Study characteristics
Regarding to aforementioned keywords total of 1873 studies were initially retrieved. Of these studies, 219 publications were duplicate, 1496 and 134 publications excluded by title & abstract and full text examination, respectively. Finally, twenty-four studies qualified for quantitative analysis Figure 1. The eligible studies were published from 2000 to 2016 and had an overall good methodological quality with NOS scores ranging from 5 to 8. Different genotyping method were used by included studies. Table 1 and 2 summarized the characteristics and genotype frequency of the included studies.
Table 1: Characteristics of studies included in meta-analysis of overall asthma.
Study author
|
Year
|
Country
|
Ethnicity 1
|
Ethnicity 2
|
Sex
cases/controls
|
Total cases/control
|
Age case/control
|
Genotyping method
|
Quality
score
|
Suzuki et al.
|
2000
|
Japan
|
Asian
|
Caucasian
|
M= 50/60
F= 100/150
|
120 / 120
|
Adult
|
PCR-RFLP
|
6
|
Beghe et al.
|
2003
|
UK
|
European
|
Caucasian
|
M= 88/99
F= 93/89
|
187 / 182
|
Adult
|
PCR-RFLP
|
7
|
Basehore et al.(i)
|
2004
|
USA
|
American (USA white)
|
Caucasian
|
M= 93/140
F= 98/147
|
233 / 245
|
Adult
|
PCR
|
7
|
Basehore et al.(ii)
|
2004
|
USA
|
American
|
American- African
|
M= 77/91
F= 121/148
|
168 / 269
|
Adult
|
PCR
|
6
|
Basehore et al.(iii)
|
2004
|
USA
|
American (USA Hispanic)
|
Caucasian (Hispanic)
|
M= 54/62
F= 41/89
|
116 / 130
|
Adult
|
PCR
|
6
|
Park et al.
|
2004
|
Korea
|
Asian
|
Caucasian
|
M= 248/302
F= 85/86
|
532 / 170
|
Mixed
|
SnaP shot
|
8
|
Donfack et al.(i)
|
2005
|
USA
|
American
|
Caucasian
|
M=NR
F=NR
|
126 / 205
|
Mixed
|
LAS
|
6
|
Donfack et al.(ii)
|
2005
|
USA
|
American
|
American- African
|
M=NR
F=NR
|
205 / 183
|
Mixed
|
LAS
|
6
|
Garcia et al.
|
2005
|
Spain
|
European
|
Caucasian
|
M=NR
F=NR
|
133 / 79
|
Mixed
|
TaqMan
|
6
|
Battle et al.
|
2007
|
USA
|
American
|
American- African
|
M= 105/156
F= 67/109
|
261 / 176
|
Mixed
|
PCR-RFLP
|
6
|
Amirzargar et al.
|
2009
|
Iran
|
Asian
|
Caucasian
|
M=NR
F=NR
|
59 / 139
|
Mixed
|
PCR-SSP
|
5
|
Jiang et al.
|
2009
|
China
|
Asian
|
Caucasian
|
M= NR
F= NR
|
24 / 24
|
Adult
|
PCR-RFLP
|
5
|
Daley et al.
|
2009
|
Australia
|
Australian
|
Caucasian
|
M= NR
F= NR
|
643 / 751
|
Mixed
|
Illumina Bead array system
|
8
|
Haller et al.
|
2009
|
USA
|
American
|
American- African
|
M=NR
F=NR
|
72 / 70
|
Adult
|
PCR-RFLP
|
6
|
Wang et al.
|
2009
|
Taiwan
|
Asian
|
Caucasian
|
M= 299/147
F= 245/265
|
446 / 510
|
Children
|
TaqMan
|
7
|
Berce et al.
|
2010
|
Slovenia
|
European
|
Caucasian
|
M=NR
F=NR
|
106 / 89
|
Children
|
PCR-RFLP
|
6
|
Undarmaa et al.(i)
|
2010
|
Japan
|
Asian
|
Caucasian
|
M=NR
F=NR
|
324 / 336
|
Children
|
TaqMan-ASA
|
7
|
Undarmaa et al.(ii)
|
2010
|
Japan
|
Asian
|
Caucasian
|
M=NR
F=NR
|
367 / 676
|
Adult
|
TaqMan-ASA
|
8
|
Wu et al.
|
2010
|
China
|
Asian
|
Caucasian
|
M= 138/114
F= 118/109
|
252 / 227
|
Children
|
PCR-RFLP
|
7
|
Michel et al.
|
2010
|
Germany
|
European
|
Caucasian
|
M=NR
F=NR
|
703 / 658
|
Children
|
Illumina Sentrix Bead chip
|
8
|
Huang et al.
|
2011
|
China
|
Asian
|
Caucasian
|
M= 51/49
F= 70/52
|
100 / 122
|
Children
|
PCR-RFLP
|
6
|
Yang et al.
|
2011
|
China
|
Asian
|
Caucasian
|
M= 101/101
F= 155/50
|
202 / 205
|
Adult
|
MALDI-TOF
|
6
|
Chen et al.
|
2011
|
China
|
Asian
|
Caucasian
|
M=NR
F=NR
|
202 / 191
|
Children
|
MALDI-TOF
|
7
|
Micheal et al.
|
2013
|
Pakistan
|
Asian
|
Caucasian
|
M=NR
F=NR
|
108 / 120
|
Mixed
|
PCR-RFLP
|
6
|
Miyake et al.
|
2013
|
Japan
|
Asian
|
Caucasian
|
M= 0/89
F= 0/1281
|
89 / 1281
|
Adult
|
TaqMan
|
6
|
Davoodi et al.
|
2013
|
India
|
Asian
|
Caucasian
|
M= 45/55
F= 21/29
|
100 / 50
|
Adult
|
Mass Array
|
5
|
Wang et al.
|
2015
|
China
|
Asian
|
Caucasian
|
M=NR
F=NR
|
392 / 849
|
Children
|
Mass Array
|
8
|
Li et al.
|
2016
|
China
|
Asian
|
Caucasian
|
M= 134/183
F= 151/200
|
317 / 351
|
Children
|
PCR-RFLP
|
7
|
NR, not reported; M, male; F, female
Table 2. Distribution of genotype and allele among asthma patients and controls.
Study author
|
Asthma cases
|
Healthy control
|
P-HWE
|
MAF
|
CC
|
CT
|
TT
|
C
|
T
|
CC
|
CT
|
TT
|
C
|
T
|
|
|
|
Suzuki et al.
|
11
|
56
|
53
|
78
|
162
|
10
|
59
|
51
|
79
|
161
|
0/21
|
0/67
|
|
Beghe et al.
|
140
|
41
|
6
|
321
|
53
|
132
|
48
|
2
|
312
|
52
|
0/29
|
0/142
|
|
Basehore et al.(i)
|
153
|
72
|
8
|
378
|
88
|
185
|
56
|
4
|
426
|
64
|
0/91
|
0/13
|
|
Basehore et al.(ii)
|
51
|
83
|
34
|
185
|
151
|
87
|
132
|
50
|
306
|
232
|
0/99
|
0/431
|
|
Basehore et al.(iii)
|
48
|
53
|
15
|
149
|
83
|
60
|
57
|
13
|
177
|
83
|
0/92
|
0/319
|
|
Park et al.
|
19
|
164
|
349
|
202
|
862
|
7
|
57
|
106
|
71
|
269
|
0/84
|
0/791
|
|
Donfack et al.(i)
|
83
|
37
|
6
|
203
|
49
|
150
|
50
|
5
|
350
|
60
|
0/73
|
0/146
|
|
Donfack et al.(ii)
|
68
|
107
|
30
|
243
|
167
|
70
|
86
|
27
|
226
|
140
|
0/94
|
0/382
|
|
Garcia et al.
|
93
|
39
|
1
|
225
|
41
|
64
|
15
|
0
|
143
|
15
|
0/35
|
0/094
|
|
Battle et al.
|
85
|
128
|
48
|
298
|
224
|
57
|
87
|
32
|
201
|
151
|
0/9
|
0/428
|
|
Amirzargar et al.
|
2
|
56
|
1
|
60
|
58
|
61
|
78
|
0
|
200
|
78
|
<0.001
|
0/28
|
|
Jiang et al.
|
0
|
9
|
15
|
9
|
39
|
2
|
10
|
12
|
14
|
34
|
0/96
|
0/708
|
|
Daley et al.
|
481
|
150
|
12
|
1112
|
174
|
555
|
181
|
15
|
1291
|
211
|
0/95
|
0/14
|
|
Haller et al.
|
21
|
36
|
15
|
78
|
66
|
27
|
33
|
10
|
87
|
53
|
0/98
|
0/378
|
|
Wang et al.
|
22
|
147
|
277
|
191
|
701
|
16
|
186
|
308
|
218
|
802
|
0/05
|
0/786
|
|
Berce et al.
|
67
|
31
|
8
|
165
|
47
|
51
|
35
|
3
|
137
|
41
|
0/3
|
0/23
|
|
Undarmaa et al.(i)
|
27
|
142
|
155
|
196
|
452
|
37
|
144
|
155
|
218
|
454
|
0/68
|
0/675
|
|
Undarmaa et al.(ii)
|
28
|
154
|
185
|
210
|
524
|
64
|
286
|
326
|
414
|
938
|
0/91
|
0/693
|
|
Wu et al.
|
6
|
83
|
163
|
95
|
409
|
11
|
87
|
129
|
109
|
345
|
0/44
|
0/759
|
|
Michel et al.
|
458
|
210
|
35
|
1126
|
280
|
474
|
173
|
11
|
1121
|
195
|
0/28
|
0/148
|
|
Huang et al.
|
1
|
23
|
76
|
25
|
175
|
3
|
49
|
70
|
55
|
189
|
0/09
|
0/774
|
|
Yang et al.
|
14
|
56
|
132
|
84
|
320
|
7
|
67
|
131
|
81
|
329
|
0/65
|
0/802
|
|
Chen et al.
|
6
|
72
|
124
|
84
|
320
|
6
|
62
|
123
|
74
|
308
|
0/58
|
0/806
|
|
Micheal et al.
|
77
|
31
|
0
|
185
|
31
|
93
|
27
|
0
|
213
|
27
|
0/16
|
0/112
|
|
Miyake et al.
|
12
|
33
|
44
|
57
|
121
|
160
|
604
|
517
|
924
|
1638
|
0/42
|
0/639
|
|
Davoodi et al.
|
65
|
31
|
4
|
161
|
39
|
36
|
14
|
0
|
86
|
14
|
0/24
|
0/14
|
|
Wang et al.
|
49
|
176
|
167
|
274
|
510
|
102
|
410
|
337
|
614
|
1084
|
0/18
|
0/638
|
|
Li et al.
|
147
|
170
|
0
|
464
|
170
|
170
|
181
|
0
|
521
|
181
|
<0.001
|
0/257
|
|
P-HWE, p-value for Hardy–Weinberg equilibrium; MAF, minor allele frequency of control group
Meta-analysis of IL-4 C33T polymorphism and the risk of asthma
Twenty-four studies with 6587 cases and 8408 controls were included in final analysis of overall population. Among these studies, 15 publications were carried out in Asian countries, 5 publications were in American countries and 4 publications were in Europe. Our results showed that IL-4 C33T polymorphism increase risk of asthma across all genotype models including dominant model (OR= 1.15, 95% CI= 1.04-1.26, P= ≤0.001, FEM), recessive model (OR= 1.16, 95% CI= 1.06- 1.28, P= ≤0.001, FEM), allelic model (OR= 1.14, 95% CI= 1.07-1.21, P= ≤0.001, FEM), CC vs. TT model (OR= 1.21, 95% CI= 1.02-1.43, P= 0.02, FEM) and CT vs. TT model (OR= 1.10, 95% CI= 1-1.22, P= 0.05, FEM) Figure2. The results of pooled ORs, heterogeneity tests and publication bias tests for different analysis models are shown in Table 3.
Table 3. Main results of pooled ORs in meta-analysis of IL-4 (C33T) gene polymorphisms.
Test of publication bias (Egger’s test)
|
Test of publication bias (Begg’s test)
|
Test of heterogeneity
|
Test of association
|
Sample size
|
|
Subgroup
|
P
|
t
|
P
|
z
|
P
|
I2 (%)
|
95% CI (p-value)
|
OR
|
Case/Control
|
Genetic model
|
0.37
|
0.90
|
0.44
|
0.77
|
0.16
|
20.9
|
1.04 – 1.26 (≤0.001)
|
1.15
|
6587 / 8404
|
Dominant model
|
Overall
|
0.001
|
2.88
|
0.001
|
2.68
|
0.56
|
0
|
1.06 – 1.28 ((≤0.001)
|
1.16
|
6587 / 8404
|
Recessive model
|
|
0.02
|
2.39
|
0.01
|
2.53
|
0.08
|
27.8
|
1.07 – 1.21 (≤0.001)
|
1.14
|
6587 / 8404
|
Allelic model
|
|
0.13
|
1.57
|
0.07
|
1.78
|
0.58
|
0
|
1.02 – 1.43 (0.02)
|
1.21
|
6587 / 8404
|
CC vs. TT
|
|
0.58
|
0.56
|
0.81
|
0.23
|
0.17
|
20.5
|
1 – 1.22 (0.05)
|
1.10
|
6587 / 8404
|
CT vs. TT
|
|
Age groups
|
0.19
|
-1.43
|
0.32
|
-0.98
|
0.54
|
0
|
0.97 – 1.40 (0.10)
|
1.16
|
1678 / 3252
|
Dominant model
|
Adults
|
0.007
|
3.57
|
0.01
|
2.41
|
0.93
|
0
|
0.99 – 1.39 (0.06)
|
1.17
|
1678 / 3252
|
Recessive model
|
|
0.18
|
1.44
|
0.31
|
1.01
|
0.75
|
0
|
1.02 – 1.26 (0.02)
|
1.14
|
1678 / 3252
|
Allelic model
|
|
0.54
|
0.64
|
0.40
|
0.83
|
0.72
|
0
|
0.93 – 1.61 (0.15)
|
1.22
|
1678 / 3252
|
CC vs. TT
|
|
0.12
|
-1.72
|
0.24
|
-1.16
|
0.43
|
0.2
|
0.90 – 1.32 (0.91)
|
1.09
|
1678 / 3252
|
CT vs. TT
|
|
0.01
|
3.47
|
0.02
|
2.23
|
0.02
|
55.8
|
0.96 – 1.34 (0.13)
|
1.14
|
2067 / 1823
|
Dominant model
|
Mixed
|
0.67
|
0.47
|
0.62
|
-0.49
|
0.89
|
0
|
0.84 – 1.40 (0.53)
|
1.08
|
2067 / 1823
|
Recessive model
|
|
0.02
|
3.11
|
0.007
|
2.72
|
0.02
|
56.9
|
1.01 – 1.29 (0.03)
|
1.14
|
2067 / 1823
|
Allelic model
|
|
0.13
|
2.01
|
0.14
|
1.47
|
0.88
|
0
|
0.77 – 1.57 (0.60)
|
1.10
|
2067 / 1823
|
CC vs. TT
|
|
0.01
|
3.38
|
0.01
|
2.47
|
0.03
|
53.7
|
0.95 – 1.35 (0.15)
|
1.13
|
2067 / 1823
|
CT vs. TT
|
|
0.64
|
-0.48
|
0.67
|
-0.42
|
0.33
|
12.4
|
0.99 – 1.34 (0.07)
|
1.15
|
2842 / 3333
|
Dominant model
|
Children
|
0.05
|
2.34
|
0.21
|
1.24
|
0.03
|
54
|
1.03 – 1.35 (0.01)
|
1.18
|
2842 / 3333
|
Recessive model
|
|
0.65
|
0.47
|
0.67
|
0.42
|
0.07
|
44.6
|
1.04 – 1.24 (≤0.001)
|
1.13
|
2842 / 3333
|
Allelic model
|
|
0.36
|
0.97
|
0.32
|
0.99
|
0.09
|
42.3
|
0.97 – 1.65 (0.08)
|
1.27
|
2842 / 3333
|
CC vs. TT
|
|
0.54
|
-0.64
|
0.67
|
-0.42
|
0.39
|
4.5
|
0.92 – 1.26 (0.33)
|
1.08
|
2842 / 3333
|
CT vs. TT
|
|
Ethnicity1
|
0.17
|
1.44
|
0.41
|
0.81
|
0.13
|
29.3
|
0.93 – 1.130 (0.25)
|
1.10
|
3634 / 5371
|
Dominant model
|
Asia
|
0.24
|
1.24
|
0.39
|
0.85
|
0.56
|
0
|
1.02 – 1.26 (0.01)
|
1.14
|
3634 / 5371
|
Recessive model
|
|
0.01
|
2.70
|
0.02
|
2.31
|
0.08
|
34.9
|
1.03 – 1.21 (≤0.001)
|
1.12
|
3634 / 5371
|
Allelic model
|
|
0.93
|
0.09
|
1
|
0
|
0.77
|
0
|
0.87 – 1.33 (0.49)
|
1.08
|
3634 / 5371
|
CC vs. TT
|
|
0.36
|
0.93
|
0.65
|
0.45
|
0.12
|
29.6
|
0.89 – 1.18 (0.76)
|
1.02
|
3634 / 5371
|
CT vs. TT
|
|
0.58
|
-0.64
|
0.49
|
-0.68
|
0.12
|
48.3
|
1.01 – 1.50 (0.03)
|
1.23
|
1129 / 1008
|
Dominant model
|
Europe
|
0.55
|
-0.85
|
0.60
|
-0.52
|
0.95
|
0
|
1.54 – 5.62 (≤0.001)
|
2.94
|
1129 / 1008
|
Recessive model
|
|
0.56
|
-0.68
|
1
|
0
|
0.22
|
31.2
|
1.10 – 1.54 (≤0.001)
|
1.30
|
1129 / 1008
|
Allelic model
|
|
0.44
|
-1.20
|
0.60
|
-0.52
|
0.87
|
0
|
1.56 – 5.76 (≤0.001)
|
3
|
1129 / 1008
|
CC vs. TT
|
|
0.61
|
-0.59
|
0.49
|
-0.68
|
0.09
|
52.8
|
0.92 – 1.38 (0.24)
|
1.13
|
1129 / 1008
|
CT vs. TT
|
|
0.61
|
0.54
|
0.65
|
0.45
|
0.76
|
0
|
1.05 – 1.51(≤0.001)
|
1.26
|
1181 / 1278
|
Dominant model
|
America
|
0.06
|
6.60
|
0.01
|
2.55
|
0.89
|
0
|
0.88 – 1.52 (0.29)
|
1.16
|
1181 / 1278
|
Recessive model
|
|
0.04
|
2.71
|
0.09
|
1.65
|
0.55
|
0
|
1.03 – 1.33 (0.01)
|
1.17
|
1181 / 1278
|
Allelic model
|
|
0.05
|
3.17
|
0.02
|
2.25
|
0.84
|
0
|
0.93 – 1.71 (0.14)
|
1.26
|
1181 / 1278
|
CC vs. TT
|
|
0.86
|
0.18
|
0.65
|
0.45
|
0.85
|
0
|
1.02 – 1.49 (0.03)
|
1.23
|
1181 / 1278
|
CT vs. TT
|
|
Ethnicity 2
|
0.50
|
0.69
|
0.43
|
0.78
|
0.08
|
30.7
|
1.04 – 1.28 (0.008)
|
1.15
|
5881 / 7710
|
Dominant model
|
Caucasian
|
0.02
|
2.44
|
0.04
|
1.98
|
0.3
|
6.7
|
1.06 – 1.30 (0.002)
|
1.17
|
5881 / 7710
|
Recessive model
|
|
0.08
|
1.79
|
0.06
|
1.87
|
0.04
|
35.7
|
1.07 – 1.22 (≤0.001)
|
1.14
|
5881 / 7710
|
Allelic model
|
|
0.48
|
0.72
|
0.25
|
1.14
|
0.4
|
4.6
|
1.01 – 1.49 (0.03)
|
1.23
|
5881 / 7710
|
CC vs. TT
|
|
0.61
|
-0.59
|
0.49
|
-0.68
|
0.08
|
30.6
|
0.98 – 1.22 (0.09)
|
1.1
|
5881 / 7710
|
CT vs. TT
|
|
0.61
|
0.54
|
0.65
|
0.45
|
0.76
|
0
|
0.89 – 1.45 (0.29)
|
1.14
|
706 / 698
|
Dominant model
|
African-American
|
0.06
|
6.60
|
0.01
|
2.55
|
0.87
|
0
|
0.80 – 1.46 (0.6)
|
1.08
|
706 / 698
|
Recessive model
|
|
0.04
|
2.71
|
0.09
|
1.65
|
0.74
|
0
|
0.93 – 1.27 (0.32)
|
1.08
|
706 / 698
|
Allelic model
|
|
0.05
|
3.17
|
0.02
|
2.25
|
0.79
|
0
|
0.82 – 1.62 (0.4)
|
1.16
|
706 / 698
|
CC vs. TT
|
|
0.86
|
0.18
|
0.65
|
0.45
|
0.8
|
0
|
0.87 – 1.45 (0.35)
|
1.13
|
706 / 698
|
CT vs. TT
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Subgroup analysis
We categorized studies into different subgroups on the basis of age, continent and ethnicity. The results of pooled ORs, heterogeneity tests and publication bias tests for different analysis models are shown in Table 3.
Subgroup analysis by age
In this group, we stratified eligible articles into three groups including: adult (8 articles), children (7 articles) and mixed (cover both ranges; 9 articles). Overall, the results rejected significant association between IL-4 C33T polymorphism and risk of asthma in different age group except for allelic model [adults (OR= 1.14, 95% CI= 1.02-1.26, P= 0.02, FEM), mixed (OR= 1.14, 95% CI= 1.01-1.29, P= 0.03, REM), children (OR= 1.13, 95% CI= 1.04-1.24, P= ≤0.001, FEM)] and recessive model (just in children (OR= 1.18, 95% CI= 1.03-1.35, P= 0.01, REM)) Figure 3.
Subgroup analysis by continent
Our included studies performed in Asia (15 articles), Europe (4 articles), America (4 articles), and Oceania (1 article). Since there was only one study for Oceania, we exclude it. The final results revealed strong significant association between IL-4 C33T polymorphism and asthma risk in European population across dominant model (OR= 1.23, 95% CI= 1.01- 1.50, P= 0.03, FEM), recessive model (OR= 2.94, 95% CI= 1.54- 5.62, P= ≤0.001, FEM), allelic model (OR= 1.30, 95% CI= 1.10-1.54, P= ≤0.001, FEM) and CC vs. TT (OR= 3, 95% CI= 1.56- 5.76, P= ≤0.001, FEM). Moreover, there was a significant association between IL-4 C33T polymorphism and risk of asthma in American population under dominant model (OR= 1.26, 95% CI= 1.05 -1.51, P= ≤0.001, FEM), allelic model (OR= 1.17, 95% CI= 1.03- 1.33, P= 0.01, FEM), and CT vs. TT model (OR= 1.23, 95% CI= 1.02-1.49, P= 0.03, FEM). Eventually, Significant positive association was revealed in Asians just in recessive model (OR= 1.14, 95% CI= 1.02- 1.26, P= 0.01, FEM), and allelic model (OR= 1.12, 95% CI= 1.03-1.21, P= ≤0.001, FEM) Figure 3.
Subgroup analysis by ethnicity
Finally, we stratified eligible articles according ethnicity including Caucasians (20 articles), and African-Americans (4 articles). The results showed significant association between IL-4 SNP (C33T) and asthma risk in Caucasians under dominant model (OR= 1.15, 95% CI= 1.04- 1.28, P= 0.008, FEM), recessive model (OR= 1.17, 95% CI= 1.06- 1.30, P= 0.002, FEM), allelic model (OR= 1.14, 95% CI= 1.07-1.22, P= ≤0.001, FEM),and CC vs. TT model (OR= 1.23, 95% CI= 1.01-1.49, P= 0.03, FEM) but not CT vs. TT model (OR= 1.1, 95% CI= 0.98 -1.22, P= 0.09, FEM). However, there was no significant association between IL-4 C33T polymorphism and risk of asthma in American-African population across all genotype models Figure 3.
Evaluation of heterogeneity
No significant heterogeneity was detected for IL-4 C33T polymorphism neither in overall population nor subgroup analysis (Table 3).
Publication bias
Publication bias was estimated by using funnel plot, Begg's and Egger's tests. No evidence of publication bias was seen for overall population and subgroup analysis under all genetic models. Additionally, the shape of the funnel plot appeared to be symmetrical which demonstrated that there was no significant publication bias Figure 4.
Sensitivity analysis
Sensitivity analysis was conducted after sequentially removing each eligible study. This approach is to enumerate as an inevitable step for analyzing multiple criteria. The significance of the pooled ORs was not affected by any single study in the dominant model for IL-4 SNPs, indicating that our results were statistically robust Figure 5.