Micro-CT provides 3D volume imaging with spatial resolution at the micrometre scale. We investigated the optimal human placenta tissue preparation (contrast agent, perfusion pressure, perfusion location and perfusion vessel) and imaging (energy, target material, exposure time and frames) parameters.



Microfil (Flow Tech, Carver, MA) produced better fill than Barium sulphate (84.1%(±11.5%)vs70.4%(±18.02%) p = 0.01). Perfusion via umbilical artery produced better fill than via chorionic vessels (83.8%(±17.7%)vs78.0%(±21.9%), p < 0.05), or via umbilical vein (83.8%(±16.4%)vs69.8%(±20.3%), p < 0.01). Imaging at 50 keV with a molybdenum target produced the best contrast to noise ratio. We propose this method to enable quantification and comparison of the human fetoplacental vascular tree

Arthurs, OJ

David, AL

Hutchinson, JC

Melbourne, A

Ourselin, S

Pratt, R

Sebire, NJ

Vercauteren, T

Virasami, A

Zuluaga, MA

UCL Discovery

lable at ScienceDirectPlacenta 60 (2017) 36e39Contents lists avaiPlacentajournal homepage: www.elsevier .com/locate/placentaTechnical noteImaging the human placental microcirculation with micro-focuscomputed tomography: Optimisation of tissue preparation and imageacquisitionRosalind Pratt a, b, *, J. Ciaran Hutchinson c, d, Andrew Melbourne a, Maria A. Zuluaga a,Alex Virasami d, Tom Vercauteren a, e, Sebastien Ourselin a, e, Neil J. Sebire c, d,Owen J. Arthurs c, f, 1, Anna L. David b, e, 1a Translational Imaging Group, Dept. Medical Physics and Biomedical Engineering, University College London, Malet Place Engineering Building, GowerStreet, London WC1E 6BT, UKb Institute for Women's Health, University College London, 86-96 Chenies Mews, London WC1E 6HX, UKc University College London Great Ormond Street Institute of Child Health, University College London, 30 Guilford Street, London, WC1N 1EH, UKd Department of Histopathology, Great Ormond Street Hospital for Children NHS Trust, Great Ormond Street, London, WC1N 3JH London, UKe Wellcome / EPSRC Centre for Surgical and Interventional Sciences, University College London, London, UKf Paediatric Radiology, Great Ormond Street Hospital for Children NHS Trust, Great Ormond Street, London, WC1N 3JH London, UKa r t i c l e i n f oArticle history:Received 26 June 2017Received in revised form22 September 2017Accepted 25 September 2017Keywords:Micro-CTOptimisationPerfusionContrast* Corresponding author. Translational Imaging GroGower Street, London WC1E 6BT, UK.E-mail address: Rosalind.pratt@ucl.ac.uk (R. Pratt)1 ALD and OJA contributed equally in their oversighttps://doi.org/10.1016/j.placenta.2017.09.0130143-4004/© 2017 The Authors. Published by Elseviea b s t r a c tMicro-CT provides 3D volume imaging with spatial resolution at the micrometre scale. We investigatedthe optimal human placenta tissue preparation (contrast agent, perfusion pressure, perfusion locationand perfusion vessel) and imaging (energy, target material, exposure time and frames) parameters.Microﬁl (Flow Tech, Carver, MA) produced better ﬁll than Barium sulphate (84.1%(±11.5%)vs70.4%(±18.02%) p ¼ 0.01). Perfusion via umbilical artery produced better ﬁll than via chorionic vessels(83.8%(±17.7%)vs78.0%(±21.9%), p < 0.05), or via umbilical vein (83.8%(±16.4%)vs69.8%(±20.3%), p < 0.01).Imaging at 50 keV with a molybdenum target produced the best contrast to noise ratio. We propose thismethod to enable quantiﬁcation and comparison of the human fetoplacental vascular tree.© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).1. IntroductionFetal health and development is intricately bound with humanplacental circulation, yet there is no validated quantitative methodwith which to assess vascularisation of the human placenta.Developing a quantitative method may improve our ability toinvestigate, and therefore understand, normal placental functionand pathologies such as fetal growth restriction, stillbirth and twin-to-twin transfusion syndrome.Micro-focus Computed Tomography (micro-CT) provides three-dimensional volume imaging with spatial resolution at the micro-metre scale. The technique has been used to investigate thebranching structure and tortuosity of the fetoplacental circulationup, Dept. Medical Physics and Biom.ht and guidance of this work.r Ltd. This is an open access articleof mouse placentae [1] [2], and shown differences in vasculardensity of the human placenta between normally grown andgrowth restricted pregnancies [3], [4].This study was designed to develop optimised tissue-speciﬁcpreparation and micro-CT imaging parameters, in order to pro-vide a validated approach to human placenta micro-CT.2. MethodThis series of experiments is divided into two sections; inves-tigating tissue preparation techniques, and then micro-CT imagingparameters. The full experimental methodology is described insupplementary data.edical Engineering, University College London, Malet Place Engineering Building,under the CC BY license (http://creativecommons.org/licenses/by/4.0/).R. Pratt et al. / Placenta 60 (2017) 36e39 372.1. Tissue acquisitionExperimental procedures were approved by Bloomsbury Na-tional Research Ethics Service Committee and by University CollegeLondon Hospital Research and Development (REC Referencenumber 133888). Placentas delivered by elective term caesareansection were taken directly to the laboratory, had the membranestrimmed, and the amnion removed.Vascular Fill ð%Þ ¼Total Perfused Vessel AreaTotal Perfused Vessel Areaþ Total Unperfused Vessel Area 100 (1)2.2. Tissue preparation comparatorsWe designed experiments to compare (Table 1): Contrast agent e comparing Barium sulphate with Microﬁl(Flow Tech, Carver, MA.). Perfusion pressure e comparing manual pressure with noquantiﬁcation of perfusion pressure, with controlled pressure of60 mmHg, physiologically relevant to fetal life [5e7]. Cannulation location e comparing perfusion via the umbilicalartery with perfusion via a chorionic artery. Arterial or Venous Cannulation e comparing perfusion viacannulation of the umbilical artery with perfusion via the um-bilical vein.The fetal vessel of interest was cannulated, and a cutmade in themain draining vessel close to the point of cannulation, to create aﬂuid exit vent. 0.9% sodium chloride solution with 5IU heparin/mlwas perfused until the outﬂow ran clear, then contrast agent wasperfused until the chorionic vasculature was fully perfused andcontrast agent was seen in the draining vessel. The vessel wasTable 1Comparison of placental tissue preparation and micro-CT imaging parameters used in thresults of this study. SNR ¼ signal to noise ratio.Langheinrich [4](Human)Rennie et al.[10](Mouse)AssessmeTissue PreparationContrast Agent Microﬁl and BaSO4 ingelatinMicroﬁl Microﬁl aPerfusion Pressure(mmHg)74 Not reported Manual pPerfusion Location Chorionic (peripheral)perfusionUmbilical (central)PerfusionChorionic(central)Perfusion Vessel Chorionic plate artery Umbilical Artery/Umbilical VeinChorionicveinTissue samplingtechnique8  2 mm full thicknessblocksWhole placenta 8  2 cmMicro CT ImagingCone-beam energy(keV)60 80 30-100 inTarget material Not reported Not reported TungstenIsotropic voxel size (mm) 13 and 4 13 13Radiograph exposuretime (ms)2400 Not reported 500/1000Number of projections 400 720 3141/628occluded and the contrast agent was left to set. The placenta wasdissected into 2 2cm full thickness blocks, which were ﬁxed in 4%formalin for a minimum of 48 h. One full thickness section stainedwith hematoxylin and eosin (H&E) was cut for every block and 6micrographs at x100 magniﬁcation taken (see supplementarymaterial).Histological analysis was done in FIJI (ImageJ Version 2.0.0-rc-54/1.51f) [8]. Vascular ﬁll was calculated for each micrograph asshown in equation one.2.3. Micro-CT imaging comparatorsWe designed experiments to compare (Table 1). Energy level e from 30 to 100 keV in 10 keV increments. Target material e comparing Tungsten, Copper andMolybdenum. Exposure timee500 and 1000 ms Averaged frames per projectione1 and 2A 2  2cm full thickness block of human placenta was repeat-edly imaged (XT H 225 ST Micro-CT, Nikon Metrology, Tring, UK)adjacent to a 3 mm internal diameter tube ﬁlled with Microﬁl.Scans were reconstructed using a modiﬁed Feldkamp ﬁltered backprojection algorithm with proprietary software (CTPro3D; NikonMeterology), and the average greyscale values of recorded areas ofinterest drawn over placenta, Microﬁl and air were calculated. Thecontrast to noise ratio was calculated as shown in equation two.is study and in two previous studies, and optimised protocol as determined by thent Parameters Optimised Protocolnd BaSO4 in gelatin Microﬁlressure and 60 No difference Manual and 60 mmHg giveequivalent results(peripheral) and umbilicalperfusionCentral vessel, ideally umbilical vesselartery/Umbilical artery/Umbilical Arteryfull thickness blocks Dependent on magniﬁcation and ﬁeld of viewrequired10 keV increments 50, Molybdenum, Copper MolybdenumDependent on magniﬁcation and ﬁeld of viewrequiredBalance with throughput 1000 gives good SNR2/12564 Balance with throughput3141 gives good SNRContrast to Noise ratio ðCNRÞ ¼ ðPlacenta Grey Scale ValueMicrofil Grey Scale ValueÞStandard Deviation of Signal of Air(2)R. Pratt et al. / Placenta 60 (2017) 36e39382.4. Statistical analysisData is presented as mean ± SD. Statistical analysis was done inSPSS Statistics (IBM version 23). Group comparison was performedusing independent sample t-tests with signiﬁcance set at 95%.3. Results3.1. Tissue preparation comparatorsThere was lower mean vascular ﬁll with barium sulphate thanMicroﬁl (70.4% (±18.02%) BaSo4 vs 84.1% (±11.5%) Microﬁl,(p¼ 0.01)) and barium sulphatewas seen in the extravascular spacein all three blocks sampled (47% of micrographs), whereas Microﬁlwas never seen in the extravascular space.There was no signiﬁcant difference in vascular ﬁll betweenmanual or controlled 60 mmHg perfusion pressure (77.8%(±13.9%)manual vs 78.0%(±21.9%) controlled pressure p ¼ 0.95). Perfusionvia an umbilical artery achieved higher vascular ﬁll than perfusionvia a more peripheral chorionic vessel (83.8%(±17.7%) umbilicalartery vs 78.0%(±21.9%) chorionic artery, p < 0.05). Umbilicalarterial perfusion produced higher vascular ﬁll than umbilicalvenous perfusion (83.8%(±16.4%) umbilical artery vs 69.8%(±20.3%)Fig. 1. Identifying optimal micro-CT imaging parameters for Contrast to Noise Ratio (CNR)arbitrary units) between placenta and Microﬁl grey scale value with increasing energy for Mimage noise, with increasing energy for Molybdenum, Tungsten and Copper target. C: ContrD: Effect of increasing the exposure time and the averaged frames per projection on the CNumbilical vein p < 0.01) (see Table 1 for summary, and supple-mentary data Table 1 for full results).3.2. Micro-CT imaging parametersContrast and noise were both greatest at the lower energy levels(Fig. 1A/B). The optimal CNR was with Molybdenum target at50 keV (Fig. 1C). Increasing exposure time from 500 ms to 1000 msand averaged frames per projection reduced the noise andimproved the CNR (Fig. 1D) at the cost of imaging time andthroughput (Table 1).4. DiscussionWe have established optimal tissue and imaging parameters forplacental angiographic micro-CT (Table 1). Our studies show thatMicroﬁl is a superior contrast agent to barium suphate, and thatcentral and arterial perfusion are superior to peritheral and venousperfusion. Contrast to noise ratio is optimal when imaging with50 keV energy, with a Molybdenum target. Increasing the numberof projection and exposure time improves CNR at the cost ofthroughput. Our studies found 1000 ms exposure time and 3141projections over 360 rotation produced good CNR with a 54 min. A: Contrast (deﬁned as Microﬁl Grey Scale value e placental issue Grey Scale value,olybdenum, Tungsten and Copper target. B: Standard deviation of the signal in air, theast to noise ratio with increasing energy for Molybdenum, Tungsten and Copper target.R.R. Pratt et al. / Placenta 60 (2017) 36e39 39scan time.This approach can be used to investigate the microcirculation ofthe human placenta. The technique beneﬁts from its high resolu-tion and large ﬁeld of view, allowing images of the vascular tree tobe captured from the chorionic plate to the intermediate villousvessels (see supplementary data for images).Micro-CT allows measurement of vascular density and analysisof the structure of the vascular trees, which could improve ourunderstanding of the heterogeneity within normal placentae, andthe structural changes associated with diseases such as early andlate intrauterine growth restriction.FundingALD and SO are supported at UCLH/UCL by funding from theDepartment of Health NIHR Biomedical Research Centre's fundingscheme. OJA is funded by a National Institute for Health Research(NIHR) Clinician Scientist Fellowship (NIHR-CS-012-002), NJS isfunded by an NIHR Senior Investigator award, Great Ormond StreetChildren’s Charity and the Great Ormond Street Hospital NIHRBiomedical Research Centre [ORMBRC-2012-1].This paper presents independent research funded by the Na-tional Institute for Health Research (NIHR). The views expressed arethose of the author(s) and not necessarily those of the NHS, theNIHR or the Department of Health.Statements of contributionRosalind PrattI declare that I have contributed to the design, acquired the dataand performed the analysis of this study, that I am the primarycontributor to the manuscript, and that I have seen and approvedthe ﬁnal version. I have no conﬂicts of interest to declare.J. Ciaran HutchinsonI declare that I have contributed to the design, execution andanalysis of this study and that I have seen and approved the ﬁnalversion. I have no conﬂicts of interest to declare.Andrew MelbourneI declare that I have contributed to the design, execution andanalysis of this study and that I have seen and approved the ﬁnalversion. I have no conﬂicts of interest to declare.Maria Zuluaga ValenciaI declare that I have contributed to the automated FIJI analysis ofhistology, and that I have seen and approved the ﬁnal version. Ihave no conﬂicts of interest to declare.Alex VirasamiI declare that I have contributed to histological analysis in thisstudy and that I have seen and approved the ﬁnal version. I have noconﬂicts of interest to declare.Tom VercauterenI declare that I have contributed to the design, execution andanalysis of this study and that I have seen and approved the ﬁnalversion. I have no conﬂicts of interest to declare.Sebastien OurselinI declare that I have contributed to the design, execution andanalysis of this study and that I have seen and approved the ﬁnalversion. I have no conﬂicts of interest to declare.Neil SebireI declare that I have contributed to the design, execution andanalysis of this study and that I have seen and approved the ﬁnalversion. I have no conﬂicts of interest to declare.Owen J ArthursI declare that I have contributed to the design, execution andanalysis of this study and that I have seen and approved the ﬁnalversion. I have no conﬂicts of interest to declare.Anna L DavidI declare that I have contributed to the design, execution andanalysis of this study and that I have seen and approved the ﬁnalversion. I have no conﬂicts of interest to declare.Conﬂicts of interestNone.AcknowledgmentsThis work was supported through an Innovative Engineering forHealth award by theWellcome Trust [WT101957]; Engineering andPhysical Sciences Research Council (EPSRC) [NS/A000027/1], andthe Wellcome/EPSRC Centre EPSRC [NS/A000050/1] Wellcome[203145Z/16/Z].Appendix A. Supplementary dataSupplementary data related to this article can be found athttps://doi.org/10.1016/j.placenta.2017.09.013.References[1] M.Y. Rennie, K.J. Whiteley, S.L. Adamson, J.G. Sled, Quantiﬁcation of gestationalchanges in the uteroplacental vascular tree reveals vessel speciﬁc hemody-namic roles during pregnancy in mice, Biol. Reprod. 95 (2016), 43e43.[2] M.Y. Rennie, J. Detmar, K.J. Whiteley, J. Yang, A. Jurisicova, S.L. Adamson,J.G. Sled, Vessel tortuousity and reduced vascularization in the fetoplacentalarterial tree after maternal exposure to polycyclic aromatic hydrocarbons, Am.J. Physiol. Heart Circ. Physiol. 300 (2) (2011) 675e684.[3] A.C. Langheinrich, J. Wienhard, S. Vormann, B. Hau, R.M. Bohle, M. Zygmunt,Analysis of the fetal placental vascular tree by X-ray micro-computed tomog-raphy, Placenta 25 (2004) 95e100.[4] A.C. Langheinrich, S. Vormann, J. Seidenstucker, M. Kampschulte, R.M. Bohle,J. Wienhard, M. Zygmunt, Quantitative 3D micro-CT imaging of the human feto-placental vasculature in intrauterine growth restriction, Placenta 29 (2008)937e941.[5] P.C. Struijk, V.J. Matthews, T. Loupas, P.A. Stewart, E.B. Clark, E.A.P. Steegers,J.W. Wladimiroff, Blood pressure estimation in the human fetal descendingaorta, Ultrasound Obstet. Gynecol. 32 (2008) 673e681.[6] B. Castle, I.Z. Mackenzie, In vivo observations on intravascular blood pressure inthe fetus during mid-pregnancy, in: P. Rolfe Fetal Physiological Measurements,Elsevier, 1986, pp. 65e69.[7] P. Johnson, D.J. Maxwell, M.J. Tynan, L.D. Allan, Intracardiac pressures in thehuman fetus, Heart 84 (2000) 59e63.[8] J. Schindelin, C.T. Rueden, M.C. Hiner, K.W. Eliceiri, The ImageJ ecosystem: anopen platform for biomedical image analysis, Mol. Reprod. Dev. 82 (2015)518e529.[10] M.Y. Rennie, K.J. Whiteley, S. Kulandavelu, S.L. Adamson, J.G. Sled, 3D visual-isation and quantiﬁcation by microcomputed tomography of late gestationalchanges in the arterial and venous feto-placental vasculature of the mouse,Placenta 28 (2007) 833e840.

Imaging the human placental microcirculation with micro-focus computed tomography: Optimisation of tissue preparation and image acquisition

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Imaging the human placental microcirculation with micro-focus computed tomography: Optimisation of tissue preparation and image acquisition

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