Atrial Fibrillation Burden and Early Mitral Valve Remodelling in Patients Without Significant Mitral Regurgitation: A 3D Transesophageal Echocardiographic Study

Abstract

Background

The progression of mitral valve (MV) remodelling in atrial fibrillation (AF) patients without significant mitral regurgitation (MR) remains poorly characterized. We aimed to determine whether AF burden—paroxysmal versus persistent—differentially affects MV annular geometry, leaflet morphology, and functional dynamics using three‐dimensional transesophageal echocardiography (3D‐TEE).

Methods

In prospective cross‐sectional observational study, 46 consecutive AF patients without significant MR (paroxysmal AF: n = 18; persistent AF: n = 28) underwent comprehensive 3D transesophageal echocardiography. Offline semi‐automated quantitative analysis (4D MV Assessment, TomTec) was performed to assess mitral annular dimensions and geometry, leaflet morphology (area and angulation), and functional parameters including tenting volume, coaptation depth, and dynamic annular motion.

Results

Despite comparable demographics, persistent AF patients had larger LA diameter (45.4 ± 7.1 vs. 40.3 ± 6.3 mm; p = 0.05), lower LVEF (45.5 ± 15.4 vs. 56.5% ± 15.2%; p = 0.042), and greater LV end‐systolic dimension (41.3 ± 9.7 vs. 33.1 ± 9.4 mm; p = 0.018). 3D‐TEE demonstrated significant annular enlargement in persistent AF: larger AP diameter (3.92 ± 0.53 vs. 3.60 ± 0.47 cm; p = 0.042), AL‐PM diameter (3.74 ± 0.50 vs. 3.49 ± 0.34 cm; p = 0.048), 3D annular area (11.98 ± 3.19 vs. 10.34 ± 2.05 cm²; p = 0.038), and 2D annular area (9.78 ± 2.83 vs. 8.27 ± 1.87 cm²; p = 0.035). Anterior leaflet area was larger (8.93 ± 2.07 vs. 7.60 ± 2.02 cm²; p = 0.036) and distal anterior leaflet angle was reduced (16.97 ± 5.03 vs. 20.76 ± 6.40°; p = 0.042). Annular shape, tenting parameters, coaptation depth, and dynamic motion were preserved in both groups.

Conclusion

Persistent AF was associated with early mitral annular enlargement, anterior leaflet remodelling, impaired LV function, and LA dilation, even in the absence of significant MR. However, the higher heart rate in persistent AF, unrecorded AF duration, and lack of data on prior rhythm‐control therapy limit attribution of these changes to AF burden alone. These findings are hypothesis‐generating and require confirmation in prospective studies accounting for these potential confounders.

Persistent atrial fibrillation is associated with early mitral valve remodeling, characterized by annular enlargement and anterior leaflet adaptation despite the absence of significant mitral regurgitation. These subclinical structural changes may represent a pre‐regurgitation substrate, highlighting the potential value of early rhythm control to prevent disease progression.

1. Introduction

Atrial fibrillation (AF), the most prevalent sustained cardiac arrhythmia, poses a significant global health burden associated with substantial morbidity and mortality [1, 2]. Beyond its established clinical consequences, AF has a complex bidirectional relationship with mitral regurgitation (MR), particularly in atrial‐functional and ventricular‐functional MR. In these conditions, MR develops secondary to atrial or ventricular remodelling despite the absence of primary structural abnormalities of the mitral leaflets, chordae tendineae, or papillary muscles [3]. Progressive remodelling of the atria and ventricles may establish a self‐perpetuating cycle in which AF promotes MR, while MR further exacerbates AF [4].

Recent studies suggest that AF itself, independent of hemodynamic consequences, may initiate adverse cardiac remodelling through mechanisms like atrial cardiomyopathy, inflammation, and altered shear stress [5]. Consistent with this concept, Ortiz‐Leon et al. demonstrated using three‐dimensional echocardiography that patients with progressive AF exhibit significant mitral annular and leaflet remodelling compared with those in sinus rhythm, even in the absence of clinically significant MR, indicating that structural alterations of the MV apparatus may precede overt valvular dysfunction [6]. However, whether AF burden influences the extent of these early MV changes remains unclear. In particular, the differential effects of paroxysmal and persistent AF on mitral annular geometry, leaflet morphology, and valve dynamics have not been well defined. Therefore, this study used three‐dimensional transesophageal echocardiography (3D‐TEE) to evaluate the association between AF burden and early MV remodelling in patients with AF without significant MR.

2. Methods

We conducted a prospective cross‐sectional observational study enrolling 46 consecutive AF patients without significant mitral regurgitation (≤ mild grade) referred for clinically indicated 3D transesophageal echocardiography. MR severity was assessed according to contemporary echocardiographic recommendations using an integrated multiparametric approach including vena contracta width, color Doppler jet characteristics, effective regurgitant orifice area when feasible, regurgitant volume, pulmonary vein flow pattern, and quantitative chamber remodelling. Mild MR was defined by findings consistent with guideline‐recommended criteria [7], whereas patients meeting criteria for moderate or greater MR were excluded.

Participants were stratified by AF burden according to current ESC/EHRA guidelines into (i) paroxysmal AF (n = 18): AF episodes that self‐terminate within 7 days; (ii) persistent AF (n = 28): AF episodes lasting longer than 7 days or requiring intervention for termination. The duration of AF prior to enrollment was not used as an inclusion or exclusion criterion and was not consistently available in all patients. Exclusion criteria encompassed moderate/severe mitral regurgitation, primary mitral valve pathology (e.g., prolapse, rheumatic disease), prior mitral interventions, left ventricular ejection fraction <30%, or suboptimal TEE image quality.

All subjects underwent standardized transthoracic echocardiography followed by TEE using a fully sampled matrix‐array probe (Philips Epic CVXi with X8‐2t TEE probe, Philips Ultrasound Inc, Bothell, Washington, USA). 3D TEE image acquisition from the mid‐esophageal level using 3D‐Zoom mode was performed acquiring a volume including the whole mitral valve, annulus, aortic valve.

Offline three‐dimensional transesophageal echocardiographic (3D TEE) data sets were analyzed using dedicated software (4D MV Assessment, 2.2, TomTec Imaging systems, Munich, Germany). The end‐diastolic frame was identified based on mitral valve closure, and the end‐systolic frame was demarcated as the frame preceding mitral valve opening. Landmarks were manually placed along both the anterior and posterior mitral annulus, as well as the coaptation point. The software then semi‐automatically tracked the movement of these landmarks throughout the cardiac cycle, generating a dynamic three‐dimensional model of the mitral valve. When necessary, the software's automated tracking was overridden with manual adjustments. Upon approval of the tracking, the software provided measurements of the mitral valve geometry to assess the following parameters (See Figure 1):

• Mitral Annular Dimensions: (i) Anteroposterior diameter; defined as the distance from the anterior to posterior annular points. (ii) Anterolateral‐Posteromedial (AL‐PM) diameter; defined as the measurement across the annulus from the anterolateral to the posteromedial commissure. (iii) 3D Saddle‐shaped annulus area; defined as the surface area of the annulus measured in three dimensions, reflecting its natural saddle shape. (iv) 2D D‐shaped annulus area; defined as the area measured in a two‐dimensional plane, representing the annulus as a D‐shape. (v) 3D saddle‐shaped perimeter; circumference of the annulus in three dimensions. (vi) 2D D‐shaped perimeter: Circumference measured in the two‐dimensional D‐shaped plane.

• Leaflet Morphology: (i) Anterior leaflet area; which means the surface area of the anterior mitral leaflet. (ii) Distal Anterior leaflet angle; which means the angle formed by the distal part of the anterior leaflet, reflecting its spatial orientation and mobility.

• Functional Parameters: (i) Tenting volume/area; defined as the space and area between the leaflets and the annular plane during systole, indicating leaflet tethering. (ii) Coaptation depth; defined as the vertical distance between the leaflet coaptation point and the annular plane, assessing the adequacy of leaflet closure. (iii) Dynamic annular motion; defined as the assessment of annular movement throughout the cardiac cycle, reflecting the flexibility and contractility of the mitral annulus.

FIGURE 1.

Three‐Dimensional Measurements of the Mitral Annulus. AML, anterior mitral leaflet. PML, posterior mitral leaflet. AO, aortic annulus. Diagram created using ChatGPT (accessed June 2026) to generate an initial draft, with subsequent modifications by the authors. All content was reviewed for accuracy by the clinical team.

2.1. Statistical Analysis

Continuous variables are presented as mean ± standard deviation or median (interquartile range), as appropriate. Categorical variables are reported as frequencies and percentages. Comparisons of quantitative variables between patients with persistent and paroxysmal atrial fibrillation (AF) were performed using the independent samples t‐test. A p‐value < 0.05 was considered statistically significant.

3. Results

3.1. Demographics, clinical and 2D Echocardiographic parameters

Baseline demographic characteristics were comparable between the paroxysmal and persistent AF groups, with no significant differences in age, weight, height, body mass index, or body surface area. Hypertension was more prevalent in patients with paroxysmal AF than in those with persistent AF (77.8% vs. 42.9%, p = 0.02), whereas the prevalence of smoking, diabetes mellitus, ischemic heart disease, cerebrovascular stroke, and chronic kidney disease was similar between groups.

Patients with persistent AF had a significantly higher resting heart rate than those with paroxysmal AF (100.9 ± 20.5 vs. 86.3 ± 17.5 beats/min, p = 0.017). They also exhibited more advanced cardiac remodelling, characterized by lower left ventricular ejection fraction (45.5% ± 15.4% vs. 56.5% ± 15.2%, p = 0.042), larger left ventricular end‐systolic dimension (41.3 ± 9.7 vs. 33.1 ± 9.4 mm, p = 0.018), and greater left atrial diameter (45.4 ± 7.1 vs. 40.3 ± 6.3 mm, p = 0.05). Left ventricular end‐diastolic dimension did not differ significantly between the groups (p = 0.49). A detailed comparison of demographic, clinical, and two‐dimensional echocardiographic characteristics is presented in Table 1.

TABLE 1.

Demographic and 2D echocardiographic comparison between paroxysmal and persistent AF groups.

Variable	Paroxysmal AF (n = 18) (Mean ± SD)	Persistent AF (n = 28) (Mean ± SD)	p‐value
Age, years	51.2 ± 15	56.9 ± 13.8	0.204
Weight, kg	80.6 ± 21.5	84.9 ± 19.2	0.49
Height, cm	162.8 ± 11.3	165.8 ± 9.3	0.35
BMI, kg/cm2	30 ± 6.6	31 ± 7	0.62
BSA, m2	1.87 ± 0.3	1.94 ± 0.2	0.42
Smoking, n (%)	13 (16.7%)	11 (39.3%)	0.1
Hypertension, n (%)	14 (77.8%)	12 (42.9%)	0.02
Diabetes, n (%)	3 (16.7%)	7 (25%)	0.71
Ischemic heart disease, n (%)	4 (22.2%)	5 (17.9%)	0.72
Cerebrovascular stroke, n (%)	1 (5.6%)	2 (7.1%)	0.9
Chronic kidney disease, n (%)	2 (11.1%)	1 (3.6%)	0.55
Average heart rate, beat per minute	86.3 ± 17.5	100.9 ± 20.5	0.017
EF, %	56.5 ± 15.2	45.5 ± 15.4	0.042
LVEDD, mm	49.4 ± 8.3	51.5 ± 8.9	0.49
LVESD, mm	33.1 ± 9.4	41.26 ± 9.7	0.018
LA diameter, mm	40.3 ± 6.3	45.4 ± 7.1	0.05

3.2. 3D Mitral Valve Quantification

Quantitative 3D analysis demonstrated substantial mitral annular remodelling in persistent AF (illustrated in Table 2), with significantly larger anteroposterior diameters (3.92 ± 0.53 cm vs 3.60 ± 0.47 cm, p = 0.042), anterolateral‐posteromedial diameters (3.74 ± 0.50 cm vs 3.49 ± 0.34 cm, p = 0.048), 3D saddle‐shaped areas (11.98 ± 3.19 cm² vs 10.34 ± 2.05 cm², p = 0.038), and 2D D‐shaped areas (9.78 ± 2.83 cm² vs 8.27 ± 1.87 cm², p = 0.035). Annular perimeters were similarly increased in both 3D (12.74 ± 1.56 cm vs 11.94 ± 1.14 cm, p = 0.05) and 2D configurations (11.51 ± 1.47 cm vs 10.69 ± 1.15 cm, p = 0.042).

TABLE 2.

Comparison of mitral valve annulus measurements between paroxysmal and persistent AF groups.

Variable	Paroxysmal AF (n = 18) (Mean ± SD)	Persistent AF (n = 28) (Mean ± SD)	p‐value
Annular dimensions:
AP Diameter, cm	3.60 ± 0.47	3.92 ± 0.53	0.042
AL‐PM Diameter, cm	3.49 ± 0.34	3.74 ± 0.50	0.048
Inter‐trigonal Distance, cm	2.74 ± 0.33	2.92 ± 0.36	0.09
Commissural Diameter, cm	3.44 ± 0.32	3.67 ± 0.55	0.08
Annulus Height, cm	1.02 ± 0.22	1.03 ± 0.20	0.97

Annular geometry and shape:
Sphericity Index (AP / AL‐PM)	1.03 ± 0.09	1.05 ± 0.10	0.48
Saddle Shaped Annulus Area (3D), cm2	10.34 ± 2.05	11.98 ± 3.19	0.038
Saddle Shaped Annulus Perimeter (3D), cm	11.94 ± 1.14	12.74 ± 1.56	0.05
D‐Shaped Annulus Area (2D), cm2	8.27 ± 1.87	9.78 ± 2.83	0.035
D‐Shaped Annulus Perimeter (2D), cm	10.69 ± 1.15	11.51 ± 1.47	0.042
Non‐planar Angle, °	143.11 ± 14.92	146.16 ± 7.60	0.43

Leaflet Morphology:
Anterior Leaflet Area, cm2	7.60 ± 2.02	8.93 ± 2.07	0.036
Posterior Leaflet Area, cm2	4.88 ± 1.40	4.99 ± 2.22	0.85
Distal Anterior Leaflet Angle, °	20.76 ± 6.40	16.97 ± 5.03	0.042
Posterior Leaflet Angle, °	48.93 ± 12.52	43.79 ± 9.63	0.15
Anterior Leaflet Length, mm	3.04 ± 0.41	3.26 ± 0.42	0.08
Posterior Leaflet Length, mm	1.43 ± 0.27	1.37 ± 0.37	0.55

Functional parameters:
Tenting Volume, mL	3.69 ± 1.70	4.05 ± 2.28	0.54
Tenting Area, cm2	2.35 ± 0.71	2.56 ± 0.95	0.41
Coaptation Depth, mm	10.14 ± 2.64	9.10 ± 2.87	0.22
Angle AAo‐AP, °	108.53 ± 9.02	107.17 ± 7.95	0.61
Maximum Prolapse Height, mm	2.14 ± 1.53	1.98 ± 1.33	0.72
Maximal Open Coaptation Width, mm	33.33 ± 4.45	34.96 ± 8.02	0.38

Dynamic annular motion:
Annular Displacement (max), mm	6.74 ± 3.55	5.43 ± 2.65	0.19
Annular Displacement Velocity (max), mm/s	29.69 ± 15.30	27.34 ± 13.10	0.59

Leaflet analysis showed significantly enlarged anterior leaflet areas (8.93 ± 2.07 cm² vs 7.60 ± 2.02 cm², p = 0.036) and reduced distal anterior leaflet angles (16.97 ± 5.03° vs 20.76 ± 6.40°, p = 0.042) in persistent AF. Posterior leaflet parameters and all functional metrics (tenting volume/area, coaptation depth, prolapse height) showed no significant intergroup differences, as did dynamic annular motion parameters (maximal displacement and velocity).

4. Discussion

This study provides novel insights into the differential impact of AF burden on early MV remodelling in patients without significant MR. Employing quantitative three‐dimensional transesophageal echocardiography (3D‐TEE), we demonstrate that persistent AF is associated with significant adverse structural changes in the mitral annulus and anterior leaflet compared to paroxysmal AF. These changes occur alongsid worse LV systolic function and greater LA dilation in the persistent AF group.

4.1. Mitral Annular Dilation in Persistent AF

Patients with persistent AF exhibited significant enlargement of the mitral annulus across multiple dimensions, including the AP and AL‐PM diameters, 3D saddle‐shaped area and perimeter, and 2D D‐shaped area and perimeter. These findings are consistent with prior three‐dimensional echocardiographic data demonstrating that chronic AF drives progressive atrial and ventricular dilation, which in turn propagates adverse changes in mitral annular geometry [6, 8]

From a mechanistic standpoint, the mitral annulus is an integral component of the fibrous skeleton and is subject to mechanical and neurohormonal forces that accompany sustained atrial arrhythmia [9, 10]. Chronic irregular, rapid atrial activation during persistent AF increases atrial wall stress, promotes collagen crosslinking, and upregulates inflammatory mediators and matrix metalloproteinases (MMPs), particularly MMP‐1 and MMP‐9, which are central drivers of extracellular matrix (ECM) degradation and annular dilation [11]. These pathways are further amplified by the neurohormonal milieu of persistent AF—including elevated angiotensin II and endothelin‐1 activity—which promotes myofibroblast activation, progressive interstitial fibrosis, and structural remodelling of the atrial wall and annular fibrous skeleton [12]. Karakasis et al. provided a comprehensive mechanistic overview describing how AF perpetuates atrial cardiomyopathy through interconnected pathways involving oxidative stress, inflammation, calcium dysregulation, gap junction remodelling, and ECM disruption [5]. While atrial cardiomyopathy likely contributes substantially to the annular structural changes observed in our cohort, the concurrent presence of reduced LVEF and increased LVESD in the persistent AF group suggests that ventricular remodelling may also play an important role. Therefore, the observed remodelling pattern likely reflects a continuum involving both atrial‐functional and ventricular‐functional mechanisms rather than a purely atrial process. Importantly, patients with persistent AF demonstrated not only greater LA enlargement but also significantly lower LVEF and larger LVESD. These findings indicate the coexistence of ventricular remodelling, which may alter annular geometry through changes in ventricular shape, papillary muscle mechanics, and annular‐ventricular coupling. Consequently, the observed mitral annular remodelling cannot be exclusively attributed to atrial cardiomyopathy. Rather, our findings support a multifactorial remodelling process involving both atrial‐functional and ventricular‐functional components, potentially representing an intermediate stage before the development of clinically significant functional MR. Additionally, the possibility that higher ventricular rates may contribute to ventricular dysfunction and remodelling through tachycardia‐mediated cardiomyopathy cannot be excluded and may partially account for the observed differences in LV dimensions and function.

The preservation of the sphericity index and non‐planar (saddle) angle in the setting of annular enlargement is an important and nuanced finding. It indicates that while the annulus dilates in a size‐dependent manner with increasing AF burden, its fundamental three‐dimensional saddle‐shaped geometry is maintained. This geometric preservation may represent an adaptive response that partially offsets leaflet stress and coaptation compromise [13]

4.2. Anterior Leaflet Remodelling

The significant enlargement of the anterior mitral leaflet area combined with a reduced distal anterior leaflet angle in persistent AF patients represents one of the most clinically compelling findings of this study. Anterior leaflet area expansion is consistent with a well‐characterized adaptive response: when the mitral annulus dilates, the anterior leaflet enlarges to maintain adequate coaptation surface area—a phenomenon coined “leaflet adaptation” as characterized by Kim et al. using echocardiographic and biomechanical modelling [14]. This biologically active process is mediated by serotonin signalling through 5‐HT2B receptors on interstitial cells, triggering de novo leaflet matrix synthesis and cell proliferation, and highlights that the mitral valve is not a passive structure but an actively remodelling tissue [15]

The concomitant reduction in the distal anterior leaflet angle suggests that leaflet expansion is accompanied by geometric flattening—a finding that reflects a shift in the biomechanical environment imposed by both annular dilation and the altered tethering forces arising from LA and LV remodelling. In the context of atrial functional mitral regurgitation (AFMR), “atriogenic leaflet tethering”—as described by Silbiger and subsequently substantiated by 3D echocardiographic studies—involves displacement of the posterior annulus and inward bending of the basal LV segments, causing posterior leaflet tethering and counterclockwise‐directed torque on the anterior annulus, which in turn tethers the anterior leaflet [16, 17].

The selective vulnerability of the anterior leaflet—compared to the posterior leaflet, which showed no significant changes in area, angle, or length—may reflect its unique structural and anatomical properties. The anterior leaflet is attached to the left and non‐coronary fibrous trigones and is in direct fibrous continuity with the aortic annulus. Its attachment to the fibrous cardiac skeleton renders it more susceptible to tensile forces transmitted from an enlarged atrial chamber and to the geometric perturbations imposed by progressive mitral annular dilation [16]. In contrast, the posterior leaflet, anchored more peripherally along the muscular annulus, may be partially shielded from these forces at earlier stages of AF‐related remodelling.

4.3. Functional Preservation Despite Structural Remodelling

A particularly important observation is the absence of significant differences in functional MV parameters—tenting volume, tenting area, coaptation depth, and maximum prolapse height—between paroxysmal and persistent AF groups, despite the structural annular and leaflet differences described above. This functional preservation in the setting of ongoing structural remodelling reinforces the concept of a “silent phase” in MV valvulopathy, where compensatory mechanisms maintain valvular competence until a critical threshold is reached [18].

This observation aligns with the framework of AFMR as an evolving and underrecognized entity. Pagnesi et al. and a growing body of literature characterize AFMR as a condition rooted primarily in annular enlargement rather than LV tethering, closely linked to chronic AF and heart failure with preserved ejection fraction (HFpEF), and associated with poor long‐term outcomes if unaddressed [19]. Abdelgawad et al. further demonstrated in a 3D echocardiography study of AFMR that the leaflet‐annular remodelling pattern in this entity is distinct from ventricular functional MR, with annular geometry changes—rather than tethering forces—being the primary driver of regurgitation [20].

The trend toward reduced annular displacement and velocity in persistent AF, though not reaching statistical significance in our sample, warrants particular attention. Impaired annular dynamics reflect diminished atrial and ventricular contractile contribution to valve closure, and progressive loss of annular dynamism is an established precursor to MR development [21, 22]. If left untreated, this functional deterioration, combined with structural annular dilation, creates a biomechanical environment in which leaflet coaptation becomes increasingly precarious.

4.4. Clinical Implications and Future directions

Our findings emphasize that adverse MV remodelling begins early in the AF disease spectrum, particularly with persistent forms, before the onset of significant MR. This underscores the importance of timely rhythm control strategies (e.g., catheter ablation) in persistent AF patients, not only to alleviate symptoms but potentially to mitigate or reverse these structural changes and prevent future MR. Although several annular measurements reached statistical significance, the absolute differences between groups were relatively modest. Therefore, these findings should be interpreted cautiously and viewed primarily as evidence of early remodelling rather than large structural alterations.

Future directions should include: (1) prospective longitudinal cohort studies examining MV structural evolution in AF patients before and after rhythm control interventions; (2) studies correlating quantitative 3D MV parameters with biomarkers of atrial cardiomyopathy (e.g., NT‐proBNP, galectin‐3, TGF‐β1) to mechanistically link molecular and structural remodelling; (3) larger multicenter registries to establish normative 3D MV geometry data stratified by AF type and burden; and (4) investigation of whether early 3D‐TEE‐based MV remodelling indices predict future MR development, guiding the optimal timing of prophylactic rhythm control or structural interventions.

5. Conclusions

Persistent AF was associated with early mitral annular enlargement, anterior leaflet remodelling, impaired LV function, and LA dilation, even in the absence of significant MR. However, the higher heart rate in persistent AF, unrecorded AF duration, and lack of data on prior rhythm‐control therapy limit attribution of these changes to AF burden alone. These findings are hypothesis‐generating and require confirmation in prospective studies accounting for these potential confounders. Future longitudinal studies are warranted to determine if reversing AF burden (e.g., via ablation) can halt or reverse these early changes.

6. Limitations

The moderate sample size and single‐center design may limit subgroup analyses and generalizability. The cross‐sectional design precluded assessment of causality and temporal changes in mitral valve remodelling. AF duration before enrollment and prior rhythm‐control interventions were not systematically recorded, limiting evaluation of their independent effects. Left atrial diameter, rather than the guideline‐preferred left atrial volume index, was used because it was consistently available. Echocardiographic analyses were performed locally without independent core laboratory adjudication and formal inter‐observer or intra‐observer reproducibility assessment. Inherent technical limitations of three‐dimensional transesophageal echocardiography should also be acknowledged. Future longitudinal studies are needed to determine whether rhythm‐control strategies can prevent or reverse early mitral valve remodelling.

Ethics Statement

The study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Institutional Ethics Committee.

Consent Statement

Written informed consent was obtained from all participants prior to their enrollment in the study.