ABSTRACTObjectivesThe introduction of three‐dimensional (3D) analysis of the puborectalis muscle (PRM) for diagnostic purposes into daily practice is hindered by the need for appropriate training of observers. Automatic segmentation of the PRM on 3D transperineal ultrasound may aid its integration into clinical practice. The aims of this study were to present and assess a protocol for manual 3D segmentation of the PRM on 3D transperineal ultrasound, and to use this for training of automatic 3D segmentation method of the PRM.MethodsThe data used in this study were derived from 3D transperineal ultrasound sequences of the pelvic floor acquired at 12 weeks' gestation from nulliparous women with a singleton pregnancy. A manual 3D segmentation protocol was developed for the PRM based on a validated two‐dimensional segmentation protocol. For automatic segmentation, active appearance models of the PRM were developed, trained using manual segmentation data from 50 women. The performances of both manual and automatic segmentation were analyzed by measuring the overlap and distance between the segmentations. Intraclass correlation coefficients (ICCs) and their 95% CIs were determined for mean echogenicity and volume of the puborectalis muscle, in order to assess inter‐ and intraobserver reliabilities of the manual method using data from 20 women, as well as to compare the manual and automatic methods.ResultsInterobserver reliabilities for mean echogenicity and volume were very good for manual segmentation (ICCs 0.987 and 0.910, respectively), as were intraobserver reliabilities (ICCs 0.991 and 0.877, respectively). ICCs for mean echogenicity and volume were very good and good, respectively, for the comparison of manual vs automatic segmentation (0.968 and 0.626, respectively). The overlap and distance results for manual segmentation were as expected, showing an average mismatch of only 2–3 pixels and reasonable overlap. Based on overlap and distance, five mismatches were detected for automatic segmentation, resulting in an automatic segmentation success rate of 90%.ConclusionsThis study presents a reliable manual segmentation protocol and automatic 3D segmentation method for the PRM, which will facilitate future investigation of the PRM, allowing for the reliable measurement of potentially clinically valuable parameters such as mean echogenicity. © 2017 The Authors. Ultrasound in Obstetrics & Gynecology Published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.