Women in healthy transition deep phenotyping cohort study protocol (KiSO-DP): a within-participant multidisciplinary longitudinal cohort study across the menopausal transition.

Cardiovascular disease remains the leading cause of premature mortality in Western countries 1 underscoring the need for effective prevention strategies to ensure that increased lifespan is accompanied by sustained health and quality of life. However, the trajectories of cardiovascular disease development differ between men and women. In men, the cardiovascular risk increases progressively with age, whereas in women, it remains relatively low until midlife, after which the risk increases substantially, coinciding with the menopausal transition. 2 3 The menopausal transition is marked by the cessation of endogenous ovarian hormone production and is thus characterised by a profound hormonal shift with widespread physiological changes across multiple organ systems, including the cardiovascular system. 2 4 5 Within the cardiovascular system, the vascular endothelium is a key regulator of vascular homeostasis and blood flow, and endothelial dysfunction represents an early and critical event in the development of vascular disease. 6 The endothelium regulates arterial tone and tissue perfusion through vasoactive mediators such as nitric oxide and prostacyclin. 7 Oestrogen supports endothelial signalling through both genomic and non-genomic pathways, and reduced oestrogen availability is associated with impaired vascular function. 4 5 Endothelial dysfunction is therefore frequently implicated in menopause-related cardiovascular changes; however, the timing and progression of vascular alterations across the menopausal transition remain insufficiently characterised. In parallel, prognostically important structural and functional cardiac changes, including alterations in ventricular dimensions and diastolic function, have been observed after menopause, 8 – 10 and the temporal relationship between vascular and cardiac changes remains unclear. Therapeutic modulation of the hormonal shift has therefore been proposed to influence health trajectories in midlife women. Menopause hormone therapy (MHT) alleviates menopausal symptoms and exerts systemic effects on the vascular system, including modulation of endothelial function. 11 12 While experimental studies support potentially beneficial vascular effects of oestrogen, particularly when initiated close to the menopausal transition, clinical evidence has predominantly focused on cancer and thrombotic risk, which varies by formulation, route of administration and timing of exposure. 13 – 15 Consequently, the long-term cardiovascular impact of MHT remains incompletely understood, and longitudinal data capturing early cardiovascular changes across the menopausal transition and the subsequent postmenopausal years are limited. With women spending an increased proportion of life after menopause, defining the mechanisms and temporal sequence of cardiovascular ageing in women is essential to identify strategies and modifiable factors that may preserve long-term health. Beyond cardiovascular changes, menopause is also associated with a wide range of vasomotor, physical and psychological symptoms 16 – 21 leading to risk of psychological challenges 22 23 and reduced quality of life. 11 24 25 However, the menopausal transition is highly heterogeneous, with substantial interindividual variation in both symptom burden and physiological adaptations. 16 26 – 28 This variability reflects the interplay between biological, behavioural and social factors. 29 In addition to hormonal changes, lifestyle factors such as physical activity may influence cardiovascular adaptations during this period. While regular physical activity improves vascular function, 30 31 evidence suggests that vascular responses to exercise may be attenuated with increasing time since menopause. 32 33 Lifestyle is associated with menopausal symptoms, 34 35 but whether habitual physical activity influences the onset or progression of endothelial dysfunction during and after the menopausal transition remains unclear. 36 Understanding how ageing and menopause impair cardiovascular function across this pivotal period requires longitudinal studies with repeated deep cardiovascular phenotyping across the menopausal transition and into postmenopausal years. However, such large-scale longitudinal studies remain limited. Strengthening the evidence base on women’s health, including the cardiovascular consequences of menopause, aligns with broader global calls for more rigorous sex-specific research, as persistent data gaps continue to contribute to sex inequalities in health outcomes. 37 The Women in Healthy Transition Deep Phenotyping (Danish: Kvinder i Sund Overgan, KiSO DP) study is a multidisciplinary prospective longitudinal cohort study designed to determine the mechanism underlying cardiovascular changes from the late reproductive stage through 20 years of postmenopause. By providing deep mechanistic, longitudinal insight into menopause-related cardiovascular ageing, the study will contribute to improved understanding of female physiology and inform future prevention strategies for cardiovascular disease in women. Vascular function is assessed as changes in brachial artery vascular conductance with intra-arterial acetylcholine (ACh) infusion, measured at timepoints: pre, 1, 3, 5, 10 and 20 years postmenopause. Change in brachial vascular conductance with intra-arterial epoprostenol (EPO) infusion; non-invasive assessment of vascular function with flow-mediated dilation, brachial artery and carotid artery compliance, cardiac parameters measured with echocardiography at rest and during exercise, arterial blood pressures, cardiorespiratory fitness (maximal pulmonary oxygen uptake (VO 2max )), skeletal muscle properties (mitochondrial function, fibre-type, capillary density), skeletal muscle and circulating in serum and urine multi-omics outcomes, whole blood rheological properties, body composition, hormonal levels, self-reported measures of physical activity, socioeconomic factors, supplement use, menopausal symptoms, as well as qualitative assessments of women’s experiences of the menopausal transition obtained through focus group interviews and individual in-depth interviews.

The KiSO DP study is a multidisciplinary prospective longitudinal cohort study. All data collected throughout the study are administered by the University of Copenhagen. The study protocol structure and content were informed by Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines, where applicable. 38 All participants are enrolled into the KiSO DP cohort when clinically characterised as premenopausal in the late reproductive stage, according to the Stages of Reproductive Aging Workshop (STRAW)+10 criteria, 39 assessed by menstrual cycle patterns, circulating follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH) concentrations and antral follicle count. Participants in the KiSO DP cohort undergo two experimental test days at each time point. The KiSO DP cohort experimental days are conducted at six time points over approximately 25 years, beginning in the late reproductive stage ( pre ) and repeated at the first postmenopausal assessment, defined as 12 months after the final menstrual bleeding or an equivalent hormonally confirmed postmenopausal stage in participants using hormonal/levonorgestrel-releasing intrauterine devices (LNG-IUD), this is referred to as postmenopause through the protocol. Assessments are subsequently repeated at 3, 5, 10 and 20 years postmenopause, as illustrated in figure 1 . Additional subcohorts of the KiSO DP cohort will be established for echocardiography, mitochondrial function, rheology and individual and focus group interviews. Protocols for these sub cohorts will be described in detail in the specific publications. Any substantial amendments to the study protocol will be submitted for approval to the regional ethics committee prior to implementation. Approved amendments will be updated in the trial registration (ClinicalTrials.gov) and reported in subsequent publications when relevant. The KiSO DP cohort will include 200 Danish women aged 43–55 years. Recruitment began in December 2022 and is expected to conclude in 2026. Participants are recruited via randomly distributed Digital Post invitations, a national secure digital communication system, used by Danish public authorities and citizens, 40 local newspaper advertisements, social media and participant referral. Initial contact with potential participants includes a telephone-based eligibility assessment, followed by an invitation to a health and menopausal status screening. Written and oral information is provided, and written informed consent is obtained prior to the examination. Screenings are scheduled within the first to fifth day of the participant’s reported menstrual cycle; for amenorrhoeic participants using LNG-IUD, screening is not conducted according to menstrual cycle. 41 Screenings consist of questionnaires on anamnesis and menopausal status (questionnaire developed based on STRAW+10 criteria) and health examination measures consist of height, weight, resting blood pressure (7-series; Omron, Japan), 12-lead ECG (electrocardiograph model ECG-2150, Nihon Kohden, Cardiofax, 2017, China) and venous blood samples from the antecubital vein. Predetermined exclusion criteria are: Essential hypertension defined according to current European guidelines as systolic and/or diastolic blood pressures of ≥140 mm Hg and/or ≥90 mm Hg, respectively (European Society of Cardiology/European Society of Hypertension Guidelines), cardiovascular chronic disease, renal or kidney dysfunction, insulin resistance, diabetes mellitus type 1 and 2 (glycated haemoglobin >40), body mass index >28 kg/m², hypercholesterolaemia (low-density lipoprotein cholesterol >5), polycystic ovary syndrome (PCO(S)), endometriosis, ovariectomy, cancer (active or within the past 5 years), ECG deviations, smoking within the last 10 years or an alcohol intake >7 units per week. All inclusion and exclusion criteria will be reassessed at each follow-up visit. Participants who develop medical conditions during follow-up that meet predefined exclusion criteria will be evaluated on an individual basis. In general, participants developing conditions that represent a contraindication to the invasive vascular procedures will be excluded from further experimental testing but will remain in the observational cohort where possible. Incident comorbidities will be recorded systematically during follow-up and incorporated into the statistical analyses as time-varying covariates when relevant. This approach allows preservation of longitudinal information while accounting for changes in health status over time. To ensure enrolment of participants in the premenopausal stage, menopause-related exclusion criteria are applied during screening. Exclusion criteria are: use of systemic hormonal contraception (except LNG-IUD), menopause hormone therapy or amenorrhoea suggestive of perimenopausal or postmenopausal status. Participants meeting all screening criteria undergo reproductive staging based on circulating hormone concentrations measured in venous blood samples along self-reported menstrual cycle bleeding pattern determination. In cases where reproductive staging based on circulating hormones and age is indecisive, their reproductive stage is further confirmed through transvaginal ultrasound assessment of antral follicle count ( figure 2 ). Accurate reproductive staging is essential in longitudinal assessment across the menopausal transition and proximity to menopause is estimated using established endocrine markers. AMH, a validated indicator of ovarian reserve that declines with reproductive ageing and predicts time to menopause, 42 – 44 is evaluated together with FSH, which increases during late reproductive stages due to reduced ovarian feedback. 45 Combined AMH–FSH assessment improves classification of reproductive stage compared with single-marker approaches. 44 46 AMH is measured using a 2-site ELISA (MenoCheck picoAMH ELISA, Ansh Labs) with intra-assay and interassay coefficient of variances in our laboratory ranging from 0.9% to 7.2% and 3.1% to 8.0%, respectively, at levels of 204±12 and 748±31 pg/mL. The limit of detection of this kit is 6.8 pg/mL in our laboratory. Thresholds are prespecified based on population reference distributions and predictive modelling from prior cohort studies, 44 47 with cut points selected to correspond to hormonal ranges associated with proximity to menopause of 1–3 years. Detailed classification algorithm with criteria and decision rules are provided in figure 2 . Participants who meet general eligibility criteria, but whose hormonal profile does not yet indicate late reproductive stage are followed prospectively with annual blood sampling. These women enter the KiSO DP cohort once hormonal concentrations meet the inclusion thresholds specified in the classification algorithm ( figure 2 ). To determine the timing of the first postmenopausal follow-up, all included participants will undergo monitoring after completing pre round testing. The included participants are divided according to menstrual cycle patterns. Participants with regular bleeding pattern/eumenorrhoea are instructed to monitor bleeding patterns, note irregularities and to report amenorrhoeic periods ≥5 months. They are further monitored annually, where they report for a short visit to the laboratory consisting of a venous blood sample, questionnaires for determination of menopausal stage and symptoms, anamneses follow-up and resting blood pressure measurements, allowing for monitoring changes in hormonal status. This monitoring continues until 12 months of amenorrhoea have been reported, after which the first postmenopausal test round ( 1 year post ) is conducted. For amenorrhoeic participants due to the use of LNG-IUD, monitoring will include biannual short visits to the laboratory, which will consist of the same test measurements and questionnaires as the annual visits for eumenorrheic participants. Once an LNG-IUD amenorrhoeic participant demonstrates hormonal levels consistent with postmenopause (FSH >30 IU/L, AMH <200 pg/mL and estradiol <0.09 nmol/L), the participant is scheduled for follow-up short visit after 6 and 12 months. If all blood samples obtained, during this 12-month period, indicate postmenopause, the participant will be invited to undergo the first postmenopausal experimental test round, see figure 3 for details. Flowchart outlining the monitoring procedures used to determine the timing of post visits for participants with and without IUD use. For participants without IUDs, post-visit eligibility is based on menstrual history, whereas for LNG-IUD users, eligibility is determined by repeated biochemical assessments, including FSH and AMH, and confirmation of postmenopausal status. Monitoring continues at predefined intervals until criteria for post visit are met. Time-based control group: Participants under active monitoring who remain non-perimenopausal at predefined follow-up time points are allocated to 3-year and eventually 5-year time control groups. Monitoring procedures are stratified by IUD status. Included participants who have not reached perimenopausal status 3 years after study inclusion will undergo repeated follow-up assessments under premenopausal conditions. These assessments will be repeated every second year until perimenopausal status is reached. This design enables the establishment of a time control group independent of menopausal status, allowing assessment of temporal stability in vascular measurements and ensuring that observed changes are not solely attributable to the passage of time or repeated testing. For analytical purposes, participants contributing multiple premenopausal measurements will be handled in two complementary ways. In primary analyses, all available observations will be included in the longitudinal mixed-effects models, allowing appropriate modelling of within-subject trajectories over time. In addition, for specific comparisons requiring a single reference value, the measurement closest to the menopausal transition (ie, the last premenopausal assessment) will be used. Based on conservative computational modelling of expected menopausal timing, incorporating population-based age at menopause distributions and baseline age and hormonal information from the KiSO DP cohort (n=175), we estimate that approximately n=39 will be eligible for a 3-year time control group and n=18 at a 5-year time control group ( figure 3 ). It is acknowledged that participants remaining premenopausal over longer follow-up periods may represent a selected subgroup. Therefore, baseline characteristics of the time control group will be compared with the overall cohort, and sensitivity analyses will be performed to evaluate potential selection bias. Participants report to the laboratory in the morning from 08:00 to 10:00, in a fasted state, and having refrained from caffeine, alcohol and strenuous physical activity for 24 hours prior. Participants provide a mid-beam urine sample, before determination of body composition and bone mineral density by whole-body dual-energy X-ray absorptiometry (DXA) scan (iDXA, Lunar Corporation, Madison, Wisconsin, USA). A whole-body scan is performed, with the region of interest (ROI) automatically generated by enCORE Software (V.18), and verified by a trained assessor. Height is measured using a stadiometer and used for the subsequent scan. From the scan, lean body mass (LBM) is used to determine the workload during the subsequent incremental bike test. Further, total right arm mass is quantified and used to calculate infusion rates for experimental day 2. Shortly after, participants receive a standardised light meal. 1 hour later, a venous blood sample is obtained to assess microstructure of an incipient blood clot (marker of thrombogenicity) via rheometry 48 , followed by 10 min of rest. VO 2max is then assessed using a gas-exchange analyser (Vyntus CPX; Vyaire, Mettawa, Illinois, USA), during an incremental exercise test on a mechanically braked cycle ergometer (Monark Ergomedic LC6 Novo Duo; Monark, Vansbro, Sweden). In short, participants undergo 2 min of resting baseline measurements followed by 8-minute warm-up: 50 W for the first 4 min, followed by an increase to 1.5 × LBM for the final 4 min. The workload is increased by 20 W per minute until exhaustion. After a 10-minute break, participants perform a two-scale verification phase 49 50 ( online supplement 1 ). For both tests, oxygen uptake (VO 2 ) is measured as 30-second rolling averages to determine VO 2max . The test is accepted if the participant reaches exhaustion and is unable to maintain a pedalling cadence of ≥70 RPM. Verbal encouragement is given during both tests. Immediately after the verification test ends, a post-exercise venous blood sample is obtained to repeat measurement of clot microstructure. Participants report to the laboratory in the morning from 08:00 to 10:00 or afternoon 12:00 and report having refrained from caffeine, alcohol and strenuous physical activity for 24 hours prior. A percutaneous skeletal muscle biopsy is obtained from the right muscle vastus lateralis under local anaesthesia (xylocaine 20 mg/mL; Aspen Pharma Trading Limited, South Africa), using a Bergström needle technique with manual suction. 51 Part of the sample is used for immediate analysis of mitochondrial function and the remaining tissue is stored for subsequent histological and molecular analyses. An arterial catheter (20 Ga; Arrow International, Reading, Pennsylvania, USA) is then placed in the participant’s right brachial artery under local anaesthesia, followed by the placement of a brachial venous catheter (20 Ga, Venflon, Becton Dickinson Therapy System, UT, USA), enabling intra-arterial infusions and continuous blood pressure monitoring. Microvascular function is assessed during intra-arterial infusion of ACh and EPO, with infusions separated by 15 min. After another 15 min rest, brachial arterial function in the left arm is assessed by reactive hyperaemia using the flow-mediated dilation (FMD) technique. Arterial and venous blood samples are obtained at rest and at the highest dose of infusion for both ACh and EPO ( online supplement 2 ). Bilateral carotid arterial compliance and intima media thickness are assessed during 30s two-dimensional (2D) images with concomitant intra-arterial pressure recordings. Both test days are conducted in the laboratory at the August Krogh Section of Human and Molecular Physiology, at the University of Copenhagen, Denmark. To explore enabling and constraining cultural norms and powerful societal ideologies of womanhood during menopause in depth, 52 a part of the KiSO DP cohort participants are interviewed in focus groups to explore how social expectations, cultural norms, gendered ideals and dominant societal discourses are negotiated, reproduced, and re-interpreted. To explore how menopause influences lived experiences within the contemporary Danish welfare society and reveal sensitive, nuanced and often hidden dimensions of women’s personal lives during menopause some participants will be invited to in-depth individual interviews to provide deep sociological understanding of emotions, embodiment and everyday experiences before, during and after menopause. Vascular function is assessed using invasive intra-arterial infusions and non-invasive FMD. All measurements are performed with participants in the supine position under resting conditions. Intra-arterial infusion of ACh consists of three infusion rates (10, 25 and 100 µg/min/kg total arm mass), (ACh 10 mg/mL, Fidia Farmaceutici S.p.A., Italy). Intra-arterial infusion of EPO consists of three infusion rates (25, 50 and 100 µg/min/kg total arm mass), (Flolan 0.5 mg, GlaxoSmithKline Pharma A/S, Denmark). Each infusion rate is maintained for 2 min and 30 s to reach steady state, with haemodynamic measurements obtained during the final 30 s of each stage. Vascular responses are expressed as changes in brachial arterial blood flow and conductance, and dose–response curves are constructed for each infusion. 53 54 A washout period of 15 min is included between infusion protocols. FMD is assessed in the brachial artery of the non-catheterised arm, to avoid influence of the catheterisation with the cuff movement, according to the gold standard guidelines. 55 56 Arterial diameter is analysed offline using automated edge-detection software, and FMD is expressed as the relative change from baseline diameter, with baseline diameter accounted for in statistical analyses where appropriate. Brachial artery diameter and blood flow velocity are measured using Doppler ultrasound (Vivid E9, GE Healthcare) with a linear-array probe (L11, 8–12 MHz). The probe is positioned longitudinally over the brachial artery, proximal to the arterial and venous catheters (≈3–5 cm), and maintained at a constant position throughout each protocol. The position is noted and repeated at each visit. 2D imaging and Doppler recordings are obtained with an insonation angle ≤60° and synchronised to a continuous ECG. Brachial artery blood flow (Q) is calculated as: Q=V mean × π × (D/2)² × 60, where V mean is mean blood velocity and D is peak systolic arterial diameter. Continuous intra-arterial and intravenous blood pressure are measured via arterial and venous catheters (20 Ga; Arrow International and 20 Ga, Venflon, BD) connected to pressure transducers (Truwave, Edwards Lifesciences), recorded using Power laboratory (AD Instruments, Sydney, Australia) and displayed and analysed offline via LabChart 8.1 (AD Instruments). Arterial compliance is assessed in the two brachial and two common carotid arteries by recording a 30 s 2D video of the arteries with simultaneous measurement of intra-arterial blood pressure. Compliance is estimated based on the relationship between beat-to-beat changes in arterial cross-sectional diameter and the corresponding intra-arterial pulse pressure, such that arterial compliance is calculated as ΔD/ΔP, where D is the arterial diameter and P is the change in pressure. 57 Vascular ultrasound measurements are acquired using study-specific standardised acquisition and analysis protocols. Flow-mediated dilation of the brachial artery is assessed in accordance with published expert consensus recommendations, including standardised participant preparation, image acquisition and diameter analysis procedures. 58 Blood flow during pharmacological infusion is derived from duplex ultrasound measurements of arterial diameter and Doppler-derived blood velocity. Given the sensitivity of flow calculations to small differences in diameter, analysis is performed using predefined study protocols, and inter-analyst reproducibility of ultrasound-derived diameter measurements has been formally evaluated using intraclass correlation coefficients 59 and Bland-Altman analyses. 60 Across infusion conditions, interanalyst (n=50) intraclass correlation coefficients (ICCs) demonstrated good agreement (ICC: 0.85±0.02), with small absolute measurement error (standard error of mean (SEM): 0.009±0.001 mm; minimal detectable change at the 95% threshold (MDC95): 0.026±0.002 mm). Intra-analyst repeatability assessed using triplicate analyses demonstrated excellent agreement (ICC: 0.94±0.03) and low measurement error (SEM: 0.007±0.002 mm). To limit operator-related variability over the long-term study period, new ultrasound operators undergo structured, protocol-specific training. Competency is assessed using resting baseline and FMD measurements analysed against reference operators prior to independent data acquisition. Ongoing quality control is performed through periodic re-analysis of stored images, repeat reliability assessments and cross-validation between operators. To ensure long-term consistency across the study duration, ultrasound equipment is subject to regular calibration and maintenance in accordance with manufacturer recommendations and institutional standards. Measurement protocols, including participant preparation, probe positioning and analysis procedures, are fixed and documented in detailed standard operating procedures, which are maintained throughout the study period. In the event of changes in personnel or ultrasound equipment, dedicated bridging procedures will be implemented. These include parallel measurements, re-analysis of archived data and repeat reliability testing to ensure comparability across time. Any systematic differences introduced by equipment upgrades or software changes will be evaluated and, if necessary, accounted for in subsequent analyses. Venous blood is collected by venipuncture into Vacutainer tubes containing EDTA, citrate or no additive (serum). Whole blood samples for standard clinical biochemical analyses are sent to the clinical biochemistry unit at the local hospital (Rigshospitalet, Denmark). EDTA and citrate plasma tubes are immediately centrifuged at 2200 g for 5 min at 4°C, and serum samples are allowed to coagulate at room temperature for 40–60 min before centrifugation at 1300 g for 10 min at 18°C. Plasma and serum are transferred into aliquots and stored at −70°C until further analysis. These samples are used for multiple analyses, including targeted biochemical markers, transcriptomics and exploratory biomarker discovery. A mid-beam urine sample is collected on the first experimental day following an overnight fast. Urine is transferred into three 5 mL Eppendorf tubes and stored at −70°C until further analysis. Urine samples are analysed to complement blood-based biomarkers and to explore potential non-invasive markers for future studies. Biopsies are taken from close proximity to previous sampling sites on the same leg when repeated measures are required. Each biopsy is divided into three portions: one portion is mounted in Tissue-Tek OCT (Sakura Chemicals, The Netherlands) and frozen in isopentane cooled with liquid nitrogen for microscopic analysis, another portion is immediately placed in BIOPS buffer (100 mM CaK2EGTA, 100 mM K2EGTA, 5.77 mM Na2ATP, 6.56 mM MgCl2·6H2O, 20 mM Taurine, 15 mM Na2Phospho-creatine, 20 mM Imidazole, 0.5 mM DTT, 50 mM MES) for mitochondrial analyses via high resolution respirometry and fluorometry and the last portion is snap frozen immediately in liquid nitrogen. All skeletal muscle samples are stored in cryotubes at −70°C until further analysis. Biopsies are used to evaluate cellular target proteins involved in vascular mechanisms in skeletal muscle. Participants are asked to answer the following questionnaires during experimental day 1. Menopausal stage will be determined based on both circulating hormonal levels and self-reported menstrual bleeding patterns in accordance with the STRAW+10 criteria. 39 The STRAW+10 staging system is considered the gold standard for classification of reproductive ageing. In the present protocol, classification based on self-reported data is defined as follows: regular menstrual cycles indicate premenopause, changes in menstrual cycle with bleeding within the past year indicate perimenopause and absence of menstrual bleeding for ≥12 months defines postmenopause. 16 Menopausal symptoms will be assessed using the Menopause Rating Scale (MRS), comprising 11 items covering psychological, somato-vegetative and urogenital domains. 61 Each item is scored on a 5-point Likert scale ranging from 0 (‘no complaints’) to 4 (‘extremely severe’), and both domain-specific and total scores will be calculated according to established guidelines. In addition to the validated MRS instrument, five supplementary symptoms (night sweats, skin changes, headache, rage and dizziness) will be included based on previous KiSO survey data. 16 These additional items will be treated as exploratory and analysed separately from the validated MRS total and domain scores. All questionnaires will be completed at baseline and during perimenopausal monitoring. Quality of life will be assessed using validated questionnaire instruments. Particular attention will be given to domains relevant to the menopausal transition, including physical, emotional, sexual and social well-being, as done similarly in the KiSO survey. 16 While not included as a primary outcome in the present protocol, these aspects will be addressed through validated instruments where available. In particular, the Utian Quality of Life scale, which includes a specific sexual quality-of-life domain, will be included in follow-up assessments 62 Self-reported habitual physical activity is assessed using the long-form International Physical Activity Questionnaire, capturing activity across work, transport, domestic and leisure domains over the preceding 7 days. 63 Weekly metabolic equivalent task scores are calculated from reported activity duration and intensity and used to classify participants as having low, moderate or high habitual physical activity levels. This questionnaire collects information on demographics, occupation, marital status, educational attainment, smoking and alcohol consumption, sleep duration and quality, self-perceived body weight, supplement use, reproductive history and status. To determine sample size, a priori power calculation was performed on the chosen primary outcome with the largest variance; change (Δ) in brachial vascular conductance during intra-arterial infusion of ACh at the first post 1 year follow-up. This calculation was informed by previous results from cross-sectional data obtained in the laboratory. 64 To detect a clinically relevant effect size of 25% (mean 2.8 mm Hg/mL/min, SD 1.65, α-level 0.05 and statistical power of 80%) 174 subjects are required. It should be noted that the primary outcome in the KiSO DP cohort is longitudinal within-subject changes across the menopausal transition, which is expected to present lower variability compared with cross-sectional comparisons which laid the foundation of the calculations of sample size. In addition, the changes in primary outcome can be expected to increase across the later follow-up time points. Consequently, the applied sample size is considered conservative. To account for potential dropout, 200 participants will be included. Primary analyses will address predefined objectives by evaluating longitudinal changes in cardiovascular outcomes across the menopausal transition. The primary endpoint is defined as the change (Δ) in brachial vascular conductance during intra-arterial ACh infusion from baseline to follow-up assessments. Longitudinal analyses will be conducted using linear mixed-effects models, with time, relevant exposures and their interactions specified as fixed effects, and participant included as a random effect to account for repeated measurements within-individuals. This within-subject design allows each participant to serve as her own control across the menopausal transition. Baseline analyses will be performed to characterise the cohort and explore associations between vascular function and key factors including menopausal status, hormonal profile, contraceptive use, menopausal hormone therapy (MHT) and habitual physical activity. These variables will be considered as covariates in longitudinal models to assess their influence on vascular trajectories over time. Exposure to MHT will be explicitly accounted for during follow-up. MHT use will be treated as a time-varying variable and categorised as current use, previous use or non-use, plus route of administration. Given the potential influence of MHT on vascular outcomes, MHT will be included as a covariate in the mixed models, and interactions with time and relevant exposures will be explored where appropriate. Sensitivity analyses will be performed, including stratification by MHT use and exclusion of participants initiating MHT during follow-up. Missing data are expected in a long-term follow-up study. Linear mixed-effects models inherently allow inclusion of participants with incomplete observations under the assumption of missing at random, thereby reducing bias and preserving statistical power. The pattern and extent of missing data will be evaluated, and sensitivity analyses will be conducted if necessary. For secondary outcomes, adjustments for multiple comparisons will be performed using Tukey post-hoc procedures or false discovery rate correction, as appropriate. These approaches aim to balance control of type I error while retaining statistical power across multiple endpoints. Model assumptions will be assessed using standard diagnostic approaches, including residuals versus fitted values and Q–Q plots. Data will be presented as mean±SD with individual values where appropriate, or as median (IQR) for non-normal or heteroscedastic data. Statistical modelling of menopausal transition will be performed using an epidemiological simulation framework based on the population distribution of age at natural menopause. 65 Individual-level menopause timing will be simulated using Monte Carlo methods conditional on current age and censoring, ensuring that menopause can only occur after study inclusion and only among participants still at risk. Baseline hormonal information (AMH, FSH and estradiol) will be incorporated conservatively as partial modifiers of menopausal timing. During follow-up period, prediction models will be updated and refined as additional study-specific data is available. Model outputs will be used to estimate participant flow across follow-up visits and to derive conservative projections of stable pre-menopausal time control groups at fixed time points (3 and 5 years). Data handling will comply with data protection laws; General Data Protection Regulation, Danish national legislation, including the Danish Data Protection Act. The University of Copenhagen is the data responsible authority and is responsible for all data processing. Enrolled participants will be assigned a unique pseudo-anonymous study specific ID. Identifiable data will be stored in secure, access-restricted databases on the University’s approved servers, located in Denmark. The study was further registered at ClinicalTrials.gov (ID: NCT05647876 ) before enrolment of the first participant. Any data sharing will be limited to de-identified datasets, and with the written consent from all included participants. Prior to the start of the study, the Danish Data Protection Agency approved the project. De-identified participant data will be available on reasonable request from the corresponding author after publication of the primary results. Data will be shared subject to approval by the relevant ethics committee and in accordance with applicable data protection regulations. No public data repository is planned for individual-level data due to the sensitive and identifiable nature of the physiological and clinical data collected. The overall risk of adverse events in this study is considered low. Venous blood samples are obtained by venipuncture from the median cubital vein during screening and follow-up visits, and from an indwelling venous catheter on the experimental day following completion of infusions. Minor discomfort at the puncture site may occur, with a low risk of bruising or infection. Brachial arterial and venous catheterisation for intra-arterial infusion of vasoactive agents is performed under sterile conditions with local anaesthesia by a medical doctor. The procedure is associated with a low risk of minor complications, including local haematoma, bleeding, infection, thrombosis or transient nerve irritation, with serious adverse events occurring in fewer than 1 in 1000 procedures. ACh and EPO are administered at low doses for short durations and are rapidly metabolised, resulting in transient physiological effects only. Flow-mediated dilation and Doppler ultrasound assessments are non-invasive and pose no known risk to participants, although transient discomfort may be experienced during forearm cuff occlusion. Muscle biopsies are obtained under sterile conditions with local anaesthesia using a percutaneous needle technique and are performed by a medical doctor. The procedure is generally well tolerated, with potential risks including minor bleeding, infection and transient muscle soreness; rare cases of temporary localised sensory disturbance may occur, while overall muscle function is unaffected. Standardised post-procedural care instructions are provided. Whole-body DXA scans performed during experimental visits result in a maximal radiation exposure of approximately 0.0006 mSv, which is considered negligible and well below thresholds associated with long-term health risk. The study is conducted in accordance with the Declaration of Helsinki and has been approved by the regional ethics committee (Ethics Committee of Copenhagen, H-22025286) and registered at ClinicalTrials.gov ( NCT05647876 ). All adverse effects (AEs) will be logged in the participants case report file and reported to the regional ethics committee annually while all serious AEs (SAEs) will be reported to the regional ethics committee no later than 7 days after the principal investigator becomes aware of the incident. The medically responsible investigator will, alongside the principal investigator, provide all the necessary information for the ethics committee and the participants private practitioner for appropriate safety assessment. Annually the principal investigator will submit a cumulative report of all SAEs to assess the overall safety and ethics of the continuation of the study. All clinical procedures will be carried out by physicians or under their direct supervision, and all procedures in the study will be performed by trained personnel with extensive experience in the procedures involved, drawn from prior studies conducted in the research group. The overall risk associated with participation in the study is thus deemed minimal. The study is expected to generate valuable novel knowledge on the cardiovascular physiology of women undergoing the menopausal transition. The menopausal transition is a hallmark in the cardiovascular ageing in women, as cardiovascular risk is accelerated in the years following the menopausal transition. This study will longitudinally follow the changes within the cardiovascular system in a large cohort of women, enabling the study to potentially elucidate several cardiovascular domains, including potential targets for prevention and treatment of the increased risk of cardiovascular disease observed in postmenopausal women. This is a population that has been largely under-represented in physiological research, and knowledge in this field is thus crucial. It is therefore considered that the potential risk, inconvenience and discomfort associated with participation in the study are overweighed by the potential scientific and societal benefits. Findings from the study will be disseminated through publication in peer-reviewed scientific journals, presentations at national and international conferences and through PhD theses. Study findings will also be communicated to participants and relevant stakeholders to support future research and potential clinical and preventive strategies in women’s cardiovascular health. Moreover, the KiSO umbrella also has a branch dedicated to work on translating research knowledge into usable formats directed towards women, policymakers, workplaces and healthcare professionals. Patients or the public were not involved in the design of the study, the selection of outcome measures or the conduct of the research. Participants were not consulted on the research question or study design prior to enrolment. Participants are continuously consulted on when and how new knowledge is translated into publicly available material and learnings from the interviews are used to guide any future updates to the protocol.

To our knowledge, the KiSO study represents a novel longitudinal cohort combining large-scale recruitment with in-depth physiological phenotyping and repeated measurements from late premenopause through up to 20 years postmenopause. This long-term design enables detailed characterisation of vascular and endothelial change across the menopausal transition and into later postmenopausal life, a period that remains sparsely examined in existing deep physiological research. The study is designed to fill the gap between large scale epidemiological cohort studies 66 and smaller physiological longitudinal 67 or cross-sectional studies 68 by combining a larger prospective cohort with deep cardiovascular phenotyping over an extended follow-up. Vascular function assessed by intra-arterial ACh infusion was selected as the primary outcome because of its established validity in assessing resistance vessel endothelial function, with activation of multiple vasodilator pathways, including both nitric oxide and prostacyclin. 69 Emerging evidence suggests that the relative contribution of these pathways may be altered across the menopausal transition. Studies have demonstrated pronounced prostacyclin-mediated vasodilatory responses in postmenopausal women, indicating that prostacyclin signalling may play an increasingly important role in vascular regulation in this context. 70 71 On this basis, the KiSO study additionally includes intra-arterial infusion of EPO, a stable prostacyclin analogue, to specifically investigate prostacyclin-mediated vasodilator function. By combining invasive vascular assessments with non-invasive flow-mediated dilation 56 and detailed cardiovascular phenotyping, the KiSO study is designed to examine if impairments in vascular function emerge early during the menopausal transition and precede later structural or functional cardiovascular changes. Secondary outcomes allow investigation of factors hypothesised to modify vascular trajectories, including habitual physical activity, hormonal profiles, contraceptive use and sociological factors. The inclusion of women with and without LNG-IUD reflects common contraceptive practices in the background population and strengthens external validity, while limiting systemic hormonal confounding compared with other contraceptive methods. 72 73 It is acknowledged that self-reported assessments of habitual physical activity are subject to measurement bias, which may attenuate observed associations with vascular outcomes. 74 An accelerometer was implemented in a subset of participants (n=15) to validate self-reported activity levels; however, based on data completeness and concordance with self-reported measures, accelerometer variables were not continued for the full cohort. Inclusion criteria are intentionally stringent to isolate menopause-related vascular changes, acknowledging that the cohort may represent a healthier and more physically active subset of the background population. The long-term follow-up inherent to the KiSO study introduces methodological challenges, including changes in personnel and equipment over time. To address this, standardised protocols, centralised operator training and validated acquisition and analysis procedures are implemented, particularly for ultrasound-based outcomes, to minimise measurement variability. Further time of day for pre-tests will be respected for future post-test days. The planned sample size provides sufficient statistical power to allow stratified analyses and adjustment for time-varying exposures, including initiation of menopausal hormone therapy during follow-up. The KiSO study is expected to provide novel insight into the temporal development of vascular dysfunction across the menopausal transition and to identify early markers and modifiers of cardiovascular risk in women. Recruitment commenced in December 2022 and is ongoing. Inclusion is expected to be finalised in 2026, with longitudinal follow-up continuing according to the predefined study schedule.