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Background:Uterine peristalsis occurs in the endometrial–myometrial junctional zone. It is involved in sperm transport and embryo implantation in spontaneous and in vitro fertilization (IVF) cycles. We investigated the reproducibility of transvaginal assessment of peristalsis and endometrial combined thickness (ECT) as a surrogate marker for contraction frequency and directionality. Methods:We studied 74 women undergoing IVF between 2015 and 2018. On day 9 of stimulation, participants had a transvaginal ultrasound to assess follicular development and ECT. Women proceeding to fresh embryo transfer had an ultrasound preprocedure. A videoclip was analyzed for frequency (total contractions per minute) and directionality of contractions (cervical to fundal [CF] and fundal to cervical [FC]). Anonymized images were reviewed on three separate occasions by a single observer. Intraclass correlation (ICC with 95% CI) and Bland–Altman plots were constructed to assess intraobserver agreement. Secondary analysis was performed to assess peristalsis with ECT and pregnancy rates. Results:Between the first and second observations, there was suboptimal correlation between visual assessment of contraction frequency and directionality (ICC–total 0.67 (0.43–0.80) p < 0.001, ICC–CF 0.62 (0.35–0.78), p < 0.001, ICC–FC 0.74 (0.55–0.85), p < 0.001. Correlation for both frequency and directionality significantly improved between second and third viewing (ICC–total 0.97 (0.95–0.98), p < 0.001, ICC–CF 0.84 (0.73–0.91), p < 0.001, ICC–FC 0.89 (0.81–0.94), p < 0.001). Good agreement was seen on Bland–Altman plots with narrower limits of agreement on second and third viewing. Median ECT was 10 mm (interquartile range [IQR] 8–12 mm). Women with ECT [Formula: see text] 10 mm versus <10 mm demonstrated more contractions per minute (2.2 ± 0.7 vs. 1.7± 0.6, p = 0.02). The clinical and ongoing pregnancy rates were 33% (20/60) and 22% (13/60), respectively. There was no association between pregnancy and peristalsis frequency or directionality. Conclusion:Uterine peristalsis is a reproducible observation displaying a learning curve to achieve excellent agreement. Endometrial thickness [Formula: see text]10 mm was associated with increased contraction frequency. There was no association between contraction frequency and/or directionality and clinical pregnancy rates.