This retrospective study enrolled patients who were admitted to the Department of Cardiology at Xiangtan Central Hospital between October 2018 and June 2023 for elective or urgent PCI targeting bifurcation lesions in the LAD artery, including presentations such as stable angina, unstable angina, non–ST-elevation myocardial infarction (NSTEMI), and ST-elevation myocardial infarction (STEMI). Prior to undergoing PCI, all participants provided written informed consent. The research protocol was approved by the Ethics Committee of Xiangtan Central Hospital and complied with the principles outlined in the Declaration of Helsinki (2013 revision). Ethical approval number: X201807352-1. Inclusion criteria were as follows: patients who underwent PCI for LAD bifurcation lesions with CAG confirming ≥70% diameter stenosis and a vessel diameter ≥2.5 mm; in addition, the implanted stent in the MV had to extend across the ostium of the SB. CBL were defined as atherosclerotic narrowing involving or located adjacent to the origin of a functionally significant SB. Exclusion criteria included: left main coronary bifurcation lesions; chronic total occlusion (CTO) of the LAD; isolated septal branch lesions; bifurcation lesions in the LCX-OM (obtuse marginal), RCA-RV (right ventricular), or PDA-PL (posterior descending artery- posterolateral branch) regions; procedures where SB stenting was performed electively before MV stenting; SB with pre-stenting TIMI (thrombolysis in myocardial infarction) flow grade ≤2; and patients with a known allergy to contrast media (Figure 1).

Baseline demographic data, comorbid conditions, and laboratory parameters were systematically documented in a dedicated research database. The choice of vascular access for PCI (either radial or femoral), procedural strategy, device selection, and application of intracoronary imaging were left to the discretion of the interventional cardiologist, guided by current clinical practice guidelines and expert recommendations (11). All main vessel stents implanted were second-generation drug-eluting stents (DES), including everolimus-eluting and zotarolimus-eluting stents. Bare metal stents were not used in this cohort. Provisional stenting was the primary strategy utilized in this cohort, with SB stenting performed only if there was significant compromise following MV stenting. All participants received a loading dose of clopidogrel (300 mg) and aspirin (300 mg) within 24 h prior to the intervention. For P2Y12 inhibitor therapy, the majority of patients received clopidogrel (300 mg loading dose followed by 75 mg/day maintenance), while ticagrelor (180 mg loading dose followed by 90 mg twice daily maintenance) was administered to some acute coronary syndrome patients at the discretion of the treating physician. Prasugrel was not used in this cohort. During the procedure, unfractionated heparin was administered to maintain an activated clotting time within the therapeutic range of 250–300 s. Post-PCI, patients were prescribed dual antiplatelet therapy for at least 12 months, consisting of clopidogrel (75 mg once daily) or ticagrelor (90 mg twice daily) in combination with lifelong aspirin (100 mg/day).

CAG was independently reviewed by two experienced investigators selected from a larger pool of qualified analysts. Offline assessment of the baseline angiographic images, obtained prior to PCI, was conducted using the QAngio software system (version 2.1.9, Medis, Leiden, the Netherlands). Quantitative coronary angiography (QCA) of both the MV and SB was performed according to standardized protocols previously reported in the literature (12).

The variables collected in this study encompassed four major categories: (1) Angiographic and procedural characteristics of the target bifurcation lesion, including the lesion's location within the main vessel lesion (ML), Medina classification, identification of true bifurcation lesions, stenosis severity at the bifurcation core, baseline bifurcation angle prior to PCI, and the total length of implanted stents. (2) ML–specific angiographic and procedural parameters, such as reference vessel diameter (RVD), degree of stenosis before the procedure, presence of moderate-to-severe calcification or angulation, evidence of thrombus, pre-PCI TIMI flow grade, plaque irregularity, dissection following pre-dilation, lesion preparation strategy (e.g., semi-compliant or non-compliant balloon use, with no systematic use of scoring balloons, cutting balloons, or intravascular lithotripsy recorded), post-dilation TIMI flow grade, and residual stenosis after pre-dilation. (3) SB–related angiographic and procedural characteristics, including reference diameter, pre-procedural stenosis severity, presence of moderate-to-severe calcification or angulation, thrombus involvement, TIMI flow grade before PCI, plaque irregularity, whether balloon pre-dilation was performed, use of the jailed wire technique, and residual stenosis after pre-dilation. (4) Primary outcome, defined as the occurrence of SBO following MV stent implantation.

In cases of SBO without a jailed wire in place, the side branch was first rewired, followed by balloon dilatation using semi-compliant or non-compliant balloons to restore flow. In cases with a jailed wire in place, the jailed wire was removed after the side branch was then rewired prior to balloon dilatation. If flow could not be adequately restored despite these measures, bailout side branch stenting was performed as a final strategy.

Bifurcation classification: Among the various classification systems available for CBL, the Medina classification remains the most commonly adopted due to its simplicity, consistency, and ease of clinical application (13). A true bifurcation lesion was defined as one that met any of the following Medina criteria: 1,1,1; 1,0,1; or 0,1,1. MV plaque location: When atherosclerotic plaque was confined to the side of the MV opposite the SB ostium, it was classified as plaque opposite to the SB. If the plaque was situated on the same side as the SB ostium or simultaneously present on both sides, it was categorized as plaque on the SB side. Assessment of coronary artery calcification: The extent of calcification was evaluated based on standardized criteria previously established in the literature (14). Bifurcation angulation: The angle at the bifurcation site was categorized as follows: mild (<45°), moderate (>45° and <90°), or severe (>90°), based on the measured angle between the MV and SB. Coronary blood flow evaluation: Coronary perfusion was assessed using the TIMI flow grading system, in accordance with the definitions outlined by Gibson et al. (15). Coronary dissection was classified according to the criteria established by the National Heart, Lung, and Blood Institute (NHLBI) (16). SB pre-dilation referred to the use of balloon angioplasty in the SB prior to the implantation of the MV stent. Jailed wire technique was defined as the placement of a guidewire in the SB during MV stenting to safeguard patency at the SB ostium (2). The bifurcation core (carina region) was defined as the anatomical zone located within 5 mm proximal to the carina point, where the distal MV and SB intersect; this region was evaluated visually. Diameter stenosis (%): calculated as [RVD−minimal lumen diameter(MLD)]/RVD, representing the most severe luminal narrowing within the analyzed segment. MV/SB RVD ratio: calculated as (proximal MV reference diameter + distal MV reference diameter)/(2× SB reference diameter). Bifurcation angle (°) was defined as the angle formed between the central axis of the distal MV and that of the SB. RVD was visually estimated based on the dimensions of the proximal and distal artery segments not affected by atherosclerotic plaque, representing the presumed normal vessel caliber. SBO was defined as a complete or significant reduction in SB blood flow—either temporary or persistent—following MV stent implantation, characterized by a decline in TIMI flow grade (17). A SB was considered significant if its RVD, as assessed by QCA, was ≥1.5 mm. The Visual estimation-based Risk prEdiction of Side branch OccLusion in coronary bifurcation interVEntion (V-RESOLVE) score for each patient was calculated in accordance with the methodology and criteria established in prior studies (5). Derived from the Risk prEdiction of Side branch OccLusion in coronary bifurcation intervention (RESOLVE) study, the V-RESOLVE score is a simplified angiographic-based scoring system incorporating six bifurcation lesion characteristics to predict the risk of SOB during PCI. This scoring system was originally designed to estimate the likelihood of SBO in BCL. Technical success was defined by achieving TIMI grade 3 flow and a residual stenosis of less than 30% in both the MV and SB when SB stenting was performed. In cases where no SB stent was attempted, technical success was defined as TIMI grade 3 flow in the MV with residual stenosis <30%, and in the SB either (1) TIMI 3 flow with residual stenosis less than or equal to its baseline severity, (2) residual stenosis <50%, or (3) physiologically normal flow parameters. Procedural success was defined as the attainment of technical success without the occurrence of in-hospital MACE.

In-hospital MACE included any of the following events occurring before discharge: cardiac death, MI, acute stent thrombosis, recurrence of symptoms requiring urgent repeat target-vessel revascularization (TVR) via PCI or coronary artery bypass grafting (CABG), and cardiac tamponade necessitating either pericardiocentesis or surgical intervention. All clinical endpoints and adverse outcomes were defined based on criteria provided by the Academic Research Consortium (18).

Statistical analyses were conducted using SPSS software version 26.0 (IBM Corp., Armonk, NY, USA). Categorical variables were summarized as frequencies and percentages, and intergroup comparisons were assessed using either the chi-square test or Fisher's exact test, depending on data distribution and sample size. Continuous variables were reported as mean ± standard deviation (SD) for normally distributed data, and as median with interquartile range (IQR) for data not following a normal distribution. For comparisons involving continuous variables, the independent samples t-test was applied when the assumption of normality was met; otherwise, the Mann–Whitney U-test was employed. To identify independent predictors of SBO, multivariate logistic regression analysis was performed. Variables included in the multivariate model were those demonstrating statistical significance in univariate analysis or deemed clinically important based on prior literature. A P-value less than 0.05 was considered to indicate statistical significance.

A total of 2,182 patients were initially screened, of whom 553 met the inclusion criteria and were included in the final analysis (Figure 1). Among these, 41 cases (7.4%) experienced SBO. As presented in Table 1, baseline demographic characteristics and comorbid conditions were comparable between the SBO and without-SBO groups. The mean age did not differ significantly between the groups (57.3 ± 10.6 vs. 58.1 ± 11.6 years, P = 0.674), and the majority of patients in both cohorts were male (95.1% vs. 86.1%, P = 0.162). No statistically significant differences were observed in other clinical parameters, including body mass index, presence of diabetes mellitus, hypertension, dyslipidemia, smoking history, previous MI, stroke, peripheral artery disease, or left ventricular ejection fraction(LVEF) (all P > 0.05). Similarly, the distribution of clinical presentations—such as stable angina, unstable angina, STEMI, and NSTEMI—did not show a significant difference between the groups (P = 0.469).

As shown in Table 2, the SBO group demonstrated significantly greater lesion complexity and procedural difficulty. True bifurcation lesions were markedly more prevalent among patients who developed SBO (68.3% vs. 35.5%, P < 0.001), with a higher incidence of Medina 1,1,1 configurations (53.7% vs. 25.4%, P = 0.004). Baseline angiographic assessments of both the MV and SB revealed notable disparities between the groups, particularly in terms of calcification severity, bifurcation geometry, and degree of ostial narrowing (Table 2). Within the MV, patients in the SBO group exhibited a higher frequency of moderate-to-severe calcification (41.5% vs. 29.5%, P = 0.034), thrombus-laden lesions (9.8% vs. 4.7%, P = 0.029), and irregular plaque morphology (14.6% vs. 6.1%, P = 0.028). Atherosclerotic plaque was significantly more likely to be situated on the same side as the SB ostium in these patients (90.2% vs. 35.2%, P < 0.001). Additionally, greater stenosis was observed in the bifurcation core (median 51.7% vs. 35.5%, P = 0.027), alongside narrower bifurcation angles (57.1° vs. 62.8°, P = 0.015), and elevated MV/SB reference diameter ratios (1.72 vs. 1.35, P = 0.041). In terms of SB characteristics, patients with SBO had smaller reference diameters (2.28 mm vs. 2.36 mm, P = 0.018) and more severe ostial stenosis (43.4% vs. 15.2%, P = 0.032). However, there were no significant group differences in SB calcification, angulation, thrombus presence, or pre-procedural TIMI flow grade (all P > 0.05). Procedural data further underscored the technical challenges associated with SBO. Dissection prior to MV stent implantation was more frequent in the SBO group (7.3% vs. 1.2%, P = 0.023). Notably, balloon pre-dilation of the SB was performed significantly less often in this group (4.9% vs. 25.4%, P = 0.005), and use of the jailed wire technique was considerably lower (14.6% vs. 43.0%, P < 0.001). Furthermore, patients who developed SBO had significantly higher V-RESOLVE scores (median 20.7 vs. 10.1, P < 0.001), and were substantially less likely to undergo pre-stenting intravascular imaging with intravascular ultrasound (IVUS) or optical coherence tomography (OCT) (2.4% vs. 33.2%, P < 0.001). Among the 41 cases defined as SBO, 39 patients (95.1%) successfully achieved TIMI grade 3 flow following side branch balloon dilatation. Bailout side branch stenting was required in 4 cases (9.8%) due to persistent flow compromise despite balloon dilatation, and 2 patients (4.9%) did not achieve full restoration of TIMI 3 flow even after stenting or balloon dilatation.

As detailed in Table 3 and Figure 2, the occurrence of SBO was significantly associated with a reduced rate of procedural success (82.9% vs. 94.7%, P = 0.007) and a higher incidence of in-hospital MACE (17.1% vs. 5.3%, P = 0.007). The increased MACE rate in the SBO group was primarily driven by a significantly elevated incidence of periprocedural MI (14.6% vs. 3.5%, P = 0.003). Other complications, including cardiac tamponade, acute stent thrombosis, and TVR, were observed infrequently and did not differ significantly between the two groups.

Univariate logistic regression analysis (Table 4) identified several factors significantly associated with the occurrence of SBO, including the presence of a true bifurcation lesion (P < 0.001), a higher MV/SB diameter ratio (P < 0.001), absence of jailed wire technique (P = 0.037), lack of SB pre-dilation (P = 0.022), and an elevated V-RESOLVE score (P = 0.001). Subsequent multivariate logistic regression analysis confirmed that three variables remained independent predictors of SBO: true bifurcation lesion [odds ratio [OR] 1.221, 95% confidence interval [CI] 1.052–1.522, P < 0.001], MV/SB reference diameter ratio (OR 1.431, 95% CI 1.333–2.727, P < 0.001), and V-RESOLVE score (OR 3.736, 95% CI 1.227–8.665, P = 0.001). These results emphasize the critical role of anatomical complexity and the importance of strategic procedural planning in reducing the risk of SBO during bifurcation PCI.

In this single-center cohort study evaluating PCI for LAD artery bifurcation lesions, the incidence of SBO was 7.4%, consistent with previous studies such as the V-RESOLVE trial (7.4%) (5) and the COBIS II registry (8.4%) (8). These findings reinforce the reproducibility and clinical relevance of SBO as a common complication in bifurcation interventions. Although the PROGRESS-BIFURCATION registry reported a higher SBO incidence of 13%, the variation likely reflects differences in lesion complexity and inclusion criteria, with broader anatomical patterns represented in that cohort (19).

Several anatomical characteristics were found to be significantly associated with SBO in our study. True bifurcation lesions—especially those classified as Medina 1,1,1—were notably more common among patients who experienced SBO (68.3% vs. 35.5%, P < 0.001). This observation is consistent with prior research suggesting that true bifurcations are more vulnerable to plaque or carina shift during stent implantation in the MV (20). Additionally, atherosclerotic plaques positioned on the same side as the SB ostium were significantly more prevalent in the SBO group (90.2% vs. 35.2%, P < 0.001), a finding supported by OCT study showing that layered or bulky plaques at the SB origin increase the likelihood of occlusion (21).

Geometrical considerations also played a key role. A narrower bifurcation angle was independently associated with SBO in our population (57.1° vs. 62.8°, P = 0.015), likely due to increased susceptibility to carina displacement during stent expansion. Although the impact of bifurcation angle on SB outcome remains a topic of debate, several investigations support the notion that narrower angles elevate the risk of carina shift and subsequent SB occlusion (22). Conversely, some studies have suggested that wider bifurcation angles may also compromise SB outcomes by reducing ostial area or shortening ostial length (23). One analysis even found that the SBO group exhibited larger bifurcation angles than non-SBO counterparts (23). Nonetheless, our results align more closely with studies that highlight mechanical carina shift—more pronounced at narrower angles—as a key mechanism of SBO development (21).

Another important predictor was the MV/SB reference diameter ratio, which was significantly higher in the SBO group (1.72 vs. 1.35, P = 0.041). This metric reflects the relative vessel size mismatch and has been implicated in plaque redistribution and hemodynamic compromise at the SB ostium during stent deployment, as documented in IVUS-based investigations (24). Furthermore, a higher degree of stenosis within the bifurcation core was observed in SBO cases (51.7% vs. 35.5%, P = 0.027), emphasizing the contribution of lesion burden at the carina to impaired SB perfusion (20). Taken together, these anatomical risk factors highlight the critical importance of thorough preprocedural angiographic assessment in bifurcation PCI. They also provide a mechanistic framework suggesting that SBO is not merely a procedural mishap, but rather a foreseeable event stemming from adverse bifurcation geometry and complex plaque distribution.

Beyond anatomical complexity, procedural techniques and operator-driven decisions play a pivotal role in determining the risk of SBO during bifurcation PCI. Our findings highlight several procedural characteristics that were significantly associated with SBO and may be amenable to optimization through refined interventional strategies.

Notably, SB pre-dilation was performed far less frequently in the SBO group compared to patients without SBO (4.9% vs. 25.4%, P = 0.005). Pre-dilation may enhance ostial lesion compliance and facilitate favorable plaque redistribution, thereby improving SB preservation during MV stenting. This protective role has been emphasized in prior studies, particularly for lesions with high plaque burden or tight ostial narrowing (20, 24). Similarly, use of the jailed wire technique—an established and technically straightforward approach to maintain SB access—was significantly lower in the SBO cohort (14.6% vs. 43.0%, P < 0.001). The presence of a jailed wire enables re-access and bailout maneuvers in cases of SB flow compromise and has been shown to lower the incidence of complete SB occlusion in high-risk bifurcation anatomy (24, 25).

Procedural complications also appeared to influence SBO risk. Dissections occurring prior to MV stent deployment were significantly more common among SBO cases (7.3% vs. 1.2%, P = 0.023), potentially contributing to altered flow dynamics or formation of a false lumen that impairs SB perfusion. These observations reinforce the importance of meticulous lesion preparation and conservative balloon sizing during pre-dilation and modification procedures (20, 24). A particularly striking finding was the markedly low rate of intravascular imaging use in the SBO group. Only 2.4% of these patients underwent IVUS or OCT guidance, compared to 33.2% in the without-SBO group (P < 0.001). Intravascular imaging provides valuable insights into lesion morphology, plaque eccentricity, and carina shift potential—factors that are critical in guiding stent deployment and minimizing SB compromise. Several studies have advocated for routine use of IVUS/OCT in complex bifurcation interventions to enhance procedural precision and outcomes (20, 24, 26). Taken together, these data suggest that SBO is not solely determined by anatomical constraints but is also significantly influenced by modifiable, operator-dependent factors. Inadequate SB preparation, failure to employ protective techniques, and underutilization of intravascular imaging are all preventable contributors. These findings support the need for greater procedural standardization and broader integration of evidence-based bifurcation techniques to reduce the incidence of SBO in contemporary practice.

Our study demonstrated a strong and statistically significant association between the V-RESOLVE score and the occurrence of SBO in patients undergoing PCI for LAD bifurcation lesions. Patients who experienced SBO had substantially higher V-RESOLVE scores compared to those without SBO (median 20.7 vs. 10.1, P < 0.001). Furthermore, multivariate logistic regression confirmed the V-RESOLVE score as an independent predictor of SBO (OR 3.736, 95% CI: 1.227–8.665, P = 0.001). These results support the V-RESOLVE scoring system's discriminatory power and validate its clinical applicability for peri-procedural risk stratification in real-world settings. This finding is consistent with the original V-RESOLVE study, which demonstrated that visual estimation of lesion characteristics could effectively substitute for QCA in predicting SBO, with a c-statistic of 0.76 (95% CI: 0.71–0.80)—a performance nearly equivalent to that of the QCA-based RESOLVE score (c-statistic: 0.77, 95% CI: 0.72–0.81) (5). Simulation analyses from the original study further showed acceptable inter-observer consistency (c-statistic range: 0.65–0.77), highlighting the score's robustness across different operators and institutions (5). Importantly, our analysis extends the validation of V-RESOLVE specifically to LAD bifurcation lesions—a subset characterized by higher SBO risk due to frequent involvement of critical diagonal or septal branches. While earlier studies have evaluated bifurcation PCI in general, few have focused exclusively on LAD-specific anatomy or validated risk assessment tools in this context. Our findings thus address a notable gap in the current evidence base. Given its predictive strength and practical simplicity, we advocate incorporating the V-RESOLVE score into routine pre-procedural assessments for bifurcation PCI. The score offers a rapid, visually assessed, and non-QCA-dependent tool to identify high-risk patients. This early stratification could inform the use of protective strategies—such as jailed wire or balloon techniques—or prompt the deployment of intravascular imaging to better characterize lesion morphology. When employed proactively, these measures have the potential to mitigate SBO risk and improve procedural outcomes (20).

Incorporating the V-RESOLVE score into standardized clinical workflows—potentially as part of a consensus-driven PCI decision-making pathway—could enhance both individualized treatment planning and institutional protocol consistency. While prior expert consensus documents have advocated for structured bifurcation PCI strategies, few have formally integrated validated predictive scores into these frameworks (20). Nevertheless, despite its utility, the V-RESOLVE score is subject to variability due to its reliance on visual assessment. To ensure consistency, operator training and standardized interpretation protocols are essential. Prospective, multicenter trials are warranted to further validate the prognostic impact of the V-RESOLVE score on long-term outcomes and to explore its integration within algorithmic, guideline-based approaches to bifurcation PCI.

SBO during PCI for bifurcation lesions carries significant clinical consequences and should no longer be viewed as a minor angiographic inconvenience. In our cohort of 553 patients undergoing PCI for LAD bifurcation lesions, SBO occurred in 7.4% of cases and was associated with notably lower procedural success (82.9% vs. 94.7%, P = 0.007), higher rates of periprocedural MI (14.6% vs. 3.5%, P = 0.003), and increased in-hospital MACE (17.1% vs. 5.3%, P = 0.007). These results highlight the immediate prognostic significance of SBO, even when the affected SB appears angiographically small or non-dominant.

Pathophysiologically, SBO impairs myocardial perfusion in the territory supplied by the occluded SB, potentially leading to ischemia, infarction, and microvascular dysfunction—particularly in cases involving functionally important branches such as diagonals or septals. Furthermore, the abrupt cessation of flow can result in endothelial damage and activate inflammatory cascades, exacerbating myocardial injury (11). Our findings align with previous research by Strepkos et al., who examined 933 bifurcation PCI cases across six centers. Their analysis demonstrated that SBO was linked to significantly lower procedural success (73.5% vs. 92.2%, P < 0.001), a greater need for unplanned two-stent strategies (24.8% vs. 6.0%, P < 0.001), and higher rates of dissection and plaque modification interventions (19). Importantly, patients with untreated SBO exhibited increased long-term MACE and mortality, emphasizing the enduring consequences of unaddressed SB compromise (19). Similarly, Guo et al. reported that in a cohort of 245 patients with chronic total occlusion and bifurcation lesions (CTO-BFL), SBO- defined as post-recanalization TIMI flow <3—was significantly associated with periprocedural MI and composite procedural complications. Key independent predictors included the absence of SB protection, ostial stenosis of the SB, and use of dissection-reentry techniques (27). Although some literature has suggested that occlusion of small or non-dominant SBs may be clinically inconsequential, our results and those from large registries challenge this assumption. Even SBs with reference diameters ≥1.5 mm can supply critical myocardial territories—particularly in LAD bifurcations—and their occlusion may initiate a cascade of ischemic injury (27). These findings reinforce the view that SBO is a clinically meaningful complication with both procedural and prognostic implications. Furthermore, periprocedural MI itself has been shown to be independently associated with increased short- and long-term all-cause mortality in patients undergoing PCI, underscoring its prognostic importance (28). It not only reflects underlying anatomical complexity but also acts as a direct mediator of myocardial injury and adverse outcomes. As such, prevention, early detection, and effective management of SBO should be prioritized in bifurcation PCI. Further prospective, large-scale studies are needed to investigate the long-term effects of SBO, including its role in target vessel failure, progression of heart failure, and mortality. Incorporating SBO risk assessment into pre-procedural planning and post-PCI surveillance protocols may contribute to improved short- and long-term cardiovascular outcomes.

Several limitations of this study should be acknowledged. First, this was a single-center, retrospective cohort study, which may introduce inherent selection bias and limit the generalizability of our findings. Although multivariate analyses were performed to adjust for confounding variables, residual confounders cannot be fully excluded. Second, the assessment of lesion characteristics and V-RESOLVE scoring was based on angiographic visual estimation, which, despite standardization and operator training, is subject to inter-observer variability. Third, our study focused exclusively on LAD bifurcation lesions and may not be fully extrapolatable to left main or other coronary bifurcations. Fourth, the lack of long-term follow-up data precludes definitive conclusions regarding the chronic impact of SBO on clinical outcomes such as mortality, target vessel failure, or late stent thrombosis. Finally, although intravascular imaging was underused in the SBO group, its limited overall usage prevented subgroup analysis regarding its potential protective role. Future multicenter, prospective studies with comprehensive imaging and long-term outcome data are warranted to validate and extend these findings.