 weeks old (Janvier), housed in temperature-controlled facilities with a 12:12-h light cycle, were employed for the study. The rats had access to water and food ad libitum and were randomly assigned to experimental groups. Samples were collected during the light phase  and in the dark phase . The time points were chosen according to Borjigin and Liu , where tn = 23, 11 light phase and 12 dark phase) was carried out by MS-Omics ApS  using an untargeted metabolomic approach. Semipolar metabolite separation was achieved with a slightly modified version of the protocol described by Doneanu et al.  and library preparation was performed by Novogene Company Limited with NEB Next Ultra RNA Library Prep Kit (NEB) prior to RNA sequencing  on an Illumina NovaSeq 6000 (Illumina). Raw data are available at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (GSE228866). Quality control and adapter removal were done by Novogene. The 150 base paired-end reads were mapped to rat reference genome Rnor_6.0.104 (Rattus norvegicus) using Spliced Transcripts Alignment to a Reference (STAR) RNA-seq aligner (v. 2.7.9a) .RNA extraction from the excised choroid plexus  2.7.9a) . The mape GSE2288. Quality 2.7.9a) , and raw 2.7.9a) . Gene in 2.7.9a) . PCA plo 2.7.9a) . Differedatabase  with a cdatabase  and genedatabase  to ascria . The mR. norvegicus and parameters were set to hypergeometric, relative-betweenness centrality and combined P value. Significant pathways were defined as having a FDR < 0.10, with the perturbation determined according to the combination of significance and impact factor  Database.Metabolic pathways were identified with pathway, and joint pathway, analysis employing MetaboAnalyst , using “t factor , 75. ForPNAS Nexus online.pgad262_Supplementary_DataClick here for additional data file.


Elateriospermum tapos (E. tapos) is proven to improve cognition and modulate the stress hormone due to its high concentration of flavonoids. However, the effects of medicinal plant integrated probiotics in F1 generations warrants further investigation. Thus, this study aimed to study the effect of E. tapos yoghurt on the maternal obesity induced cognitive dysfunction and anxiety in female offspring. In this study, female Sprague Dawley rats were fed with normal chow (n = 8) or high fat diet (n = 40) across pre-pregnancy, gestation, and weaning. The treatment with different concentrations of E. tapos yoghurt  were initiated in the obese dams upon post coitum day 0 up to postnatal day 21 (PND 21). Female offspring were weaned on PND 21 and body mass index, waist circumference, lee index, behavior, metabolic parameter, and antioxidant status were analyzed. The result shows that the female offspring of the 500 mg/kg E. tapos yoghurt supplemented group shows a decreased level of insulin, fasting blood glucose, cholesterol, triglycerides, LDL, low fat tissue mass with a high level of HDL, and an increased level of antioxidant status in the hypothalamus. The behavioral assessment proves that the female offspring of the 500 mg/kg E. tapos yoghurt supplemented group exhibits a high recognition index on novel object/place with low anxiety-like behavior in an open field test. In conclusion, our data signify the beneficial effect of early intervention in obese dams on the transgenerational impact on female offspring’s metabolic profile, cognitive performance, and anxiety-like behavior.Pre-pregnancy weight gain induces dysregulation in the metabolic profile of the offspring, thereby serving as a key factor for cognitive decline and anxiety status in the offspring. However, early probiotic supplementation during the gestational period is linked with improved metabolic health. At the same time, a natural plant known as  Maternal obesity has raised the concern of public health as it is currently shows an increasing trend similar to the obesity prevalence rate globally. A study based on panel data model on global maternal obesity shows that there were more than 14 million pregnant obese women worldwide in the year of 2014 . There iElateriospermum tapos (E. tapos), comprises of high levels of different types of flavonoids, which are proven to improve endogenous antioxidants in the brain [E. tapos seed is known for its high level of protein, unsaturated fatty acids, oleic acid, linoleic acid, omega-3 essential fatty acid [E. tapos seed have a molecular weight of less than 600 daltons, implying its capacity to cross the placenta and blood brain barrier to exhibit its effects. In addition, preliminary studies on E. tapos extract have shown that it possess the ability to prevent fat absorption by inhibiting the action of pancreatic lipase [E. tapos extract could inhibit the activity of α-amylase, α-glucosidase, and lipoprotein lipase [E. tapos seed as an effective anti-obesity agent. In fact, E. tapos seed is scientifically proven to modulate the stress hormone in F1 generation of obese dams [E. tapos into a probiotic could be more effective in inhibiting transgenerational effects of maternal obesity. Thus, this study aimed to study the effect of E. tapos yoghurt on the maternal obesity induced cognitive dysfunction and anxiety in female offspring. Accordingly, the outcome from this study could provide new insight into the beneficial effect of novel formulated first medicinal plant integrated yoghurt (E. tapos yoghurt) on modulating female offspring’s metabolic profile, cognitive performance, and anxiety-like behavior.Thus, our recent discovery demonstrates that probiotic intake during the gestational period modulates the metabolic profile of pregnant obese women and the offspring . Concurrhe brain . Besidestty acid , saponintty acid  which poc lipase . A follon lipase , demonstese dams . Hence, Elateriospermum tapos (E. tapos) was supplied by the Research Centre of Forest Research Institute of Malaysia and was sent to Herbarium Biodiversity Unit at Universiti Putra Malaysia (UPM) for confirmation under the voucher code of UPM SK 3154/17. Upon confirmation, the extraction of E. tapos seed was done using 2 L of 95% of ethanol. The seed was soaked in ethanol for 7 days, filtered using rotary evaporator. The crude extract was mixed with maltodextrin powder and oven dried overnight until the powder form of E. tapos was obtained. The E. tapos powder was then refrigerated at −20 °C until further usage [er usage . E. tapos powder. For this, 100 mL of full cream supplied by Dutch Lady Purefarm UHT was boiled for 30 min, cooled down at room temperature until the temperature dropped to 40–45 °C. Starter culture obtained from a New England cheesemaking supplier was then added to the pre-boiled milk and incubated in a yoghurt maker (Pensonic PYM-700) overnight. The following day, the yoghurt was refrigerator for 12 h and 2 g of E. tapos powder was added to the yoghurt [Plain yoghurt was formulated as an initial method before being integrated with  yoghurt . n = 48) were obtained from Saintifik Entreprise, and were housed in an animal facility in UPM, with a controlled 12 h light and dark cycle (22 ± 3 °C). All rats were fed with standard rat pellet (Gold Coin Feedmills (M) Sdn Bhd, Selangor, Malaysia) with free access to water (ad libitum) [n = 40) were fed a high fat diet (HFD) pellet comprising of 43% of carbohydrates, 17% of protein, and 40% of fats for 16 weeks to induce obesity [n = 8) were fed a standard rat pellet with ad libitum. The obesity was confirmed by measuring the significant increase of 13% of mean value compared to the control group [All animal protocols were conducted in accordance with the guideline approved by the Institutional Animal Care and Use Committee (IACUC), UPM (UPM/IACUC/AUP-R025/2022). The ethics was approved by IACUC, UPM on 24 June 2022. In this, female rats (libitum) . After ool group . E. tapos yoghurt concentrations were given through oral administration to the obese dams up to the postnatal day (PND 21). The litters were adjusted to at least 8 female offspring per dams prior to the treatment. The treatment groups are as follows: normal chow and saline (NS), HFD and saline (HS), HFD and yoghurt (HY), HFD and 5 mg/kg of E. tapos yoghurt (HYT5), HFD and 50 mg/kg of E. tapos yoghurt (HYT50), and HFD and 500 mg/kg of E. tapos yoghurt (HYT500).Upon successful obesity induction, all female rats were mated with standard rat pellet fed male Sprague Dawley rats by co-housing 2 female rats with one male rat for 5–7 days. During the mating period, the female rats were checked for pregnancy daily at 8 am by manual palpation followed by vaginal smears. The presence of sperms were observed using a microscope  and the first day of sperm detection was recorded as post coitum day 0 . Upon coOn PND 21, the body length, body weight, and waist circumference of the female offspring were recorded. The BMI and lee index were calculated using the formula below. 2 (cm2), in which the BMI > 0.687 g/cm2 was considered as obese [(a) Body mass index (BMI) = body weight (g)/lengthas obese . (b) Lee index = cube root of body weight (g)/length (cm), in which the Lee index value > 310 g was considered obese . The anxiety-like behavior of female offspring was accessed using an open field test (OFT) during the light phase of the illumination cycle. In this, a PVC box, measuring 80 cm width × 80 cm length with a 50 cm of height, provided by Muromachi Kikai Co., Tokyo, Japan, was used. The box was divided into two regions by an imaginary line  drawn in the recording software . Upon setting up the apparatus for OFT, the rats were dropped from one particular area in the box for 5 min. The total distance traveled (m) in the central region, time of exploration in the central and outer region, were recorded using the recording software . For the novel object recognition test (NORT) and place recognition test (PRT), a PVC box, measuring 80 cm width × 80 cm length with a 50 cm of height, provided by Muromachi Kikai Co., Tokyo, Japan, was used. For NORT and PRT, the rats were allowed to explore the empty box for 10 min for 2 days continuously. On the 3rd day, during the trial phase, the rats were allowed to explore two identical objects (mugs) for 5 min, followed by another 5 min of a retention phase. During the test phase, a new object was replaced with one of the mugs and the explorative time at the novel object by the rats was recorded using the ANY-maze™ Video Tracking System. A similar protocol was used for PRT, however, in PRT, two identical objects were placed in one selected location (north and south) initially. During the test phase, one of the identical objects was transferred to the new location (west). In PRT, the explorative time at the new location by the rats was recorded using the ANY-maze™ Video Tracking System . On PND 21, all female offspring were fasted for 12 h prior to the fasting blood glucose (FBG) analysis. For this, the tail of the rats was pricked and the blood was sucked using a glucose strip, and the reading was recorded using a glucometer  . The ratThe hypothalamus was minced into small pieces and diluted in 1:15 of phosphate buffer saline prior to homogenization . The samples were then sonicated 3 times continuously using an ultrasonic cell disrupter  in which each cycle lasted for 20 s. The final samples were collected, centrifuged for 20 min at 5000 rpm, and the level of the ferric reducing ability of plasma (FRAP) and glutathione (GSH) were measured using double-antibody sandwich enzyme-linked immunosorbent assay ELISA kits by Cayman Chemical Company, Ann Arbor, MI, USA . MeanwhiSPSS version 27.0  was used to analyze data obtained from this study. The normality test was completed on all data to ensure normal distribution. Two-way ANOVA was used to compare between all 6 groups for BMI, lee index, waist circumference, recognition index, antioxidant status, and metabolic parameter. This was followed by post hoc to determine the significance among different groups through Tukey HSD correction.p < 0.05) compared to NS. The BMI of HYT5, HYT50, and HYT500 is significantly low (p < 0.05) compared to HS. There is no significant difference (p > 0.05) in BMI between HYT5 and HYT50 compared to HY. The BMI of HYT500 is significantly low (p < 0.05) compared to HS, HY, HYT5, and HYT50 with a mean BMI value similar to NS.p < 0.05) with a mean value > 310 g compared to NS. There is no significant difference (p > 0.05) in lee index between HY, HYT5, and HYT50 compared to HS and NS. Meanwhile, the lee index of HYT500 is significantly low (p < 0.05) compared to HS, with a similar mean value as in NS.p < 0.05) compared to NS. There is no significant difference (p > 0.05) in waist circumference between HYT5 and HYT50 compared to HY and HS. Meanwhile, the waist circumference of HYT500 is significantly low (p < 0.05) compared to HS and HY, while there is no significant difference (p > 0.05) of waist circumference among HYT500, HYT50, and HYT5. However, the mean value of HYT500 for waist circumference is comparable to NS.p < 0.05) duration of time near the wall (thigmotactic area) compared to female offspring of NS. There is no significant difference (p > 0.05) in the time spent at the thigmotactic region of HY compared to HS and NS. Female offspring of HYT5, HYT50, and HYT500 spend significantly low (p < 0.05) duration in the thigmotactic area compared to female offspring of HS. The mean value of HYT5, HYT50, and HYT500 for the explorative duration in the thigmotaxis is comparable to NS. The data in p < 0.05) duration of time at the central region compared to female offspring of NS. There is no significant difference (p > 0.05) in the time spent at the central area of HY compared to HS and NS. Female offspring of HYT5, HYT50, and HYT500 spend significantly high (p < 0.05) duration in the central region compared to female offspring of HS. The mean value of HYT5, HYT50, and HYT500 for the explorative duration in the central region is similar to NS. Meanwhile, the data from p < 0.05) compared to female offspring of NS. There is no significant difference (p > 0.05) in the total distance traveled HY in the central zone compared to HS and NS. The total distance traveled female offspring of HYT5, HYT50, and HYT500 in the central zone is significantly high (p < 0.05) compared to female offspring of HS. The mean value of HYT5, HYT50, and HYT500 for the total distance traveled in the central zone shows no significant difference (p > 0.05) compared to NS, while the mean value of HYT500 is identical to the NS group.p < 0.05) compared to NS in NORT. There is no significant difference (p > 0.05) in the recognition index of HYT5 compared to HS and NS in NORT. Female offspring of HYT50 and HYT500 demonstrate a significantly high (p < 0.05) level of the recognition index recognizing novel objects in NORT compared to female offspring of HS. Thus, as shown in p < 0.05) compared to NS in PRT. Female offspring of HYT5, HYT50, and HYT500 demonstrate a significantly high (p < 0.05) level of the recognition index in PRT compared to female offspring of HS and HY. Thus, as shown in p < 0.05) compared to NS. There is no significant difference (p > 0.05) in the FBG level between HY, HYT5, and HYT50. The FBG level of HYT50 and HYT500 is significantly low (p < 0.05) compared to HS. However, the mean value of HYT500 for the FBG level is comparable to NS.p < 0.05) compared to NS. There is no significant difference (p > 0.05) between HS, HY, HYT5, and HYT50. The total cholesterol of female offspring in HYT500 is significantly decreased (p < 0.05) compared to HS and HY, with a similar mean value as in the NS group. As demonstrated in p < 0.05) in HS compared to NS. Meanwhile, the HDL in HY, HYT5, HYT50, and HYT500 is significantly high (p < 0.05) compared to HS. There is no significant difference (p > 0.05) in the plasma HDL level of female offspring in HY, HYT5, HYT50, and HYT500 compared to NS. However, the mean value for HYT500 for plasma HDL concentration is similar to NS group. As shown in p < 0.05) compared to NS. Meanwhile, the plasma LDL level of HYT5, HYT50, and HYT500 is significantly low (p < 0.05) compared to HS and HY, but there is no significant difference (p > 0.05) in HYT5, and HYT50 for the plasma LDL level, while HYT50 and HYT500 show no significant difference (p > 0.05) compared to NS for plasma LDL concentration. Similarly, the triglycerides content in plasma of female offspring in HS and HY groups is significantly high (p < 0.05) compared to NS. In this, HYT5 and HYT50 show gradual reduction in plasma triglycerides, however, no significant difference (p > 0.05) in HYT5 and HYT500 compared to HS, HY, and NS. Meanwhile, the triglycerides content in female offspring’s plasma of HYT500 is significantly decreased (p < 0.05) compared to HS and HY, with a similar mean value as in the NS group as demonstrated in p < 0.05) compared to NS. In this, significant reduction (p < 0.05) in female offspring of HYT5, HYT50, and HYT500 for visceral, RpWAT, BAT, and gonadal fat percentage is seen compared to HS and HY groups. There is no significant difference (p > 0.05) in fat percentage of visceral, RpWAT, BAT, and gonadal fat of female offspring in HYT5, HYT50, and HYT500 compared to NS. However, the mean value of HYT500 for visceral, RpWAT, BAT, and gonadal fat percentage is comparable to NS.p < 0.05) compared to NS. The plasma insulin concentration of HY, HYT5, HYT50, and HYT500 is significantly low (p < 0.05) compared to HS and there is no significant difference (p > 0.05) in plasma insulin concentration between HY, HYT5, HYT50, and HYT500 compared to NS. However, the mean value of HYT500 for the plasma insulin level is identical to NS.p < 0.05) compared to NS. The serum level in HY, HYT5, and HYT50 is significantly high (p < 0.05) compared to HS and shows no significant difference (p > 0.05) compared to NS. Meanwhile, the serum FRAP level of female offspring in HYT500 is significantly high (p < 0.05) compared to NS, HS, HY, HYT5, and HYT50 on PND 21. As shown in p < 0.05) compared to NS. Meanwhile, the FRAP level in the hypothalamus of female offspring in HYT50 and HYT500 is significantly high (p < 0.05) compared to HS, HY, and HYT5. There is no significant difference (p > 0.05) in the hypothalamic FRAP level in HYT50 and HYT500 compared to NS.p < 0.05) compared to NS. A significant increase (p < 0.05) in the serum GSH level in HY, HYT5, and HYT50 compared to HS is recorded on PND 21, while the serum GSH level of HYT500 is significantly high (p < 0.05) compared to HS, HY, HYT5, and HYT500 on PND 21. As shown in p < 0.05) compared to NS. There is a slight increase of the GSH level in the hypothalamus of HY and HYT5, however, there is no significant difference in the GSH level in the hypothalamus of HY and HYT5 compared to HS and NS. Meanwhile, the hypothalamic GSH level in HYT50 and HYT500 is significantly high (p < 0.05) compared to HS, with a similar mean value to the NS group.E. tapos yoghurt on the metabolic profile of maternal obesity induced cognitive dysfunction and anxiety-like behavior in female offspring. This study specifically focuses on metabolic parameter changes, such as in FBG, insulin, lipid profile, fat concentration in different fat tissue, and how it affects the antioxidants status as well as cognitive function and thigmotactic behavior. The key findings from this study prove that the E. tapos yoghurt intervention during gestation in obese dams successfully reversed the maternal obesity inherited cognitive decline, anxiety-like behavior, and antioxidants status in the serum and hypothalamus of the female offspring.Human and animal study proves that excessive weight gain during pregnancy has a detrimental effect on the child’s metabolic profile . Such chAnimal studies have demonstrated that excessive intake of HFD by dams during pregnancy leads to a high level of adiposity and cholesterol on PND 21 ,38. SimiE. tapos yoghurt treated dams show similar fat percentage, cholesterol, LDL, and triglycerides level as in the NS group. This could be due to the high concentration of flavonoids and phenols in the E. tapos extract. Phytochemical assessment on E. tapos seed further proves that the high composition of flavonoids could potentially stop the action of α-amylase, α-glucosidase, and pancreatic lipase [E. tapos seed [E. tapos is capable of suppressing the activity of PPARy and stimulates the activity of lipolysis, thereby inhibiting adipogenesis [E. tapos yoghurt in obese dam’s female offspring give a conceivable reason for low BMI, lee index, and waist circumference in HYT5, HYT50, and HYT500 compared to the HS group.Anyhow, female offspring of c lipase . In thisc lipase . Likewisc lipase . Therefopos seed  favors tpos seed  and HDL pos seed . In addiogenesis . This coE. tapos administered obese dams show a gradual increase of FRAP and GSH in the serum and hypothalamus, with a similar recognition index for novel objects and place recognition. This is mainly due to the existence of strong antioxidants in E. tapos seed as observed by Tisadondilok et al., 2018 in their study [E. tapos yoghurt is a strong antioxidant. This is because antioxidants could exhibit protective effects against oxidative stress and block the maternal obesity induced anxiety phenotype in the offspring. Simultaneously, antioxidants prevent the shortening of dendrites, and reduction of gamma-aminobutyric acid subunit receptors in the hippocampus, thereby inhibiting the development of anxiety-like behavior in the offspring [E. tapos intervene groups show more prominent and consistent positive outcome throughout all the parameters assessed in this study. The integration of natural extract (E. tapos) into yoghurt is proven to stimulate probiotic growth [E. tapos into yoghurt prevents the placental barrier crossing of HFD induced oxidative stress from maternal into the fetus [E. tapos yoghurt is a beneficial supplement that can re-modulate the metabolic profile of maternal obesity induced cognitive dysfunction and anxiety-like behavior in female offspring.In addition, offspring of HFD induced maternal obesity tend to suppress the level of endogenous antioxidants. A study conducted by Moraes-Souza et al., 2021 demonstrated that maternal intake of HFD induced oxidative stress on the fetus, subsequently initiating abnormal expressions of the gene associated with antioxidant defense of the offspring, causing the level of antioxidants to decrease . In parair study . Apace wffspring . A similffspring . Hence, c growth  and intehe fetus . This fuE. tapos yoghurt reverses the action of HFD induced maternal obesity inherited metabolic dysregulation, cognitive performance, and anxiety-like behavior in female offspring at the dose of 500 mg/kg/day.In conclusion, this experimental study demonstrates that HFD supplementation during pregnancy is the root cause for metabolic profile dysregulation in the female offspring. However, the supplementation of


However, issues like electrocatalyst stability under continuous operation and cost minimization through a reduction in the catalyst loading are of great importance to the research community. In this study, unsupported IrO2 of various particle sizes  were evaluated for the OER and as anode electrodes for PEM water electrolyzers. The electrocatalysts were synthesized by the modified Adams method, and the effect of calcination temperature on the properties of IrO2 electrocatalysts is investigated. Physicochemical characterization was conducted using X-ray diffraction (XRD), Brunauer–Emmett–Teller (BET) surface area measurement, high-resolution transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS) analyses. For the electrochemical performance of synthesized electrocatalysts in the OER, cyclic voltammetry (CV) and linear sweep voltammetry (LSV) were conducted in a typical three-cell electrode configuration, using glassy carbon as the working electrode, which the synthesized electrocatalysts were cast on in a 0.5 M H2SO4 solution. The materials, as anode PEM water electrolysis electrodes, were further evaluated in a typical electrolytic cell using a Nafion®115 membrane as the electrolyte and Pt/C as the cathode electrocatalyst. The IrO2 electrocatalyst calcined at 400 °C shows high crystallinity with a 1.24 nm particle size, a high specific surface area (185 m2 g−1), and a high activity of 177 mA cm−2 at 1.8 V for PEM water electrolysis.Polymer electrolyte membrane (PEM) water electrolyzers suffer mainly from slow kinetics regarding the oxygen evolution reaction (OER). Noble metal oxides, like IrO Hydrogen, owing to its high mass–energy density (the highest of any fuel) and its clean combustion in air, may be considered the ultimate clean energy carrier. Hydrogen can be produced through water electrolysis using surplus electricity from variable renewable energy sources and thus become a solution for storing electricity, which can be further used as a carbon-free fuel. Among the different electrolysis technologies, proton-exchange membrane water electrolyzers (PEMWEs) are one of the most attractive and efficient technologies for water electrolysis due to (i) wide-operational range of current densities; (ii) high energy efficiency (40–50%); (iii) excellent dynamic response to variable input and on/off cycling; (iv) the ability to operate at high operating pressure values; (v) high-purity hydrogen production; and (vi) safety, non-pollution, and long lifetimes ,2.+ and molecular O2 in the anode side . Proton ions pass through the polymer electrolyte membrane and reach the cathode electrocatalyst, where their reduction takes place toward the production of molecular H2 . In conventional PEM water electrolysis cells, platinum black (unsupported Pt particles) is used as an electrocatalyst at the cathode for HER, as it takes place with low overpotential values. In contrast, the OER involves a much more complex mechanism, resulting in slow kinetics and high electrode overpotentials [In a PEMWE, liquid water is generally introduced in the electrolysis cell from the anode side, where it passes through the anode porous current collector and reaches the anode electrocatalyst by enriching the polymer membrane as well. Water is electrolyzed (oxidized) towards the production of Htentials . Studiestentials .2 and IrO2 exhibit high metallic conductivity and efficiency. A major drawback of RuO2 is that it is sensitive to corrosion and lacks stability. Besides Ru, Ir is the metal that exhibits the best catalytic properties for the OER compared to other noble metals, giving rise to a great number of studies on PEM water electrolysis concentrated on Ir-based catalysts and particularly pure IrO2 which is usually employed as an OER catalyst with typical loadings of 2–4 mg·cm−2 [Various precious metals have been tested, such as Pt, Rh, Ru, Ir, and Ni . However mg·cm−2 . Based o2 nanoparticles with particle sizes ranging down to ~2 nm has been shown to effectively increase the number of surface atoms with respect to bulk and thus increase the OER activity per gram of catalyst [x electrocatalysts, including the Adams method [2 has been synthesized with a crystallite size varying between 4 and 9 nm and a BET surface area in the range of 68–113 m2 g−1, depending on calcination temperature [The synthesis of IrOcatalyst . A majorcatalyst . Towardss method , the sols method , the suls method , the mags method , the sons method , and thes method . Among ts method , presentperature ,16,17.2 was investigated by Reier et al. [2 thin films prepared by the thermal decomposition of iridium Ir chloride precursors and by Silva et al. [2 synthesized according to the hydrothermal method. Both studies revealed that there is a strong relationship between structural properties and electrocatalytic performance/stability, which can be attributed to the presence of a mixture of amorphous and crystalline IrO2. Specifically, the high crystallinity of high-temperature Ir oxide species is detrimental, whereas low-temperature amorphous Ir oxyhydroxides are highly active and efficient catalysts for the OER. On the other hand, higher stabilities were achieved for the materials with increased crystallinity. In order to reveal the effect of calcination temperature on IrOx activity and stability for OER, online inductively coupled plasma mass spectrometry (ICP-MS) experiments were conducted by Cherevko et al. [x catalysts strongly depend on the chemical and structural nature of the Ir oxide species and their synthesis conditions. Specifically, the calcination temperature resulting in different crystallinity of the IrO2 film affects the Ir dissolution rate, with the stability essentially increasing with increasing calcination temperature and crystallinity. Therefore, regardless of the IrOx synthesis method, most studies agree that regarding the activity of the OER, amorphous iridium oxide is regarded to be more active than the crystalline version, while the latter is considered to be more stable.The effect of calcination temperature on the OER performance of IrOr et al.  for IrO2a et al.  for IrO2o et al. . They co2 electrocatalysts with high specific surface area and high OER activity have been synthesized by the modified Adams method [2 electrocatalysts were calcined at 400, 500, and 600 °C and systematically studied the effect of calcination temperature on the morphology, structure, and performance. The catalysts were characterized using several techniques, such as specific surface area (BET) analysis, energy dispersive X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM). The prepared materials were electrochemically characterized, and their OER activities, as well as stabilities, were evaluated in half-cell configurations in an acidic electrolyte. The synthesized materials were used as anode electrocatalysts to fabricate membrane electrode assemblies (MEAs), which were studied in a single-cell electrolysis configuration regarding their activity and short-term stability.In this study, nano-sized unsupported IrOs method . IrO2 el2-x °C”, where x is the sintering temperature) and on the commercial catalyst (“IrO2-c”). The corresponding diffraction patterns are shown in 2-400 °C catalyst, two broad peaks (2θ = 35 and 59°) are clearly visible in both XRD patterns. The XRD results for these catalysts are characteristic of materials that are amorphous and/or have small-sized crystallites, while in the case of IrO2-c, some additional sharp peaks appear , which are assigned to the metallic Ir. The characteristic peaks of the rutile tetragonal IrO2 started to develop in the diffraction patterns of the catalysts calcined at 500 °C and the peaks were fully developed in the diffraction patterns of catalyst calcined at 600 °C. In addition, by increasing the calcination temperature to 500 °C, the amorphicity of the electrocatalyst was observed to decrease. After raising the temperature to 600 °C, these reflection peaks became thinner and sharper, indicating a higher degree of crystallinity for this material. The XRD pattern also confirmed the absence of other phases, such as metallic iridium, which is present in the commercial IrO2 catalyst.XRD analysis was used to investigate the evolution in the crystalline structure of the unsupported catalysts with increasing calcination temperature. The XRD analysis was performed on the calcined catalysts  reflection peak at 2θ = 36° was used to define the full width at half maximum (FWHM) intensity and to calculate the crystallite size of IrO2. Two different behaviors were observed. The XRD spectra of the IrO2-400 °C and IrO2-c materials present only small and broad diffraction peaks, while well-defined diffraction peaks are missing, which is a typical characteristic of amorphous catalysts. Consequently, the absence of defined diffraction peaks makes it impossible to use the Debye–Scherrer analysis, which meant that the mean crystallite sizes of the materials calcined at 400 °C, as well as the commercial catalyst, could not be determined. On the contrary, the materials calcined at higher temperatures, IrO2-500 °C and IrO2-600 °C, exhibited pronounced and resolved diffraction peaks that evolved with the increase in calcination temperature. For these catalysts, the mean crystallite size was determined to be 5.18 nm for the IrO2-500 °C, while that for the material obtained at 600 °C is 7.77 nm. Furthermore, the lattice parameters for all IrO2 catalysts were determined. The calculated parameters are summarized in 2-rutile crystallographic pattern (JCPDS No. 15-871). A slight increase in unit cell volume is observed for IrO2-600 °C compared to IrO2-500 °C (62.4 Å3 vs. 62.0 Å3), which implies the introduction of tensile strain and possible generation of oxygen vacancies. Theoretical computations reveal that this leads to the reduction in the thermodynamic energy barrier for hydroxyl adsorption and a concomitant improvement in alkaline OER on perovskite oxides [2-500 °C and IrO2-600 °C, this is not expected to significantly affect their OER activity since hydroxyl adsorption is not a rate-determining step (rds) for any of the proposed reaction mechanisms regarding acidic OER [The average crystal size and the lattice parameter for each IrOe oxides . Althougidic OER ; however2 catalysts are presented in 2-400 °C catalysts reveal spherical particles with sizes in the 1–2 nm range; however, the mean particle size could not be safely determined due to the agglomeration of these nanoparticles. For the catalysts obtained at higher temperatures  for the IrOeratures b,c, the  results . The res7/2) and 65.1 eV (Ir 4f5/2), and it can be attributed to Ir4+ (IrO2). All spectra appear to have an almost equal contribution from another doublet (blue curve) at binding energies 63.8 and 66.9 eV, which is attributed to Ir at a higher oxidation state, i.e., IrOx (x > 2). The presence of surface Ir in two oxidative states and the corresponding binding energy values are close to the reported ones for chemically produced IrO2 [0), in agreement with the XRD data. The surface composition of the catalysts, expressed in % percentages of Ir4+, are 41, 55, and 52% for IrO2-400 °C, IrO2-500 °C, and IrO2-600 °C catalysts, respectively. It should be noted that the above values refer to the surface composition of the IrO2 catalysts, which in general, may significantly deviate from the bulk composition. Nevertheless, as electrochemical reactions take place on the surface, it is particularly important to consider the surface state of the catalysts in relation to their electrochemical behavior.The chemical state of Ir in the catalysts was examined by XPS analysis. ced IrO2 ,23. In aHigh-resolution oxygen 1s-orbital spectra were also acquired and are displayed in 2, revealing again the co-existence of iridium oxides at higher oxidation states. Furthermore, the O:Ir ratio did not exhibit a specific trend with sintering temperature, indicating that thermal treatment did not result in an oxygen loss on the surface due to the conversion of Ir4+ into Ir3+.The oxygen-to-iridium atomic ratios were calculated using the signal intensity from O 1s and Ir 4f spectra, normalized for the different element sensitivity factors. It was found that the O:Ir ratio is between 2.41 to 2.82 for all three samples, which is slightly higher than the theoretical value of 2 for IrO2 g−1 for IrO2-400 °C, 127 m2 g−1 for IrO2-500 °C, and reduced to 50% of its initial value (66 m2 g−1) when the catalyst was calcined at 600 °C. Despite the calcination temperature, these values are higher than the reported values in the literature [2 g−1) for commercial IrO2. It is observed that the modified Adams method results in the formation of nanosized particles with high surface areas. In TEM images, it was shown that the iridium-based species grew with calcination temperature. Therefore, the reduction in the BET surface area was sharper when the calcination temperature was increased from 400 to 600 °C. This is in agreement with TEM images, where the increase in the size of iridium-based species was more evident when the calcination temperature was increased from 400 to 600 °C.The BET surface areas of the calcined catalysts were determined using nitrogen physisorption isotherms and the BET equation . The meaterature  . This redox peak corresponds to the redox reaction of iridium, where the oxidation state changes between Ir3+ and Ir4+. Additionally, one reduction peak was observed at 1.30 V vs. RHE, which is attributed to the change in the Ir4+ to Ir5+ transition. The oxidation peaks for IrO2-500 °C, IrO2-600 °C, and IrO2-c catalysts corresponding to the above oxidation state transitions were not clearly observed. This was most likely due to an overlap with the OER. The OER starts around 1.43 V vs. RHE, where the oxidation peak was supposed to be seen. The recorded current for the CVs decreased with calcination temperature, and it was accompanied by less pronounced redox peaks and a smaller surface area under the voltammograms.Cyclic voltammetry was performed on the calcined catalysts and the commercial IrOThe surface area under the voltammograms divided by the scan rate corresponds to the voltammetric charge. The oxidation voltammetric charges were obtained from CVs over the whole anodic potential window. The charge transferred during the iridium redox reaction is proportional to the number of iridium active sites. It is frequently used as an estimation of electrochemically active surface area (ECSA). The ECSA reduced with the increase in calcination temperature . This wa−1 Ir). As depicted in this figure, the catalysts’ performance decreased as the calcination temperature increased. The performance of the commercial IrO2 is also measured and added to the figure for comparison. The mass activities at 1.70 V vs. RHE are summarized in the inset table , as shown in et table . 2-400 °C) synthesized by the Adams method is superior to the commercial IrO2. The amorphous IrO2-400 °C catalyst is the most active, and its corresponding starting potential of oxygen evolution is the lowest. With the increase in calcination temperature, the starting potential of oxygen evolution increases since the IrO2-400 °C is more electrocatalytically active (more active sites) than IrO2-500 °C and IrO2-600 °C in OER, resulting in lower current densities at any potential. This decrease to the current (in the performance) with the increase in calcination temperature was due to the reduction in the available ECSA, as previously discussed. This is comparable with the result discussed in the surface electrochemistry section regarding cyclic voltammograms. Moreover, it could be concluded that the amorphous iridium-based species have higher activity toward OER compared to crystalline IrO2 [It is evident that the performance of the calcined catalyst . This step follows the water discharge step  and involves the activation of chemisorbed OH* groups to reactive OH species. However, at higher current densities, a Tafel slope of 120 mV dec−1 indicates that the rate-determining step moves to the oxidative adsorption of water [2-600 °C and ΙrO2-c are observed to be higher both of low and high overpotentials and are attributed to the formation of oxides on the surface which is in agreement with the results of XPS analysis.Two distinguishable linear regions can be observed, with Tafel slopes close to 60 mV decred IrO2 ,28. A Taof water . Finally2 oxides under typical water electrolysis conditions. Each experiment was repeated three times and then characterized using polarization curves. All parameters  were kept constant except the calcination temperature of the IrO2 catalyst. Electrolysis performances were measured at a constant temperature of 50 °C at atmospheric pressure (1 bar). Liquid water was fed at the anode with a mass flow rate of 300 g h−1, while He saturated with vapor (58 kPa H2O) was supplied at the cathode. The corresponding steady-state polarization curves for all catalyst formulations are shown in 2-400 °C as the anodic electrocatalyst exhibited the highest performance among all samples, achieving 177 mA cm−2 at 1.8 V. It should be noted that the reported values are not corrected for the ohmic losses. The superior performance of the MEA with the IrO2 calcined at 400 °C is in agreement with the findings of the intrinsic OER activity measurements performed in the RDE configuration.A single-cell PEM water electrolysis cell was used to assess the performance of the synthesized IrOIn order to evaluate the stability of the MEAs, short-term testing was carried out for 8 h continuous operation under constant applied potential (1.8 V), and the results are summarized in 2-400 °C as anode electrocatalyst, showed remarkable stability reaching 200 mA cm−2 after the initial stabilization period. These results are very promising regarding the stability of the synthesized electrocatalysts and MEAs.The current density for all MEAs decreased quickly during the first minutes (~50 min) of potential application and then reached a steady-state value during the remaining time of testing. The initial drop in current is a common feature of MEAs subjected to a constant voltage (or current) switch. This rapid drop occurring during the first ~10 min of potential application (stabilization period) could be possibly ascribed to two main phenomena occurring under electrolysis conditions, i.e., a mass-transfer polarization or a modification of the oxidation state at the anode surface . The bes2IrCl6.xH2O  was dissolved in isopropanol with an excess of NaNO3 , and a homogeneous mixture was prepared. The metal concentration in the solution was 0.07 M. The salt mixture was heated at 60 °C under continuous stirring until isopropanol evaporated and the mixture was rather dry. Following, the mixture was allowed to dry completely for 30 min in an oven at 80 °C until a dry salt mixture was obtained. This dry salt mixture was then finally introduced into a ceramic furnace and sintered at 400, 500, and 600 °C for 30 min. After cooling to room temperature, the mixture was washed with distilled water in order to remove all Cl− ions and remaining solvable salts, and finally dried in air at 80 °C overnight [A series of unsupported nanoparticle iridium oxide electrocatalysts were synthesized using a modified Adams method ,3. First−1. Τhe data were collected for 2θ values between 20 and 80°. The characteristic reflection peaks  were matched with JCPDS data files, and the crystalline phases were identified. The average crystallite size was calculated by means of the Scherrer formula after Warren’s correction for instrumental broadening. The unit cell parameters were refined with the CrystalSleuth (version: 19 May 2008) software.The crystalline structures of the synthesized electrocatalysts were analyzed by X-ray diffraction (XRD) using a Siemens D500 X-ray diffractometer with auto divergent slit and graphite monochromator using KaCu radiation (1.5418 Å) having a scanning speed 1.2° minThe specific surface areas and pore volumes of the catalysts were determined using an Autosorb-1, Quantachrome instrument. The specific surface area was evaluated using the Brunauer–Emmett–Teller (BET) method.The morphology, particle sizes, and microstructure of the metal oxides were studied with transmission electron microscopy (TEM). TEM images were obtained on a JEOL JEM 2010 high-resolution transmission electron microscope coupled with an Oxford INCA X-ray EDS for elemental analysis and surface mapping of the catalyst surface. Initial TEM images of the catalysts were collected at different regions.p < 10−9 mbar), which consists of a fast-entry specimen assembly, sample preparation, and an analysis chamber. The analysis chamber is equipped with a SPECS LHS-10 hemispherical electron analyzer and a twin-anode X-ray gun, operating under the following conditions: unmonochromatized MgKa radiation at 1253.6 eV, analyzer pass energy of 97 eV, imposing a full width at half maximum (FWHM) of 1.7 eV for the Au 4f7/2 peak. The accuracy for binding energies assignments is ~0.1 eV, while in all samples, the main C 1s peak was at 284.6 eV. The XPS core level spectra were analyzed with a fitting routine, which decomposes each spectrum into individual, mixed Gaussian–Lorentzian peaks using a Shirley background subtraction over the energy range of the fit.The surface composition and metal oxidation state of the electrocatalysts were evaluated by X-ray photoelectron spectroscopy (XPS). The photoemission experiments were carried out in a commercial ultra-high vacuum system . The working electrode consisted of a catalyst layer loading 3.25 mg cm−2. This layer was prepared by dispersing 10 mg of the catalyst powder in 1 mL of ethanol, and 80 μL of Nafion solution  was also added to the catalyst ink solution as the binder. After that, the catalyst ink solution was sonicated for 30 min at room temperature to ensure uniform dispersion, and consequently, an amount of 25 μL was cast on the clean, glassy carbon substrate using a micropipette. The formatted electrode was dried in air for 1 h.Electrochemical experiments were performed using a standard three-electrode electrochemical cell in helium-purged 0.5 M H2 catalysts were characterized by repetitive cyclic voltammetry (CV) in the three-electrode cell between 0 and 1.45 V vs. RHE with scan rates of 20, 50, 75, and 100 mV s−1. The electrochemically active surface areas (ECSAs) were assumed to be proportional to the anodic redox charge of the CVs, as proposed by Ardizone et al. [Ιnitially, the prepared ΙrOe et al. . Here thRHE at a scan rate of 1 mV s−1 (near steady state). To avoid the accumulation of oxygen bubbles, a rotation speed of 2500 rpm was applied using a Metrohm rotator system. The electrolyte was deaerated via bubbling with He prior to and during all measurements. All reported measurements were repeated at least three times to ensure the reproducibility of the results.The OER activities of the catalysts were evaluated by linear sweep voltammetry (LSV) within the potential range between 1.30 and 1.70 V®115  membrane with a typical thickness of 127 μm was used as a solid polymer electrolyte. IrO2 oxides powders, prepared as described above, were used as OER electrocatalyst while Pt/C catalyst  was used as hydrogen evolution electrocatalyst at the cathode. Electrodes were prepared as follows: catalysts and Nafion ionomer  mixtures were first ultrasonically suspended in a mixture of deionized water and isopropanol. Catalytic suspensions thus obtained were then brushed directly onto the Nafion®115 membrane . The catalyst loadings were 0.5 mg cm−2 Pt/C for the hydrogen side and 1.5 mg cm−2 IrO2 for the oxygen side. Electrode layers (with 25 cm2 active area) were bonded by placing the catalyst-coated membrane between carbon paper for the oxygen side and carbon cloth for the hydrogen side and then by hot pressing at 130 °C and 10 MPa for 3 min. The Nafion content of the electrocatalytic layers was set to 30 wt% for the anode. Finally, the single-cell tests were carried out with a PEM water electrolysis cell. MEAs were clamped between a porous carbon paper at the anode and a carbon cloth as a gas diffusion layer at the cathode side. The cell body was made of end plates (stainless steel), graphite bipolar plates with serpentine flow fields (suitable for electrolysis), and the MEA between them. The cell was tightened between two stainless steel plates, using a dynamometric wrench to set the fastening screws to 40 cNm.A Nafion2 catalysts were synthesized by the modified Adams method. The effect of the calcination temperature, in the range between 400 and 600 °C, on the morphological/surface characteristics and the electrochemical performance of the catalysts for the oxygen evolution reaction (OER) was studied.IrO2 electrocatalysts with small, nano-scale particle size (1.24–7.77 nm) and high BET surface area (185–66 m2g−1). The structures, activities, and stabilities of Adams’ fusion method prepared iridium oxide nanomaterials as catalysts for the OER were studied. Heat treatment at different temperatures provided IrO2 catalysts with varying degrees of crystallinity. The unsupported prepared materials exhibit outstanding activity for oxygen evolution reaction (OER) compared to commercial IrO2. To achieve both high OER activity and stability, three key factors have been identified: (i) high anodic charge, (ii) high surface area due to nano-size IrOx particles well dispersed in the Nafion ionomer electrolyte, and (iii) homogeneous layer morphology. The best-performing material was the IrO2 calcined at 400 °C.The application of a modified Adams method resulted in the synthesis of unsupported nanostructured IrO2 catalyst. In terms of stability, the tests of four MEAs at steady operation stability (1.8 V) after 8 h continuous operation perform excellent stability. Further studies are required to examine the long-term  performance of the catalyst in electrolyzer conditions.The performances of the unsupported catalysts were also studied in MEA configuration under practical water electrolysis conditions. In agreement with the OER activity measurements, the best-performing MEA was the one employing the 400 °C calcined IrO


To quantify cardiovascular health (CVH), the American Heart Association (AHA) recently launched an updated construct of the “Life's Simple 7” (LS7) score, the “Life's Essential 8” (LE8) score. This study aims to analyse the association between both CVH scores and carotid artery plaques and to compare the predictive capacity of such scores for carotid plaques.Randomly recruited participants aged 50–64 years from the Swedish CArdioPulmonary bioImage Study (SCAPIS) were analysed. According to the AHA definitions, two CVH scores were calculated: i) the LE8 score  and two different versions of the LS7 score . Ultrasound-diagnosed carotid plaques were classified as no plaque, unilateral, and bilateral plaques. Associations were studied by adjusted multinomial logistic regression models and adjusted  prevalences, while comparison between LE8 and LS7 scores was performed through receiver operating characteristic (ROC) curves.P = 0.578, respectively; and for any carotid plaque, 0.602 (95% CI: 0.596–0.609) vs. 0.600 (95% CI: 0.593–0.607), P = 0.194, respectively.After exclusions, 28,870 participants remained for analysis (50.3% women). The odds for bilateral carotid plaques were almost five times higher in the lowest LE8 (<50 points) group  compared to the highest LE8 (≥80 points) group . Also, the odds for unilateral carotid plaques were more than two times higher in the lowest LE8 group  compared to the highest LE8 group . The areas under ROC curves were similar between LE8 and LS7 (0–14) scores: for bilateral carotid plaques, 0.622 (95% CI: 0.614–0.630) vs. 0.621 (95% CI: 0.613–0.628), The new LE8 score showed inverse and dose-response associations with carotid plaques, particularly bilateral plaques. The LE8 did not outperform the conventional LS7 score, which showed similar ability to predict carotid plaques, especially when scored as 0–14 points. We conclude that both the LE8 and LS7 may be useful in clinical practice for monitoring CVH status in the adult population. Cardiovascular disease, including coronary heart disease and cerebrovascular disease, continues to be the leading cause of morbidity and mortality globally . The AmeAtherosclerosis is firmly established as a leading cause of clinical cerebrovascular events , and carConsequently, to combat the cardiovascular burden, public health policies based on primordial prevention have been advocated. In this sense, the AHA defined the construct “ideal cardiovascular health” or “Life's Simple 7” (LS7) in 2010, which meant a crucial shift from cardiovascular disease management to positive cardiovascular health (CVH) promotion . During In the last decade, LS7 has accumulated evidence of its association with functional and structural indicators of carotid atherosclerosis both in cross-sectional and prospective studies –16, espeDetailed design and methods of the Swedish CArdioPulmonary bioImage Study (SCAPIS) have been published elsewhere . In brieThis study included participants from SCAPIS with necessary data to calculate LE8 and LS7 scores, and with available ultrasound images in both left and right carotid arteries. We constructed a LE8 score to include the 8 components as defined by the AHA . SupplemBriefly, LE8 factors were measured using standardized clinical and laboratory techniques. Regarding LE8 behaviors, questionnaires were used to measure nicotine exposure, sleep health  and diet [web-based MiniMeal-Q to calculate adherence to the Mediterranean Eating Pattern for Americans ]. PhysicIn accordance with the AHA recommendations, a total score from 0 to 100  was calculated based on 7 or 8 components within LE8  through the unweighted average of all present components. To aid clinical interpretation, the total LE8 score was categorized in 5 groups as follows: <50, 50–59.9, 60–69.9, 70–79.9, and 80–100 points. This categorization is comparable to that proposed by the AHA  , but we Similarly, two separate scores ranging from 0 to 100 were calculated and categorized for LE8 factors and LE8 behaviors.Measurements used in LS7 score were similar to those used in LE8 score . Two different LS7 scores were derived: the LS7 (0–7) score, which is the sum of the number of LS7 components at ideal level, producing a total 0–7 score ; and the LS7 (0–14) score, which is the sum of the scores of LS7 components when rated as: 0 (poor CVH), 1 (intermediate CVH), and 2 , generating a total 0–14 score .The detailed imaging protocol for SCAPIS, which meets relevant guidelines, has been published elsewhere . All theIn consonance with the Mannheim consensus, carotid plaque was defined as “any focal structure that encroaches into the arterial lumen of at least 0.5 mm or 50% of the surrounding intima media thickness value or demonstrates a thickness >1.5 mm as measured from the media-adventitia interface to the intima-lumen interface” . ParticiMeans and standard deviations were presented for continuous variables, and frequencies and percentages were presented for categorical variables. Multinomial logistic regression models were used to analyse the associations between LE8 and LS7 scores and carotid plaques: Model 1, unadjusted; Model 2, adjusted for age, sex, and study site ; and Model 3, adjusted for age, sex, study site, alcohol intake , educational status (attained highest level of education), current marital status, and presence of cardiovascular disease . Model 2 was chosen as the main analysis, since it includes only basic demographic details, and may therefore be easily replicated.To improve clinical interpretation, we estimated the adjusted  prevalence of carotid plaques indicators across CVH scores, which will be henceforth referred as “adjusted prevalence”. The capacity to predict carotid plaques for LE8 and LS7 [using both the LS7 (0–7) and the LS7 (0–14) scores] was analysed by comparing unadjusted receiver operating characteristic (ROC) curves through DeLong tests. Reclassification and discrimination were examined with the categorical Net Reclassification Index (NRI) and Integrated Discrimination Improvement (IDI) considering covariates in Model 2. For NRI, we set the threshold level for the risk categories at 50%.The relationship between LE8, LS7 (0–7) and LS7 (0–14) was also modelled by restricted cubic splines of binary logistic models adjusted by age, sex and site.In the primary analysis, we performed a complete case analysis on all included 28,870 participants. In addition, we performed an extreme scenario sensitivity analysis to corroborate the robustness of our findings. As such, we considered participants with missing data on either LE8 score or carotid plaque as having the best or the worst possible result, i.e., 0 (worst) or 100 (best) for LS8 score, and “no plaque” (best) or “bilateral plaques” (worst) for carotid plaques. In addition, we performed a secondary sensitivity analysis considering: (1) exclusion of participants with previous stroke, (2) exclusion of participants with previous cardiovascular diseases , and (3) inclusion of participants with data on all 8 components of the LE8 .P < 0.05 was considered statistically significant. Analyses were conducted using IBM-SPSS-28  and Stata 17 (StataCorp. 2021).All statistical tests were two-sided and Of a total sample of 30,154 participants, 4.3% were excluded due to a lack of data, leaving 28,870 participants for the analysis . In geneOverall, having low LE8 scores was strongly and inversely associated with the presence of carotid plaques, particularly bilateral plaques . Thus, tP < 0.05), , . In a se< 0.05), .As in LS8, there were strong and inverse associations between LS7 (0–7) scores and carotid plaques, particularly for bilateral plaques . The oddP < 0.001]; and similar to LS7 0–14 points , respectively  , respectively ] compared to LS7 (0–7) [0.594 (95% CI: 0.587–0.600), ectively . Similarectively . SupplemIn an extreme scenario sensitivity analysis, when all participants with missing data in either LS8 or carotid plaques were considered as having the best or the worst possible result, LS8 maintained the strong, dose-response and inverse associations with carotid plaques, though such associations were somewhat attenuated . In a seThe present study provides evidence of a strong, inverse and dose-response associations between the novel LE8 score and the presence of carotid plaques, especially bilateral ones, both in terms of relative and absolute measures. Similar results were found for the conventional LS7 score, showcasing that the LS7 (0–14) score had similar capacity to predict carotid plaques compared to LS8.To our knowledge, this is the first study analysing the association of the novel LE8 score with carotid plaques. Our study builds on the previous literature by demonstrating the strong and inverse associations between both LS8 and LS7 scores and carotid plaques. Particularly, in our study, after adjustments, the odds for unilateral and bilateral carotid plaques were roughly two and five times higher in those with poor LE8 (<50 points) compared to those with ideal LE8 (≥80 points) scores, respectively, finding similar associations for the appurtenant LS7 score.Although there are no previous studies to allow comparison with our LE8 results, as the construct was recently developed, our findings for LS7 are in line with those previously published in the literature. For instance, cross-sectional data from Europe and China have demonstrated inverse associations between the number of LS7 components at ideal level and carotid plaques , 24. SimIn our study, in comparison to the LE8 score, the LS7 score exhibited similar capacity to predict carotid plaques, especially when LS7 was scored as 0–14 points. As such, beyond sex, site, and age, the capacity for reclassification between LE8 and LS7 (0–14) scores seems very comparable, with marginal improvements of the LE8 score to reclassify the presence of bilateral plaques and the absence of any carotid plaque/s. That is, despite the difference between LE8 and LS7 performance being statistically significant, it is not clear that this difference would have a noticeable impact on public health surveillance. This is surprising seeing as, in theory, LE8 seems more advantageous, since small changes in LS7 components or in the sleep health would go unnoticed using LS7 for follow-up in individual patients. However, in our study, sleep health was not associated with carotid plaques, which may partially explain the similar capacity of prediction between LS7 and LE8. Similarly, we did not observe any association between diet and the presence of carotid plaques. Since LE8 is a novel metric introduced in June 2022, there are no previous studies specifically analyzing its association with carotid plaques. However, a prospective cohort study investigating the association of LE8 and cardiovascular disease mortality reported that sleep health and diet had the lowest population attributable fraction among the LE8 components, which aligns with our current findings .A previous study from SCAPIS showed that a Swedish version of the Systematic Coronary Risk Evaluation (SCORE) risk chart was useful to predict carotid plaques in the adult population . SpecifiIn the present study, more than 55% of the SCAPIS population had carotid plaques and 25% had bilateral plaques. Due to these high prevalences, the strong relative associations (expressed as ORs) were also followed by important absolute risk metrics . Altogether, this created a particular trade-off, with stable adjusted prevalences in unilateral plaques but dose-response prevalences in absent and bilateral plaques. This apparent discrepancy between the relative and absolute measures of association raises intriguing considerations. We have hypothesized that individuals with high and low LE8 scores may undergo opposite redistributions among the plaque groups . This would result in similar prevalences of unilateral plaques among LE8 groups, but different ORs . Specifically, compared to participants with ideal LE8, those with poor LE8 had adjusted prevalences that were 25.5% units lower for absent plaques, 23.4% units higher for bilateral plaques, but a neglectable 2.1% units higher for unilateral plaques. Therefore, from a clinical practice perspective, LE8 seems to be a feasible tool to monitor the absence of carotid plaques and the presence of bilateral plaques, but with poor utility to predict unilateral plaques. Nevertheless, it should be noted that bilateral plaques are more strongly associated with both stroke  and coroA main strength of this study is its size. The SCAPIS project comprises more than four times the population of relevant studies such as the MESA study  or the BHowever, some limitations should be recognized. First, carotid plaques were only categorized as absent/unilateral/bilateral plaques, not analysing neither the different “plaque phenotypes” nor the plaque burden. In this sense, some novel studies have proved that certain characteristics of the atherosclerotic plaques suggesting their vulnerability could improve the cardiovascular risk prediction . Second,In conclusion, this large population-based study showed inverse and dose-response associations between the novel LE8 score and the presence of carotid plaques, especially bilateral ones. The conventional LS7 score showed similar capacity to predict carotid plaques, particularly when scored in a 0–14 increment scale. Both scores proved to be powerful tools to measure, monitor and promote CVH in an adult population in the clinical setting.