Long-term results of percutaneous coronary intervention in no-touch vein grafts are significantly better than in conventional vein grafts.

INTRODUCTION: Conventional vein grafts have a high risk of thrombosis and early atherosclerosis. Percutaneous coronary intervention (PCI) in conventional vein grafts is associated with a higher incidence of late adverse cardiac events. The aim of this study was to evaluate the long-term results after PCI in saphenous vein grafts (SVG) harvested with the no-touch technique compared to the conventional technique. METHODS: This was a single-center, retrospective, cohort study, based on data from the Swedeheart register. The inclusion criterion was individuals who underwent CABG using different vein graft techniques between January 1992 and July 2020, and who required a PCI in SVGs between January 2006 and July 2020. The primary end point was long-term in-stent restenosis. The secondary endpoints were long-term major adverse cardiac events (MACE) and 1-year re-hospitalization rates. The associations between the graft types and the endpoints were evaluated using the Fine and Gray competing-risk regression analysis. RESULTS: The study included 346 individuals (67 no-touch, 279 conventional). The mean clinical follow-up time was 6.4 years with a standard deviation of 3.7 years. The long-term in-stent restenosis rate for the no-touch grafts was 3.2% compared to 18.7% for the conventional grafts (p < .01), with a subdistribution hazard ratio (SHR) of 0.16 (p = .010). The long-term MACE rate was 27.0% in the no-touch group and 48.3% in the conventional group (p < .01) with a SHR of 0.53 (p = .017). The short-term results were similar in both groups. CONCLUSIONS: Percutaneous coronary intervention in a no-touch vein graft was associated with statistically significantly fewer in-stent restenoses and MACE at long-term follow-up compared to a conventional SVG.

Five-Year Follow-Up After Transcatheter Aortic Valve Implantation in Patients with Severe Aortic Stenosis and Concomitant Coronary Artery Disease: A Single-Center Experience

ABSTRACT Introduction: There is no consensus on the impact of coronary artery disease in patients undergoing transcatheter aortic valve implantation. Therefore, the objective of this study was, in a single-center setting, to evaluate the five-year outcome of transcatheter aortic valve implantation patients with or without coronary artery disease. Methods: All transcatheter aortic valve implantation patients between 2009 and 2019 were included and grouped according to the presence or absence of coronary artery disease. The primary endpoint, five-year all-cause mortality, was evaluated using Cox regression adjusted for age, sex, procedure years, and comorbidities. Comorbidities interacting with coronary artery disease were evaluated with interaction tests. In-hospital complications was the secondary endpoint. Results: In total, 176 patients had aortic stenosis and concomitant coronary artery disease, while 170 patients had aortic stenosis only. Mean follow-up was 2.2±1.6 years. There was no difference in the adjusted five-year all-cause mortality between transcatheter aortic valve implantation patients with and without coronary artery disease (hazard ratio 1.00, 95% confidence interval 0.59-1.70, P=0.99). In coronary artery disease patients, impaired renal function, peripheral arterial disease, or ejection fraction < 50% showed a significant interaction effect with higher five-year all-cause mortality. No significant differences in complications between the groups were found. Conclusion: Five-year mortality did not differ between transcatheter aortic valve implantation patients with or without coronary artery disease. However, in patients with coronary artery disease and impaired renal function, peripheral arterial disease, or ejection fraction < 50%, we found significantly higher five-year all-cause mortality.

Five-Year Follow-Up After Transcatheter Aortic Valve Implantation in Patients with Severe Aortic Stenosis and Concomitant Coronary Artery Disease: A Single-Center Experience.

INTRODUCTION: There is no consensus on the impact of coronary artery disease in patients undergoing transcatheter aortic valve implantation. Therefore, the objective of this study was, in a single-center setting, to evaluate the five-year outcome of transcatheter aortic valve implantation patients with or without coronary artery disease. METHODS: All transcatheter aortic valve implantation patients between 2009 and 2019 were included and grouped according to the presence or absence of coronary artery disease. The primary endpoint, five-year all-cause mortality, was evaluated using Cox regression adjusted for age, sex, procedure years, and comorbidities. Comorbidities interacting with coronary artery disease were evaluated with interaction tests. In-hospital complications was the secondary endpoint. RESULTS: In total, 176 patients had aortic stenosis and concomitant coronary artery disease, while 170 patients had aortic stenosis only. Mean follow-up was 2.2±1.6 years. There was no difference in the adjusted five-year all-cause mortality between transcatheter aortic valve implantation patients with and without coronary artery disease (hazard ratio 1.00, 95% confidence interval 0.59-1.70, P=0.99). In coronary artery disease patients, impaired renal function, peripheral arterial disease, or ejection fraction < 50% showed a significant interaction effect with higher five-year all-cause mortality. No significant differences in complications between the groups were found. CONCLUSION: Five-year mortality did not differ between transcatheter aortic valve implantation patients with or without coronary artery disease. However, in patients with coronary artery disease and impaired renal function, peripheral arterial disease, or ejection fraction < 50%, we found significantly higher five-year all-cause mortality.

Quality of Life After Percutaneous Coronary Intervention in No-Touch Saphenous Vein Grafts is Significantly Better Than in Conventional Vein Grafts.

OBJECTIVE: To compare health-related quality of life (HRQoL) of patients primarily treated with a no-touch saphenous vein graft with that of patients who received a conventional graft. METHODS: The study included all individuals treated with a percutaneous coronary intervention (PCI) on a saphenous vein graft (SVG) between January 2006 and June 2020. The RAND-36 health survey was used to assess HRQoL. The Mann-Whitney U test was used to test differences in HRQoL between the two groups. Effect size was estimated via Cohen's d. The average treatment effect between the groups was tested by propensity score matching (PSM). RESULTS: Of the 346 patients treated with a PCI in a stenosed or occluded SVG, 165 responded to RAND-36 (no-touch: n=48; conventional: n=117). Patients with a no-touch graft reported better mean values on seven of the eight health survey domains. Statistically significant differences were observed for four of the domains, all in favour of the no-touch group. The effect size estimates indicated a small difference for five domains, with the highest values (>0.40) seen for the general health and energy/fatigue domains. PSM confirmed a statistically significant difference for the physical functioning and general health domains. CONCLUSION: At a mean follow-up of 5.4 years, patients who received a PCI in no-touch vein grafts showed significantly better HRQoL than those who received a PCI in conventional vein grafts.

Quality of Life After Percutaneous Coronary Intervention in No-Touch Saphenous Vein Grafts is Significantly Better Than in Conventional Vein Grafts

ABSTRACT Objective: To compare health-related quality of life (HRQoL) of patients primarily treated with a no-touch saphenous vein graft with that of patients who received a conventional graft. Methods: The study included all individuals treated with a percutaneous coronary intervention (PCI) on a saphenous vein graft (SVG) between January 2006 and June 2020. The RAND-36 health survey was used to assess HRQoL. The Mann-Whitney U test was used to test differences in HRQoL between the two groups. Effect size was estimated via Cohen's d. The average treatment effect between the groups was tested by propensity score matching (PSM). Results: Of the 346 patients treated with a PCI in a stenosed or occluded SVG, 165 responded to RAND-36 (no-touch: n=48; conventional: n=117). Patients with a no-touch graft reported better mean values on seven of the eight health survey domains. Statistically significant differences were observed for four of the domains, all in favour of the no-touch group. The effect size estimates indicated a small difference for five domains, with the highest values (>0.40) seen for the general health and energy/fatigue domains. PSM confirmed a statistically significant difference for the physical functioning and general health domains. Conclusion: At a mean follow-up of 5.4 years, patients who received a PCI in no-touch vein grafts showed significantly better HRQoL than those who received a PCI in conventional vein grafts.

Calcium Load in the Aortic Valve, Aortic Root, and Left Ventricular Outflow Tract and the Risk for a Periprocedural Stroke.

Background: Periprocedural stroke during transcatheter aortic valve implantation is a rare but devastating complication. The calcified aortic valve is the most likely source of the emboli in a periprocedural stroke. The total load and distribution of calcium in the leaflets, aortic root, and left ventricular outflow tract varies from patient to patient. Consequently, there could be patterns of calcification that are associated with a higher risk of stroke. This study aimed to explore whether the pattern of calcification in the left ventricular outflow tract, annulus, aortic valve, and ascending aorta can be used to predict a periprocedural stroke. Methods: Among the 3282 consecutive patients who received a transcatheter aortic valve implantation in the native valve in Sweden from 2014 to 2018, we identified 52 who had a periprocedural stroke. From the same cohort, a control group of 52 patients was constructed by propensity score matching. Both groups had one missing cardiac computed tomography, and 51 stroke and 51 control patients were blindly reviewed by an experienced radiologist. Results: The groups were well balanced in terms of demographics and procedural data. Of the 39 metrics created to describe calcium pattern, only one differed between the groups. The length of calcium protruding above the annulus was 10.6 mm (interquartile range 7-13.6) for patients without stroke and 8 mm (interquartile range 3-10) for stroke patients. Conclusions: This study could not find any pattern of calcification that predisposes for a periprocedural stroke.

Five-year patency for the no-touch saphenous vein and the left internal thoracic artery in on- and off-pump coronary artery bypass grafting.

BACKGROUND: Randomized trials show high long-term patency for no-touch saphenous vein grafts in coronary artery bypass grafting. The patency rate in off-pump coronary bypass surgery for these grafts has not been investigated. Our center participated in the CORONARY randomized trial, NCT00463294. This is a study aimed to assess the patency of no-touch saphenous veins in on- versus off-pump coronary bypass surgery at five-year follow-up. METHODS: Fifty-six patients were included. Forty of 49 patients, alive at 5 years, participated in this follow-up. There were 21 and 19 patients in the on- and off-pump groups respectively. No-touch saphenous veins were used to bypass all targets and in some cases the left anterior descending artery. Graft patency according to distal anastomosis was evaluated with computed tomography angiography. RESULTS: The five-year patency rate was 123/139 (88.5%). The patency for the no-touch vein grafts was 57/64 (89.1%) in the on-pump versus 37/45 (82.2%) in the off-pump group. All left internal thoracic arteries except for one, 29/30 (96.6%), were patent. All vein grafts used to bypass the left anterior descending and the diagonal arteries were patent 32/32. The lowest patency rate for the saphenous veins was to the right coronary territory, particularly in off-pump surgery (80.0% vs. 62.5% for the on- respective off-pump groups). CONCLUSIONS: Comparable 5-year patency for the no-touch saphenous veins and the left internal thoracic arteries to the left anterior descending territory in both on- and off-pump coronary artery bypass grafting. Graft patency in off-pump CABG is lower to the right coronary artery.

What is the impact of preserving the endothelium on saphenous vein graft performance? Comments on the 'NO' touch harvesting technique.

Saphenous veins used for coronary artery bypass surgery are subjected to considerable vascular trauma when harvested by conventional methods. This vascular damage is responsible, at least in part, for the inferior patency of the saphenous vein when compared with the internal thoracic artery. The performance of saphenous vein grafts is improved when this conduit is harvested atraumatically using the no-touch technique. There is growing evidence that the success of the no-touch technique is due to the preservation of a number of vascular structures including the endothelium, vasa vasorum and perivascular fat. There is conflicting evidence regarding the degree of endothelial damage to the endothelium of conventional versus no-touch saphenous vein grafts. In general, it has been shown that this single layer of cells lining the lumen exhibits considerable damage associated with a combination of vascular trauma and high pressure intraluminal distension. Increased platelet aggregation and thrombus formation at the exposed subendothelial membrane is due to a local reduction of endothelium-derived factors including nitric oxide. In addition, damage to the vasa vasorum of conventionally-harvested veins will reduce transmural blood flow, a condition shown to promote neointimal hyperplasia and atheroma formation. By stripping off the perivascular fat during conventional harvesting, mechanical support of the graft is reduced and the source of adipocyte-derived factors potentially beneficial for graft patency removed. While most agree that endothelial damage to the saphenous vein affects graft patency, the contribution of other tissue-derived factors affected by vascular damage at harvesting need to be considered.

Percutaneous coronary intervention in saphenous vein grafts after coronary artery bypass grafting: a systematic review and meta-analysis.

To investigate the results of percutaneous coronary intervention (PCI) in saphenous vein grafts after coronary artery bypass grafting (CABG). Design. MEDLINE, Embase, and the Cochrane library were searched for relevant articles published between 1 January 2000 and 29 February 2020. The PICO (population, intervention, comparison, outcome) model was applied in constructing the clinical question. Two independent researchers performed the literature search. Thirty-six articles were identified and subjected to a quality assessment. The primary outcomes of the meta-analysis were long-term in-stent restenosis and long-term major adverse cardiac events (MACE). Results. In-stent restenosis was 9.4% (95% CI: 4.2-14.7%) and MACE was 35.3% (95% CI: 27-43.7%) at mean time 2.7 ± 1.0 years. The secondary outcomes were the unsuccessful PCI rate (7.7%; 95% CI: 2.9-12.5%), 30-day MACE (4.3%; 95% CI: 2.5-6.1%), and 1-year MACE (15.5%; 95% CI: 11.7-19.3%). The use of drug-eluting stents resulted in better outcomes at least in term of in-stent restenosis, while the benefit of using embolic protection devices was questionable. Conclusions. PCI of a stenosed or occluded saphenous vein graft is a challenge for interventional cardiologists, and is still associated with relatively high rates of restenosis, MACE, and procedural failure. All efforts to enhance the results are warranted, including improved quality of the venous grafts used during CABG.

Pedicled versus skeletonized internal thoracic artery grafts: a randomized trial.

OBJECTIVE: Concerns have been raised regarding whether skeletonization of the internal thoracic artery could damage the graft and thereby reduces its patency. The objective of this study was to compare patency rates at mid- and long-term follow-up between pedicled and skeletonized left internal thoracic artery grafts. METHODS: This randomized controlled trial included 109 patients undergoing coronary artery bypass surgery. The patients were assigned to receive either one pedicled or one skeletonized left internal thoracic artery graft to the left anterior descending artery. Follow-up was performed at 3 years with conventional angiography, and at 8 years with computed tomography angiography. Differences between patency rates were analyzed with Fisher's exact test and a generalized linear model. RESULTS: The patency rates for pedicled and skeletonized left internal thoracic artery grafts were 46/48 (95.8%) versus 47/52 (90.4%), p = 0.44 at 3 years, and 40/43 (93.0%) versus 37/41 (90.2%), p = 0.71 at 8 years, respectively. The difference in patency rates for pedicled and skeletonized grafts was 5.4% (95% confidence interval: -4.2-14.5) at 3 years and 2.8% (95% confidence interval: -9.9-14.1) at 8 years. All failed grafts, except for one with a localized stenosis, were anastomosed to native coronary arteries with a stenosis less than 70%. Three patients suffered sternal wound infections (two in the pedicled group, one in the skeletonized group). CONCLUSIONS: The skeletonization technique can be used without jeopardizing the patency of the left internal thoracic artery. The most important factor in graft failure was target artery stenosis below 70%.

Saphenous veins in coronary artery bypass grafting need external support.

The saphenous vein is the most commonly used conduit for coronary artery bypass grafting. Arterial grafts are harvested with the outer pedicle intact whereas saphenous veins are harvested with the pedicle removed in the conventional graft harvesting technique. This conventional procedure causes considerable vascular damage. One strategy to improve vein graft patency has been to provide external support. Ongoing studies show that fitting a metal external support improves conventionally harvested saphenous vein graft patency. On the other hand, the no-touch technique of harvesting the saphenous vein provides an improved graft with long-term patency comparable to that of the internal mammary artery. This improvement is suggested to be due to preservation of vessel structures. Interestingly, many of the mechanisms proposed to be associated with the beneficial actions of an artificial external support on saphenous vein graft patency are similar to those underlying the beneficial effect of no-touch saphenous vein grafts where the intact outer layer acts as a natural support. Additional actions of external supports have been advocated, including promotion of angiogenesis, increased production of vascular-protective factors, and protection of endothelial cells. Using no-touch harvesting, normal vascular architecture is maintained, tissue and cell damage is minimized, and factors beneficial for graft patency are preserved. In this review, the significance of external support of saphenous vein grafts in coronary artery bypass grafting is discussed.

Expression of Paracrine Effectors in Human Adipose-Derived Mesenchymal Stem Cells Treated With Plasma From Brown Bears (Ursus arctos).

Adipose-derived mesenchymal stem cells (ADSCs) are promising candidates for novel cell therapeutic applications. Hibernating brown bears sustain tissue integrity and function via unknown mechanisms, which might be plasma borne. We hypothesized that plasma from hibernating bears may increase the expression of favorable factors from human ADSCs. In an experimental study, ADSCs from patients with ischemic heart disease were treated with interventional media containing plasma from hibernating and active bears, respectively, and with control medium. Extracted RNA from the ADSCs was sequenced using next generation sequencing. Statistical analyses of differentially expressed genes were performed using fold change analysis, pathway analysis, and gene ontology. As a result, we found that genes associated with inflammation, such as IGF1, PGF, IL11, and TGFA, were downregulated by > 10-fold in ADSCs treated with winter plasma compared with control. Genes important for cardiovascular development, ADM, ANGPTL4, and APOL3, were upregulated in ADSCs when treated with winter plasma compared with summer plasma. ADSCs treated with bear plasma, regardless if it was from hibernating or active bears, showed downregulation of IGF1, PGF, IL11, INHBA, IER3, and HMOX1 compared with control, suggesting reduced cell growth and differentiation. This can be summarized in the conclusion that plasma from hibernating bears suppresses inflammatory genes and activates genes associated with cardiovascular development in human ADSCs. Identifying the involved regulator(s) holds therapeutic potential.

Twenty-Five Years of No-Touch Saphenous Vein Harvesting for Coronary Artery Bypass Grafting: Structural Observations and Impact on Graft Performance.

The saphenous vein is the most common conduit used in coronary artery bypass grafting (CABG) yet its failure rate is higher compared to arterial grafts. An improvement in saphenous vein graft performance is therefore a major priority in CABG. No-touch harvesting of the saphenous vein is one of the few interventions that has shown improved patency rates, comparable to that of the left internal thoracic artery. After more than two decades of no-touch research, this technique is now recognized as a Class IIa recommendation in the 2018 European Society of Cardiology and the European Association for Cardio-Thoracic Surgery guidelines on myocardial revascularization. In this review, we describe the structural alterations that occur in conventional versus no-touch saphenous vein grafts and how these changes affect graft patency. In addition, we discuss various strategies aimed at repairing saphenous vein grafts prepared at conventional CABG.

Bone marrow- and adipose tissue-derived mesenchymal stem cells from donors with coronary artery disease; growth, yield, gene expression and the effect of oxygen concentration.

Mesenchymal stem cells (MSCs) for cardiovascular cell therapy are procured from different sources including bone marrow and adipose tissue. Differently located MSCs differ in growth potential, differentiation ability and gene expression when cultured in vitro, and studies show different healing abilities for different MSC subgroups. In this study, bone marrow derived MSCs (BMSCs) and adipose tissue derived MSCs (ADSCs) from six human donors with coronary artery disease were compared for growth potential and expression of target genes (Angpt1, LIF, HGF, TGF-ß1 and VEGF-A) in response to exposure to 1% and 5% O2, for up to 48 h. We found greater growth of ADSCs compared to BMSCs. ADSCs expressed higher levels of Angpt1, LIF and TGF-ß1 and equal levels of VEGF-A and HGF as BMSCs. In BMSCs, exposure to low oxygen resulted in upregulation of TGF-ß1, whereas other target genes were unaffected. Upregulation was only present at 1% O2. In ADSCs, LIF was upregulated in both oxygen concentrations, whereas Angpt1 was upregulated only at 1% O2. Different response to reduced oxygen culture conditions is of relevance when expanding cells in vitro prior to administration. These findings indicate ADSCs as better suited for cardiovascular cell therapy compared to BMSCs.

Twenty-five years of no-touch saphenous vein harvesting for coronary artery bypass grafting: structural observations and impact on graft performance

Abstract The saphenous vein is the most common conduit used in coronary artery bypass grafting (CABG) yet its failure rate is higher compared to arterial grafts. An improvement in saphenous vein graft performance is therefore a major priority in CABG. No-touch harvesting of the saphenous vein is one of the few interventions that has shown improved patency rates, comparable to that of the left internal thoracic artery. After more than two decades of no-touch research, this technique is now recognized as a Class IIa recommendation in the 2018 European Society of Cardiology and the European Association for Cardio-Thoracic Surgery guidelines on myocardial revascularization. In this review, we describe the structural alterations that occur in conventional versus no-touch saphenous vein grafts and how these changes affect graft patency. In addition, we discuss various strategies aimed at repairing saphenous vein grafts prepared at conventional CABG.

Surgical Aspects of No-Touch Saphenous Vein Graft Harvesting in CABG: Clinical and Angiographic Follow-Up at 3 Months.

With more than 800,000 coronary artery bypass grafting (CABG) operations annually worldwide and the saphenous vein being the most common conduit used, there is no question that improving saphenous vein graft patency is one of the most important tasks in CABG. This video describes the no-touch harvesting procedure of the saphenous vein on an 80-year old man with hypertension, hyperlipidemia and a previous myocardial infarction with percutaneous coronary intervention to the right coronary artery. He was complaining of exertional chest pain and was diagnosed with stable angina pectoris. The coronary angiography showed advanced three vessel disease with significant stenoses in the left anterior descending (LAD) artery, two marginal arteries (MAs) and the posterior descending artery (PDA), in addition to an occluded diagonal artery (DA). The patient received a triple sequential no-touch vein graft to the PDA and two MAs together with a double sequential no-touch vein graft to the DA and LAD. A vein graft was used to bypass the LAD due to the age of the patient and the low degree of stenosis in the LAD. The no-touch harvesting technique is described in detail in the film with complete narration. A follow-up of this patient was performed at three months both clinically and with a computed tomography angiography (CTA). No angina pectoris symptoms were reported by the patient and the wounds in the chest and lower limb were completely healed. The CTA showed patent no-touch saphenous vein grafts to all the distal anastomoses.

Surgical aspects of no-touch saphenous vein graft harvesting in cabg: clinical and angiographic follow-up at 3 months

Abstract With more than 800,000 coronary artery bypass grafting (CABG) operations annually worldwide and the saphenous vein being the most common conduit used, there is no question that improving saphenous vein graft patency is one of the most important tasks in CABG. This video describes the no-touch harvesting procedure of the saphenous vein on an 80-year old man with hypertension, hyperlipidemia and a previous myocardial infarction with percutaneous coronary intervention to the right coronary artery. He was complaining of exertional chest pain and was diagnosed with stable angina pectoris. The coronary angiography showed advanced three vessel disease with significant stenoses in the left anterior descending (LAD) artery, two marginal arteries (MAs) and the posterior descending artery (PDA), in addition to an occluded diagonal artery (DA). The patient received a triple sequential no-touch vein graft to the PDA and two MAs together with a double sequential no-touch vein graft to the DA and LAD. A vein graft was used to bypass the LAD due to the age of the patient and the low degree of stenosis in the LAD. The no-touch harvesting technique is described in detail in the film with complete narration. A follow-up of this patient was performed at three months both clinically and with a computed tomography angiography (CTA). No angina pectoris symptoms were reported by the patient and the wounds in the chest and lower limb were completely healed. The CTA showed patent no-touch saphenous vein grafts to all the distal anastomoses.

No-touch vein grafts and the destiny of venous revascularization in coronary artery bypass grafting-a 25th anniversary perspective.

Ischemic heart disease is currently the leading cause of death globally, with coronary artery bypass grafting among the most common operations performed worldwide. More extensive use of arterial grafts has been advocated because of their high long-term patency, long-term survival benefit, and freedom from reinterventions. Despite this, the saphenous vein is the most frequently used conduit in patients undergoing coronary artery bypass surgery since its introduction over 50 years ago. Consequently, the saphenous vein remains an indispensable conduit in coronary artery bypass grafting and maintaining its long-term patency is one of the most crucial challenges in cardiovascular surgery. This situation led to the development of the no-touch saphenous vein harvesting technique, where the vein is harvested completely with its pedicle of surrounding tissue. Several studies report a superior long-term patency rate, slower progression of atherosclerosis, and better clinical outcomes whilst employing no-touch harvesting technique. The success of the technique is multifactorial, including the decreased risk for graft spasm-and the need for manual distension-preservation of the vaso vasorum and an intact endothelium, reducing neointimal hyperplasia and subsequent atherosclerosis. Furthermore, the intact perivascular tissue, including the surrounding cushion of fat, may act as a "natural external stent", providing mechanical support preventing the graft from kinking. We are convinced that the use of arterial grafts, in combination with the no-touch saphenous vein graft, will significantly improve the results of coronary artery bypass grafting. This is important for achieving a comprehensive and evidence-based balance between the major treatment strategies of ischemic heart disease, explicitly coronary artery bypass grafting and percutaneous coronary intervention. The no-touch technique is becoming increasingly popular among surgeons, with further studies to be initiated worldwide.