Abstract Study question What is the impact of prolonged downregulation with GnRH analogue (GnRHa) on uterine peristalsis in women with moderate/severe adenomyosis undergoing frozen-thawed embryo transfer? Summary answer Women with moderate/severe adenomyosis have lower coordination of uterine peristalsis and myometrial contractility; prolonged downregulation with GnRHa appears to offer benefit. What is known already Adenomyosis can adversely reduce the chance of pregnancy in women undergoing assisted conception, with altered uterine peristalsis proposed as a mechanism. We aimed to i) develop an objective tool to quantify uterine peristalsis on transvaginal sonography(TVS) using an algorithm based on optical flow for wave motion quantification, ii) compare uterine peristalsis between women with a normal uterus and women with moderate/severe adenomyosis, iii) evaluate the impact of prolonged downregulation with GnRH analogue for 6 weeks vs 1 week on uterine peristalsis in women with moderate/severe adenomyosis undergoing frozen-thawed embryo transfer within the MODA RCT (Modified Downregulation for Adenomyosis). Study design, size, duration Prospective observational cohort study(July 2021–Jan 2024) in women with moderate/severe adenomyosis(n = 26) and a normal uterus(n = 40) having TVS during assisted conception. Participants/materials, setting, methods We included women with moderate/severe adenomyosis or a normal uterus undergoing assisted conception.TVS was performed at baseline(BL),day 6-10 of ovarian stimulation(OS) and day of embryo transfer(ET)by three observers(A/B/C)to assess inter-/intra-observer agreement. TVS recordings(n = 146) were stored offline for optical flow analysis using 3D Slicer(Ver. 4.11). Pixel-level displacements for points at endometrial-myometrial junction(EMJ)and myometrium were used to compute frequency(f) and amplitude(u) by fitting to the 2D-wave equation, fit error was taken as a measure of coordination. Main results and the role of chance We recruited 66 women with moderate/severe adenomyosis (n = 26) or a normal uterus (n = 40) undergoing assisted conception. Uterine peristalsis at the EMJ in women with adenomyosis showed a significantly poorer fit with the ideal wave equation and a significantly higher fit error compared to women with normal uterus at all time points (BL (+ 0.649), OS (+ 0.630), ET (+ 0.685) (n = 66, p<0.001), indicating lower coordination of uterine peristalsis in women with adenomyosis compared to women with a normal uterus. Fit error of myometrial contractility was also significantly lower in women with adenomyosis at all time points (BL (+ 0.656), OS (+ 0.510) and ET (+ 0.639) (n = 66, p < 0.001). There was no significant difference in wave amplitude or frequency in women with adenomyosis compared to women with a normal uterus (p > 0.06). Women with moderate/severe adenomyosis exposed to prolonged downregulation with GnRH analogue for 6 weeks had lower fit error of uterine peristalsis (UP) and myometrial contractility (MC) on the day of embryo transfer compared to women receiving 1 week GnRH analogue (UP ET(+0.615) (n = 19, p = 0.058) (MC ET(+4.28) (n = 19, p < 0.05). Inter- and intra-observer agreement was found to be moderate to substantial across all wave features(ICC 0.58-0.79). Limitations, reasons for caution There is a need to recruit a larger cohort of patients and correlate wave features with clinical outcomes to increase the robustness of the conclusions drawn in this study. Wider implications of the findings Our algorithm for wave motion quantification offers an objective,reproducible measuring tool to quantify uterine peristalsis with potential for wide clinical application.Women with adenomyosis have higher fit error and lower coordination of uterine peristalsis and myometrial contractility,which may account for their reduction in chances of conception.Prolonged GnRHa appears to offer benefit. Trial registration number N/A