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Abstract: Twin-to-twin transfusion syndrome (TTTS) is a multifactorial disorder that develops in 9–15% of diamniotic–monochorionic twin gestations. While the pathogenesis of TTTS remains poorly understood, unbalanced deep artery-to-vein (AV) anastomoses have traditionally been implicated in the gradual shift of blood from donor to recipient. The aim of this study was to define the placental markers of twin-to-twin transfusion syndrome, with special emphasis on the deep AV anastomoses. A prospective cohort of 284 consecutive diamniotic/monochorionic twin placentas was examined at Women and Infants Hospital between 2001 and 2008. Following exclusion of monoamniotic, multiple, disrupted and laser-treated placentas, 218 twin placentas (21 TTTS and 197 non-TTTS controls) formed the subject of this study. Placentas were injected with color-coded dyes. Anatomic characteristics and choriovascular anastomotic patterns of TTTS placentas were compared with non-TTTS controls. The TTTS placentas showed significantly higher frequencies of velamentous cord insertion, magistral vascular distribution patterns, uneven placental sharing, absence of AA anastomoses and presence of VV anastomoses. Deep AV anastomoses were identified in ≥95% of TTTS and non-TTTS placentas and were overall more abundant than previously reported. The total and net numbers of AV anastomoses were similar in both groups. However, the net cross-sectional area of AV anastomoses, which also takes into account the caliber of the vessels, was significantly smaller in TTTS placentas. There was no correlation between the direction of the AV imbalance and the twin donor/recipient status. In conclusion, TTTS has distinct placental characteristics, warranting their routine inclusion in the diamniotic–monochorionic placental pathology report. Our findings suggest imbalance of AV anastomoses is not required for the development for TTTS, although their presence, whether balanced or unbalanced, may contribute to the creation or perpetuation of the syndrome. Elucidation of the role of the various placental determinants in diamniotic–monochorionic twin gestations may lead to further refinement of therapeutic strategies. [Copyright &y& Elsevier]