The effect of smoking on early chorionic villous vascularisation R.H.F. van Oppenraaij a, * , A.H.J. Koning b , M.J.B. van den Hoff c , P.J. van der Spek b , E.A.P. Steegers a , N. Exalto a a Department of Obstetrics and Gynecology, Division of Obstetrics and Prenatal Medicine, Erasmus MC, University Medical Center Rotterdam, Postbox 2040, 3000 CA, Rotterdam, The Netherlands b Department of Bioinformatics, Erasmus MC, University Medical Center Rotterdam, The Netherlands c Department of Anatomy, Embryology and Physiology, Academic Medical Center Amsterdam, The Netherlands article info Article history: Accepted 16 May 2012 Keywords: Placenta vascularisation First trimester Vasculogenesis Virtual reality Optical Projection Tomography Tobacco exposure abstract The aim of the study was to investigate whether first trimester chorionic villous vascularisation is different in women who smoked cigarettes before and during pregnancy in comparison with women who did not smoke. Placentas of smoking (>10 cigarettes/day, n ¼ 13) and non-smoking women (n ¼ 13), scheduled for a legal termination of a viable first trimester pregnancy for social indications, were retrieved. Placental tissues of 3e5 mm 3 were whole mount CD31 immunofluorescence stained. Images of the CD31 immunofluorescence and contour of the villi were captured using an Optical Projection Tomography scanner. An immersive BARCO virtual reality system was used to create an enlarged interactive 3- dimensional hologram of the reconstructed images. Automatic volume measurements were performed using a flexible and robust segmentation algorithm that is based on a region-growing approach in combination with a neighbourhood variation threshold. The villous volume, vascular volume and vascular density were measured for the total chorionic villous tree as well as for its central and peripheral parts. No differences in maternal age and gestational age were found between non-smoking and smoking women. No differences were found in the total, central and peripheral villous tree volume and vascular volume. The central (13.4% vs. 9.5%, p¼0.03) and peripheral (8.4% vs. 6.4%, p¼0.02) villous tree vascular densities were increased in the smoking women as compared with the non-smoking women. In conclusion, chorionic villous vascularisation is already altered in first trimester of pregnancy in women who smoked cigarettes before and during pregnancy. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Prenatal maternal smoking is one of the most important modifiable risk factors for pregnancy complications. The prevalence of prenatal smoking is 15e25% [1e3]. About 30e40% cease smoking in the first trimester of pregnancy, therefore 10e15% will continue smoking throughout pregnancy [2,3]. Smoking is associated with increased risks of adverse obstetric outcome such as intrauterine fetal growth restriction, preterm delivery and fetal death [4,5]. Although the association with adverse obstetric outcome is clear, the precise pathological mechanism is not yet completely under- stood. Studies on the effects of cigarette smoking on early first trimester placental development are sparse [6] and various histo- morphometric studies on term placentas of smoking and non- smoking mothers show contrasting effects of smoking on the villous vascularisation [7e13]. The differences in methology between the studies might explain the controversy in results found. Villous vascularisation was primarily investigated by the use of conventional 2D microscopy [14]. Recently, vascularisation of chorionic villi was visualized in 3D by immunofluorescent staining of endothelial cells (CD31) and Confocal Laser Scanning Microscopy (CLSM) [15e18]. The data were analyzed in 2D [17] and 3D [18]. Although the latter technique improved the possibility to investi- gate the spatial arrangement of the vessels, it was hampered by the limited penetration depth of 1 mm and the marked difference between the lateral and axial resolution. Optical Projection Tomography (OPT scanner 3001, Bioptonics) does not have these limitations. Transferring these OPT dataset to the I-Space Virtual Reality (VR) system offers an excellent opportunity to study vasculature of the villi in 3D. * Corresponding author. Tel.: þ31 647 100 562. E-mail addresses: robbertoppen@hotmail.com, r.vanoppenraaij@erasmusmc.nl (R.H.F. van Oppenraaij). Contents lists available at SciVerse ScienceDirect Placenta journal homepage: www.elsevier.com/locate/placenta 0143-4004/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2012.05.007 Placenta 33 (2012) 645e651