Statistical analysis:

Continuous variables are presented as mean ± SD or median (interquartile range). Categorical variables are presented as percentages. The t-test was used for normally distributed data, the Mann–Whitney U test was used for non-normally distributed data, and the χ2-test was used for categorical variables. Multivariate logistic regression analyses were conducted to assess the anatomical characteristics of the PFO concerning the incidence rate of CS. Odds ratios are reported with 95% confidence intervals. Independent risk factors for CS were determined based on the results of multivariate analysis. Receiver operating characteristic (ROC) curves and area under the ROC curves (AUC) were studied and calculated. For each participant, demographic data and clinical data were analyzed using SPSS 27.0 software, with statistical significance defined as P < 0.05.

Results:

1.Study cohort

This study was an observational study. In total, 134 patients were included (male patients n=55, 40.7%), with an average age of years (38±11.2) (Table 1). There were 50 patients with severe RLS (right-to-left shunts) and 84 patients with mild-to-moderate RLS (right-to-left shunts). In the massive shunt group, cerebral infarction accounted for 39.6% and migraine accounted for 64%.

2. Severe RLS vs. mild-to-moderate RLS

There was no significant difference in terms of gender (42% vs. 38.8%,p=0.383), age (39.76±11.38 vs. 37.98±11.10, p=0.373), previous hypertension (18% vs. 14.1%,p=0.625), diabetes (6% vs. 2.4%, p=0.359), smoking (16% vs. 15.3%, p=1.000), the incidence of cerebral infarction (39.6 vs. 38.5, p=1.000 ), migraine (64% vs. 63.5%, p=1.000), the presence of the Eustachian valve or a Chiari’s network (10% vs. 5.9%, p=0.499), left funnelform (28% vs. 31.8% p=0.701), right funnelform (26.0% vs. 21.2%, p=0.532), the length of the tunnel (10.2±3.7 vs. 9.7±4.6, P=0.585), the thickness of the primary septum and the secondary septum (1.2±0.3 vs. 1.3±0.4, 3.7±1.0 vs. 3.5±1.0, P=0.172, P=0.369), and the RoPE score and the multiple exits of the tunnel of LA (6.6±1.5 vs. 6.8±1.7, 28% vs. 27.1%, P=0.463, P=1.000). However the long diameter of FO (1.74±0.3 vs. 1.60±0.4, P=0.039), short diameter of FO (1.12±0.3 vs. 1.00±0.3, P=0.036), perimeter of FO (4.62±0.7 vs. 4.22±1.0, P=0.026) and area (1.80±0.8 vs. 1.35±0.8, P=0.05) of FO were significantly larger in the severe RLS group.

There were 49 cases of cerebral infarction, 69 cases of migraine, and 16 cases of dizziness. In table 2, compared with the migraine group, the cerebral infarction group had a higher proportion of male participants (59.2% vs. 30.25%, p=0.002), a higher age (41.9±8.6 vs. 36.8±12.1, P=0.01), a larger proportion of hypertension (26.5% vs. 9.3% P=0.012), a larger number of smokers (30.6% vs. 3.0%, P<0.001), a larger proportion of Eustachian valve or a Chiari’s network (14.3% vs. 3.5%, P=0.036), a larger proportion of left funnelform (55.1% vs. 16.3%, P<0.001), a longer length of the tunnel (13.4±4.4 vs. 7.8±2.5, P<0.001), a smaller angle (16.4±3.4 vs. 20.3±7.7, P=0.001), a higher proportion of vascular stenosis in the brain and neck (8.2% vs. 0%, 53.1% vs. 20.9%, P=0.016, P=0.001), a higher RoPE score (7.6±1.1 vs. 6.3±1.4, P<0.001), and a higher proportion of multiple exits of the tunnel of LA (46.9% vs. 14.3%, P<0.001). However, there was no significant difference in shunt between the two groups (36.7% vs. 37.6, P=0.969).

3.Multivariate regression analysis showed that male (HR:4.026,95%CI:0.883~18.361, P=0.072), age (HR:1.076,95%CI:1.002~1.155, P=0.045), left funnelform (HR:7.299, 95%CI:1.585~33.618, P=0.011), long tunnel (HR:1.843, 95% CI: 1.404~2.418, P<0.001) and multiple exits of the tunnel of LA (HR: 8.544, 95% CI: 1.595~45.754, P=0.012) were positively associated with the risk of cerebral infarction (Table 3).

4.The diagnostic performance of high-risk PFO was assessed by using receiver operating characteristic (ROC) curves and the area under the ROC curve analysis show in figure 9 and figure10. The cut-off value calculated by ROC for the diagnosis of high-risk PFO was that the length of the PFO tunnel was 10.4 mm (sensitivity was 90%, specificity was 87%). Left funnelform combined with multiple exits of left atrial (sensitivity was 89%, specificity was 91%). The area under the curve of PFO tunnel length VS left funnelform + multiple exits of left atrial VS PoPE score (0.921 VS 0.932 VS 0.736) relative to the RoPE score was statistically significant. The cut-off value calculated by ROC for the diagnosis of high-risk PFO was that the length of the PFO tunnel was 12 mm and Left funnelform combined with multiple exits of left atrial (sensitivity was 92%, specificity was 90%). The area under the curve of combine index VS PoPE score (0.932 VS 0.736) relative to the RoPE score was statistically significant.

Discussions:

PFO can serve as a conduit for paradoxical embolism, allowing venous thrombi to enter the arterial circulation, avoiding filtration by the lungs, and causing ischemic stroke. The anatomical structure of PFO indicates that in addition to thrombi, it allows harmful circulatory factors to circulate directly from veins to arteries [4]. There are many theories about the mechanism of stroke in patients with PFO: 1) Abnormal embolism, one type of venous thrombosis (also known as air, fat, and infectious substances) enters the arterial circulation through the PFO, avoiding the filtration of the lungs and leading to cerebral embolism; and 2) due to various high-risk anatomical features, primary thrombosis occurs in the PFO lumen.

PFO itself has been suspected as the site of thrombus formation [2, 16]. Consistent with previous studies, this study found that the proportion of PFO long tunnel and the left funnelform was higher in CS.

The potential mechanisms underlying the link between PFO and migraine [17] micro embolus-induced cortical spreading depression (CSD)[18], the vasoactive substance hypothesis [19] and shared genetic predisposition [20]. Migraine is a common and recurrent disease, often confined to one side, manifested as paroxysmal pain. The symptoms of migraine often include nausea, vomiting, dizziness, photophobia and phonophobia [21]. Studies have shown that migraine can lead to various neurological diseases, including stroke, cerebral infarction, and long-term ischemia-reperfusion injury. Therefore, migraine is considered to be the third most frequent chronic disabling disease worldwide [22]. PFO-related right-to-left shunting allows potentially vasoactive substances to bypass the lung, which may aggravate cerebral injury [23]. 5-HT has been linked to the pathogenesis of migraine [24]. This study found that severe right-to-left shunt was relatively common among patients with severe migraine and stroke. PFO and migraine are both prevalent and have highly heterogeneous presentations. Their coincidence remains a debating issue, but it is less likely a coincidental finding, since experimental micro-embolism can depress cortical spreading [25].

C-TCD diagnosis of RLS mainly relies on the Valsalva maneuver. It is not possible to stimulate right-to-left shunting using Faraday’s maneuver under TEE; thus, we used abdominal compression to stimulate right-to-left shunting. Our findings were highly consistent with the results of the C-TCD stimulation test. The blood flow returning from the vena cava to the right atrium increases and RAP instantly increases after the release of abdominal pressure. When the right atrial pressure increases and exceeds the left atrial pressure, PFO may re-open, and microbubbles can be observed to enter the left atrium through the PFO tunnel.

PFO is a dynamic and three-dimensional structure as it originates from the embryonic septum primum and secondum. The structure of PFO is best described as a dynamic tunnel (not just a “hole”) outlined by opposing myocardial flaps remaining from the fetal septum primum and secondum, which never fully fused to wall off the right atrium from the left atrium. Since the IAS is a complex, dynamic, and 3D anatomic structure, 2D echocardiography cannot comprehensively evaluate it.

Current treatments of PFO include medical treatment utilizing anticoagulants and/or antiplatelet agents, endovascular PFO closure by meshed devices, or even surgical closure. Even “asymptomatic” PFO leads to pulmonary gas exchange inefficiency. Endovascular PFO closure improves exercise capacity and reduces exercise-induced hypoxemia. In clinical settings, patients frequently report improved general exercise tolerance and better vascular perfusion to the extremities after the closure of PFO.

Conclusion

Even if a quarter of people have PFO, or asymptomatic PFO, but prone to right to left PFO there is a risk of unexplained stroke. PFO with high-risk features may lead to clinically significant and clinically asymptomatic events. The evaluation of PFO dynamics using three-dimensional combined two-dimensional echocardiography can help better identify high-risk PFO.

Limitation:

Our study had some limitations as follows:

1. This was a single-center study.

2. Neurologically asymptomatic subjects who have a PFO on TEE would be the ideal participants for the control or comparison group, but for this study, we used patients with migraine as the control group.

3. The grade of RLS might be underestimated because it depends on the degree of Valsalva maneuver.

Author contribution:

Li Wang: Data collation and analysis, methodology, writing the first draft

Haibo Sun: Data collation and analysis

Han Shen: Project management, methodology, monitoring, writing, and editing. All authors studied and approved the final manuscript.

Funding:

This study was specifically funded by The Bo Xi Youth Natural Science Foundation (NO.BXQN2023016)

Acknowledgments:

We would like to thank all patients and researchers involved in this study.

Conflict of interest

None

Competing interests

The authors have no known competing financial interests or personal relationships that could affect the work reported in this paper.

Ethics approval

The Ethics Committee of The Affiliated Hospital of Soochow University (No.235/2024) approved the protocol of this study.

Availability of data and materials

The research data is confidential.

References:

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Table 1: The characteristics of patients and PFO.

Note: Values are presented as mean±SD or n (%).

Abbreviation: PFO, patent foramen ovale. RoPe, risk of paradoxical embolism. BMI, body mass index. FO, fossa ovale. RLS, right-to-left shunts. LA, left atrium.

Table 2: Comparison of basic characteristics and echocardiographic features between the cryptogenic group and the migraine group.

Note: Values are mean±SD or n (%).

Abbreviation: PFO, patent foramen ovale. RoPe, risk of paradoxical embolism. BMI, body mass index. FO, fossa ovale. RLS, right-to-left shunts. LA, left atrium. TCD, transcranial doppler. IVC, inferior vena cava.

Table 3: Multivariate logistic regression analysis for the echocardiographic characteristics of the cryptogenic group and the migraine group.

Note:

Abbreviation: PFO, patent foramen ovale. LA, left atrium.

Figure 1: Fossa Ovalis and PFO.

Figure 2: The thickness of the primary septum and the thickness of the secondary septum.

Figure 3: The height and length of the PFO tunnel.

Figure 4: Left funnelform and right funnelform.

Figure 5: The exit of the PFO tunnel by three-dimensional TEE.

Figure 6: Measurement of the oval fossa.

Figure 7: The presence of PFO was confirmed by actual visualization of microbubbles passing through the separation between the septum primum and septum secundum.

Figure 8: Measurement of IVC-PFO angle.

Figure 9. The diagnostic performance of high-risk PFO was assessed by using receiver operating characteristic (ROC) curves

Figure 10. The combine index diagnostic performance of high-risk PFO was assessed by using receiver operating characteristic (ROC) curves

Supplementary Figure: Trial flowchart.