Characteristics of participating NTD cases and controls are presented in Table 1. Virtually (99.5%) of all participants were of Han ethnicity. Generally, study subjects were young, with a mean age of 26.9 years in cases and 26.6 years in the controls. Compared to controls, cases were less likely to complete high school education and to report folic acid supplementation during the periconceptional period, more likely to work as a farmer, to report a history of pregnancy affected by birth defects, to have a fever or influenza, and to report passive smoking during the periconceptional period. Over half (56.4%) of the cases had a gestational age at placenta collection of fewer than 28 weeks, compared to only 2.4% in controls, reflecting the impact of elective termination following prenatal diagnosis in the case group. Most of the women had a normal prepregnancy body mass index (approximately 62%), with no difference being observed between the two groups. Approximately half of the participants (49%) were primigravida, and there was no difference in parity between the cases and controls.
Table 1. Characteristics of neural tube defects and healthy controls in northern China, 2003 to 2016.
Characteristics
|
Neural tube defects (N=408)
|
Controls
(N=593)
|
Pa
|
n (%)
|
n (%)
|
Age (years)
|
|
|
0.667
|
<25
|
168 (41.2)
|
240 (40.5)
|
|
25–29
|
117 (28.7)
|
182 (30.7)
|
|
30–34
|
79 (19.4)
|
119 (20.1)
|
|
≥35
|
42 (10.3)
|
47 (7.9)
|
|
Prepregnancy body mass index (kg/m2)
|
|
|
0.804
|
<18.5
|
31 (7.6)
|
48 (8.1)
|
|
18.5–24.9
|
252 (61.8)
|
372 (62.7)
|
|
≥25.0
|
114 (27.9)
|
162 (27.3)
|
|
Occupation, farmer
|
315 (77.2)
|
443 (74.7)
|
0.019
|
Education, ≥high school
|
73 (17.9)
|
173 (29.2)
|
<0.001
|
Gestational age at sample collection, <28 weeks
|
230 (56.4)
|
14 (2.4)
|
<0.001
|
Parity, 1
|
283 (47.7)
|
205 (50.2)
|
0.066
|
History pregnancy affected by birth defects, yes
|
21 (5.1)
|
9 (1.5)
|
0.003
|
Periconceptional affected by fever or influenza, yes
|
138 (33.8)
|
75 (12.6)
|
<0.001
|
Periconceptional folic acid supplementation, yes
|
130 (31.9)
|
264 (44.5)
|
<0.001
|
Periconceptional passive smoking, ever
|
248 (60.8)
|
234 (39.5)
|
<0.001
|
a Comparisons between NTD cases and controls were used the Chi-square test.
Descriptive data of metal concentrations in the placental tissue of the study subjects are presented in Table 2. Median concentrations of copper and silver were significantly higher in NTD cases than in controls, while the median concentration of titanium did not show a significant difference. When presented in geometric parameters, similar profiles were exhibited for the two groups. Anencephaly and spinal bifida, the two major subtypes of NTDs, showed a similar pattern for the three metals with total NTDs (Table S1).
Table 2. Concentrations of metals in placental tissue (dry weight) in neural tube defects and healthy controls in northern China, 2003 to 2016.
Metal
|
Median (Interquartile range)a
|
Geometric mean±Standard deviationb
|
NTDs (N=408)
|
Controls (N=593)
|
NTDs (N=408)
|
Controls (N=593)
|
Cu (μg/g)
|
4.16 (3.65–4.91)*
|
3.91 (3.47–4.49)
|
4.42±1.30*
|
4.10±1.09
|
Ag (ng/g)
|
0.96 (0.56–1.57)*
|
0.71 (0.16–1.29)
|
1.41±1.84*
|
0.98±1.06
|
Ti (μg/g)
|
0.65 (0.57–0.74)
|
0.63 (0.57–0.71)
|
0.69±0.40*
|
0.65±0.12
|
NTD, neural tube defect, Cu, copper, Ag, silver, Ti, titanium, *P<0.001.
a Comparisons between NTDs and controls were used Mann-Whitney U test.
b Comparisons between NTDs and controls were used Student's t-test.
Variations in metal concentrations were observed among the five counties where the study subjects were recruited (Table S2). The variations by region were then considered with multilevel mixed-effects logistic regression in the following analyses.
The associations between tertile concentrations of each metal and risk for NTDs are displayed in Table 3. In single metal regression models, the highest tertile of copper was associated with a 2.28-fold (1.64–3.17) higher risk for NTDs in univariate analyses. However, this association disappeared when confounding factors were adjusted. For silver, the risk for NTDs increased by 2.02-fold (95% CI 1.45–2.82) and 2.19-fold (95% CI 1.55–3.11) in the second and the highest tertiles compared to the lowest tertile. These associations remained after adjustment for potential confounders, and a dose-response relationship remained with an adjusted OR of 1.78 (95% CI 1.04–3.06) and 1.92 (95% CI 1.11–3.32) for the second and highest tertiles, respectively. No association between titanium levels and NTD risk was observed in either unadjusted or adjusted models. In multiple-metal regression models, however, the association between silver concentrations and NTD risk turned to non-significant after adjustment for potential confounders. Similar patterns of associations were presented for anencephaly and spina bifida (Table S3).
Table 3. Associations between placental tertile concentrations of metals (dry weight) and risk for neural tube defects in northern China, 2003 to 2016.
Metal
|
OR (95% CI) a,
|
Adjusted OR (95% CI) a, b
|
One-metal model
|
Three-metal model
|
One-metal model
|
Three-metal model
|
Cu (μg/g)
|
1.00 (Reference)
|
1.00 (Reference)
|
1.00 (Reference)
|
1.00 (Reference)
|
|
1.34 (0.96–1.87)
|
1.24 (0.89–1.71)
|
0.66 (0.39–1.11)
|
0.64 (0.38–1.08)
|
|
2.28 (1.64–3.17)*
|
1.93 (1.40–2.67)*
|
1.24 (0.74–2.06)
|
1.17 (0.70–1.96)
|
Ptrend
|
<0.001
|
<0.001
|
0.451
|
0.582
|
Ag (ng/g)
|
1.00 (Reference)
|
1.00 (Reference)
|
1.00 (Reference)
|
1.00 (Reference)
|
|
2.02 (1.45–2.82)*
|
2.02 (1.46–2.80)*
|
1.78 (1.04–3.06)*
|
1.69 (0.98–2.92)
|
|
2.19 (1.55–3.11)*
|
1.97 (1.41–2.74)*
|
1.92 (1.11–3.32)*
|
1.74 (0.99–3.06)
|
Ptrend
|
<0.001
|
<0.001
|
0.023
|
0.063
|
Ti (μg/g)
|
1.00 (Reference)
|
1.00 (Reference)
|
1.00 (Reference)
|
1.00 (Reference)
|
|
0.88 (0.64–1.21)
|
0.85 (0.61–1.17)
|
0.77 (0.46–1.29)
|
0.76 (0.45–1.28)
|
|
1.09 (0.79–1.50)
|
1.01 (0.73–1.40)
|
1.00 (0.61–1.67)
|
0.95 (0.57–1.59)
|
Ptrend
|
0.608
|
0.686
|
0.970
|
0.859
|
Cu, copper, Ag, silver, Ti, titanium, OR, odds ratio, CI, confidence interval, *P<0.05.
a Calculated by multilevel mixed-effects logistic regression, geographical region as a random effect.
b Adjusted for maternal occupation, education, gestational age at sample collection, history of pregnancy affected by birth defects, have had a fever or influenza, passive smoking, and folic acid supplementation during the periconceptional period.
In BKMR, silver was the only metal that shown risk effects on NTDs, as indicated by its three point estimates, which are greater than the null value of zero and their lower boundaries of credible intervals exclude the null. Moreover, the three point estimates of silver are almost identical, suggesting that silver's effect is independent of the other two metals (Figure 1A). No associations between concentrations of copper and titanium and NTD risks were observed (Figure 1A). As Figure 1B indicates, NTD risks increased linearly with silver concentrations, although the curve flatted at the highest silver concentrations. No clear evidence of association was shown for copper and titanium concentrations and risk for NTDs (Figure 1B). No interaction between metals was observed because the slopes of a specific metal were similar when the other metal was set at the 25th, 50th, or 75th percentile while the third one being held at its median (Figure 1C), and none of the interactive effects was significant as the credible intervals for copper, silver, and titanium encompass the zero null value (Figure 1D). When metals were treated as a mixed exposure mixture, NTD risk increased almost linearly across the whole range of mixture concentrations from the 25th percentile through the 75th percentile (Figure S1).
Correlations between frequencies of maternal food groups during the periconceptional period and placental metal concentrations are shown in Table 4. No correlation was found between maternal diet and copper levels, except for the consumption of meat or fish. Higher consumption frequencies of meat or fish, egg or milk, fresh vegetables and fruits, beans or its products, and tea-drinking were positively correlated with concentrations of silver. In addition, increased intake of egg or milk and fresh vegetables and fruits was positively correlated with titanium concentrations.
Table 4. Correlations between placental metal concentrations (dry weight) and frequencies of maternal food consumption in northern China, 2003 to 2016.
Dietary intakea
|
Copper (μg/g)
|
Silver (ng/g)
|
Titanium (μg/g)
|
r
|
P
|
r
|
P
|
r
|
P
|
Meat or fish
|
–0.066
|
0.037
|
0.189
|
<0.001
|
0.511
|
0.991
|
Egg or milk
|
–0.029
|
0.364
|
0.217
|
<0.001
|
0.094
|
0.003
|
Fresh vegetables
|
–0.033
|
0.304
|
0.110
|
0.001
|
0.069
|
0.030
|
Fresh fruits
|
–0.007
|
0.823
|
0.177
|
<0.001
|
0.100
|
0.002
|
Bean or its products
|
–0.007
|
0.836
|
0.219
|
<0.001
|
0.038
|
0.232
|
Tea drinking
|
–0.062
|
0.056
|
0.111
|
0.001
|
0.056
|
0.080
|
a Frequency of dietary intake was classified into three levels, i.e., <1, 1 to 6, and >6 times per week.
Ninety-two pairs were obtained when cases and controls were matched by the length of gestation. Similar patterns of placental concentrations of silver in cases and controls were present with the overall analyses (Table S4). Although the dose-response relationship for silver and NTD risk was not significant, largely because of the reduced sample size, the direction of the association was the same as the total sample (Table S5), suggesting that the observed associations were unlikely to be resulted by confounding of gestation. The results did not change meaningfully when NTDs complicated with other malformations were excluded (Table S6 and Figure S2).