This study aimed to determine risk factors associated with the failure of uterine artery ligation at its origin following development of the retroperitoneal space (UALr) and evaluated its efficacy in decreasing estimated blood loss (EBL) during single-port total laparoscopic hysterectomy (SP-TLH). This study includes patient data collected prospectively from May 1st, 2013 to establish a registry for single-port surgery. Data for the present study were collected retrospectively from May 1st,

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... to August 30th, 2016. Patients who underwent SP-TLH for a symptomatic benign disease. When bilateral UALr was performed successfully, the case was classified as part of the UALr success group. When only unilateral UALr was completed or UALr failed, the case was classified as part of the failure group. We compared patients' baseline characteristics and surgical outcomes between the two groups. Bilateral UALr was successfully performed in 155 cases and failed in 64 patients. Body Mass Index (BMI) was significantly different between the two groups (24.1 kg/m2 vs. 22.86 kg/m2, p = 0.025). A BMI higher than 23.6 kg/m2 was a risk factor for UALr failure in a multivariate analysis (odds ratio = 2.42, p = 0.004). EBL was significantly lower in the UALr success group compared to the UALr failure group (100 [100.0-200.0] vs. 200 [100.0-250.0], p < 0.001), and incidence of Hb decrease of more than 2 g/dl was higher in the UALr failure group (36.1% vs. 54.7%, p = 0.017). We identified higher BMI as a risk factor for UALr failure and demonstrated the safety and efficacy of UALr in reducing blood loss during SP-TLH.