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Uterine rupture is a potential life-threatening complication during a trial of labor after cesarean delivery. Single-layer closure of the uterus at cesarean delivery has been associated with an increased risk of uterine rupture compared with double-layer closure. Lower uterine segment thickness measurement by ultrasound has been used to evaluate the quality of the uterine scar after cesarean delivery and is associated with the risk of uterine rupture.To estimate the impact of previous uterine closure on lower uterine segment thickness.Women with a previous single low-transverse cesarean delivery were recruited at 34−38 weeks' gestation. Transabdominal and transvaginal ultrasound evaluation of the lower uterine segment thickness was performed by a sonographer blinded to clinical data. Previous operative reports were reviewed to obtain the type of previous uterine closure. Third-trimester lower uterine segment thickness at the next pregnancy was compared according to the number of layers sutured and according to the type of thread for uterine closure, using weighted mean differences and multivariate logistic regression analyses.Of 1613 women recruited, with operative reports available, 495 (31%) had a single-layer and 1118 (69%) had a double-layer closure. The mean third-trimester lower uterine segment thickness was 3.3 ± 1.3 mm and the proportion with lower uterine segment thickness <2.0 mm was 10.5%. Double-layer closure of the uterus was associated with a thicker lower uterine segment than single-layer closure (weighted mean difference: 0.11 mm; 95% confidence interval [CI], 0.02 to 0.21 mm). In multivariate logistic regression analyses, a double-layer closure also was associated with a reduced risk of lower uterine segment thickness <2.0 mm (odd ratio [OR], 0.68; 95% CI, 0.51 to 0.90). Compared with synthetic thread, the use of catgut for uterine closure had no significant impact on third-trimester lower uterine segment thickness (WMD: −0.10 mm; 95% CI, −0.22 to 0.02 mm) or on the risk of lower uterine segment thickness <2.0 mm (OR, 0.95; 95% CI, 0.67 to 1.33). Finally, double-layer closure was associated with a reduced risk of uterine scar defect (RR, 0.32; 95% CI, 0.17 to 0.61) at birth.Compared with single-layer closure, a double-layer closure of the uterus at previous cesarean delivery is associated with a thicker third-trimester lower uterine segment and a reduced risk of lower uterine segment thickness <2.0 mm in the next pregnancy. The type of thread for uterine closure has no significant impact on lower uterine segment thickness.