Hormonal Regulation of Cardiometabolic Health Study

Overview

This observational longitudinal study investigates how the abrupt loss of ovarian function following bilateral (or unilateral, if only one ovary remains) oophorectomy affects the health and well being of women aged 35-50 years. Surgical menopause induces rapid endocrine, metabolic, vascular, neural, and psychosocial changes. To capture these adaptations comprehensively, the study evaluates four interconnected domains: (1) quality of life and embodied experience, (2) cardiometabolic health, (3) central nervous system function, and (4) adipose tissue health and cellular function.

Participants undergo assessments at three time points: pre surgery, 1-2 months post surgery, and 12 months post surgery. These visits include validated questionnaires, physiological and neuromuscular tests, vascular and exercise assessments, and the collection of blood samples at rest and after a standardised cycle ergometer test. A subgroup participates in qualitative interviews at all visits, and another subgroup donates adipose tissue samples during surgery for exploratory cellular analyses.

Some participants may begin hormone replacement therapy (HRT) after the 1-2 month visit as part of their clinical care. Because HRT initiation is outside the study protocol, HRT related comparisons (e.g., whether 12 month outcomes or change from baseline differ between HRT starters and non starters) will be conducted exploratorily, following definitions and modelling approaches described in the Statistical Analysis Plan.

Blood samples are analysed for a broad panel of metabolic, inflammatory, hormonal, and exercise responsive biomarkers to characterise systemic adaptations to surgical menopause. These biomarker analyses are exploratory and intended to provide mechanistic insight; they are not registered as outcome measures.

This four domain structure enables the study to integrate patient reported outcomes, whole body physiology, neural activation, vascular regulation, exercise metabolism, and adipose tissue biology, offering a multidimensional understanding of how surgical menopause impacts women's health over time.

Domain 1: Quality of Life and Embodied Experience

Surgical menopause can cause a variety of symptoms and affect daily functioning, health behaviours, and overall quality of life. We collect patient-reported outcomes using validated questionnaires covering quality of life, menopausal symptoms, sleep, pelvic floor symptoms, sexual function, psychosocial dimensions, and health behaviours such as physical activity and eating patterns. Participants also complete a food diary and wear a 7-day accelerometer to provide objective measures of dietary intake and physical activity. In addition, a subgroup participates in qualitative interviews, which help deepen our understanding of the lived experience of surgical menopause.

Primary outcomes in this domain are the RAND-36 total score and the Menopause Rating Scale (MRS) total score, which capture changes in broad health-related quality of life and menopausal symptom burden following oophorectomy.

Secondary outcomes include the Women's Health Questionnaire (WHQ), Pelvic Floor Distress Inventory-20 (PFDI-20), and the Athens Insomnia Scale-5 (AIS-5), providing a more detailed understanding of psychosocial health, pelvic floor symptoms, and sleep quality.

Other prespecified outcomes contribute additional context on general well-being, emotional functioning, health behaviours, and embodied experiences. These include the 15D, Female Sexual Function Index (FSFI), Center for Epidemiological Studies Depression Scale (CES-D), Satisfaction With Life Scale (SWLS), Positive and Negative Affect Schedule (PANAS), Eating Disorder Examination Questionnaire (EDE-Q), Three-Factor Eating Questionnaire-R18 (TFEQ-R18), the International Physical Activity Questionnaire-Short Form (IPAQ-SF), and a 4-item Finnish physical activity questionnaire. These measures extend the scope of quality-of-life assessment and support interpretation of primary and secondary outcomes.

Finally, qualitative interviews conducted with a subgroup of participants at each time point provide rich insights into bodily changes, identity, emotions, and the lived experience of surgical menopause. These interviews enhance the interpretation of questionnaire-based findings and allow a more integrated understanding of women's experiences during the transition to surgical menopause.

Domain 2: Cardiometabolic Health

Loss of ovarian function following oophorectomy may influence whole-body metabolism, body composition, vascular regulation, and exercise capacity. To characterise these possible adaptations, cardiometabolic health is assessed at each visit with standardized protocols that include indirect calorimetry, body composition measurements, vascular assessments, and exercise physiology testing using standardised cycle ergometer tests. Together, these measures help us evaluate whether endocrine changes are associated with alterations in metabolic rate, adiposity, cardiovascular function, and exercise-related responses over time.

Primary outcomes in this domain are resting energy expenditure (REE), total fat mass (FM), and aortic pulse wave velocity (PWV). These endpoints capture core aspects of metabolic function, whole body adiposity, and arterial stiffness-parameters that may change after the loss of ovarian function and are relevant to longer-term cardiometabolic risk.

Secondary outcomes include fat-free mass (FFM), augmentation index (AIx) as an additional marker of arterial wave reflection, and peak fat oxidation during exercise, offering insight into potential shifts in metabolic flexibility and substrate use at submaximal workloads.

Other prespecified outcomes provide supporting information on anthropometry, muscular performance, vascular regulation, and exercise responses. These include body mass index (BMI), waist circumference, handgrip strength, knee extension strength, and countermovement jump (CMJ) height. Additional central hemodynamic measures-aortic systolic and diastolic blood pressure, aortic pulse pressure, and reflection time-further characterise vascular load.

Microvascular and tissue-level parameters include forearm Laser Doppler readings (baseline perfusion, endothelium-dependent vasodilation, post-occlusive reactive hyperemia) and quadriceps near-infrared spectroscopy (NIRS) measures (resting StO2, minimum StO2 during exercise, StO2 recovery slope, total hemoglobin). Exercise metabolism is additionally profiled using peak oxygen uptake (VO2peak), resting fat oxidation rate, and maximal fat oxidation rate (MFO).

Exploratory blood biomarkers (not registered as outcomes) At each visit, venous blood samples are collected at rest and after the standardised cycle ergometer test. These samples will be analysed for exploratory biomarkers, including a broad panel of metabolic, inflammatory, and hormonal markers, to characterise systemic adaptations to the loss of ovarian function after oophorectomy and to examine potential changes in exercise responses. These biomarker analyses are exploratory and intended to provide mechanistic insight; they are not registered as outcome measures and follow definitions and procedures described in the Statistical Analysis Plan.

Domain 3: Central Nervous System Function

Loss of ovarian function following oophorectomy may influence neural activation, neuromuscular performance, and corticospinal and reticulospinal responsiveness. To characterise these possible adaptations, a subsample of participants completes a set of standardised neurophysiological assessments, including isometric strength testing, reaction time tasks, and transcranial magnetic stimulation (TMS). These tests provide complementary insight into neural drive to muscle and the excitability of corticospinal and cortico reticulospinal pathways during the first postoperative year.

No primary outcomes are defined for this domain. Secondary outcomes in this domain quantify neural activation and corticospinal responsiveness using complementary physiological measures. Maximum isometric voluntary contraction force (MVC) is assessed to evaluate volitional muscle activation during isometric elbow flexion. A StartReact test assesses reaction time and the StartReact effect, defined as the difference in reaction time between quiet and loud sound conditions. Cortical excitability is assessed using single pulse TMS, with the main analysis variable being motor evoked potential (MEP) amplitude normalised to the maximal compound muscle action potential (Mmax), elicited via peripheral nerve stimulation.

Other prespecified outcomes further characterise neuromuscular and corticospinal function. Rate of torque development (RTD) and voluntary electromyography (EMG) amplitude (0-50 ms) are derived from force and EMG signals recorded during the StartReact test. Paired pulse TMS methods are used to quantify short interval intracortical inhibition (SICI), long interval intracortical inhibition (LICI), and intracortical facilitation (ICF) relative to single pulse comparator responses. StartleTMS, which pairs a loud acoustic stimulus with TMS, provides an additional measure of startle related facilitation of corticospinal output. Silent period duration is measured during single pulse TMS and then normalised to MEP amplitude to enable comparison across time points.

Together, these measures provide a multidimensional characterisation of neural responsiveness, including voluntary activation, intracortical inhibitory and facilitatory circuits, and the excitability of corticospinal and cortico reticulospinal pathways.

DOMAIN 4: Adipose Tissue Health and Cellular Function

Loss of ovarian function following oophorectomy may be associated with changes in adipose tissue biology, including cellular composition, lipid handling, inflammatory signalling, and gene expression. To explore these potential mechanisms, a subsample of participants donates subcutaneous and, where available, visceral adipose tissue during surgery. From these tissues, adipose organoids ("Adipoids") are developed and exposed to controlled hormonal environments (e.g., defined hormones or conditioned media supplemented with pre and postmenopausal sera) to model post oophorectomy conditions in vitro.

This domain is considered exploratory; thus, no primary or secondary outcomes are described. We describe one other prespecified outcome (exploratory mechanistic readout): Hormonal Regulation of Adipose Tissue Health and Cellular Function, which refers to a change in Adipoid cellular content/cellularity (e.g., nuclei count or cells/mL) in response to a change in hormonal environment. We will also perform additional mechanistic assays (exploratory; not registered as outcome measures), including

• Lipid uptake/handling
• Responses to inflammatory stimuli
• Gene expression profiles
• Secretome characteristics

These laboratory analyses aim to reveal molecular pathways through which the loss of ovarian hormones may affect adipose biology. They are exploratory, not intended as clinical endpoints, and follow laboratory Standard Operation Protocols and the Statistical Analysis Plan.

Exploratory analyses of Hormone Replacement Therapy (HRT)

Some participants may begin hormone replacement therapy (HRT) after the 1-2 month visit as part of their routine clinical care. Because HRT initiation is outside the study protocol, comparisons involving HRT are conducted exploratorily. These analyses examine whether 12 month outcomes-or the change from baseline to 12 months-differ between HRT starters and non starters. HRT exposure will be defined using time varying information collected at each visit, and sensitivity analyses will explore alternative exposure windows. All analytic approaches, including covariate selection and handling of missing data, are prespecified in the Statistical Analysis Plan. These exploratory analyses do not constitute registered outcome measures for this study.