Morphometric Analysis of Aortic Media in PatientsWith Bicuspid and Tricuspid Aortic ValveMatthias Bauer, MD, Miralem Pasic, MD, PhD, Rudolf Meyer, MD, PhD,Nadine Goetze, MD, Ulrike Bauer, MD, Henryk Siniawski, MD, andRoland Hetzer, MD, PhDDeutsches Herzzentrum Berlin, Berlin, Germany

Background. Patients with bicuspid aortic valves tendto develop dilatation of the ascending aorta. The aim ofthis study was to analyze whether or not there is anyhistologic difference in the aortic media of patients witha bicuspid aortic valve or a tricuspid aortic valve.

Methods. A morphometric analysis of the wall of theascending aorta was performed in 107 patients withbicuspid aortic valves undergoing aortic valve opera-tions. The thickness of the elastic lamellae of the aorticmedia and the distances between the elastic lamellaewere measured with the use of an image analysis system.The histologic specimens of the ascending aorta from 61surgical patients with tricuspid aortic valve diseaseserved as a control.

Results. The patients with bicuspid aortic valves hadthinner elastic lamellae of the aortic media (2.71 �

0.23 �m) of the ascending aortic wall than the patientswith tricuspid aortic valve disease (2.83 � 0.23 �m) (p �0.006). The patients with bicuspid aortic valves also hadgreater distances between the elastic lamellae (27.21 �8.69 �m) of the ascending aortic wall in comparison withthe patients with tricuspid aortic valve disease (24.34 �5.32 �m) (p � 0.033). There was no difference in the totalthickness of the aortic media between the groups (p �0.62).

Conclusions. Patients with a bicuspid aortic valve hadthinner elastic lamellae of the aortic media and greaterdistances between the elastic lamellae than patients witha tricuspid aortic valve.

(Ann Thorac Surg 2002;74:58–62)© 2002 by The Society of Thoracic Surgeons

Bicuspid aortic valve is one of the most frequentcongenital heart defects. Its prevalence in the gen-

eral population is approximately 1% to 2% [1]. Bicuspidaortic valve also can occur in combination with othercongenital heart defects [2]; individuals with these con-ditions tend to have aortic valve stenosis or regurgitation,or both, develop early in life [3]. A bicuspid aortic valve(even when it is functioning normally) is frequentlyassociated with an enlargement of the ascending aorta.This aortic wall dilatation is typically located on theconvexity of the ascending aorta, although diffuse aorticenlargement also can occur. The presence of a bicuspidaortic valve carries a risk of severe complications such asaortic rupture or dissection [4, 5]. It is unknown whetherthis abnormality is caused by altered hemodynamicstress or by a developmental defect. There are only fewhistologic data from patients with bicuspid aortic valve,and these are predominantly from patients with aorticdissection already present [5–9]. In some studies thenumber of patients included was very small [10].

The aim of our study was to analyze the morphometricfeatures of the aortic wall in patients with bicuspid andtricuspid aortic valves undergoing open heart operations.

Material and Methods

Study GroupWe investigated aortic wall segments of the ascendingaorta of 107 patients with bicuspid aortic valves who wereundergoing aortic valve operations. There were 31 fe-males and 76 males with a mean age of 60.9 � 12.8 years.Only patients with a clearly congenital bicuspid valve asassessed by both intraoperative anatomical examinationand patient history were included. The patients whoseaortic valves became functionally bicuspid during theirlifetime were not included in this group. Patients withtricuspid aortic valve disease combined with a connectivetissue disorder also were not considered for this group.The main indications for valve operation were aorticvalve stenosis in 50 patients (47%), regurgitation in 28(26%), and combined aortic valve disease in 25 (23%).

Control GroupThe aortic wall segments of the ascending aorta of 61surgical patients with tricuspid aortic valve disease (24females, 37 males; mean age 59.5 � 14.3 years) served asthe control group. Patients with tricuspid aortic valvedisease combined with a connective tissue disorder werenot considered for this group. The main indications forvalve operation were aortic valve stenosis in 35 patients(57%), regurgitation in 18 (29.5%), and combined aorticvalve disease in 8 (13%).

There were no statistically significant differences be-

Accepted for publication March 28, 2002.

Address reprint requests to Dr Pasic, Deutsches Herzzentrum Berlin,Augustenburger Platz 1, Berlin D-13353, Germany; e-mail: pasic@dhzb.de.

© 2002 by The Society of Thoracic Surgeons 0003-4975/02/$22.00Published by Elsevier Science Inc PII S0003-4975(02)03650-0

tween the mean ages of the study patients and the controlgroup (p � 0.141).

Preoperative and Intraoperative Examinations andAortic Wall ExcisionThe diagnosis of a bicuspid or tricuspid aortic valve wasestablished by echocardiography and angiography andwas confirmed intraoperatively. The surgical protocolconsisted of aorta–atrial cannulation for extracorporealcirculation and the use of cold crystalloid cardioplegicsolution and moderate hypothermia of 30°C. The aorticcannula was placed in the proximal transverse aorticarch. The aorta was opened with an incision directedalong the anterior aortic aspect into the noncoronarysinus of Valsalva. The aortic wall specimen taken at thistime point was removed. At least a 3 mm � 8 mm pieceof the aortic wall was excised from the incision at theconvexity of the ascending aorta 2 to 4 cm above the levelof the aortic valve annulus. The excised parts of the aorticwall were fixed in 10% formalin in buffered saline andthen transferred for further processing and histologicexaminations. If the aortic diameter was enlarged, thereduction aortoplasty to normal diameter was performedby removal of an elliptical portion of aortic wall along theaortotomy incision [11]

According to the diameter of the ascending aorta thepatients with bicuspid and tricuspid aortic valve weredivided into three subgroups: (1) patients without dilata-tion of the ascending aorta (diameter less than 38 mm),(2) patients with moderate dilatation of the ascendingaorta (diameter between 39 and 49 mm), or (3) patientswith severe dilatation of the ascending aorta (diameter50 mm and more).

A normal ascending aorta without dilatation (diameterless than 38 mm) was found in 34 patients with bicuspidaortic valves and 29 patients with tricuspid aortic valves.A moderate dilatation of the ascending aorta (diameterbetween 39 and 49 mm) was found in 43 patients withbicuspid aortic valves and 20 patients with tricuspidaortic valves. A severe dilatation of the ascending aorta(diameter of the ascending aorta equal to 50 mm andmore) was found in 30 patients with bicuspid aorticvalves and 12 patients with tricuspid aortic valves.

Histologic ExaminationsWe measured the total thickness of the aortic media, thethickness of the elastic lamellae of the aortic media, andthe distances between the elastic lamellae. After histo-logic preparation of the specimen, morphometric mea-surements were done using an automatic microscopeimage analysis system (KS 400, release 3.2, Vision; CarlZeiss, Jena, Germany). The histologic specimens wereprepared with elastica van Gieson’s stain (Fig 1a). Theelastic lamellae of the aortic media was color-marked (Fig1b) and selected (Fig 1c) by removing the thin supportingmembranes. Then measurements of the thickness ofelastic lamellae of the aortic media and the distancesbetween the elastic lamellae were performed at multiplemeasuring points (about 200 measuring points) at 73 sites

for each specimen (Fig 1d). The multiple values wererecorded as one single average value for each patient.

Patient Informed Consent and Ethical CommitteeApprovalInformed consent was obtained from the patients. Thestudy was approved by the Institutional EthicalCommittee.

StatisticsThe student’s t test was used for statistical analysis. A pvalue of less than 0.05 was considered significant.

Results

Differences Between the GroupsTHICKNESSES OF AORTIC MEDIA. There was no difference (p �0.62) in the total thickness of the aortic media betweenthe patients with bicuspid and tricuspid valves (Fig 2).

THICKNESS OF ELASTIC LAMELLAE OF THE AORTIC MEDIA. Patientswith bicuspid aortic valves had significantly thinnerelastic lamellae of the aortic media (2.71 � 0.23 �m) of theascending aortic wall than patients with tricuspid aorticvalve disease (2.83 � 0.23 �m) (p � 0.006) (Fig 3).

DISTANCES BETWEEN ELASTIC LAMELLAE OF THE AORTIC MEDIA.

The distances between the elastic lamellae of the aorticmedia of the ascending aortic wall were significantlygreater in patients with bicuspid aortic valves (27.21 �8.69 �m) than patients with tricuspid aortic valve disease(24.34 � �m) (p � 0.033) (Fig 4).

Differences Between the Subgroups According to AorticDiameterTHICKNESS OF ELASTIC LAMELLAE OF THE AORTIC MEDIA. Patientswith bicuspid aortic valves had thinner elastic lamellae ifthe ascending aorta was dilated (both moderately andseverely) in comparison with patients with bicuspidvalves and normal diameter of the aorta. In contrast,patients with tricuspid aortic valves had no changes inthe thickness of the elastic lamellae in regard to the aorticdiameter (Fig 5). Only when the aorta was severelydilated, the difference between the patients with bicuspidand tricuspid aortic valves was statistically significant(Fig 5).

DISTANCES BETWEEN ELASTIC LAMELLAE OF THE AORTIC MEDIA.

Patients with bicuspid aortic valves and moderate aorticdilatation had a statistically significant (p � 0.039) in-crease of the distances between the elastic lamellae incomparison with patients with bicuspid aortic valves andnormal aortic diameter. In patients with severe aorticdilatation, this difference was not statistically significant(p � 0.59). In contrast, patients with tricuspid aorticvalves had a statistically significant increase of the dis-tances only when the aorta was severely dilated (Fig 6).

Univariate logistic regression was applied to test dif-ferent variables for the influence on the presence ofbicuspid or tricuspid aortic valves. The variables were

59Ann Thorac Surg BAUER ET AL2002;74:58–62 MORPHOMETRIC AORTIC MEDIA ANALYSIS

patient age, gender, ascending aortic diameter, thicknessof the aortic media, maximal pressure gradient across theaortic valve, mean pressure gradient across the aorticvalve, aortic valve stenosis, aortic valve regurgitation,thickness of the elastic lamellae, and distances betweenthe elastic lamellae. There were only statistically signifi-cant differences for the thickness of the elastic lamellae

(p � 0.008; odds ratio 8.69; 90% confidence interval, 1.76 to42.85) and for the distances between the elastic lamellae(p � 0.037; odds ratio 0.95; 90% confidence interval, 0.9 to0.99).

Fig 1. Preparation process for measurementswith the microscope picture analyzing system.The histologic specimens were prepared withelastica van Gieson’s stain (a). Then the elasticlamellae of the aortic media were color-marked(b) and selected (c) by removing the thin sup-porting membranes. The fine white lines werethen set in the field for measurements (d). Themeasurements included the thickness of the elas-tic lamellae and the distances between the la-mellae. The arrows indicate two elastic lamellaeand the distance between them that wasmeasured.

Fig 2. Thickness of the aortic media (mm) (� 95% confidence inter-val) in patients with bicuspid and tricuspid aortic valves.

Fig 3. Thickness (� 2 standard error) of the elastic lamellae (�m) ofthe aortic media in patients with bicuspid and tricuspid aorticvalves. Patients with a bicuspid aortic valve had significantly thin-ner elastic lamellae of the aortic media than patients with a tricus-pid aortic valve.

60 BAUER ET AL Ann Thorac SurgMORPHOMETRIC AORTIC MEDIA ANALYSIS 2002;74:58–62

Comment

Our study showed differences in histologic findings ofthe ascending aortic wall between the patients withbicuspid and tricuspid aortic valves. Although there wasno difference in the total thickness of the aortic mediabetween the groups, patients with a bicuspid aortic valvehad thinner elastic lamellae of the aortic media andgreater distances between the elastic lamellae than pa-tients with a tricuspid aortic valve. The second mainfinding of our study was an increase of the distancesbetween elastic lamellae in correlation with increasingdiameter of the ascending aorta in patients with bothbicuspid and tricuspid aortic valves.

There has been controversial discussion concerningwhether aortic wall alterations in patients with bicuspidaortic valves are caused by a congenital wall defect of theaorta [3, 12–15] or whether the alterations are due to theabnormal hemodynamic stresses on the aortic wall

caused by valve malformation [6, 16, 17]. As early as 1928,Abbott [18] theorized that bicuspid aortic valve, coarcta-tion of the aorta, and aortic wall thinning and rupturewere related to a common developmental abnormality.Schievink and Mokri [19] assumed a connection betweena bicuspid aortic valve and aorto-arterial abnormalities,because the semilunar valves and the medial layer of theaortic arch and its branches are embryologically derivedfrom the cells of the neural crest. Therefore, bicuspidaortic valves are considered part of a common develop-mental defect that also causes coarctation and aortic wallabnormalities. On the other hand, it is presumed that ifthe pressure gradient across the aortic valve is signifi-cantly high, which is frequently observed in patients withbicuspid aortic valves, the resulting hemodynamic stressalso may play an important role in bringing about thechanges that we saw in the aortic wall.

Systematic histologic examinations of the aortic wallfrom patients with bicuspid aortic valve do not exist.With the recently available microscope picture analyzingsystem it was possible to make systematic measurementsof the thickness of and distances between the elasticlamellae of the aortic media from the ascending aortasegments. Parai and colleagues [10], who used morphom-etry to analyze the proportional target area for elastictissue in Movat stained histologic slides, also showed asignificantly smaller area of elastic lamellae per micro-scopic field in patients with bicuspid aortic valves than inpatients with tricuspid aortic valve disease. However,most of the reports are from patients in whom an aorticdissection had already occurred [5, 7–9] or the patientnumbers were very small [10].

The results of our study cannot resolve the question ofwhether this abnormality is caused by altered hemody-namic stress or by a developmental defect. Further he-modynamic studies in combination with investigationsthat analyze the ultrastructure of the elastic lamellae ofthe aortic media and the other constituents of the aortic

Fig 4. Distances (� 2 standard errors) between the elastic lamellae(�m) of the aortic media in patients with bicuspid and tricuspid aor-tic valves. The distances between the elastic lamellae of the aorticmedia of the ascending aortic wall were significantly greater in pa-tients with bicuspid aortic valves than in patients with tricuspid aor-tic valve disease.

Fig 5. Thickness (� 2 standard error) of the elastic lamellae (�m) ofthe aortic media in patients with bicuspid and tricuspid aortic valvesaccording to the ascending aortic diameter.

Fig 6. Distances (� 2 standard error) of the elastic lamellae (�m) ofthe aortic media in patients with bicuspid and tricuspid aortic valvesaccording to the ascending aorta diameter.

61Ann Thorac Surg BAUER ET AL2002;74:58–62 MORPHOMETRIC AORTIC MEDIA ANALYSIS

wall are necessary in patients with a bicuspid aortic valveto elucidate the specific underlying defect.

We thank Ms Anne Gale for her editorial assistance and Ms JuliaStein for the statistical calculations.

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