Positron emission tomography (PET) with radiotracers that bind to synaptic vesicle glycoprotein 2A enables quantification of synaptic density in vivo. This study used [11C]UCB‐J PET, structural magnetic resonance imaging, and a neuropsychological battery, including the revised Addenbrooke Cognitive Examination, and INECO frontal screening. General linear models compared [11C]UCB‐J binding potential maps and gray matter volume between groups, and assessed associations between synaptic density and clinical severity in patients. Analyses were also performed using partial volume corrected [11C]UCB‐J binding potential from regions of interest (ROIs).Eleven participants with clinically probable bvFTD and 25 age‐ and sex‐matched healthy controls were included. Participants underwent dynamic [11C]UCB‐J binding was reduced bilaterally in medial and dorsolateral frontal regions, inferior frontal gyri, anterior and posterior cingulate gyrus, insular cortex, and medial temporal lobe. Synaptic loss in the frontal and cingulate regions correlated significantly with cognitive impairments. Synaptic loss was more severe than atrophy. Results from ROI‐based analyses mirrored the voxelwise results.Patients with bvFTD showed severe synaptic loss compared to controls. [11C]UCB‐J PET could support translational studies and experimental medicine strategies for new disease‐modifying treatments for neurodegeneration. ANN NEUROL 2023;93:142–154In accordance with preclinical models, and human postmortem evidence, there is widespread frontotemporal loss of synapses in symptomatic bvFTD, in proportion to severity. [ Frontotemporal dementia is a clinically and pathologically heterogeneous group of neurodegenerative conditions. Similar clinical phenotypes arise from different pathologies,Our hypothesis was that in people with frontotemporal dementia, synaptic loss is severe and proportionate to clinical severity. Regionally specific and clinically relevant loss of synapses has been shown in the tauopathies of Alzheimer disease,18F]FDG positron emission tomography (PET)Previous evidence for synaptic loss in frontotemporal dementia has been indirect, including reduced synaptic density in carriers of mutations associated with frontotemporal dementia,11C]UCB‐J to assess the regional distribution of synaptic loss in patients with a clinical diagnosis of probable bvFTD, compared to age‐ and sex‐matched controls. Synaptic density was estimated using nondisplaceable binding potential (BPND), a metric proportional to binding site density. In this context, if proven useful, [11C]UCB‐J PET could become an important tool for patient stratification and a surrogate endpoint in clinical trials and early interventions. PET measures for synaptic density would give additive and complementary information to fluid marker evidence, which does not elucidate in vivo spatial distributions of brain changes and may be less sensitive in prodromal stages. These characteristics of [11C]UCB‐J therefore permit its use as an in vivo marker for synaptic loss in frontotemporal dementia, irrespective of whether the underlying pathology is tau or TDP‐43. We predicted substantial synaptic loss in frontotemporal regions across all patients, and a correlation with clinical severity.Here, we used PET with the SV2A radioligand [Eleven patients with a clinical diagnosis of probable bvFTD11C]UCB‐J), history or magnetic resonance imaging (MRI) evidence of ischemic or hemorrhagic stroke, any severe physical illness or comorbidity that would limit the ability to fully and safely participate in the study, and any contraindications to MRI. Eligible volunteers underwent a clinical and neuropsychological assessment including the Mini‐Mental State Examination (MMSE), revised Addenbrooke Cognitive Examination (ACE‐R), INECO frontal screening (IFS), Frontotemporal Dementia Rating Scale (FTD‐RS), and the Clinical Dementia Rating Scale (CDR). FTD‐RS was only acquired for patients, as it is a caregiver‐based assessment of patients' performance in activities of daily living. Within 3 months, all participants underwent brain imaging with 3T MRI and PET scanning with [11C]UCB‐J ((R)‐1‐((3‐(methyl‐11C)pyridin‐4‐yl)methyl)‐4‐pyr‐rolidin‐2‐one).Participants were screened for exclusion criteria: current or recent history (within the past 5 years) of cancer, current use of the anticonvulsant medication levetiracetam  and the Administration of Radioactive Substances Advisory Committee. All participants provided written informed consent in accordance with the Declaration of Helsinki.11C]UCB‐J synthesis, data acquisition, image reconstruction, and kinetic analysis have been published elsewhere.11C]UCB‐J injection, with attenuation correction including the use of a multisubject atlas methodFull details of the protocol for [11C]UCB‐J BPND map was determined from dynamic images corrected for partial volume effects at the voxel level using the iterative Yang method.ND was calculated using a basis function implementation of the simplified reference tissue model, with centrum semiovale as the reference tissue.ND map was warped to the ICBM 152 2009a asymmetric magnetic resonance (MR) template using parameters from the spatial normalization of the coregistered T1 MR image with Advanced Normalization Tools (http://www.picsl.upenn.edu/ANTS/). For voxelwise analyses, the spatially normalized [11C]UCB‐J BPND maps were spatially smoothed with a full width at half maximum (FWHM) Gaussian kernel to an effective estimated smoothness of 10mm, prior to statistical analysis. T1‐weighted MRIs were segmented using the Computational Anatomy Toolbox 12 (http://www.neuro.uni-jena.de/cat/), and total intracranial volume (TIV) was estimated as the sum of gray matter, white matter, and CSF segmentations. Gray matter maps were warped to Montreal Neurological Institute space and smoothed with a 10mm FWHM Gaussian kernel prior to statistical analysis.For each subject, a UCB‐J BPND, age, and symptom duration. All results were tested at an uncorrected voxel height threshold of p < 0.001 combined with a familywise error (FWE) corrected cluster threshold of p < 0.05; peak voxels in clusters that reached the conservative voxel‐level FWE threshold (FWE p < 0.05) are also indicated.Voxelwise analyses were performed using SPM12. First, a 2‐sample https://www.r-project.org/) and JASP . [11C]UCB‐J BPND values from regions of interest were aggregated into left and right frontal lobe, temporal lobe, parietal lobe, occipital lobe, and cingulate cortex, and included in the analyses alongside insular cortex, hippocampus, amygdala, and thalamus. First, 2‐sample t tests were performed in each region to compare the bvFTD group to controls. Across all brain regions, p values from t tests were corrected with false discovery rate correction, and Cohen d effect sizes were calculated. As explorative analyses, we also performed 2‐sample t tests on [11C]UCB‐J BPND values of bvFTD group versus controls considering smaller regions, obtained from the Hammers atlas with and without partial volume correction. Second, Spearman correlation analyses were performed to test the association between cognitive performance and [11C]UCB‐J BPND in cortical regions. In addition to the above frequentist statistics, we also present Bayesian analogous tests for regional effects (https://osf.io/gny35/), complementing the strength of the evidence for the null versus alternate hypotheses.We performed complementary analyses on regions of interest, using R version 4.0.0 , using 10mm‐smoothed gray matter images, including TIV as covariate . Second, we considered regional partial volume corrected [11C]UCB‐J BPND and gray matter volumes (divided by TIV) extracted using the Hammers atlas. We calculated regional z scores for each patient and modality using regional means and standard deviations from the controls. We compared regional z scores between the two modalities, identifying regions where patient‐average z score values for [11C]UCB‐J BPND were lower than z scores for gray matter volumes. We ran a further explorative analysis to assess the association between regional gray matter z scores and regional [11C]UCB‐J BPNDz scores , using a linear mixed effects model (lmer function in R) with uncorrelated random intercept and slope terms, to take into account the variability between subjects in relation to the modalities  and atrophy (MRI). The analysis of gray matter volume loss, at voxelwise and regional levels, was analogous to the [11C]UCB‐J PET) is attributable to the associated gray matter atrophy, we ran 2 additional analyses. First, we ran region‐specific logistic regression models, with group as the dependent variable, and either gray matter volumes as the predictor or both gray matter volumes and [11C]UCB‐J binding potential. The first model tests whether individuals can be classed as patient versus control based on their atrophy alone, whereas the second considers the additional effect of synaptic density. We compared the models by analysis of variance and Akaike information criterion. Second, we repeated the regional group comparison analyses for [11C]UCB‐J PET after regressing out the region‐specific gray matter volumes. We used partial volume noncorrected binding potential as the dependent variable and gray matter volume as the predictor in a linear regression model and took the resulting residuals to t test between groups.Finally, to test whether the apparent effect of bvFTD on synaptic loss  represent higher synaptic density, and lower values (blue/green areas) represent lower synaptic density. People with bvFTD showed mild (Patient 1) to severe (Patient 11) regional synaptic loss compared to controls.Single‐subject 1CUCB‐J Bp < 0.05), the 2‐sample t test on [11C]UCB‐J BPND maps revealed synaptic loss in the bvFTD group compared to controls (5.1 ≤ t ≤ 9.3) in frontal, temporal, insular, and cingulate cortex bilaterally . The bvFTD group did not show significantly higher binding than controls in any area (p < 0.001 uncorrected).At the FWE corrected voxel‐level threshold  between cognition and [11C]UCB‐J BPND in the anterior cingulate gyrus bilaterally, the left superior and middle frontal gyri, and the medial anterior temporal lobe  with [11C]UCB‐J BPND in the superior and middle frontal gyri bilaterally, left cingulate cortex and inferior frontal gyrus, and left superior parietal gyrus and anterior medial temporal lobe . Voxelwise correlation analyses did not reveal significant associations between [11C]UCB‐J BPND, age, and symptom duration (p < 0.001 uncorrected).For voxelwise correlation analyses between UCB‐J BPND and ACE‐R scores in the left frontal lobe (Bayes factor [BF] = 56.2) and cingulate cortex (BF = 10.7), with positive evidence in the parietal lobe (BF = 3.9). Bayesian correlation analyses of IFS indicated strong evidence for positive correlation in the left frontal lobe (BF = 23.4) but a lack of positive evidence for or against a correlation in the left cingulate cortex (BF = 2.3) and parietal lobe (BF = 0.8). In Supplementary Table 11C]UCB‐J BPND with MMSE, ACE‐R, and IFS subscores, and FTD‐RS are reported.Considering cortical regions of interest, Spearman correlation between partial volume corrected [11C]UCB‐J BPND and gray matter volumes show an extensive pattern of synaptic loss, and a more restricted pattern of gray matter atrophy in patients compared to controls . The regions of significant difference from both comparisons include medial frontal and orbitofrontal cortex, anterior cingulate gyrus, insula, anterior and medial temporal lobe, and striatum. Comparing regional z scores between the two modalities, we found that 84% of the regions have larger negative [11C]UCB‐J BPND effect sizes than gray matter volume effect sizes . The strength of this association (slope) varied between patients, but was positive in all cases , even after penalization for increased model complexity. Group comparison of regional residuals of uncorrected [11C]UCB‐J BPND after regressing out region‐specific gray matter volumes is described in Supplementary Table The results of logistic regression analyses are described in Supplementary Table 11C]UCB‐J BPND correlates with patients' cognitive performance. [11C]UCB‐J BPND was not related to age, in line with previous evidence.11C]UCB‐J PET raises 3 points of particular importance.This study confirms the hypothesis that people with a clinical diagnosis of bvFTD have severe and widespread synaptic loss over frontotemporal cortex, including cingulum and insula. The synaptic density in frontal and cingulate regions as quantified with UCB‐J may now be used in autoradiography or in vivo PET11C]UCB‐J therefore offers a bridge between preclinical and clinical models, where quantitative whole brain models of pathogenesis in dementia, including frontotemporal dementia, can be improved by the inclusion of synaptic density estimates in addition to structural connectivity11C]UCB‐J PET in preclinical models and people with genetically determined familial frontotemporal dementia,Second, quantifying the degree and distribution of synaptic loss can enrich models of frontotemporal dementia pathogenesis, in preclinical and clinical settings. For example, in preclinical tauopathy models, connectivity more than proximity has been shown to influence the spread of diverse toxic oligomeric tau species.Third, the ability to quantify the degree and distribution of synaptic loss can enhance novel experimental medicine studies,11C]UCB‐J is not the only potential marker of synaptic health in frontotemporal dementia and related disorders. A previous study using [18F]UCB‐H PET to assess synaptic loss did not identify a deficit in frontotemporal dementia versus controls, nor was there a difference between frontotemporal dementia and Alzheimer's disease.18F]UCB‐H compared to [11C]UCB‐J may also have contributed to their null result. Furthermore, blood‐based kinetic analysis was used to determine total volume of distribution (VT) for [18F]UCB‐H, which includes nonspecific binding and free tracer in tissue. Hence, [18F]UCB‐H VT may be less sensitive to synaptic changes than [11C]UCB‐J BPND. Results with the alternative ligand [18F]SDM8/SynVesT‐1 have not yet been reported in frontotemporal dementia. Several studies have used [18F]FDG PET to assess brain metabolism and interpreted changes as a marker of synaptic loss. Such metabolic change is disproportionate to atrophy,11C]UCB‐J and [18F]FDG PET in Alzheimer's disease indicated a high correlation in medial temporal regions, but not elsewhere18F]fluorodeoxyglucose and [11C]UCB‐J PET, synaptic function may be assessed by electro−/magnetoencephalography. For example, in Alzheimer's disease, tau burden ([18F]flortaucipir) and synaptic loss ([11C]UCB‐J) are both associated with changes in magnetoencephalography.11C]UCB‐J PET has excellent reproducibility, which is advantageous for longitudinal and interventional studies.[11C]UCB‐J binding potentials and gray matter volumes identified an extensive pattern of synaptic loss, and a more restricted pattern of gray matter loss in patients with bvFTD compared to controls. Specifically, the atrophy pattern in these patients was a subregion of the more widespread significant synaptic loss. Confirmatory analyses showed that regressing the effect of atrophy , [11C]UCB‐J PET leaves widespread significant differences in regional synaptic density between patients and controls. One could interpret [11C]UCB‐J PET as being more sensitive to the presence of brain changes associated with frontotemporal dementia, and conclude that detectable synaptic loss precedes detectable atrophy in patients with bvFTD.11C]UCB‐J PET has potential advantages over MRI for 3 main reasons. First, it allows in vivo quantification in humans of a process that in preclinical models, including transgenic animal models, has been shown to occur before neurodegeneration and cell death, and to occur in response to changes in proteostasis and inflammation. Second, it provides direct evidence for synaptic loss as one of the processes that is commonly presented in depictions of the hypothesis of cascading “multiple biomarkers,” which has to date lacked direct evidence in humans. Third, it provides a readout tool for experimental medicine studies that target processes upstream of synaptic loss.Further studies are needed to develop the utility of synaptic PET as a clinical and/or research tool in frontotemporal dementia. However, our results suggest that it may be useful for the quantification and localization of a major etiopathogenic process such as synaptic loss, and cannot be reduced to cell loss, which can be assessed with structural MRI. Our direct comparison of voxelwise and regional analyses with UCB‐J binding potential group comparisons and correlations, and to assess the variance and effects explained by synaptic loss over and above atrophy. Synaptic loss shows a more extensive pattern than gray matter loss in frontotemporal dementia, and the concordance of partial volume corrected and noncorrected analyses already indicates that the synaptic loss we observe is not simply attributable to atrophy.Our study has several limitations. We acknowledge the relatively small sample size, which does not permit statistically elaborate models (ie, with multiple covariates, such as age and sex, which have previously been shown to have minor/negligible effects on synaptic PET signal11C]UCB‐J PET can facilitate the validation of preclinical models, inform models of human pathogenesis, and inform the design of new disease‐modifying treatment strategies.To conclude, our study confirms that bvFTD is associated with significant and widespread frontotemporal loss of synapses, in proportion to disease severity. We suggest that [M.M., J.T.O., and J.B.R. contributed to the conception and design of the study. M.M., P.S.J., T.E.C, N.H., M.N., M.A.R., T.R., G.S., D.J.W., D.S., T.D.F., Y.T.H., S.M.S., and F.I.A. contributed to the acquisition and analysis of data. M.M., J.T.O., and J.B.R. contributed to drafting the text or preparing the figures.Nothing to report.APPENDIX S1. Supporting InformationClick here for additional data file.


Observational evidence suggests associations between sex hormone levels and heart failure (HF). We used sex‐specific genetic variants associated with androgenic sex hormone profiles to investigate the causal relevance of androgenic sex hormone profiles on cardiac structure and function and HF using Mendelian randomization (MR).P < 5 × 10−8) variants predicting sex hormone‐binding globulin (SHBG), total testosterone, and bioavailable testosterone were extracted from summary statistics of genome‐wide association study (GWAS) on 425 097 participants in the UK Biobank. Sex‐specific gene–outcome association estimates were computed for left ventricular ejection fraction (LVEF), left ventricular end‐diastolic and end‐systolic volumes , left ventricular stroke volume (LVSV), cardiac index, and cardiac output in 11 528 female and 14 356 male UK Biobank Imaging Study participants and for incident or prevalent HF in an external cohort of 47 309 cases and 930 014 controls. Inverse‐variance weighted MR was the primary analysis method. In females, higher genetically predicted bioavailable testosterone was associated with lower LVEDV , lower LVESV , lower LVSV , lower cardiac output , and lower cardiac index  and a higher risk of HF  on external validation analysis in larger scale, sex‐adjusted GWAS data. Higher genetically predicted SHBG was associated with higher LVEDV , higher LVESV , and higher LVSV . In males, higher genetically predicted total and bioavailable testosterone was associated with lower LVESV  and LVEF , respectively.Sex‐specific uncorrelated genome‐wide significant (This study supports a causal effect of pro‐androgenic sex hormone profiles in females on adverse markers of left ventricular structure and function typically associated with HF with preserved ejection fraction and with HF. There was weaker evidence of association in males. Heart failure (HF) is a major global health threat, affecting 64.3 million worldwide.Hyperandrogenic states, characterized by increased levels of bioavailable testosterone, commonly occur in both males and females due to physiological, pathological, and iatrogenic factors. Physiologically, females are known to switch to a proportionally more pro‐androgenic hormonal profile after the menopause,The cardiovascular associations of hyperandrogenism vary between males and females. In males, an inverse association between androgen levels and cardiovascular risk has been reported,The observational nature of currently available evidence is a major limitation to causal inference because observational associations are liable to residual confounding and bias. Mendelian randomization (MR) is a method that leverages ‘natural’ randomization to high or low genetic predisposition to risk factors within an instrumental variable framework to explore the causal relevance of risk factors on an outcome. As the allocation to genetic risk is random, this is akin to randomization in a clinical trial and thus less liable to influence by observational confounding.Improving current understanding regarding sex hormones and cardiovascular risk is a key public health priority. First, it might lead to improved life course risk stratification relating to physiological, pathological, or iatrogenic hormonal changes. Second, it may better inform care for people receiving testosterone‐based hormonal therapy by improving knowledge of cardiovascular effects.This study aims to leverage genetic variants associated with bioavailable testosterone, as well the closely related measures of sex hormone‐binding globulin (SHBG) and total testosterone, to evaluate their causal relevance on multiple cardiac magnetic resonance (CMR) imaging markers of cardiac structure and function, and HF risk.The study is reported using the methodological framework from the Strengthening the Reporting of Observational Studies in Epidemiology using Mendelian Randomization (STROBE‐MR) guidelinesSummary‐level data from publicly available genome‐wide association study (GWAS) summary statistics were used and are available to download freely from the cited publications. Relevant ethical approval and participant consent were obtained in each individual study. Individual‐level data in the UK Biobank were accessed under Application 13784. Participants provided written informed consent for data release for research purposes within approved applications. Further details on the protocol of the UK Biobank can be found in previous publications.All statistical analyses were performed using R Version 4.1.1 (2021‐02‐15)et al.'s GWAS on 425 097 UK Biobank participants of predominantly European ancestry.n = 178 782, females n = 188 507, unit = nmol/L). As testosterone exists in a physiological equilibrium between its bioavailable form and its bound form to SHBG, we additionally studied the secondary exposures of SHBG  and total testosterone . Given their equilibrium state, it would be expected that any direct association observed for the exposure of bioavailable testosterone would be reflected by an inverse association for SHBG and vice versa. Sex‐specific, uncorrelated (r2 < 0.05) single nucleotide polymorphisms (SNPs) associated with each exposure at genome‐wide significance (P < 5 × 10−8) were selected as instrumental variants. The list of instrumental variants for each exposure, with respective effect sizes and effect alleles, is provided in Supporting Information, TablesFigureSex‐specific instrumental variants were extracted from Ruth related r2 < 0.05 related r2 < 0.05 related r2 < 0.05 Outcomes included CMR measures of cardiac structure and function, including left ventricular (LV) end‐systolic volume (LVESV), LV end‐diastolic volume (LVEDV), LV ejection fraction (LVEF), LV stroke volume (LVSV), cardiac output (CO), and cardiac index (CI), and clinical diagnosis of HF.Tableet al.'s GWAS on 47 309 HF cases and 930 014 controls.The methods used to derive measures of cardiac structure and function in the UK Biobank Imaging Study have been previously described.β) and 95% confidence intervals, indicating the expected unit change in CMR parameter for each nmol/L increment in the genetically predicted exposure.Inverse‐variance weighted (IVW) MR with multiplicative random effectsSensitivity analyses were carried out to ascertain potential violations of the instrumental variable assumptions. These include that: (i) the instruments associate with exposures of interest (relevance); (ii) do not have confounders associated with the outcome (independence); and (iii) no horizontal/direct pleiotropic effects exist (exclusion restriction).F‐statistics, reported in Supporting Information, TableTo assess the relevance assumption, instrument strength was quantified using Tableet al.'sn = 806 834 participants) and Evangelou et al.'sn = 738 168 participants) GWAS summary statistics. The strength of instruments for the sex hormone trait in the modified multivariable analyses was quantified using conditional F‐statistics.Where evidence of potential pleiotropy was discovered, instrumental variants were examined in more detail through a phenome‐wide association search using PhenoScanner , P = 0.006], lower LVESV , and lower LVSV . As would be expected, the associations for SHBG were in the opposite direction: genetically predicted SHBG was associated with higher LVEDV , higher LVESV , and higher LVSV . Higher genetically predicted total testosterone was associated with higher LVEDV , but no significant difference in LVESV or LVSV.In females, higher genetically predicted bioavailable testosterone was associated with lower LVEDV , as displayed in Tableβ = −0.12 (−0.21 to −0.03), P = 0.0.013, conditional F‐statistic = 65.4]; however, adjustment for BMI did , indicating likely pleiotropy through BMI.The results are reported in β = −0.08 (−0.16 to 0.00), P = 0.046] and a lower CI . No associations were evident for the secondary exposures of genetically predicted SHBG and genetically predicted total testosterone with either outcome, and none of the exposures were associated with LVEF. The results are reported in FigureTableIn females, higher genetically predicted bioavailable testosterone was associated with a lower CO  and did not attenuate the association with CI . However, adjustment for BMI attenuated the association with both CO  and CI , indicating likely pleiotropy through BMI.Sensitivity analyses revealed potential directional pleiotropy in the association of bioavailable testosterone with CO , P = 0.147].Higher genetically predicted bioavailable testosterone in females was associated with higher odds of HF , but no associations were observed for genetically predicted bioavailable testosterone and SHBG, as reported in FigureTableFigureHigher genetically predicted total testosterone was associated with a lower LVESV in males . No associations with LVEF were observed for genetically predicted SHBG and total testosterone. Additionally, as displayed in FigureIn males, higher genetically predicted bioavailable testosterone was associated with a lower LVEF [P = 0.359], and neither were genetically predicted total testosterone or SHBG , In this study, we explored associations between androgenic sex hormone profiles and multiple markers of cardiac structure and function. A pro‐androgenic profile was associated with adverse LV remodelling with features of greater cardiac ageing broadly reflective of an HF with preserved ejection fraction (HFpEF) phenotype. These relationships appeared more prominent in females, in whom higher bioavailable testosterone (and lower SHBG) was associated with a smaller structure  and function . A pro‐androgenic profile was associated with a greater likelihood of clinical HF in females, but not in males. Thus, our results demonstrate androgenic hormones as a proponent of poorer cardiovascular health, in particular HF, which appears more important in females than males.Several observational studies have explored the association between androgenic sex hormone profiles and cardiovascular risk in females, linking to both menopausal and pathological conditions. Post‐menopausal females exhibit proportionally more androgenic sex hormone profiles, which has been, albeit inconsistently,et al. have previously described an association between higher androgen levels and worse cardiometabolic profiles in females using MR.There are several potential mechanisms behind the findings of this study. Females have a 20‐fold to 25‐fold lower circulating androgen concentration compared with males.In males, higher androgen levels were associated with reduced LVEF and LVESV. These results are contradictory with a prior MR analysisIn this study, we identified potential pleiotropic effects in the association between bioavailable testosterone and cardiac function in females. This finding requires further discussion. First, this only involves one of the exposures, bioavailable testosterone, and the specific outcomes of LVSV, CO, and CI . Given the biologically plausible and consistent results observed across other outcomes and exposures, the potential pleiotropy discovered in some specific associations does not substantially alter the broader interpretation of study findings. Second, we further explored this by highlighting potential culprit phenotypes of BMI and SBP and demonstrated a more likely role of BMI in the pleiotropic effects. This suggests that BMI might either directly or indirectly be responsible for at least part of the observed associations, highlighting it as a key target for surveillance and risk modification.These results carry important clinical implications. First, the results support a potentially adverse mechanistic role of androgens in the cardiovascular association of multiple pathological states characterized by higher androgen levels in females, such as PCOS, and cardiac disease. The results therefore encourage further research to elucidate whether androgens are an important or primary mediator of this association, as they represent therapeutic targets amenable to intervention. Second, they support further research exploring the long‐term cardiovascular health of individuals taking testosterone‐based hormone treatments. These include post‐menopausal females taking HRT preparations that contain testosterone, as well as individuals undergoing gender‐affirming hormone treatments. Gender‐affirming hormone treatment use has increased significantly in recent years, and creating a high‐quality evidence base regarding long‐term cardiovascular health should be a key priority to ensure high standards of care. Evidence among existing studies is conflicting, with some suggesting an increased risk of cardiovascular disease in transgender males undergoing hormone therapyThere are several limitations to discuss. First, magnitude of effect estimates for reported associations cannot be interpreted intuitively as real‐life unit changes in outcome expected per unit change in the exposure through pharmacological modification. Measures of exposures in MR studies reflect a lifetime exposure, rather than a time‐limited modification in the exposure secondary to pharmacotherapy. Second, we were unable to expand the analysis to other sex hormones (e.g. oestradiol and progesterone) and additional measures of cardiac structure and function (e.g. LV mass) due to the lack of sufficiently powered data available. This should be an important target for future research. Finally, in this study, we utilized two‐sample MR methods in a one‐sample setting as both gene–exposure and gene–outcome association data were derived from the UK Biobank. However, this has been shown to be unlikely to significantly bias analyses in large‐scale biobanks.In conclusion, these data support a causal association between bioavailable testosterone in females and adverse measures of cardiac structure and function in a pattern broadly consistent with HFpEF, as well as a higher risk of HF. In males, higher androgen levels were associated with reduced LVEF and LVESV. The results call for further investigation of the prognostic value of sex‐specific risk factors typically associated with pro‐androgenic states (e.g. PCOS) for cardiovascular risk stratification of females. By highlighting a likely causal role of testosterone, they also encourage exploration of whether this might be a modifiable mediator of the increased cardiovascular risk that is known to occur in PCOS. Additionally, they encourage pharmacovigilance and ongoing research to characterize the long‐term cardiovascular health of individuals undergoing testosterone‐based therapies for both gender affirmation and hormone replacement.None declared.This work was supported by core funding from the British Heart Foundation (BHF) (RG/13/13/30194 and RG/18/13/33946), BHF Cambridge Centre of Research Excellence (RE/18/1/34212), and NIHR Cambridge Biomedical Research Centre (BRC‐1215‐20014). This work was also supported by Health Data Research UK, which is funded by the UK Medical Research Council, Engineering and Physical Sciences Research Council, Economic and Social Research Council, Department of Health and Social Care (England), Chief Scientist Office of the Scottish Government Health and Social Care Directorates, Health and Social Care Research and Development Division (Welsh Government), Public Health Agency (Northern Ireland), BHF, and Wellcome. M.A. was supported by the NIHR Academic Clinical Fellowship. J.Y.C. and R.K.R. were supported by the NIHR Imperial Biomedical Research Centre HEE Specialised Foundation post. A.M.M. was supported by the EU/EFPIA Innovative Medicines Initiative Joint Undertaking BigData@Heart (Grant No. 116074). Z.R.‐E. was supported by the BHF Clinical Research Training Fellowship (Grant No. FS/17/81/33318). S.B. was supported by the Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant No. 204623/Z/16/Z). E.D.A. has no relevant funding. F.S.N. was supported by the NIHR Imperial Biomedical Research Centre funding and the BHF (RG/16/3/32175).Table S1. Genome‐wide significant single nucleotide polymophisms (SNP) used as instrumental variants for bioavailable testosterone in females.Table S2. Genome‐wide significant single nucleotide polymorphisms (SNP) used as instrumental variants for total testosterone in females.Table S3. Genome‐wide significant single nucleotide polymorphisms (SNP) used as instrumental variants for sex hormone binding globulin in females.Table S4. Genome‐wide significant single nucleotide polymorphisms (SNP) used as instrumental variants for bioavailable testosterone in males.Table S5. Genome‐wide significant single nucleotide polymorphisms (SNP) used as instrumental variants for total testosterone in males.Table S6. Genome‐wide significant single nucleotide polymorphisms (SNP) used as instrumental variants for sex hormone binding globulin in males.Table S7. Definitions for heart failure used within this study, based on the World Health Organization International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD‐10) codes.Table S8. F‐statistics for instrument strength in all analyses.Table S9. Genome‐wide significant (P < 5.10−8) associations of all instrumental variants used for bioavailable testosterone in females.Click here for additional data file.


To explore the factors affecting the prognosis of cervical cancer (CC), and to construct and evaluate predictive nomograms to guide individualized clinical treatment.The clinicopathological and follow‐up data of CC patients from June 2013 to December 2019 in Sun Yat‐sen Memorial Hospital of Sun Yat‐sen University were retrospectively analyzed. Log‐rank test was used for univariate survival analysis, and Cox multivariate regression was used to identify independent prognostic factors, based on which nomogram models were established and evaluated in multiple aspects.n = 746) and validation sets (n = 329). Survival analysis of the training set identified cervical myometrial invasion, parametrial involvement, and malignant tumor history as prognosticators of postoperative DFS and pathological type, cervical myometrial invasion, and history of STD for OS. C‐index was 0.799 and 0.839 for the nomograms for DFS and OS, respectively. Calibration curves and Brier scores also indicated high performance. Importantly, decision curve analysis suggested great clinical applicability of these nomograms.Patients were randomly assigned into the training (In this study, we analyzed a cohort of 1075 CC patients and identified DFS‐ or OS‐associated clinicohistologic characteristics. Two nomograms were subsequently constructed for DFS and OS prognostication, respectively, and showed high performance in terms of discrimination, calibration, and clinical applicability. These models may facilitate individualized treatment and patient selection for clinical trials. Future investigations with larger cohorts and prospective designs are warranted for validating these prognostic models. Two nomograms were subsequently constructed for DFS and OS prognostication, respectively, and showed high performance in terms of discrimination, calibration, and clinical applicability.  In 2020, there were approximately 604,000 CC cases and 342,000 related deaths worldwide.A handful of independent prognostic factors have been proposed to date, including tumor size, vascular invasion, and pelvic lymph node (PLN) metastasis, although consensus has not been established for all.In this retrospective study, we identified clinicopathological factors associated with overall and DFS of 1075 Chinese CC patients, based on which a nomogram model was subsequently constructed and validated. In addition, we also measure the clinical applicability of the model using decision curve analysis.22.1n = 5) or those who also had ovarian cancer (n = 1), data for 1075 cases were used for subsequent analyses , parity, history of STD, history of malignant tumor, diabetes, liver disease, cardiovascular and cerebrovascular diseases, histological type, tumor differentiation, PLN metastasis status, para‐aortic lymph node metastasis status, tumor size, vascular invasion, cervical junction invasion, cervical myometrial invasion, parametrial invasion, vaginal fornix involvement. Follow‐up data were collected regarding occurrence of and time to disease recurrence, metastasis, and death. DFS was defined as time interval from surgical excision to disease recurrence or death, and OS was defined as the interval from diagnosis to death or last follow‐up. The study was conducted in accordance with the ethical standards laid down in the Declaration of Helsinki. All participants have signed informed consent, and institutional Review Board approval was obtained from the Medical Ethics Board at the Sun Yat‐sen Memorial Hospital.2.2p‐values of <0.1 were selected for multivariate Cox regression analysis. The results of Cox regression were then used to establish prognostic nomograms.The cohort was randomly divided into training and validation sets at 7:3 ratio. The training set was subjected to Kaplan–Meier and log‐rank analyses to examine their association with DFS or OS, and factors with Nomograms were evaluated for discrimination ability with the consistency index (C‐index) and area under curve (AUC) of the corresponding receiver operating characteristic (ROC).All statistical analyses were performed with SPSS (25.0), GraphPad Prism (8.4.3) and the R programming language (4.0.5). The R statistical packages “rms,” “survival,” “foreign,” and “survivalROC” were used to calculate the C‐index and plot the calibration curves, the ROC curves. All statistical tests were two‐sided with the significance level set at 0.05.33.1n = 746) or validation set (n = 329). Age at CC diagnosis age was 22–79 (median 49) years in the intact cohort, 22–79 (median 49 years) in the training set, and 22–79 (median 48 years) in the validation set. The two sets also had similar durations of follow‐up, with a median of 34.38 (range 0.20–86.80) months for the training set and 31.03 (0.00–77.40) months for the validation set. Other important clinicopathologic features were also comparable between the two sets, including histologic subtype, metastasis to the ovaries, tumor size, rate of vascular, cervical junction, or myometrial invasion, vaginal fornix involvement, history of malignancies or sexually transmitted disease (STD), and other medical conditions , Brier score, calibration plot, and DCA analysis Table . The nomThe nomogram for postoperative OS generally showed slightly better performance in these aspects. The C‐index of 0.831 (0.768–0.894) was for the training set and 0.839 (0.756–0.921) for validation set. AUCs for 1‐ to 5‐year predictions ranged 0.736–0.860 in the training set and 0.761–0.828 in the validation set . Same method was performed in the validation set and survival differences were observed among subgroups (p < 0.05). As presented in Figure To further analyze the feasibility and validity of the prognostic nomogram, we divided the cervical cancer patients into different subgroups after sorting by total risk score. Survival analyses demonstrated significant distinctions between subgroups in the training set . This discrepancy may be attributable to the limited number of patients with tumors with diameters >4 cm , thereby reducing statistical power.Additionally, a meta‐analysis of 25 studies with a total of 6500 patients suggested prognostic relevance for tumor size in addition to lymph node metastasis and vascular invasion.NCCN guidelines for CC also recommend parametrial involvement as a high risk factor after surgery, which was supported by a study of 110 early CC patients, which showed associated parametrial involvement with poor prognosis after surgical intervention.Prognostic significance of cervical myometrial invasion remains under debate.Notably, we also identified a novel potential poor prognosticator of DFS, i.e. malignant tumor history. This may be related to the decline in physical function caused by the patient's previous suffering from other cancer.Interestingly, a history of STD was negatively associated with OS in this study. Existing research mainly focuses on the history of STD and CC incidence instead of prognosis. Patients with a history of STD in this study mostly had syphilis, followed by condyloma acuminatum. Syphilis infection has been found necessary for the CC pathogenesis following HPV infection and proposed as a predictor of invasive CC.Nomograms are an established tool for estimating cancer prognosis in quantitative terms and has been adopted in CC.Our study established a nomogram for predicting DFS based on cervical myometrial invasion, parametrial invasion and malignant tumor history and one for predicting OS based on pathological type, cervical myometrial invasion and history of STD. As far as we know, we are the first study to link the history of malignant tumor and STD with poor prognosis in CC patients and establish nomograms. In addition, both models were evaluated with C‐index, ROC curve, Brier score, and DCA analysis, all of which indicated high performance and clinical applicability. C‐index for the two models were 0.775 (0.707–0.842) for DFS and 0.831 (0.768–0.894) for OS in the training set and 0.799 (0.711–0.888) for DFS and 0.839 (0.756–0.921) for OS in the validation set. Calibration curves of 3‐year and 5‐year were also created based on internal validation of bootstrap resampling and showed remarkable accuracy.Decision curve analysis (DCA) can calculate the net benefit of a predictive model to measure its clinical utility. DCA was performed to evaluate the clinical applicability of the constructed nomograms when quantifying the net improvement benefits under different threshold probabilities.Despite having achieved prognostic accuracy, our study is not devoid of limitations. In addition to the retrospective, single‐center nature, the nomogram models, which did not undergo validation with an external cohort. Secondly, the calibration curve suggested low performance in predicting 5‐year survival rates for OS and DFS chart is poor, which may be owing to the higher proportion of censored events at this time point.5In this study, we analyzed a cohort of 1075 CC patients and identified DFS‐ or OS‐associated clinicohistologic characteristics. Two nomograms were subsequently constructed for DFS and OS prognostication, respectively, and showed high performance in terms of discrimination, calibration, and clinical applicability. Further research is warranted for validating these nomograms with larger cohorts and prospective studies.Qunxian Rao: Conceptualization ; formal analysis ; methodology ; writing – original draft . Xue HAN: Conceptualization ; project administration ; writing – original draft ; writing – review and editing . Yuan Wei: Conceptualization ; data curation ; writing – review and editing . Hui Zhou: Conceptualization ; data curation ; writing – review and editing . Yajie Gong: Conceptualization ; data curation ; writing – original draft . Mei‐mei Guan: Conceptualization ; data curation ; writing – original draft . Xiaoyan Feng: Conceptualization ; data curation ; writing – original draft . Huaiwu Lu: Conceptualization ; project administration ; resources ; supervision . Qingsong Chen: Conceptualization ; formal analysis ; methodology ; validation ; writing – review and editing .This research was supported by Medical Science and Technology Foundation of Guangdong Province (grant No.2019GCZX012 [QSC]).There is no financial support or financial agreement, bonds, or any other conflict of interest involving the authors and companies, which may have an interest in the publication of this paper.Institutional Review Board approval was obtained from the Medical Ethics Board at the Sun Yat‐sen Memorial Hospital (No.SYSEC‐KY‐KS‐2021‐336).All participants have signed informed consent.Appendix S1.Click here for additional data file.


Objective To identify risk factors related to postpartum hemorrhage (PPH) and severe PPH with blood loss quantified objectively.Methods This is a complementary analysis of a prospective cohort study that included pregnant women delivering vaginally. The total blood loss was obtained through the sum of the volume collected from the drape with the weight of gauzes, compresses and pads used by women within 2 hours. Exploratory data analysis was performed to assess mean, standard deviation (SD), frequency, percentage and percentiles. The risk factors for postpartum bleeding were evaluated using linear and logistic regression.Results We included 270 women. The mean blood loss at 120 minutes was 427.49 mL (±335.57 mL). Thirty-one percent (84 women) bled > 500 mL and 8.2% (22 women) bled > 1,000 mL within 2 hours. Episiotomy, longer second stage of labor and forceps delivery were related to blood loss > 500 mL within 2 hours, in the univariate analysis. In the multivariate analysis, only forceps remained associated with bleeding > 500 mL within 2 hours (odds ratio [OR] = 9.5 [2.85–31.53]). Previous anemia and episiotomy were also related to blood loss > 1,000mL.Conclusion Prolonged second stage of labor, forceps and episiotomy are related to increased incidence of PPH, and should be used as an alert for the delivery assistants for early recognition and prompt treatment for PPH. In spite of the efforts to decrease maternal mortality worldwide, every day 800 women die due to complications related to pregnancy and childbirth.Postpartum hemorrhage (PPH) is defined by the World Health Organization (WHO) as bleeding > 500 mL within 24 hours after delivery and severe PPH as bleeding > 1,000 mL during the same period.For the last years, PPH and severe PPH is increasing around the world, even in developed countries.Nevertheless, several studies have shown conflicting risk factors for PPH based on visual estimation of blood loss.The present study aimed to identify risk factors related to PPH and severe PPH with blood loss quantified objectively.This is a complementary analysis of a prospective cohort study designed to identify if shock index and other vital signs could be useful to predict PPH (not published). It included pregnant women with gestational age > 34 weeks delivering vaginally at the Women's Hospital  between 1 February 2015 and 31 March 2016. The exclusion criteria were the presence of one or more of these conditions: gestational age < 34 weeks, hypertension, hypo or hyperthyroidism without treatment, coagulopathy, antepartum hemorrhage, any cardiac disease and infections with fever or sepsis.During the labor, at the obstetric ward, women were invited to participate in the study, and if accepted, they signed an informed consent form. A data collection form was filled with information from the women's interview added with information from the medical records. The hemoglobin level was checked in prenatal records. If the last dosage had been made before 3 months, we collected a new blood count before delivery. Previous anemia was defined as hemoglobin levels < 11 g/dL. If the women progressed to C-section they were excluded from the study.Immediately after the fetal delivery, trained research assistants placed a calibrated drape under the women's buttocks , minimum, median, maximum, frequency, percentage and percentiles. The risk factors for postpartum bleeding were evaluated using linear and logistic regression. All statistical analysis was made using SAS 9.4  and we defined a significance level of 5%.The Institutional Review Board  approved the main study, which included evaluating the risk factors for PPH (CAEE: 26787114.3.0000.5404). Without any participation in planning, designing, implementing, collecting data, analysis and interpreting results, Centro de Pesquisas em Saúde Reprodutiva de Campinas (CEMICAMP) and Fund for Support to Teaching, Research and Outreach Activities (Faepex-UNICAMP) supported the study.From the 319 eligible women, 8 denied participation and 41 progressed to C-section. Therefore, we included 270 women. The mean blood loss at 120 minutes was 427.49 mL (±335.57 mL). Thirty-one percent (84 women) of the sample bled > 500 mL and 8.2% (22 women) bled > 1,000mL within 2 hours. On the other hand, among those who bled less, 93 women (34.4%) had blood loss ≤ 300 mL and 125 (46.3%) had blood loss ≤ 400mL, below the mean blood loss. Forty-seven women (17.4%) arrived at the hospital during the second stage of labor. Sociodemographic and obstetrics characteristics are shown inNo women in our sample had intensive care unit (ICU) admission or surgical procedures. Only four women received blood transfusions due to PPH. Among those who bled > 500 mL, 18 women (21.4%) had forceps delivery and 38 (45.2%) had an episiotomy. And among those who bled > 1,000 mL, 4 women (18.2%) had forceps delivery, 11 (50%) had an episiotomy and 6 (27.3%) had previous anemia. The logistic regression to evaluate factors related to blood loss after delivery is shown inn = 260) shows that forceps delivery had an odds ratio (OR) of 9.48  for bleeding > 500 mL within 2 hours.Episiotomy, longer second stage of labor and forceps delivery were related to blood loss > 500mL within 2 hours. The multiple analysis  was found to be a risk factor for severe PPH. This is in agreement with another study that found hemoglobin levels < 9 g/dl as a risk factor for severe PPH, OR = 2.20 (1.63–3.15).Comparing the objective method of measuring PPH, one study from Uganda assessed postpartum bleeding using a calibrated drape. The risk factors found by them were HIV positive, multiple pregnancy and macrosomia.Our data showed that prolonged second-stage labor, forceps and episiotomy, which are very linked to each other, are related to an increased incidence of PPH. In the modern assistance to labor and delivery, it is recommended to respect the obstetrical physiological variations found among women.Although our study has the strength of evaluating objectively the postpartum bleeding for 2 hours, a rare characteristic found in studies that evaluate risk factors, it has some limitations. Our sample size is limited, only 270 women were included, and we excluded pregnant women with hypertension, a known risk factor for PPH. Ideally, we should have divided the comparison of the second stage of labor between above and below 1 hour. However, our sample size was not sufficient for this. Future research with PPH should be done evaluating postpartum bleeding objectively in a large sample size and with no exclusion criteria.Prolonged second stage of labor, forceps and episiotomy are related to PPH, and should be used as an alert for the delivery assistants for early recognition and prompt treatment for PPH.


To compare sexual functioning from diagnosis to 5 years post diagnosis among breast cancer survivors (BCS) and women without cancer (controls).Analyses included 118 BCS and 1765 controls from 20 years of the longitudinal Study of Women's Health Across the Nation (SWAN), a multiracial/ethnic cohort of mid‐life women assessed approximately annually from 1995 to 2015. Pink SWAN participants reported no cancer at SWAN enrollment and developed (BCS) or did not develop (controls) incident breast cancer after enrollment. Outcomes included: being sexually active or not, intercourse frequency, sexual desire, vaginal dryness, and pain with intercourse. Using longitudinal logistic regression, we compared BCS and controls on prevalence of sexual functioning outcomes with respect to years since diagnosis. In addition, we examined whether menopause transition stage, depressive symptoms, relationship satisfaction, vaginal dryness, or pain with intercourse modified the relation between breast cancer and sexual functioning outcomes.Adjusting for partner status, both BCS and controls reported similar declines over time in being sexually active, sexual intercourse frequency, and sexual desire. Among sexually active women, more BCS than controls consistently reported vaginal dryness with significant differences between 2 and 4 years post‐diagnosis, and pain with intercourse, with statistically significant differences between 0.5 years post‐diagnosis to 2 years post‐diagnosis. Being post‐menopausal and reporting depressive symptoms were significant effect modifiers for pain with intercourse with both variables having positive and stronger associations with pain among the controls than among BCS.Except for more reporting of vaginal dryness and pain with intercourse among BCS, negative changes in sexual function during mid‐life were similar in those with and without breast cancer. This article compared sexual functioning over time from diagnosis to 5 years post‐diagnosis among breast cancer survivors (BCS) and women without cancer. Both BCS and controls reported similar declines over time in being sexually active. Among sexually active women, BCS and controls reported similar declines in frequency of sexual intercourse, and sexual desire. Except for more reporting of vaginal dryness and pain with intercourse among BCS, negative changes in sexual function during mid‐life were similar in women with and without breast cancer. After diagnosis and treatment, BCS frequently report sexual problems, which are important quality of life issues.Breast cancer is the most frequently occurring cancer among US women: about 3.8 million breast cancer survivors (BCS) are currently alive in the US  Breast Cancer Survivors study (Pink SWAN) addresses many of the limitations of existing research. Pink SWAN, a SWAN substudy, focused on women who developed incident breast cancer, aged 45–69 years at diagnosis, during 22 years of SWAN follow‐up. Pink SWAN measured sexual functioning multiple times prior to and 5 years after breast cancer diagnosis, uniquely providing data on sexual functioning over time post‐diagnosis and addresses limitations of other studies that had multiple measures of sexual functioning by having 5 years of follow‐up, a wider age range, and more generalizable sample than did a previous study that also had multiple measures.22.1SWAN is a multi‐racial/ethnic cohort study characterizing biological and psychosocial changes occurring during the MT.Eligibility criteria included: age 42–52 years; an intact uterus and at least one ovary; not pregnant, lactating, using oral contraceptives or hormone therapy; and a menstrual cycle in the 3 months before screening. Participants were assessed in‐person at baseline and approximately annually through follow‐up visit 15 in 2015–6. Measures included: medical, reproductive and menstrual history; lifestyle and psychosocial characteristics; physical and psychological symptoms; and anthropometrics. Instruments were translated into Spanish, Japanese, and Cantonese.BCS are women who developed incident breast cancer following SWAN enrollment and had no other prior cancer. Pink SWAN controls are women who never developed breast cancer (before or after SWAN enrollment) and had no prior cancer at baseline. Beginning at visit 12, we requested medical records from women reporting incident breast cancer to verify diagnosis and treatment.Pink SWAN N = 130); 109 responded (83.8%).We identified 151 BCS and 2161 controls. Medical records were obtained for 110 BCS; breast cancers were confirmed in 104 (94.5% agreement). All self‐reports of a breast cancer diagnosis were adjudicated by a medical oncologist. As we have previously described,2.2SWAN measured sexual outcome variables at each study visit from baseline to visit 6 and at alternating study visits thereafter . Women with and without partners completed a 20‐item, self‐administered questionnaire inquiring about sexual activity and function during the prior 6 months, which was returned to staff in a sealed envelope. The questionnaire was derived from several sources,Sexually active/inactive . Women were considered sexually active if they responded “yes” to the following question: “During the past 6 months, have you engaged in sexual activities with a partner?” Women responding “no” were asked the reason(s) for inactivity: no partner, partner's physical problems, own physical problems.Consistent with our previous workDesire  asked frequency of desire to engage in any form of sexual activity, either alone or with a partner (<weekly vs. ≥weekly).Sexual Intercourse  inquired about frequency of sexual intercourse (< once/week vs. ~1 time per week or more), and Pain with sexual intercourse  assessed frequency of vaginal or pelvic pain during intercourse . Frequency of arousal  assessed frequency of feeling aroused during sexual activity .Vaginal dryness was asked of all women as part of a symptom list in the main SWAN interview form and assessed frequency of vaginal dryness in the past 2 weeks (categorized as any vs. no days).To control for pre‐diagnosis sexual functioning, we created a pre‐diagnosis summary of each outcome, calculated as the percentage of visits at which the outcome was present during the 5 years prior to diagnosis.2.3Candidate variables were selected based on previous associations with sexual functioning in SWANDepressive symptoms, assessed by the Center for Epidemiologic Studies Depression (CESD) scale, was dichotomized to <16 versus ≥ 16.Additional covariates included: age at diagnosis, time‐varying marital status (married or living as married), and race/ethnicity.Cancer‐related variables, ascertained from SWAN, medical records, and BCS questionnaire data were stage , ever chemotherapy, ever radiation, and medical treatments .2.4Aim 1 analyses, we estimated a logistic regression model for each post‐diagnosis sexual functioning outcome as a function of four predictors: BCS/control status, years since diagnosis, the interaction of BCS/control status and years since diagnosis, and concurrent marital status with generalized estimating equations to accommodate within‐participant dependence.Pre‐ and post‐diagnosis characteristics of BCS and controls were compared using chi‐squared and t‐tests. For After examining LOESS plotsN = 429; 21.9% of BCS and 18.3% of controls, p = 0.28 for difference between BCS and controls) were excluded from all analyses; the proportion excluded did not differ between BCS and controls. Visits with missing data for an outcome and/or predictor were excluded from the analysis for the outcome being modeled.Analyses of sexual activity, desire, and vaginal dryness included all measurements 0.5–5 years post‐diagnosis, and analyses of other outcomes included only observations in this interval at which concurrent partnered sexual activity was reported. Additional analyses for desire and vaginal dryness included only observations with concurrent partnered sexual activity. Participants with no observed sexual functioning outcomes in the interval 0.5–5 years post‐diagnosis (Associations of outcomes with years since diagnosis were expressed as per‐year adjusted odds ratios (aOR). Model‐based estimated outcome prevalences were compared for BCS and controls at 0.5, 1, 2, 3, 4, and 5 years post‐diagnosis. Sensitivity analyses adjusted for age, pre‐diagnosis outcome summaries, pain and dryness outcomes, and lubricant use. In BCS only, we used the same methodology to estimate associations of cancer‐specific variables with outcomes.Aim 2 analyses of effect modification, we tested whether associations between selected characteristics and sexual function differed between BCS and controls. Aim 2 analyses added two additional predictors to Aim 1 models: an hypothesized effect modifier and its interaction with BCS/control status; a separate model was run for each hypothesized effect modifier and each outcome. Characteristics examined were MT stage , vaginal dryness, pain, depressive symptoms, and emotional satisfaction.In p‐Values ≤0.05 were considered statistically significant.Analyses used SAS 9.4 (SAS Institute Inc.). 33.1p < 0.01). Post‐diagnosis, BCS reported worse health (p < 0.01), and controls were almost twice as likely as BCS to be pre‐/early peri‐menopausal with no exogenous hormone use. BCS did not differ from controls in use of lubricants pre‐diagnosis but were significantly more likely to report use post‐diagnosis (p = 0.03). Most BCS were diagnosed at cancer stage I. No participants had metastatic cancer. Almost all BCS (99.0%) had surgery; 38.3% had chemotherapy, and 70.2% had radiation. The mean age at diagnosis/pseudo‐diagnosis was 56.2 years for BCS and 55.6 years for controls, p = 0.27. Of the 118 BCS, only one woman (0.9%) had a completely missed study visit post‐diagnosis, and among the 1765 controls, no one completely missed a study visit post‐pseudo‐diagnosis. Among these participants, 7.68% were missing sexual activity, and among those reporting sexual activity, the percent of missing data ranges from 0.50% (for dryness) to 6.02% (for pain). Loss to follow‐up (due to death or study drop‐out) was 20.3% (N = 24) among BCS and 10.1% (N = 178) among controls.Of the 151 BCS and 2161 controls meeting inclusion criteria, 118 BCS and 1765 controls had at least 1 visit in the interval 6 months through 5 years post‐diagnosis and were included in analyses. BCS and controls did not differ on age, race/ethnicity, or pre‐diagnosis self‐reported health (Table 3.2Table 3.2.1p = 0.71 for BCS‐control difference). BCS and controls also had similar declines in frequency of intercourse with time since diagnosis/pseudo‐diagnosis.BCS reported a steady, but non‐statistically significant, decline in being sexually active Table  from 64.3.2.2Including all visits , desire declined similarly among BCS and controls . Vaginal dryness prevalence increased for both BCS and controls, with a slightly steeper but statistically non‐significant rise and higher prevalence in BCS than in controls .3.2.3Including only visits at which participants reported being sexually active, prevalence of sexual desire was higher than for all vists, but otherwise patterns were consistent with results for all visits: BCS and controls both reported declines in desire over time. Per‐year increase in vaginal dryness was also similar for BCS and controls, and prevalence was consistently higher in BCS throughout. However, in contrast to all visits, BCS‐control differences were greater and statistically significant between 2 and 4 years post‐diagnosis. BCS also had a consistently higher prevalence of pain with intercourse than controls, differing significantly through 2 years post‐diagnosis.Results of sensitivity analyses adjusting for age, pre‐diagnosis outcome summaries, and lubricant use were consistent with those presented (data not shown).3.3p = 0.03), and receipt of radiation therapy was significantly related to greater frequency of intercourse  (data not shown).Among BCS, at visits at which women reported being sexually active, desire was lower in stages II–III than stage 0  (Table p = 0.02 and 0.03 respectively). Being very emotionally satisfied was significantly more strongly associated with arousal in controls than in BCS (p = 0.02).Previously identified correlates  were significantly associated with at least one sexual functioning outcome in analyses of main effects Table . However5) Table . Statist4This study found that some changes in sexual functioning previously attributed to breast cancer were also present in midlife women without cancer. Both groups showed a decline over time since diagnosis/pseudo‐diagnosis in being sexually active, sexual intercourse frequency, and sexual desire, and an increase in vaginal dryness. Time since diagnosis was important for BCS‐control differences for vaginal dryness and pain in visits in which women reported being sexually active. Characteristics, such as vaginal dryness, related to sexual functioning among BCS, were similarly related to sexual functioning among controls. These BCS‐control similarities highlight the importance of a non‐cancer control group and the value of multiple longitudinal assessments for at least 5 years, in understanding sexual functioning following a breast cancer diagnosis.The percentage of BCS who were sexually active over 5 years post‐diagnosis was similar to that of controls, as was the rate of decline. The per‐year adjusted odds ratios were similar in both groups, but significant only for the controls due to their larger sample size and more narrow confidence intervals. The one other study that examined being sexually active/inactive over multiple timesIn addition to frequency of sexual intercourse, we also found a non‐significant decline in desire and an increase in vaginal dryness among BCS. The specific timing of these declines varies somewhat from that of Ribi and colleagues,We found little evidence for effect modification by BCS‐control status of characteristics previously related to sexual functioning among BCS In contrast to others,These analyses had several strengths. First, results may be more generalizable than other studies because BCS were participants in a longitudinal study of mid‐aged women and not recruited for a sexual functioning study. Age at diagnosis ranged from 45.2 to 69.5, thus covering a wider aged range than studies focusing only on younger women. Although 20% of BCS and only 10% of controls were lost to follow‐up, which could have led to a healthy sample bias, this loss to follow‐up among BCS was much lower than that of prior longitudinal studies in which follow‐up rates ranged from 31% to 77.5% (most in the range of 31% to 46%).Several limitations of the study should be considered. First, the sexual functioning questionnaire was not one of the more newly developed, validated questionnaires, such as the SPEQIn conclusion, in the first few years after diagnosis, among sexually active women, significantly more BCS reported vaginal dryness and pain with intercourse than did their counterparts without cancer. Being sexually active, frequency of sexual intercourse, and frequency of desire declined following diagnosis, but not at greater rates than those of women without cancer. Recent ASCO Guidelines provide recommendations for treating symptoms of vaginal and/or vulvar atrophy, such as dryness (which may reduce pain with intercourse), beginning with lubricants and vaginal moisturizers as a first option.Nancy E. Avis: Conceptualization (lead); formal analysis (supporting); funding acquisition (lead); investigation (lead); methodology (lead); project administration (lead); writing – original draft (lead); writing – review and editing (lead). Sybil Crawford: Conceptualization (supporting); data curation (lead); formal analysis (lead); funding acquisition (supporting); investigation (supporting); methodology (supporting); writing – original draft (supporting); writing – review and editing (supporting). Ellen Gold: Conceptualization (supporting); formal analysis (supporting); funding acquisition (supporting); investigation (supporting); methodology (supporting); writing – original draft (supporting); writing – review and editing (supporting). Gail Greendale: Conceptualization (supporting); formal analysis (supporting); funding acquisition (supporting); investigation (supporting); methodology (supporting); writing – original draft (supporting); writing – review and editing (supporting).The Study of Women's Health Across the Nation (SWAN) has grant support from the National Institutes of Health (NIH), DHHS, through the National Institute on Aging (NIA), the National Institute of Nursing Research (NINR) and the NIH Office of Research on Women's Health (ORWH) . Pink SWAN is supported by the National Cancer Institute R01CA199137. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the NIA, NINR, ORWH or the NIH.The authors do not have any conflicts of interest.Table S1Click here for additional data file.


Extracorporeal cardiopulmonary resuscitation (ECPR) is an emerging strategy in highly selected patients with refractory cardiac arrest (CA). Animal models can help to identify new therapeutic strategies to improve neurological outcome and cardiac function after global ischemia in CA. Aim of the study was to establish a reproducible ECPR rat model of ventricular fibrillation CA (VFCA) that leads to consistent neuronal damage with acceptable long-term survival rates, which can be used for future research.n = 15) and 8 min (n = 16) VFCA. Animals surviving for 14 days after return of spontaneous resuscitation (ROSC) were compared with sham operated animals (n = 10); neurological outcome was assessed daily until day 14. In the hippocampal cornu ammonis 1 region viable neurons were counted. Microglia and astrocyte reaction was assessed by Iba1 and GFAP immunohistochemistry, and collagen fibers in the myocardium were detected in Azan staining. QuPath was applied for quantification.Male Sprague Dawley rats were resuscitated with ECPR from 6 min (n = 10) survived 2 weeks. The quantity of viable neurons was significantly decreased, while the area displaying Iba1 and GFAP positive pixels was significantly increased in the hippocampus across both groups that experienced CA. Interestingly, there was no difference between the two CA groups regarding these changes. The myocardium in the 8 min CA group exhibited significantly more collagen fibers compared to the sham animals, without differences between 6- and 8-min CA groups. However, this significant increase was not observed in the 6 min CA group.Of the 15 rats included in the 6 min CA group, all achieved ROSC (100%) and 10 (67%) survived to 14 days; in the 8 min CA group, 15 (94%) achieved ROSC and 5 (31%) reached the endpoint. All sham animals (Our findings indicate a uniform occurrence of neuronal damage in the hippocampus across both CA groups. However, there was a decrease in survival following an 8-min CA. Consequently, a 6-min duration of CA resulted in predictable neurological damage without significant cardiac damage and ensured adequate survival rates up to 14 days. This appears to offer a reliable model for investigating neuroprotective therapies. In EuroAnimal models have been instrumental in discovering novel therapeutic strategies to mitigate injuries caused by global ischemia and reperfusion following extended, untreated cardiac arrest (CA) , 4. In rExtracorporeal cardiopulmonary resuscitation (ECPR) has gained prominence as a rescue therapy for patients in CA, refractory to conventional measures of CPR . While rThe objective of this study was to create a reliable and replicable ECPR VFCA rat model capable of exhibiting acceptable long-term survival rates while still reflecting profound neuronal damage. We sought to pinpoint the precise duration of VFCA needed to attain this, comparing 6 and 8 min of VFCA and 14 days (d) of survival as study endpoint. Beyond resuscitation with ECPR, no additional therapeutic interventions were employed, and animals were maintained at normothermic conditions during ECPR and after ROSC. This model is anticipated to be an appropriate platform for future research on the pathophysiological mechanisms underlying ischemic damage and the exploration of corresponding therapeutic strategies. Our primary outcome focused on histological brain damage in the cornu ammonis 1 (CA1) region of the hippocampus, while secondary outcomes included survival rate, neurologic outcome, and myocardial damage resulting from global ischemia and reperfusion after a 14-day survival period.2.2.1.The experimental protocol complied with the Austrian Federal Law , Directive 2010/63/EU and the ARRIVE guidelines  of 450–600 grams, were obtained from the Division of Laboratory Animal Science and Genetics, Himberg, Austria. The animals were acclimated at the Center of Biomedical Research of the Medical University of Vienna for at least 14 d prior to the study. Rats were subjected to VFCA and resuscitated using ECPR. After a 14-day survival period, the animals were euthanized, and their brain and heart tissues were examined histologically and immunohistochemically. Details of the ECPR rat model used can be found in previous publications , 11, and2.2.2 1.0) and received piritramide  for analgesia. They were endotracheally intubated  and anesthesia continued with 3.5% sevoflurane. Mechanical ventilation  was maintained, and the animals were continuously monitored for physiological parameters . They were placed on a heated operating table to maintain a body temperature of 37 ± 0.2°C during the experiment.Rats were anesthetized using 6% inhaled sevoflurane , the venous and arterial catheter for venous drug application, hemodynamic monitoring and arterial blood gas sampling were implanted , using aseptic techniques. The venous ECMO drainage cannula  was inserted via the right jugular vein with the tip in the inferior vena cava (correct placement verified by sonography). All catheters and cannulas were heparinized and after insertion of the venous ECMO cannula a heparin bolus (500 IU/kg BW) was administered intravenously (i.v.) to avoid clotting. The ECMO set-up  was primed  and connected to the arterial ECMO cannula . In the 2.3.2 1.0) was continued and epinephrine (10 μg/kg BW) was administered i.v. every 2 min during ECPR until ROSC was achieved or resuscitation efforts were halted after a maximum of 10 min of ECPR. The protocol is outlined in Sevoflurane administration was halted 90 s prior to the end of ventilation and simultaneous initiation of VF  as previously described by Janata et al. . After r2.4.2 0.5) adapted. Prompt removal of the venous ECMO cannula occurred after ROSC, and the wound was closed aseptically. “Sustained ROSC” was defined as maintenance of mean arterial pressure (MAP) exceeding 60 mmHg for a duration of 20 min without mechanical support. Arterial blood gas analyzes were conducted at intervals of 5 min, 15 min and 60 min following ROSC, with ventilation adapted as required. Throughout this period, continuous monitoring of the rats was performed as they remained on the heated operating table until the femoral catheters were removed. The rats were then successfully weaned from the ventilator and extubated.Upon achieving ROSC  , the ECMDuring the initial 24–48 h post-resuscitation, the rats were predominantly unconscious and therefore housed individually until recovery. Analgesia was provided with piritramide (3 mg/kg BW s.c.) at 6-h intervals as long as animals showed signs of pain. Supplemental NaCl 0.9% and Glucose 10% was given s.c. if the animals were unable to drink. Unconscious rats were placed on a heating pad with an integrated control unit linked to a rectal temperature probe to maintain normothermia.For animals that had not fully recovered and required care, visits were scheduled at a minimum of 6-h intervals. These visits included administering pain medication, providing fluid therapy, and assisting with hand- or tube-feeding. Animals that were awake but too weak to eat unaided, were fed a nourishing blend of applesauce and finely ground cereals via gavage.The sham group, which underwent all surgical interventions without experiencing CA and ECPR, received the same treatment duration and medications, excluding those specific to resuscitation.Neurologic outcomes were assessed daily using the Overall Performance Category (OPC)  and the th day, the animals were anesthetized and analgesized (with sevoflurane and piritramide), endotracheally intubated and mechanically ventilated. A thoracotomy was performed, and the heart was excised from the thorax and the brain was carefully removed from the skull. One randomly selected half of the brain, along with the whole heart, were preserved in a 4% buffered formaldehyde solution for histological analysis.On the 142.5.Coronary brain sections containing the hippocampus were embedded in paraffin wax. Sections 2 μm thick were cut and stained with hematoxylin and eosin (HE) for descriptive evaluation and to count viable pyramidal neurons in two 250 μm long sectors of the hippocampal cornu ammonis 1 (CA1) region at 200x magnification.Immunohistochemistry was performed to stain microglia and astrocytes using primary antibodies against ionized calcium-binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP), respectively. A polymer detection system, consisting of a secondary antibody conjugated to an enzyme-labeled polymer, was used in an autostainer . For details regarding pretreatment, antibodies, and dilution see To assess microglia and astrocyte activation immunohistochemical stainings were evaluated in a descriptive manner and analyzed using QuPath, a software platform for bioimage analysis . For this purpose, a digital image was taken of the hippocampal CA1-region by an Olympus SC50 camera mounted on an Olympus BX53 microscope with a 20x objective. In QuPath a pixel classifier was trained and applied to detect pixels stained brown in the immunohistochemical stainings .For the histological assessment of the myocardium a coronary section of the heart at the major circumference was embedded in paraffin wax and cut at a thickness of 2 μm. These sections were stained with HE for descriptive evaluation and with Azan-staining following the Haidenhein method to identify collagen fibers. Assessment of myocardial scar formation was conducted by scanning whole slides  and analyzing with QuPath to quantify the extent of collagen fibers. The myocardium was annotated, and a pixel classifier was trained and applied to the full slide scans to detect pixels stained blue in the annotated areas. The ratio of blue-stained pixels to all stained pixels was subsequently calculated.2.6.For our statistical analysis, continuous data that were normally distributed are expressed as the mean ± standard deviation, while categorical variables are presented as counts and percentages. Ordinal data are described by the median and interquartile range.t-test, Kruskal-Wallis-Test and Mann–Whitney-U-Test, as appropriate. Multiple comparisons were adjusted using Bonferroni corrections, and survival analysis was conducted using Kaplan–Meier methods.For testing the null hypothesis of no difference between independent groups, we employed the Student’s For histological and immunohistochemical data, we first assessed normality using the Shapiro–Wilk Test. Normally distributed data were analyzed with ANOVA, while non-normally distributed data were analyzed using the Kruskal-Wallis-Test. Corrections for multiple comparisons were applied using Tukey’s range test and Dunn’s test, respectively.Sample size calculation was performed for the primary outcome of histologic damage, CA1 hippocampus at 2 weeks survival. Assuming a slightly larger standard deviation of 7 one needs 10 animals per group do detect a difference of 10 cells with a power of 80% at a significance level of 0.05 , therefop-value of less than 0.05 as indicative of statistical significance.Data management and analysis were carried out using MS Excel, GraphPad Prism (software version 10.0.1 for Windows) and SPSS . We considered a two-sided 3.3.1.n = 20; 8 min CA, n = 24), and one sham-operated group . Technical failure led to the exclusion of 13 animals, with 5 rats from the 6 min CA group and 8 rats in the 8 min CA group. In the 6 min CA group, all 15 rats included achieved ROSC (100%), and 10 (67%) survived to 14 days . Out of 16 animals included in the 8 min CA group, 15 (94%) achieved ROSC, with 5 (31%) surviving to 14 days . The NDS score was 0  in the 6 min CA group compared to 5  in the 8 min CA group (p = 0.001).Neurologic outcomes were assessed daily using NDS and OPC scores, respectively ,B. On dap = 0.227). Cumulative survival analysis is depicted in Kaplan–Meier curves in The survival rates at 14 days did not differ significantly between the 6 min and 8 min CA group (3.2.3.2.1.p < 0.001) intact neurons and in animals with 8 min CA 34 ± 17 (p < 0.001) viable neurons were detectable. No statistically significant difference was found between the two cardiac arrest groups (p = 0.90).In the hippocampus of all animals subjected to CA either multiple hypereosinophilic, necrotic neurons were present, or many neurons were missing and replaced by activated astrocytes and microglia in HE-staining ,C. In co2; 6 min CA: 5805 ± 3316 μm2, p = 0.001; 8 min CA: 5595 ± 2722 μm2, p = 0.004), but no differences were detected between the CA groups (p = 0.99)  .2, p < 0.001, 8 min CA: 3785 ± 2402 μm2, p = 0.012). No statistically significant differences were detectable between both CA groups (p = 0.97)  .3.2.2.p = 0.012). No differences were detected between both CA groups (p = 0.27) or between 6 min CA group with 0.79% of myocardium  and sham group (p = 0.34). Representative images of all groups are shown in The myocardium of sham animals exhibited normal morphology, with striated myofibers, small amounts of interstitial connective tissue present in perivascular, subendocardial, and subepicardial regions in HE staining. In contrast, myocardium after 8 min of CA showed multifocal to coalescing areas of activated fibroblasts producing eosinophilic fibrillar material associated with myofiber loss. Similar lesions were present in small, scattered foci in the myocardium of 6 min CA animals. The quantification confirmed a significant increase of Azan-stained collagen fibers in the 8 min CA group  compared to the sham group  of VFCA. The aim was to develop a model that would simultaneously create profound neuronal injury and maintain acceptable mortality rates. This would be suitable for future investigations into the pathophysiological mechanisms of ischemic damage as well as the development of therapeutic strategies.In our comparison, the 6 min CA group showed a two-thirds survival rate to the endpoint with a more favorable neurologic outcome, while two-thirds of the 8 min CA group died. There were no differences in hippocampal neuronal damage between the groups, but the longer ischemia led to increased myocardial damage.ECPR in rodent models is seldom reported in the literature and is mainly applied in asphyxia –20 or exThe successful resuscitation rates in our model (100% of the 6 min CA group and 94% for the 8 min CA group) highlight the feasibility of ECPR in achieving ROSC in most animals. The 33% pre-endpoint mortality rate in the 6 min group aligns with average drop-out rates in similar 6 min VFCA rodent models , 25, wheJanata et al.  used 6 mIn our former study, Schober et al. compared ECPR and conventional CPR after 8 min of VFCA in their rat model. In the ECPR group 5 of 8 animals had sustained ROSC. However, this was a short-time survival experiment to analyze metabolic changes during ischemia and resuscitation displayed by cerebral microdialysis. Per protocol the survival time in this study was 80 min from ROSC .The final NDS and OPC scores were significantly different between the 6 min group and 8 min group. In the 6 min CA group, animals died within the initial 48 h, but subsequently, all surviving animals recovered. Conversely, in the 8 min CA group animals died until day 5, thereby prolonging the unstable phase in this group. Throughout this critical period of pronounced impairment, the animals progressively lost motor skills, and an escalating paresis was observed in all four extremities. During this time, animals were provided with intensive care and regular pain management.In our study, animals exposed to 6- and 8-min CA exhibited a significant reduction in the number of viable neurons in the hippocampal CA1 region, relative to sham animals. This findings mirrors the results reported by Janata et al., who observed a similar decrease in neuron numbers in their ECPR rat model after 6 min of CA and a 14-day survival period . In contInterestingly, after 14 d of survival neither neuron numbers nor activation of microglia or astrocytes were different in the CA groups. An explanation for this lack of difference might be that neuronal damage and glial response after 6 min of CA are already maximized and longer duration of ischemia does not lead to increased lesions. This theory is consistent with our prior CPR model, where differences in neuronal damage were not significant between 6 min CA and 8 min CA groups, though a broader variation in intact neurons was found in the hippocampal CA1 region in the 6 min CA group  animals, which were exposed to the fibrillation catheter without current. This indicates that the cardiac arrest procedure itself is unlikely to be the sole cause of the observed myocardial damage.Our ECPR rat model presents certain limitations when juxtaposed with the clinical scenarios in humans. Clinically, ECPR is reserved for a selected group of CA patients unresponsive to standard CPR efforts. Conversely, our animal model implants the ECMO prior to inducing CA. The ECMO is then initiated immediately after a 6 or 8-min no-flow period, bypassing the simulation of conventional CPR and the consequent low-flow period. In real-world clinical settings, ECPR is only applied if standard CPR is ongoing.In our study, rats achieved ROSC within few minutes of ECPR and were promptly weaned off ECMO, followed by an immediate removal of all ECMO cannulas. This contrasts sharply with clinical procedures where weaning and decannulation are not undertaken in such early phase. Typically, ECMO management in human ECPR patients spans 3–4 days during the post-resuscitation period .Although histologic examination of the hippocampal CA1 region is a standard evaluation of neuronal damage in CA models, we cannot rule out that additional damage in other brain regions differed between 6- and 8-min CA, contributing to the survival rate and/or longitudinally assessed neurological scores.6.In this study using a VFCA ECPR model with 6- and 8- min no-flow CA durations, we demonstrated that extending the CA duration did not increase histological damage to the hippocampus in animals that survived for 14 days. However, the longer CA duration did result in a reduced survival rate. Consequently, a 6-min CA duration led to consistent neurological damage while maintaining satisfactory survival rates up to 14 days, positioning it as a stable model appropriate for studying neuroprotective treatments.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was approved by Institutional Ethics Committee for Laboratory Animal Experiments of the Medical University of Vienna. The study was conducted in accordance with the local legislation and institutional requirements.A-MS: Writing – original draft, Writing – review & editing, Data curation, Investigation, Conceptualization, Methodology, Supervision. SH: Investigation, Methodology, Writing – original draft, Writing – review & editing, Data curation, Formal analysis, Supervision, Visualization. MM: Investigation, Writing – review & editing. IM: Investigation, Writing – review & editing, Data curation. PK: Investigation, Writing – review & editing. BU: Investigation, Writing – review & editing. AS: Writing – review & editing, Investigation. FP: Writing – review & editing, Investigation. AE-S: Writing – review & editing, Data curation. JK: Writing – review & editing, Investigation. TS: Supervision, Writing – review & editing, Resources. MH: Data curation, Visualization, Writing – review & editing, Formal analysis. WW: Conceptualization, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.