Background
Methods
Study population and sample selection
Perfluoroalkyl substances (PFAS) measurements
Measurement of elements and metals
Measurement of endogenous sex hormones
Measurement of receptor transactivities induced by AF
Calculation of receptor ligand equivalent (EQ)
Abbreviations | Explanation | Example of calculation |
---|---|---|
ER-tact | Luciferase reporter gene transactivity of estrogen receptor (ER) | |
ER-EEQ | The combined effect on ER induced by the combination of endogenous estrogen and xeno-estrogens (EDCs) given as estradiol equivalent | • ER-tact of AF: 2.51 • Interpolation of ER-tact onto E2 dose-response curve • ER-EEQ = 247.60 pg E2/ml AF |
EDC-EEQ | The integrated estrogenic effect induced by xeno-estrogens (EDCs) alone given as estradiol equivalent | • Endogenous E2: 230.23 pg/ml • (ER-EEQ) − E2 = 247.60 − 230.32 = 17.28 E2 pg/ml AF |
AR-tact | Luciferase reporter gene transactivity of androgen receptor (AR) | |
AR-AEQ | The combined effect on AR induced by the combination of endogenous androgens and xeno-androgens (EDCs) given as androgen equivalent | • AR-tact of AF: 3.30 • Interpolation of AR-tact onto DHT dose-response curve • AR-AEQ = 63.50 pg DHT/ml AF |
EDC-AEQ | The integrated androgenic effect induced by xeno-androgens (EDCs) alone given as androgen equivalent | • Testosterone: 470.12 pg/ml AF • DHT: estimated DHT = 10% × 470.12 = 47.01 pg/ml AF • (AR-AEQ) − DHT = 63.50 − 47.01 = 16.49 pg DHT/ml AF |
AR-AEQ/ER-EEQ | The proportion of total combined androgenic effect to the total combined estrogenic effect | • AR-AEQ = 63.50 pg DHT/mlAF • ER-EEQ = 247.60 pg E2/mlAF • AR-AEQ/ER-EEQ = 63.50/247.60 = 0.256 |
EDC-AEQ/EDC-EEQ | The proportion of EDC-induced androgenic effect to EDC-induced estrogenic effect | • EDC-AEQ = 16.49 DHT/mlAF • EDC-EEQ = 17.28 E2/mlAF • EDC-AEQ/EDC-EEQ = 16.49/17.28 = 0.954 |
TH-PE | Thyroid hormone-related proliferative effect | |
T3-ThEQ | The combined proliferation effect (PE) of endogenous thyroid hormone and xeno-thyroid hormone-like compounds (EDCs) given as thyroid equivalent | • TH-PE of AF: 4.56 • Interpolation of TH-PE onto T3 dose-response curve • T3-ThEQ = 3.53 ng T3/ml AF |
AhR-tact | Luciferase reporter gene transactivity of aryl hydrocarbon receptor (AhR) | |
AhR-TEQ | The combined effect on AhR of dioxin-like compounds given as TCDD equivalent | • AhR-tact of AF extraction: 5.67 • AhR-tact of blank: 4.75 • Interpolation of AhR-tact onto TCDD dose-response curve = AhR-TEQ • AhR-TEQ of AF extraction = 1.46 pg TCDD/ml, • AhR-TEQ of blank = 1.22 pg TCDD/ml • Final AhR-TEQ = 1.46 − 1.22 = 0.24 pg TCDD/ml AF |
Statistical analysis
Principal component analysis
Results
Characteristics of the study population
Characteristic | ASD cases (n = 75) | Controls (n = 135) | p value |
---|---|---|---|
Mother’s age at delivery (median, min-max, year) | 34 (17–41) | 35 (21–44) | 0.218 |
Father’s age at delivery (median, min-max, year) | 35 (19–53) | 35 (20–58) | 0.362 |
Gestational week at sampling (median, min-max) | 15 (11–20) | 15 (10–29) | 0.426 |
Parity (n, %) | 0.797 | ||
1st child | 28 (37.30%) | 48 (35.6%) | |
> 2nd child | 47 (62.70%) | 87 (64.4%) | |
Gestational age at birth (n, %) | 0.424 | ||
Preterm (< 260 days) | 7 (9.30%) | 15 (11.20%) | |
Term (260 days–294 days) | 67 (89.30%) | 113 (84.30%) | |
Post-term (> 295 days) | 1 (1.30%) | 6 (4.50%) | |
Gender of the children (n, %) | 0.731 | ||
Female | 13 (17.30%) | 26 (19.30%) | |
Male | 62 (82.70%) | 109 (80.70%) | |
Birth weight of the children (n, %) | 0.521 | ||
< 2500 g | 3 (4.10%) | 10 (7.50%) | |
2500 g–4000 g | 57 (77.00%) | 95 (70.90%) | |
> 4000 g | 14 (18.90%) | 29 (21.60%) | |
Apgar score of the children (n, %) | 0.952 | ||
> 7 | 70 (98.60%) | 130 (98.50%) | |
< 7 | 1 (1.40%) | 2 (1.50%) | |
Congenital malformation of the children (n, %) |
< 0.0001
| ||
No | 53 (70.70%) | 124 (91.90%) | |
Yes | 22 (29.30%) | 11 (8.10%) |
Levels of perfluoroalkyl substances (PFAS)
ASD cases | Control | |||||||
---|---|---|---|---|---|---|---|---|
n
| Median | Min-max |
n
| Median | Min-max |
p
| FDR (qvalue) | |
PFAS (ng/mL) | ||||||||
PFOS | 37 | 0.61 | 0.61–2.98 | 51 | 1.44 | 0.61–4.22 |
0.015
|
0.189
|
PFOSA | 37 | 0.60 | 0.60–13.30 | 51 | 0.60 | 0.60–19.00 | 0.152 | 0.638 |
PFOA | 37 | 0.29 | 0.10–0.78 | 51 | 0.32 | 0.10–1.86 | 0.516 | 0.764 |
∑PFSA | 37 | 1.56 | 1.56–15.40 | 51 | 2.60 | 1.56–21.70 |
0.011
|
0.189
|
∑PFCA | 37 | 1.54 | 1.35–2.03 | 51 | 1.57 | 1.35–3.11 | 0.489 | 0.764 |
∑PFSA+∑PFCA | 37 | 3.26 | 2.91–17.37 | 51 | 4.23 | 2.91–23.26 |
0.021
|
0.189
|
Elements (μg/L) | ||||||||
Fe | 37 | 259.00 | 16.00–778.0 | 51 | 270.00 | 128.00–2575 | 0.571 | 0.764 |
Cu | 37 | 89.15 | 45.62–206.10 | 51 | 100.40 | 53.53–223.10 | 0.189 | 0.638 |
Zn | 37 | 97.90 | 34.69–754.5 | 51 | 102.00 | 30.90–1216 | 0.557 | 0.764 |
Se | 37 | 4.43 | 2.250–7.130 | 51 | 4.55 | 2.980–7.27 | 0.758 | 0.861 |
I | 37 | 1.88 | 1.88–94.68 | 51 | 1.88 | 1.88–195.40 | 0.765 | 0.861 |
Cr | 37 | 0.75 | 0.16–3.85 | 51 | 0.73 | 0.31–2.21 | 0.365 | 0.704 |
Mn | 37 | 0.65 | 0.65–25.89 | 51 | 0.65 | 0.65–5.26 | 0.826 | 0.892 |
As | 37 | 1.33 | 1.33–6.19 | 51 | 1.33 | 1.33–4.96 | 0.328 | 0.704 |
Cd | 37 | 0.02 | 0.02–1.92 | 51 | 0.02 | 0.02–0.14 | 0.648 | 0.795 |
Pb | 37 | 0.22 | 0.22–2.27 | 51 | 0.22 | 0.22–5.30 | 0.526 | 0.764 |
Levels of elements and heavy metals
Levels of endogenous sex hormones and the combined effect of endogenous hormones and EDCs on the receptor transactivation
Parameter | ASD case | Control | ||||||
---|---|---|---|---|---|---|---|---|
n
| Median | Min-Max |
N
| Median | Min-Max |
p
| FDR (qvalue) | |
E2 (pg/mL) | 68 | 287.4 | 78.72–2778 | 128 | 311.9 | 55.02–1005 | 0.337 | 0.704 |
Testosterone (pg/mL) | 67 | 383.6 | 47.30–1393 | 130 | 366.3 | 80.47–1090 | 0.173 | 0.638 |
Testosterone/E2 ratio | 66 | 1.49 | 0.21–7.41 | 128 | 1.25 | 0.14–4.310 | 0.222 | 0.666 |
ER-EEQ (pg E2/mL) | 75 | 470.9 | 204.1–1308 | 135 | 545.8 | 149.0–1308 | 0.329 | 0.704 |
EDC-EEQ (pg E2/mL) | 68 | 168.5 | 0.28–1226 | 128 | 214.4 | 2.72–1165 | 0.162 | 0.638 |
AR-AEQ (pg DHT/mL) | 75 | 62.60 | 23.90–137.3 | 135 | 62.50 | 16.90–290.4 | 0.910 | 0.910 |
EDC-AEQ (pg DHT/mL) | 67 | 22.56 | 1.000–100.4 | 130 | 28.45 | 1.34–274.0 | 0.167 | 0.638 |
AR-AEQ/ER-EEQ | 75 | 0.13 | 0.02–0.48 | 135 | 0.11 | 0.02–0.98 | 0.343 | 0.704 |
EDC-AEQ/EDCs-EEQ | 61 | 0.18 | 0.01–59.10 | 117 | 0.086 | 0.003–26.21 | 0.515 | 0.764 |
T3-ThEQ (ng T3/mL) | 74 | 2.36 | 0.10–17.14 | 133 | 2.41 | 0–6.60 | 0.594 | 0.764 |
AhR-TEQ (pg TCDD/mL) | 37 | 0.24 | 0–1.310 | 51 | 0.15 | 0–2.10 | 0.907 | 0.910 |
Association of ASD risk and levels of chemicals
Crude | Adjusteda | ||||||
---|---|---|---|---|---|---|---|
n (cases/controls) | OR (95% CI) |
p
| n (cases/controls) | OR (95% CI) |
p
| FDR (qvalue) | |
PFOS (ng/mL) | 37/51 |
0.460 (0.230, 0.921)
|
0.028
| 37/50 |
0.410 (0.174, 0.967)
|
0.042
| 0.437 |
PFOSA (ng/mL) | 37/51 | 0.913 (0.797, 1.046) | 0.189 | 37/50 | 0.918 (0.789, 1.069) | 0.272 | 0.565 |
PFOA (ng/mL) | 37/51 | 0.335 (0.044, 2.559) | 0.292 | 37/50 | 0.164 (0.013, 2.126) | 0.167 | 0.437 |
∑PFSA (ng/mL) | 37/51 | 0.894 (0.777, 1.028) | 0.115 | 37/50 | 0.900 (0.771, 1.049) | 0.178 | 0.437 |
∑PFCA (ng/mL) | 37/51 | 0.299 (0.041, 2.160) | 0.232 | 37/50 | 0.150 (0.013, 1.760) | 0.131 | 0.437 |
∑PFSA+∑PFCA (ng/mL) | 37/51 | 0.894 (0.779, 1.025) | 0.107 | 37/50 | 0.896 (0.770, 1.044) | 0.158 | 0.437 |
Crude | Adjusteda | ||||||
---|---|---|---|---|---|---|---|
n (cases/controls) | OR (95% CI) |
p
| n (cases/controls) | OR (95% CI) |
p
| FDR (qvalue) | |
Fe (μg/L) | 37/51 | 0.999 (0.996, 1.001) | 0.255 | 37/50 | 0.999 (0.997, 1.001) | 0.449 | 0.677 |
Cu (μg/L) | 37/51 | 0.991 (0.978, 1.005) | 0.211 | 37/50 | 0.997 (0.981, 1.012) | 0.685 | 0.771 |
Zn (μg/L) | 37/51 | 0.999 (0.996, 1.002) | 0.673 | 37/50 | 0.999 (0.995, 1.002) | 0.491 | 0.698 |
Se (μg/L) | 37/51 | 0.977 (0.649, 1.469) | 0.910 | 37/50 | 1.058 (0.644, 1.740) | 0.823 | 0.823 |
I (μg/L) | 37/51 | 0.996 (0.978, 1.014) | 0.674 | 37/50 | 0.981 (0.959, 1.003) | 0.087 | 0.437 |
Cr (μg/L) | 37/51 | 1.019 (0.403, 2.576) | 0.968 | 37/50 | 0.377 (0.051, 2.764) | 0.337 | 0.650 |
Mn (μg/L) | 37/51 | 1.106 (0.878, 1.394) | 0.393 | 37/50 | 1.065 (0.841, 1.347) | 0.602 | 0.771 |
As (μg/L) | 37/51 | 1.257 (0.856, 1.846) | 0.244 | 37/50 | 1.496 (0.924, 2.424) | 0.101 | 0.437 |
Cd (μg/L) | 37/51 | 4.418 (0.099, 197) | 0.444 | 37/50 | 5.935 (0.101, 349.3) | 0.392 | 0.662 |
Pb (μg/L) | 37/51 | 1.086 (0.615, 1.918) | 0.776 | 37/50 | 1.300 (0.657, 2.575) | 0.451 | 0.677 |
Association of ASD risk with the levels of sex hormones and the combined AF-induced receptor effect biomarkers
Crude | Adjusteda | ||||||
---|---|---|---|---|---|---|---|
n (cases/controls) | OR (95% CI) |
p
| n (cases/controls) | OR (95% CI) |
p
| FDR (qvalue) | |
E2 (pg/mL) | 68/128 | 1.000 (0.999, 1.001) | 0.629 | 65/125 | 1.000 (0.999, 1.001) | 0.669 | 0.771 |
Testosterone (pg/mL) | 67/130 |
1.001 (1.000, 1.003)
|
0.050
| 64/127 |
1.002 (1.000, 1.004)
|
0.050
| 0.437 |
Testosterone/E2 | 66/128 | 1.257 (0.950, 1.664) | 0.110 | 63/125 | 1.350 (0.935, 1.949) | 0.110 | 0.437 |
ER-EEQ (pg E2/mL) | 75/135 | 1.000 (0.999, 1.000) | 0.358 | 71/132 | 1.000 (0.999, 1.000) | 0.222 | 0.500 |
EDCs-EEQ (pg E2/mL) | 68/128 | 0.999 (0.998, 1.000) | 0.112 | 65/125 | 0.999 (0.998, 1.000) | 0.089 | 0.437 |
AR-AEQ (pg DHT/mL) | 75/135 | 0.995 (0.985, 1.006) | 0.389 | 71/132 | 0.997 (0.985, 1.010) | 0.650 | 0.771 |
EDCs-AEQ (pg DHT/mL) | 67/130 | 0.989 (0.976, 1.002) | 0.109 | 64/127 | 0.989 (0.974, 1.005) | 0.169 | 0.437 |
AR-AEQ/ER-EEQ | 75/135 | 1.269 (0.104, 15.479) | 0.852 | 71/132 | 2.176 (0.115, 41.15) | 0.604 | 0.771 |
EDCs-AEQ/EDCs-EEQ | 66/128 | 1.022 (0.794, 1.552) | 0.423 | 63/125 | 1.032 (0.974, 1.092) | 0.285 | 0.662 |
T3-ThEQ (ng T3/mL) | 74/133 | 1.094 (0.874, 1.369) | 0.434 | 71/130 | 1.036 (0.806, 1.332) | 0.784 | 0.822 |
AhR-TEQ (pgTCDD/mL) | 37/51 | 0.786 (0.238, 2.589) | 0.692 | 36/50 | 0.792 (0.192, 3.270) | 0.747 | 0.822 |
Correlations between PFAS levels and combined AF-induced receptor effect biomarkers
ER-EEQ | EDC-EEQ | AR-AEQ | EDC-AEQ | T3-ThEQ | AhR-TEQ | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
n
|
r
s
|
p
|
n
|
r
s
|
p
|
n
|
r
s
|
p
|
n
|
r
s
|
p
|
n
|
r
s
|
p
|
n
|
r
s
|
p
| |
ASD cases | ||||||||||||||||||
PFOS | 37 |
0.522
|
0.001
| 30 | 0.246 | 0.189 | 37 | − 0.258 | 0.124 | 30 |
− 0.466
|
0.009
| 34 | 0.145 | 0.398 | 37 | 0.097 | 0.569 |
PFOSA | 37 | 0.237 | 0.158 | 30 | 0.199 | 0.292 | 37 |
− 0.391
|
0.017
| 30 | − 0.163 | 0.390 | 36 | − 0.038 | 0.825 | 37 | 0.140 | 0.410 |
PFOA | 37 | 0.225 | 0.180 | 30 | 0.317 | 0.087 | 37 | 0.007 | 0.968 | 30 |
− 0.399
|
0.029
| 34 | 0.039 | 0.822 | 37 | 0.125 | 0.462 |
∑PFSA | 37 |
0.332
|
0.045
| 30 | 0.158 | 0.403 | 37 | − 0.305 | 0.066 | 30 | − 0.310 | 0.095 | 34 | − 0.026 | 0.882 | 37 | 0.100 | 0.555 |
∑PFCA | 37 | 0.225 | 0.180 | 30 | 0.317 | 0.087 | 37 | 0.007 | 0.968 | 30 |
− 0.399
|
0.029
| 34 | 0.039 | 0.822 | 37 | 0.125 | 0.462 |
∑PFSA+∑PFCA | 37 |
0.343
|
0.038
| 30 | 0.330 | 0.075 | 37 | − 0.194 | 0.249 | 30 | − 0.318 | 0.087 | 34 | − 0.005 | 0.978 | 37 | 0.138 | 0.417 |
Controls | ||||||||||||||||||
PFOS | 51 | 0.156 | 0.276 | 44 | 0.273 | 0.073 | 51 | − 0.082 | 0.565 | 46 | − 0.010 | 0.947 | 48 | − 0.004 | 0.979 | 51 | − 0.065 | 0.649 |
PFOSA | 51 | 0.220 | 0.121 | 44 |
0.356
|
0.018
| 51 | − 0.160 | 0.263 | 46 | − 0.108 | 0.476 | 49 | − 0.221 | 0.127 | 51 | − 0.273 | 0.1053 |
PFOA | 51 | 0.104 | 0.469 | 44 | 0.097 | 0.530 | 51 | − 0.002 | 0.989 | 46 | 0.048 | 0.751 | 48 | 0.209 | 0.150 | 51 | 0.148 | 0.300 |
∑PFSA | 51 |
0.321
|
0.021
| 44 |
0.442
|
0.003
| 51 | − 0.160 | 0.263 | 46 | − 0.071 | 0.641 | 48 | − 0.186 | 0.202 | 51 | − 0.253 | 0.074 |
∑PFCA | 51 | 0.100 | 0.487 | 44 | 0.093 | 0.548 | 51 | − 0.005 | 0.974 | 46 | 0.051 | 0.736 | 48 | 0.203 | 0.161 | 51 | 0.142 | 0.322 |
∑PFSA+∑PFCA | 51 |
0.332
|
0.017
| 44 |
0.438
|
0.003
| 51 | − 0.146 | 0.305 | 46 | − 0.047 | 0.757 | 48 | − 0.148 | 0.309 | 51 | − 0.217 | 0.125 |
All | ||||||||||||||||||
PFOS | 88 |
0.305
|
0.004
| 74 |
0.251
|
0.031
| 88 | − 0.182 | 0.090 | 76 | − 0.085 | 0.465 | 85 | 0.18 | 0.871 | 88 | 0.015 | 0.891 |
PFOSA | 88 |
0.224
|
0.036
| 74 |
0.304
|
0.009
| 88 |
− 0.258
|
0.015
| 76 | − 0.114 | 0.328 | 85 | − 0.141 | 0.197 | 88 | − 0.120 | 0.267 |
PFOA | 88 | 0.194 | 0.070 | 74 | 0.212 | 0.070 | 88 | − 0.031 | 0.777 | 76 | − 0.127 | 0.274 | 82 | 0.117 | 0.285 | 88 | 0.145 | 0.178 |
∑PFSA | 88 |
0.342
|
0.001
| 74 |
0.354
|
0.02
| 88 |
− 0.259
|
0.015
| 76 | − 0.114 | 0.328 | 82 | − 0.119 | 0.278 | 88 | − 0.106 | 0.326 |
∑PFCA | 88 | 0.191 | 0.074 | 74 | 0.210 | 0.073 | 88 | − 0.033 | 0.758 | 76 | − 0.123 | 0.288 | 82 | 0.115 | 0.294 | 88 | 0.142 | 0.187 |
∑PFSA+∑PFCA | 88 |
0.361
|
0.001
| 74 |
0.398
|
< 0.001
| 88 |
− 0.218
|
0.042
| 76 | − 0.103 | 0.374 | 82 | − 0.088 | 0.423 | 88 | − 0.065 | 0.545 |
Principal component analysis
Interpreted biomarker component | Biomarker name | Factor loadinga | Explained variance (%) | Explained variance cumulative (%) |
---|---|---|---|---|
PC-1 | PFOS | 0.853 | 12.27 | 12.27 |
PFOA | 0.696 | |||
PFOSA | 0.523 | |||
Cu | 0.452 | |||
AR-AEQ | − 0.386 | |||
ER-EEQ | 0.363 | |||
Fe | 0.358 | |||
PC-2 | AhR-TEQ | 0.834 | 10.08 | 22.35 |
Fe | 0.758 | |||
AR-AEQ | 0.658 | |||
PC-3 | I | 0.763 | 10.05 | 32.40 |
Zn | 0.706 | |||
T3-ThEQ | 0.589 | |||
Cr | 0.427 | |||
Pb | 0.326 | |||
PC-4 | Mn | 0.777 | 8.43 | 40.83 |
E2 | 0.763 | |||
ER-EEQ | 0.347 | |||
Cr | 0.316 | |||
PC-5 | Pb | 0.778 | 8.34 | 49.17 |
Cd | 0.729 | |||
Cu | 0.424 | |||
Cr | 0.327 | |||
PC-6 | Se | 0.746 | 7.76 | 56.93 |
As | 0.691 | |||
Cu | 0.480 | |||
PC-7 | testosterone | 0.818 | 7.71 | 64.63 |
ER-EEQ | 0.622 |
Crude | Adjusteda | ||||||
---|---|---|---|---|---|---|---|
n (cases/controls) | OR (95% CI) |
p
| n (cases/controls) | OR (95% CI) |
p
| FDR (qvalues) | |
PC-1 | 29/44 |
0.236 (0.092, 0.607)
|
0.003
| 29/44 |
0.100 (0.016, 0.630)
|
0.014
|
0.098
|
PC-2 | 29/44 | 0.793 (0.449, 1.398) | 0.422 | 29/44 | 0.943 (0.456, 1.948) | 0.873 | 0.961 |
PC-3 | 29/44 | 1.180 (0.733, 1.897) | 0.496 | 29/44 | 1.059 (0.538, 2.082) | 0.868 | 0.868 |
PC-4 | 29/44 | 1.167 (0.724, 1.880) | 0.526 | 29/44 | 1.027 (0.363, 2.903) | 0.961 | 0.961 |
PC-5 | 29/44 | 1.064 (0.668, 1.695) | 0.793 | 29/44 | 1.431 (0.810, 2.531) | 0.218 | 0.763 |
PC-6 | 29/44 | 1.061 (0.664, 1.697) | 0.804 | 29/44 | 1.258 (0.661, 2.393) | 0.485 | 0.849 |
PC-7 | 29/44 | 1.25 (0.773, 2.025) | 0.363 | 29/44 | 1.354 (0.662, 2.767) | 0.407 | 0.849 |