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Objective: This study aimed to clarify the relationship between fluctuations in uterine stiffness during the third stage of labor and blood loss upon placenta delivery using shear wave elastography. Methods: This prospective cohort study enrolled consecutive singleton pregnant women above 37 weeks of gestation who delivered infants transvaginally at a single perinatal center. Shear wave velocities (SWV) were continuously measured during the third stage of transvaginal labor using transabdominal ultrasound and these values were compared between groups with large (≥500 g) and small amounts of bleeding during this stage. Results: In total, 8 cases of large bleeding and 47 cases of small bleeding were compared. The large amount of bleeding group had a significantly lower median of minimum SWV values (0.97 [0.52–1.01] m/s than the small amount of bleeding group (1.25 [1.04–1.48] m/s p = 0.02). However, no significant differences were observed between the two groups in terms of median, mean, and maximum SWV values. The time from delivery of the infant to placental delivery was significantly longer in the large amount of bleeding group (median time: 370.5 s vs. 274 s, p < 0.05). Conclusion: Ultrasound quantification of uterine stiffness using shear wave elastography demonstrated that uterine contractions may influence the biological hemostasis of the uterus during the third stage of labor. Baseline uterine stiffness was weak and a longer duration of placental separation might be associated with cases of large amounts of bleeding during this stage. [ABSTRACT FROM AUTHOR]