Three-dimensional reconstruction and morphological quantization of human embryo hearts for virtual

model validation.

* Département de Cardiologie et Maladies Vasculaires. Hôpital de Pontchaillou. CHR Rennes 35033. France

** Laboratoire du Traitement du Signal et de l’Image. Université de Rennes 1. France

*** Laboratoire d’Histologie et d’Embryologie. Faculté de Médecine, Rennes. France

J-M. Schleich*, J-L. Dillenseger**, L. Loeuillet***,

J-P. Moulinoux***, C. Almange*.

Introduction (1)

• Most common congenital anomaly observed in lives-borns

• Incidence of congenital heart defects: 5-8 / 1000 newborns

• Abortus and stillbirths: incidence is 5 times higher than in

liveborn infants

• in the last decade, improvements of prenatal diagnosis methods

=> regular decreasing gestational age of abortion

• major malformations / chromosomic anomalies

• Abortion could be realized from 9-10 weeks

(cranio-caudal length : 30 mm)

Introduction (2)

• Before 13 WG, accurate in vivo diagnosis cannot currently be made in all fetuses with the imaging instrumentation available.

• Ex vivo or in vitro imaging techniques have not been adapted to the dimensions of the organs under examination

Introduction (3)

• Before 13 WG, accurate in vivo diagnosis cannot currently be made in all fetuses with the imaging instrumentation available.

• Ex vivo or in vitro imaging techniques have not been adapted to the dimensions of the organs under examination

Introduction (4) : imaging of the surgically exposed uterus

40-50 MHz / resolution of 50-100 µm / mouse embryos penetration of US between 6-10 mm

Introduction (5)

Confocal microscopy

• Problems of penetration

• especially used for very small specimens

• limited to image only fluorescent signals

Introduction (6) : µIRM

Introduction (7)

• Complexity of cardiac anatomy and wide spectrum defects of the heart

=> the anatomo-pathological examination remains the gold standard

allowing the making of accurate diagnoses, as well as appropriate

genetic counseling, and the conduct of epidemiological studies.

However, this method reaches its own limits between 9 and 13 weeks

of gestation, when the embryo is between 25 and 70 mm in length. At

this point, the examination of isolated organs is a challenge,

particularly of the heart, which measures between 3 and 8 mm.

Correlation of prenatal US diagnosis and pathologic finding in fetal anomalies

• Comparison between autopsy studies and prenatal diagnosis

(average gestational age: 23 weeks)– Pediatr Pathology 1989; 9: 1-9

– Pediatr Develop Pathol 1999; 2: 131-42

– Ultrasound Obstet Gynecol 1999; 13: 117-26

– Méd Fœtale Echogr Gynecol 2000; 41: 18-21

– Ann Pathol 2000; 20: 549-57

• main prenatal diagnosis is « modified » in 20-46% of cases

(especially with polymalformatic syndromes)

• genetic counselling / diagnosis of a syndrome /

determination of etiology or pathogenetic mechanism /

interpretation of severity of the anomaly

Introduction (8)

The aim of this report is to present a methodology which can be applied routinely,

using standard histologic section, enabling the reconstruction, visual estimate

and quantitative analysis of the human embryonic cardiac structures.

This is the first description of 3-dimensional (3-D) reconstruction allowing a

diagnostic analysis of fetal human hearts ≤ 13 weeks of age

Method (1)

• Study approved by bioethic medical committee

• abortion specimens fixed in 4% paraformaldehyde

• visual examination of embryo and length measurements

• « limited » autopsy is performed

• internal examination requires the dissecting microscope with camera

• straight midline incision / chest wall removed

• visceral situs noted : thoracic organs removed together

• heart is isolated / weight - size are mesured

Method (2)

The heart specimen used in our study

• human abortus fetus of 68 mm crown/rump length

• 11 WOA (« 13 SA ») / spontaneous miscarriage

• normal development / normal external appearance

without any malformation

• heart weight = 0.2 gr / sized = 9 X 8 mm

Method (3)

• Deshydrated with ethanol

• embedded in paraffin

• serial 10 µ-thick transversal section (Leica RM

2145 microtome) (5300 slices)

• 1 / 10 was stained with haematoxylin-eosin

• the colored slices digitalized on optical microscope

(IKAROS 3 V 4.33 / Metasystems)

Method (4): inversion-cleaning-labeling

Original picture Inverted scaleCleaned picture Labeling picture

Method (5) : calibration

digitalized scale

pixel = 22, 3 µm

Method (6) : half-automatic matching technics

1/ Matching method based on the geometrical moments2/ Interactive own-developed fitting package

Method (7) : half-automatic matching technics

1/ Matching method based on the geometrical moments2/ Interactive own-developed fitting package

Result (1)

Result (2) : Volume formation / interpolation

Stacking the sections / resampling / interpolation of grey scale slice

Result (3) : Volume formation / visualisation

Result (4) : 2D Visualisation

frontal sagittal

horizontal

Result (5) : Volume formation

Result (6) : 3D Visualisation

Interactive manipulation of the heart

(VRML format )

Result (7) : 3D Quantification

Internal volumes / voxel = 22, 3 µm

– Ventricles : 28.5 mm3

– Atria : 40.2 mm3

Conclusion (1)

• Transmission of the digitalized data

• Visual perception improved by addition of 3rd dimension

• New approach of the morphological analysis

– Cardiac volume enterely reconstruted

– Exploration of the volume even by transparency

– 2D and 3D quantifications

– Creation of sections in any plane : The hope (the dream) of being able to section

the specimen as many times as needed in all spatial dimensions, and to finally

reexamine it in its entirety has finally be fulfilled.

• Experimental modelisation

Conclusion (2) : Improvements

Episcopic Fluorescence Imaging Capturing– exact 3D reconstruction from serial sections– « on block » staining procedure– picture ’s acquisition before section– allows automatic contour finding– no disorientation and no distorsion of the

sections– but

• necessity of specific stainings

• limited final resolution

• variability of staining ‘  intensity

6- WOA embryo (lenght : 13 mm)