After receiving approvals from the regional ethics committee (H-2100186) and the Danish Medicines Agency (EudraCT number 2020–005839-76), this trial, registered at www.ClinicalTrials.gov (ID NCT05095077), was conducted at the Zelo Phase 1 Unit, Copenhagen University Hospital Bispebjerg and Frederiksberg. The trial adhered to International Council for Harmonization Good Clinical Practice (GCP) guidelines and relevant legislation, and was overseen by the GCP Unit at Copenhagen University Hospital.

Healthy female participants were recruited through advertisement on an online platform for trial participants (www.forsoegsperson.dk). Eligible participants were women between 18 and 55 years (both included) with a body weight of 50 kg or more and a body mass index (BMI) between 18.5 and 30 kg/m2. Other inclusion criteria included the use of safe contraceptive methods or sexual abstinence throughout the trial period. We excluded women with a history of urinary dysfunction, including incontinence, retention, or overactive bladder; clinically significant acute or chronic medical condition; systemic drug use within the 2 weeks before the first trial day (except for paracetamol [up to 4 g/day], hormonal contraception and hormone replacement therapy); smoking within the previous 3 months; pregnancy within the previous 6 months; and current breastfeeding. All participants provided written informed consent upon entering the trial.

This was a randomized, double-blind, placebo-controlled, two-visit crossover trial with healthy female volunteers. Participants were randomly allocated (1:1) to receive either a blinded single dose of tadalafil (40 mg) or a visually identical placebo at their first visit and then all participants crossed over and received the opposite treatment at the second visit. The production and packaging of the blinded dosing kit and the randomization process were carried out by the hospital pharmacy. In this way, investigators, outcome assessors, data managers, and participants were blinded to the random sequence allocation throughout the trial period, data management, and statistical analysis. To avoid carry-over effects, the visits were separated by a washout period of a minimum of 6 days, corresponding to approximately eight half-lives of tadalafil, giving a mean terminal half-life of 17.5 h [12].

At both clinic visits, participants underwent a human chorionic gonadotropin (hCG) test to rule out pregnancy before ingesting the study drug (40 mg of tadalafil or matching placebo). After 2 h of rest in the clinic, aligned with the median time to peak plasma concentration of tadalafil (tmax) [12], the urethral pressure was assessed using UPR. The UPR technique involves continuous measurements of pressure and cross-sectional areas using a thin, flexible polyurethane bag. The participants were evaluated as previously described [13, 14]. With participants in the lithotomy position, the bladder was emptied by sterile urethral catheterization, followed by the introduction of 150 ml isotonic saline at 37°C. Ten urethral pressure measurements were performed while the participant was resting and five measurements during voluntary squeezes. Anal pressure was assessed using anal acoustic reflectometry (AAR) after UPR (the results from AAR measurement will be published separately). Immediately following UPR and AAR measurements, another 150 ml saline was instilled in the bladder, resulting in a standardized volume of 300 ml saline in the bladder. Subsequently, urinary flow rates and voided volume were recorded in undisturbed settings using the wireless uroflowmeter Flowmaster (Laborie, Portsmouth, NH, USA). Adverse events (AEs) were documented during clinic visits and follow-up telephone visits conducted at least 6 days after the last trial day.

The primary endpoint was mean opening urethral pressure (OUP) during the resting condition of the pelvic floor for tadalafil versus placebo, whereas the secondary endpoint was mean OUP during the squeezing condition of the pelvic floor. Prespecified exploratory endpoints included peak urine flow rate (Qmax), average urine flow rate (Qave), and voided urine volume.

Study data were collected and managed using REDCap (Research Electronic Data Capture) hosted at the Capital Region of Denmark. REDCap is a secure, web-based software platform designed to support data capture for research studies [15, 16].

The sample size was calculated based on previous single-dose pharmacodynamic UPR trials [14, 17]. Assuming that the mean within-subject standard deviation (SD) was 9.7 cmH2O and using a two-tailed alpha of 0.05, a total of 24 participants would provide 99% power to detect a clinically relevant difference of 10 cmH2O in OUP.

The basic characteristics of the participants and AEs were evaluated descriptively. The differences in OUP (tadalafil versus placebo) and voiding parameters were analyzed using analysis of covariance (ANCOVA) models with participant ID, treatment, and period fitted as fixed effects. From these tests, least squares mean estimates between treatments, 95% confidence intervals (CIs), and p values are provided. All data management and statistical analyses were performed while blinded to the sequence allocation. We used SAS software, version 7.15 of the SAS system for Windows (SAS Institute, Cary, NC, USA), and GraphPad Prism version 9.4.1 for Windows (GraphPad Software, San Diego, CA, USA) for graphical plots.