Abstract
Purpose
Left ventricular ejection fraction may remain normal or even higher despite significant impairment of contractility in cases of mitral regurgitation. The aim of this study is to evaluate the changes in left ventricular function after mitral valve replacement and to study the role of global longitudinal strain in detecting early left ventricular dysfunction using speckle tracking.
Method
Study involved 31 patients who underwent mitral valve replacement for mitral regurgitation. Patient’s preoperative and postoperative echocardiography (conventional parameters and global longitudinal strain) and other parameters like functional status, radiological findings, and electrocardiogram were recorded to evaluate left ventricular function.
Results
All patients presented in advanced stage with New York heart association class III (67.7%) and IV (32.3%). There was significant decline in left ventricular ejection fraction (with the mean value from 64.58 to 40.13%) and global longitudinal strain (− 15.57 ± 4.98to − 8.97) in the immediate postoperative period (~ 7 days). However, there was a rise in both left ventricular ejection fraction (mean 52.48%) and in global longitudinal strain (mean − 14.44 ± 3.67) at 3 months. Left ventricular and atrial size decreased significantly immediately after surgery, which further declined at 3 months. We also found that patients who attained a left ventricular ejection fraction of > 50% in postoperative period had better left ventricular ejection fraction and global longitudinal strain preoperatively. In addition, they had smaller cardiac size and milder pulmonary hypertension comparatively.
Conclusions
Mitral valve replacement in mitral regurgitation results in decline in left ventricular function immediately after surgery. In patients with chronic mitral regurgitation, left ventricular ejection fraction is fallacious and global longitudinal strain can be an important tool to assess left ventricular ejection fraction.
Keywords: Mitral valve regurgitation, Mitral valve replacement, Global longitudinal strain
Introduction
Mitral regurgitation (MR) is a common valvular heart disease characterized by volume overload of left ventricle (LV). The forward cardiac output is maintained by eccentric hypertrophy of LV. However, later in the course of disease, this compensatory hypertrophy is not able to match with the progressive increase in preload, causing an increase in LV wall stress. Ultimately LV contractile dysfunction ensues with decrease in forward cardiac output. Recent guidelines recommend mitral valve replacement/repair at left ventricular ejection fraction (LVEF) > 60% and LV end diastolic diameter ≤ 40 mm [1]. However, even with these cutoff values, a relatively high incidence of postoperative LV dysfunction, heart failure, and mortality is still noted indicating that the volume-based measurements are not sensitive enough to detect subtle LV dysfunction. In fact, in chronic severe MR, LVEF may be normal or above normal despite significant impairment of contractility due to decrease in afterload. Therefore, surgery should be done before irreversible myocardial damage occurs. In early postoperative period of mitral valve replacement/repair, there is an initial decrease in LVEF which does not reflect the actual LV function. This may be due to reduction in preload and increase in afterload early after surgery. The LVEF does increase later; however, this increase in LVEF is dependent upon the preoperative LV function.
Therefore, an accurate assessment of LV contractile function in patients with severe MR before and after surgery remains challenging. Echocardiographic (ECHO) analysis of myocardial deformation with strain imaging has been recently used as a novel and more accurate measurement of LV contractility.
The aim of this study was to detect the changes in LV contractile function just postoperatively (~ 7 days) and on a short follow-up (3 months) in patient with severe MR following mitral valve replacement (MVR) and to detect patients early before significant LV dysfunction occurs using strain imaging.
Aims and objectives
In present study, early and short-term changes in LV contractile functions were evaluated in patients who underwent mitral valve replacement for chronic mitral valve regurgitation. We also evaluated the utility of left ventricular strain imaging for identifying patients of mitral regurgitation having early systolic dysfunction with normal left ventricular ejection fraction. Predictors of postoperative decline in LVEF after mitral valve replacement were also evaluated.
Material and methods
The study involved 31 patients of chronic severe MR who underwent MVR at our center over a period of 1 year (December 2017 to November 2018). The study was approved by ethics committee of the University. Informed consent was taken in all the patients. The total number of MVR done for rheumatic mitral valve disease during the study period was 152. Out of which, 42 patients had pure MR and 31 patients fulfilled the selection criteria. Patients with concomitant significant aortic valve lesion requiring aortic valve replacement, acute MR requiring emergency surgery and patients with associated coronary artery disease were excluded from the study. All patients underwent baseline demographic, clinical and echocardiographic evaluation. Patient’s functional NYHA class, electrocardiogram, and X-ray chest findings were recorded. Echocardiography was done using “GE Vivid E9, 4D-echocardiography machine. ECHO included conventional measurement and assessment of LV global longitudinal strain by speckle tracking. For strain imaging using speckle tracking, gray scale images were acquired, which included apical 4 chamber, 2 chamber and apical long axis view at a frame rate of 30–70 per seconds with electrocardiogram gating. Strain was calculated using speckle tracking using inbuilt software. Severity of MR was assessed according to the current ACC/AHA guidelines (2014, update 2017) by use of multiparameteric approach based on color-flow and continuous wave Doppler images. Same evaluation was done in immediate postoperative period (~ 7 days) and in follow-up visit at 3 months. LV dimension, volume, and ejection fraction were measured by M mode using parasternal long axis view. Forward stroke volume and ejection fraction were also calculated. Right ventricular systolic pressure (RVSP) was estimated by peak tricuspid regurgitation velocity.
Surgical technique
All patients were operated under general anesthesia. Transesophageal echocardiography was routinely used in all the cases for preoperative evaluation and completeness of operation postoperatively. Heparin was used to achieve ACT > 480 s. Median sternotomy was used in all the patients with single aortic and bicaval cannulation. Moderate hypothermia was used and myocardial preservation was achieved with antegrade cold crystalloid blood cardioplegia intermittently along with topical cooling with ice slush. All patients underwent MVR with bileaflet prosthetic mechanical (SJM™)/bio prosthetic valve (SJM™) implantation using 2/0 continuous polypropylene suture (PROLENE® Ethicon Somerville, NJ, USA). Posterior chordae were preserved in all the patients while anterior chordae were preserved in ten patients. Intra operative details were recorded which included cardiopulmonary bypass (CPB) time, aortic cross clamp time. Tricuspid valve repair was done as per requirement. With the exception of 2 patients, who required cardioversion, the heart started beating spontaneously after the removal of cross clamp. All the excised valves were sent for histopathological examination.
Statistical analysis
In our observational study, sample size (n = 22) was calculated with formula n = z2x p̂ (1-p̂)/ ε2, where confidence level = 95%, margin of error 5%, population proportion 1.4%,z is the z score (1.96 for confidence level 95%), ε is the margin of error, p̂ is the population proportion, z = 1.96, p^ = 0.01, ε = 0.05.Categorical variables were presented in number and percentage (%) and continuous variables were presented as mean ± standard deviation (SD). Quantitative variables were compared using unpaired t test between two groups. Paired t test were used to compare pre and post-surgery variables. Qualitative variables were compared using Chi-Square test/Fisher’s exact test as appropriate. A p value of < 0.05 was considered statistically significant. The data were entered in MS EXCEL spreadsheet and analysis was done using Statistical Package for Social Sciences (SPSS) version 16.0.
Results
The mean age in our study was 29.16 ± 10.88 years with female preponderance (77.4%). Atrial fibrillation was present in 17 patients (54.8%). All patients were symptomatic and all were in NYHA class III or IV. Features of right sided heart failure were present in 7 (22.6%) patients (Table 1). All patients underwent MVR with prosthetic bileaflet valve implantation. Mechanical bileaflet prosthetic valve was used in 29 patients while bio prosthetic valve was used in 2 patients. Posterior mitral chordae were preserved in all the cases. Complete chordal preservation (posterior and anterior both) was done in 10 (32.3%) patients and tricuspid annuloplasty was required in 16 (51.6%) patients who had moderate or severe tricuspid regurgitation. Mean cardiopulmonary bypass time was 62.23 min and mean cross clamp time was 31.58 min. Postoperative course is mentioned in Table 2. There was a significant reduction in left atrium (LA) and LV size immediately after surgery with further reduction at 3 months (Table 3, Fig. 1). The mean LVEF was 64.58% which declined significantly in the immediate postoperative phase (40.13%). However, mean LVEF increased to 52.48% at 3 months, but it did not touch the baseline mean LVEF. The preoperative mean Global longitudinal strain (GLS) was − 15.57 ± 4.98 (LVEF 64.58% ± 10.06) suggesting subtle LV dysfunction (Fig. 2). In comparison with baseline, a significant decrease in GLS (− 8.97) was noted immediately after MVR (p < 0.001) correlating with a fall in LVEF. However, at 3-month follow-up, there was an improvement in GLS (− 14.44 ± 3.67) similar to LVEF (Table 3).
Table 1.
Baseline characteristics of patients
| Variables | n = 31 |
|---|---|
| Demographic features | |
| Age, mean ± SD, years | 29.16 ± 10.88 (15–57) |
| Female patients, no. (%) | 24 (77.4) |
| Body surface area, mean ± SD, m2 | 1.40 ± 0.163, (1.19–2.00) |
| Investigative parameters | |
| Hemoglobin | |
| < 9 g/dl | 3 |
| >9 g/dl | 28 |
| Severity of tricuspid valve regurgitation, no. (%) | |
| Nil/mild | 15 (48.4) |
| Moderate | 10 (32.3) |
| Severe | 6 (19.4) |
| Cardiac rhythm, no. (%) | |
| Sinus rhythm | 14 (45.2) |
| Atrial fibrillation | 17 (54.8) |
| Serum creatinine | |
| < 1.4 mg/dl | 30 |
| > 1.4 mg/dl | 1 |
| Liver function test | |
| Deranged (serum aspartate transaminase >40 U/L, serum alanine transaminase >56 U/L, Bilirubin > 1.2 mg/dl) | 4 |
| Normal | 27 |
| Echocardiographic parameters, (mean ± SD), range | |
| LA size (cm) | 7.64 ± 0.02 (4.20–12.00) |
| LVEF (%) | 64.58 ± 10.06 (42.00–78.00) |
| LVESD (mm) | 41.10 ± 8.35 (28.00–60.00) |
| LVEDD(mm) | 60.42 ± 8.00 (41.00–79.00) |
| RVSP (mm Hg) | 39.74 ± 16.67 (10.00–76.00) |
| GLS (%) | − 15.57 ± 4.98 (− 5.00) – (− 25.60) |
| CT ratio, mean ± SD, range | 0.62 ± 0.11 (0.39–0.91) |
| Clinical parameters | |
| NYHA class, no. (%) | |
| Class I | 0 (0) |
| Class II | 0 (0) |
| Class III | 21(67.7) |
| Class IV | 10 (32.3) |
| Feature of right heart failure, no. (%) | |
| Pedal edema | 7 (22.6) |
| Ascitis | 3(9.7) |
Table 2.
Postoperative hospital course (N=31)
| Parameters | Mean | Std. deviation | ||
|---|---|---|---|---|
| ICU stay (in days) | N | 2.97 | 0.95 | |
| < 2 days | 10 | |||
| 3–4 days | 19 | |||
| > 5 days | 2 | |||
| Hospital stay (in days) | 7.45 | 1.31 | ||
| < 5 days | 2 | |||
| > 5 days | 29 | |||
| Blood loss (in ml) | 206.45 | 66.76 | ||
| < 300 ml | 27 | |||
| 300–500 ml | 4 | |||
| > 500 ml | 0 | |||
| Blood transfusion (in units) | 1.06 | 0.81 | ||
| 0 unit | 7 | |||
| 1 unit | 17 | |||
| 2 unit | 5 | |||
| > 2 unit | 2 | |||
| Ventilatory support (in hours) | 7.61 | 2.01 | ||
| 4 h | 1 | |||
| 4–6 h | 9 | |||
| More than 6 h | 21 | |||
| Inotropic support (in hours) | 3.35 | 1.54 | ||
| Low dose | 18 | |||
| Medium dose | 10 | |||
| High dose | 3 | |||
Table 3.
Changes in left ventricular parameters postoperatively
| Parameter | Preoperative | ~ 7 days | 3 months | ||
|---|---|---|---|---|---|
| Mean± SD | % change | Mean± SD | % change | ||
| LA size | 7.64 ± 2.02 | 6.90 ± 1.72 | 9.69 (p = 0.001) | 5.96 ± 1.57 | 21.99 (p = 0.001) |
| LVESD (in millimeter) | 41.10 ± 8.35 | 38.03 ± 8.33 | 7.47 (p = 0.002) | 32.03 ± 7.11 | 22.07 (p = 0.001) |
| LVEDD (in millimeter) | 60.42 ± 8.01 | 52.84 ± 7.95 | 12.55 (p = 0.001) | 46.35 ± 4.78 | 23.29 (p = 0.001) |
| LVEF (%) | 64.58 ± 10.06 | 40.13 ± 10.45 | 37.86 (p = 0.001) | 52.48 ± 10.49 | 18.74 (p = 0.001) |
| RVSP (in mm Hg) | 39.74 ± 16.67 | 33.26 ± 13.86 | 16.31% (p = 0.001) | 31.52 ± 11.66 | 20.68% (p = 0.002) |
| GLS (%) | − 15.57 ± 4.98 | − 8.97 ± 3.66 | 42.20% (p = 0.001) | − 14.44 ± 3.67 | 7.26% (p = 0.050) |
| CT ratio | 0.62 ± 0.11 | 0.57 ± 0.10 | 8.06% (p = 0.001) | 0.51 ± 0.09 | 17.74% (p = 0.001) |
Fig. 1.
Preoperative chest X-ray showing severe enlargement of the heart (a), postoperative chest X-ray of the same patient after 3 months showing significantly reduced cardiothoracic ratio with prosthetic valve in situ (b)
Fig. 2.
Global peak-systolic longitudinal strain (GLS) in a patient with severe mitral regurgitation (Bull’s eye display)
Out of 31 patients, only 21 patients had an LVEF ≥ 50% at 3 months (mean 58.62 ± 5.89%). In patients who had LVEF ≥ 50% at 3 months, the mean preoperative LVEF was 68.0 ± 8.22% while in those with LVEF < 50%, the preoperative mean LVEF was 57.40 ± 10.14% (p = 0.004). Patients who attained LVEF ≥ 50% at 3 months had smaller LV dimensions preoperatively with mean left ventricular end systolic diameter (LVESD) and left ventricular end systolic diameter (LVEDD) of 38.29 ± 7.35 mm and 58.38 ± 7.97 mm respectively. Similarly, LA size was significantly higher (8.95 ± 1.77 cm vs 7.01 ± 1.86 cm) in those with LVEF < 50% at 3 months. (p = 0.010). GLS is a relatively recent echocardiographic parameter to assess LV function. It was seen that the value was below normal even in patients with normal LVEF suggesting subtle LV systolic dysfunction. The normal value of GLS is − 15.9 ± 6.0%. The preoperative GLS was significantly higher in those who attained an LVEF ≥ 50% at 3 months (− 7.31 ± 4.39% vs − 11.91 ± 4.22%). The postoperative LVEF did not differ significantly in relation to CPB time and cross clamp time. Only one patient reverted back from atrial fibrillation to normal sinus rhythm postoperatively. Patients who were in sinus rhythm preoperatively had higher chances of having LVEF ≥ 50% at 3 months than those in atrial fibrillation (85.7% vs 52.9%) (Table 4). Histopathological report of excised valve revealed rheumatic etiology in all the cases.
Table 4.
Comparison of different preoperative and postoperative parameters in patients with LVEF (< 50% and ≥ 50%) at 3 months
| < 50% (n = 10) | ≥ 50% (n = 21) | p value | |
|---|---|---|---|
| Mean ± SD | Mean ± SD | ||
| LVEF (%) | |||
| Preoperative | 57.40 ± 10.14 | 68.00 ± 8.22 | 0.004 |
| At 3 months | 39.60 ± 4.17 | 58.62 ± 5.89 | 0.001 |
| LVESD (in mm) | |||
| Preoperative | 47.00 ± 7.42 | 38.29 ± 7.35 | 0.005 |
| At 3 months | 39.50 ± 8.69 | 28.48 ± 4.56 | 0.001 |
| LVEDD (in mm) | |||
| Preoperative | 64.70 ± 6.49 | 58.38 ± 7.97 | 0.037 |
| At 3 months | 46.90 ± 3.73 | 46.10 ± 5.27 | 0.669 |
| LA size (in cm) | |||
| Preoperative | 8.95 ± 1.77 | 7.01 ± 1.86 | 0.010 |
| At 3 months | 7.17 ± 1.53 | 5.38 ± 1.24 | 0.002 |
| RVSP (in mm Hg) | |||
| Preoperative | 52.20 ± 15.92 | 33.81 ± 13.71 | 0.002 |
| At 3 months | 39.40 ± 12.17 | 27.76 ± 9.56 | 0.007 |
| GLS (%) | |||
| Preoperative | − 11.91 ± 4.22 | − 17.31 ± 4.39 | 0.003 |
| At 3 months | − 10.79 ± 2.86 | − 16.18 ± 2.60 | 0.001 |
| CT ratio | |||
| Preoperative | 0.68 ± 0.11 | 0.59 ± 0.11 | 0.049 |
| At 3 months | 0.56 ± 0.11 | 0.48 ± 0.08 | 0.030 |
Discussion
Mitral valve apparatus comprises of the mitral leaflets, chordae tendineae, papillary muscles, and mitral annulus. Abnormalities of any of these structures may cause MR. In rheumatic heart disease, MR occurs as a consequence of shortening, rigidity, deformity, and retraction of one or both mitral valve cusps and is associated with shortening and fusion of chordae tendineae and papillary muscles. In most of the prior studies, commonest etiology was degenerative followed by ischemic heart disease. However, in our study, all patients had rheumatic MR due to high prevalence of rheumatic heart disease in our region. Initially in acute MR, the LV compensates by increase in preload and more complete emptying (Frank–Starling principle) and there is a fall in late LV systolic pressure which causes a decline in LV wall tension. This in turn results in increased myocardial contraction and decrease in end systolic volume. But as the MR becomes chronic, both the end systolic volume and end diastolic volume increase (MR begets MR). This causes an increase in LV wall tension and will decrease forward cardiac output. (LV wall tension is a product of intraventricular pressure and radius divided by LV wall thickness). Initially, this increase in wall tension is compensated by eccentric hypertrophy of the LV. The reduced afterload maintains LVEF in the normal or supranormal range giving the false reassurance of normal cardiac function; however, the effective cardiac output may be low. But ultimately, this compensatory mechanism will lag behind the progressive increase in wall tension and LV fails. Therefore, timely mitral valve surgery is crucial in chronic severe MR for the maintenance of normal LV contractile function.
The mean age in our study was 29.16 ± 10.88 years which is comparatively lower to the previous studies because of higher prevalence of rheumatic disease in this part of world. The mean age was 59 ± 13 years in a study done by Quintana et al., while it was above 60 years in a study done by Suri et al. [2, 3]. In addition, we had higher percentage of female patients (77.4%) in contrast to other studies which had more number of male patients. These differences may be due to the fact that rheumatic heart disease is more common in females and its presentation is earlier, whereas degenerative MR occurs in advanced age and is more common in males [4]. Our study clearly shows that LVEF decreases soon after MVR. It is seen that early decrease is more pronounced in patients with larger left heart, reduced preoperative LVEF, and advanced symptoms. Several studies have reported that MVR is associated with approximately 10 unit decrease in the LVEF postoperatively [5–7]. In a study done by Shafii et al., the postoperative LVEF initially decreased from 58 ± 7.0% to 53 ± 20%, and increased slightly during the first postoperative year. And they also suggest that the magnitude of this decrease might be attenuated by chordal preservation. However, there are conflicting results on chordal preservation [5]. Dilip et al. found that there was better postoperative LVEF in patients with chordal preservation (60.31 ± 8.22 versus 64.47 ± 7.93; p < 0.05) [8]. Coutinho et al. found that it is often not possible to do complete preservation of subvalvular apparatus in rheumatic valves and even after chordae preservation long term survival benefit is not seen [9]. In a study done by Mazine et al., there was a trend towards better LVEF and freedom from re-operation if chordae were preserved in comparison to the patients in whom chordae were resected [10]. In our study, also the decrease in LVEF early after surgery was similar to prior studies even in the patients with chordal preservation (64.58 ± 10.06 to 40.13 ± 10.45). There was a considerable amount of reverse cardiac remodeling early after MVR. We found that LVEDD (60.42 ± 8.01 to 52.84 ± 7.95) and LVESD (41.10 ± 8.35 to 38.03 ± 8.33) decreased significantly after MVR similar to other studies [11]. LA dimension also decreased (7.64 ± 2.02 to 6.90 ± 1.72) early after mitral valve surgery, similar to prior studies [12–14]. We observed that patients who had larger hearts preoperatively (larger LVEDD, LVESD, LA, and increased cardiothoracic ratio) had poor LVEF after MVR similar to study by Suri et al. [3]. Several historic studies have addressed the issue of decline in LV contractile function after mitral valve surgery. It is hypothesized that the elimination of severe chronic MR leads to an increase in LV after load and increased LV end diastolic pressure which predispose to impaired cardiac functions postoperatively. However, it is also postulated that the decrease in the cardiac functions after mitral valve surgery for chronic MR might not be fully explained by increase in after load. Intra operative injury and myocardial fibrosis may also be important contributing factors [10]. GLS is a relatively newer technique to assess global LV function, but it is under used. It can detect LV dysfunction at an early stage when conventional parameters of LV function such as LVEF are still normal. We used speckle tracking to assess GLS, which is angle independent unlike tissue Doppler. GLS might have an additional value over conventional measures to optimize the timing of surgery as it is affected much earlier than LVEF. We found that similar to other LV parameter, GLS also changed significantly early after MVR (− 15.57 ± 4.98 to − 8.97 ± 3.66). There was a significant decline in GLS in all the patients in early postoperative period suggesting impairment of LV contractility. Other studies have also demonstrated a significant decrease in GLS immediately after operation [15].
There was a trend towards improvement in LV contractile function including LVEF (52.48 ± 10.49%, change 18.74%), LV stroke volume and GLS (− 14.44 ± 3.67%, change 7.26%) at 3-month follow-up as assessed by echocardiography. There was a further decline in LV dimensions including reduction in LVESD, LVEDD, LA size and cardiothoracic (CT) ratio. This is in accordance with previous studies where they reported an early postoperative decline in LV function followed by sustained improvement in the months following surgery [16]. Ideally in patients with chronic organic MR having normal LVEF in the preoperative period, LV function parameters should return to normal after an initial decline. However, this was not consistent in all the patients suggesting that LVEF alone may not be sufficient to assess LV function in patients with chronic severe MR as LVEF may be falsely high. Therefore, some studies used LV strain to assess LV function in addition to LVEF to identify patients with subtle LV dysfunction, so as to perform MV surgery before irreversible structural and functional damage occurs [15]. Although in our study, majority of the patients had impaired GLS even in presence of normal LVEF, the patients who had near normal GLS had better postoperative LV function suggesting fallacies of LVEF in predicting LV functions in chronic severe MR. We found that patients with a mean GLS ≥ − 17.31 ± 4.39% (more negative value) had higher chances of having postoperative LVEF ≥ 50% at 3 months. This is in accordance with the study of Witkowski et al., where patients with mean GLS ≥ − 24.4% had higher chances of postoperative LVEF more than 50% [17]. Thus we can predict that operating patients with chronic severe MR when GLS is normal may result in better postoperative LV function, as there was a trend towards better LVEF in patients with mildly deranged GLS in our study. It is seen that postoperative LVEF < 50% is associated with poorer prognosis and long-term survival, so every effort should be made to identify patients at an earlier stage. In this regard, speckle tracking for strain analysis can be an attractive tool to avoid shortcomings of conventional methods of echocardiography to assess LV function [17].
Study limitations
There are few limitations in our study.
Sample size was small.
We only included patients with rheumatic heart disease and who underwent MVR.
Three-dimensional echocardiography was not systematically available which influence the global longitudinal strain measurements.
Many of our patients had atrial fibrillation but surgical ablation for AF was not done in our study.
Conclusions
Mitral valve repair is the preferred surgery for chronic severe MR as LV functions are better preserved by repair rather than replacement and there is no need for anticoagulation. However, in our region rheumatic MR is more prevalent where repair is not possible. But in all the cases, chordal preservation should be done as far as possible. Since both the surgeries (repair and replacement) cause decline in LV function, every effort should be made to identify patients at an earlier stage when LV functions are well maintained. GLS is an easy method to identify even hidden LV dysfunction and can be used in chronic MR patients, so that patient can undergo surgery timely and their cardiac function can be well preserved.
Funding
None
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Statement of human rights
All procedures performed in studies involving human participants were in accordance with the ethical standards of King George’s Medical University, U.P. Institutional ethical committee, (Ref. code: 89 ECM IIB-thesis /P51) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Statement on the welfare of animals
This article does not contain any studies with animals performed by any of the authors.
Informed consent
Informed consent was obtained from all individual participants included in the study.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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