Basic information for 3,080 pregnant women<
In this study, 3,080 pregnant women were included as subjects. Their age range was 15–46 years old, the average age was 26.94 ± 3.95 years old, and most pregnant women were younger than 35 years old (94.19%). The sampling gestational age range was 10–32 weeks, and the average age was 17.89 ± 6.60 weeks. Most pregnant women were sampled during early or mid-gestation (90.75%), and the season was distributed in spring, summer, autumn and winter, but mostly in summer and autumn, which accounted for 29.87% and 31.23%, respectively; in this study, the parity includes the first and second or more, and most of the pregnant women were pregnant with their first child (86.30%). The age, pregnancy, sampling season and parity of 3,080 pregnant women are shown in Table 1.
Table 1
Demographic characteristics of pregnant women(N = 3080)
projects | | Number(n) | percentage(%) |
Age | 15 ≤ Age༜25 | 870 | 28.25 |
| 25 ≤ Age༜35 | 2031 | 65.94 |
| 35 ≤ Age ≤ 46 | 179 | 5.81 |
Pregnancy staging | Early pregnancy(10 week ≤ GW༜13 week) | 1025 | 33.28 |
| Second trimester(13 week ≤ GW༜28 week) | 1770 | 57.47 |
| Late pregnancy(28 week ≤ GW ≤ 32 week) | 285 | 9.25 |
Season | Spring | 578 | 18.77 |
| Summer | 920 | 29.87 |
| autumn | 962 | 31.23 |
| Winter | 620 | 20.13 |
Number of fetuses | First child(p = 1) | 2658 | 86.30 |
| Second child or above(p > 1) | 422 | 13.70 |
Vitamin D Expression In Maternal Serum
The serum vitamin D test results showed that the serum 25-(OH)D level of the pregnant women included in the study ranged from 10.0-93.9 nmol/L, and the median and quartile levels were 33.7 (24.7, 44.7) nmol/L. Among them, the number of pregnant women with severe deficiency, deficiency and sufficient vitamin D levels were 2,565 cases, 473 cases and 42 cases, accounting for 83.28%, 15.36% and 1.36%, respectively. The above results showed that the majority of pregnant women in the region had 25-(OH)D levels of less than 50 nmol/L and were in a state of vitamin D deficiency (Fig. 1A, 1B).
The influence of age and other factors on the expression of serum vitamin D in pregnant women
1. Single factor analysis of the influence of age and other factors on serum vitamin D levels in pregnant women
In this experiment, we performed a single factor analysis of age, pregnancy, sampling season, parity and serum 25-(OH)D levels in pregnant women. The results showed that there was a statistically significant difference in the effect of age on serum vitamin D levels in pregnant women (H=15.677, P<0.001). The expression level of vitamin D was the lowest in the lowest age group and the highest in the highest age group. The difference in pregnancy also had a significant effect on the serum vitamin D level of pregnant women (H=12.334, P=0.002). The level of vitamin D was the lowest in the early pregnancy group but the highest in the third trimester group.
In order to investigate the effect of the season on serum vitamin D levels in pregnant women, serum was collected in different seasons, and the results showed that the season was statistically significant (H=74.736, P<0.001). We also confirmed that parity affects the expression of serum vitamin D in pregnant women (H=26.192, P<0.001). The severe deficiency rate, deficiency rate and sufficiency rate of serum vitamin D in pregnant women with different influencing factors showed that factors such as high age, third trimester, summer and autumn, and second or higher pregnancy could significantly increase the expression of vitamin D (Table 2).
Table 2
Single factor analysis of the influence of age and other factors on serum vitamin D levels in pregnant women
Groups | samples(n) | V D level(nmol/L) | Z/P | deficiency(%) | X2/P | insufficiency (%) | X2/P | sufficiency(%) | X2/P |
Age | Low(15 ≤ Age༜25) | 870 | 32.1(24.1,43.1) | 10.588/ 0.005 | 85.06 | 3.389/0.184 | 13.91 | 3.368/0.186 | 1.03 | 1.832/0.400 |
Medium(25 ≤ Age༜35) | 2031 | 33.9(24.7,45.1) | 82.77 | 15.66 | 1.58 |
High(35 ≤ Age ≤ 46) | 179 | 37.3(28.7,48.0) | 80.50 | 18.99 | 0.56 |
Period | Early pregnancy(10 week ≤ GW༜13 week) | 1025 | 32.4(24.2,42.6) | 4.771/ 0.092 | 82.63 | 0.461/0.794 | 15.71 | 0.147/0.929 | 1.66 | 1.079/0.583 |
Second trimester(13 week ≤ GW༜28 week) | 1770 | 50.7(25.0,44.8) | 83.62 | 15.20 | 1.19 |
Late pregnancy(28 week ≤ GW ≤ 32 week) | 285 | 35.1(26.7,47.4) | 83.51 | 15.09 | 1.40 |
Season | Spring | 578 | 32.3(22.5,40.5) | 44.736/ ༜0.001 | 86.16 | 4.851/0.183 | 13.15 | 3.262/0.353 | 0.69 | 9.075/0.028 |
Summer | 920 | 40.3(28.3,58.5) | 81.85 | 16.30 | 1.85 |
autumn | 962 | 39.0(27.7,56.8) | 83.06 | 15.18 | 1.77 |
Winter | 620 | 28.9(20.8,39.6) | 83.06 | 16.29 | 0.65 |
Number of fetuses | First child(p = 1) | 2658 | 35.5(24.9,46.8) | 1.064/ 0.287 | 83.52 | 0.614/0.433 | 15.35 | 0.279/0.598 | 1.13 | 0.862/0.353 |
Second child or above(p > 1) | 422 | 38.3(25.3,47.2) | 81.99 | 16.35 | 1.66 |
| Total | 3080 | | | 83.28 | | 15.36 | | 1.36 | |
2. Multivariate logistic regression analysis of the influence of age and other factors on serum vitamin D levels in pregnant women
Univariate analysis showed that age, pregnancy and other factors had significant effects on serum vitamin D levels in pregnant women. Therefore, multivariate logistic regression was used to comprehensively analyze the influence of various factors. During the analysis, the vitamin D level was used as a dependent variable, where serum vitamin D levels < 75 nmol/L (deficiency or insufficiency) were assigned a value of 0 and serum vitamin D levels ≥ 75 nmol/L were assigned a value of 1. The independent variable X1 (age) used as the reference level 15 years ≤ age < 35 years old, the independent variable X2 (pregnancy week) used as the reference level 10 weeks ≤ gestational age < 28 weeks, and the independent variable X3 (season) used as the reference winter and spring. The independent variable X4 (parity) used the first child as the reference level, and the respective variable assignment is shown in Table 3.
Table 3
Logistic regression independent variable assignment
Factor | variable | X = 0 | X = 1 |
Age | X1 | 15 ≤ Age༜35 | 35 ≤ Age ≤ 46 |
Period | X2 | 10 weeks ≤ GW༜28 weeks | 28 ≤ GW ≤ 32 |
Season | X3 | Winter/spring | Autumn/summer |
Number of fetuses | X4 | First child | Second child or above(p > 1) |
The results of stepwise regression analysis showed that there was no collinearity between the independent variables. Multivariate logistic regression analysis showed that the summer and autumn were protective factors for vitamin D in pregnant women (Table 4).
Table 4
Multivariate logistic regression analysis
Factor | Β-value | S.E-value | Wald X2-value | Sig-value | OR-value | OR-value 95%CI |
X1 | 1.251 | 0.216 | 9.132 | 0.121 | 2.872 | 1.523ཞ4.615 |
X2 | 1.204 | 0.198 | 13.085 | 0.086 | 2.675 | 1.231ཞ4.162 |
X3 | 2.326 | 0.187 | 15.732 | ༜ 0.001 | 4.383 | 2.532ཞ5.967 |
X4 | 2.051 | 0.232 | 3.824 | 0.158 | 2.796 | 1.015ཞ5.168 |
3. Linear regression analysis of the effect of temperature on serum vitamin D levels in pregnant women
In order to prove whether the effect of the season on vitamin D is due to temperature changes, we conducted a linear regression analysis of the local monthly mean temperature and the serum vitamin D level of pregnant women during the study period. The analysis showed that the serum vitamin D level of pregnant women was highly positively correlated with temperature. The higher the temperature, the higher was the serum 25-(OH)D level (R2 = 0.823, t = 6.818, P < 0.001) (Fig. 2A, 2B).
Correlation between serum vitamin D levels and common adverse pregnancy outcomes in pregnant women
In this experiment, we grouped pregnant women according to vitamin D levels, including the deficiency group, insufficient group and sufficient group. Univariate analysis showed that the incidence of preterm birth and spontaneous abortion was the highest in the deficiency group, and the difference was statistically significant (P < 0.05, Table 5).
Table 5
Univariate analysis of vitamin D levels in maternal serum and common adverse pregnancy outcomes
Serum V D level | Number | Gestational diabetes | Hypertension during pregnancy | Premature rupture of membranes |
Samples | X2/P | RR/95%CI | Samples | X2/P | RR | Samples | X2/P | RR |
Deficiency group | 2565 | 209 | 1.717/0.424 | 0.86(0.334ཞ2.193) | 192 | 0.582/0.748 | 0.79(0.306ཞ2.016) | 498 | 1.064/0.587 | 1.17(0.590ཞ2.301) |
Insufficiency group | 473 | 47 | 1.04(0.395ཞ2.755) | 32 | 0.71(0.264ཞ1.912) | 83 | 1.05(0.521ཞ2.128) |
sufficiency group | 42 | 4 | 1.00 | 4 | 1.00 | 7 | 1.00 |
To investigate the effects of different serum vitamin D levels on pregnancy outcomes, we performed a univariate analysis and found no significant effect of vitamin D expression on pregnancy outcomes, including gestational diabetes mellitus and hypertensive disorder complicating pregnancy (P > 0.05, Table 6).
Table 6
Univariate analysis of maternal serum with vitamin D levels and common adverse pregnancy outcomes
Serum V D level | Number | Cesarean section | Premature birth | Natural abortion |
Samples | X2/P | RR/95%CI | Samples | X2/P | RR | Samples | X2/P | RR |
Deficiency group | 2565 | 1202 | 2.560/0.278 | 0.90(0.669ཞ1.197) | 195 | 8.373/0.015 | 3.19(0.458ཞ22.246) | 204 | 11.727/0.003 | 3.34(0.480ཞ23.267) |
Insufficiency group | 473 | 239 | 0.97(0.713ཞ1.305) | 20 | 1.78(0.244ཞ12.906) | 18 | 1.60(0.219ཞ11.678) |
sufficiency group | 42 | 22 | 1.00 | 1 | 1.00 | 1 | 1.00 |
Correlation analysis of vitamin D levels in maternal serum and small for gestational age (SGA) neonates
In this study, 3,080 pairs of mothers and children met the criteria included in the study, of which 423 were born with SGA, for an incidence rate of 13.71%. A single factor analysis of the serum vitamin D levels and incidence of SGA in pregnant women revealed that the incidence of SGA in the deficiency group, the insufficient group and the sufficient group were 15.52%, 5.07%, and 2.38%, respectively. After statistical analysis, we found that the serum vitamin D level had a statistically significant effect on the incidence of SGA (X2 = 41.390, P < 0.001), and the incidence of SGA was highest in the deficient group. The risk of SGA in the deficient group and the insufficient group was 6.52 times and 2.13 times the sufficient group, respectively (Table 7).
Table 7
Univariate analysis of the incidence of 25-(OH)D levels in maternal serum and small for gestational age (SGA)
VD Level | Total | SGA | Incidence rate(%) | X2/P | Relative risk(RR) | 95%CI |
Deficiency group | 2565 | 230 | 8.97 | 10.827/0.004 | 3.77 | 0.541ཞ26.218 |
Insufficiency group | 473 | 23 | 4.86 | 2.04 | 0.283ཞ14.749 |
sufficiency group | 42 | 1 | 2.38 | 1.00 | --- |
Vitamin D levels inhibit the expression of inflammatory factors
In order to demonstrate the effect of vitamin D expression on inflammatory factors, RT-PCR was used to detect the gene expression of IL-6 and TNF-α. In this experiment, subjects with higher serum vitamin D levels and lower vitamin D levels were used for experimental studies, of which 46 subjects had high serum vitamin D and 42 subjects had low vitamin D, named the high VD group and low VD group, respectively. The results showed that compared with the low VD group, the expression of IL-6 and TNF-α mRNA in the high VD group was significantly decreased, indicating that the expression of inflammatory factors may be negatively correlated with the content of vitamin D (Fig. 2A, 2B).
High vitamin D expression can inhibit the production of inflammation-related proteins
To investigate the effect of vitamin D on IL-6 and TNF-α protein expression (Fig. 3A, B), we detected IL-6 and TNF-α protein by western blot. The results showed that IL-6 and TNF-α protein expression was low in the high VD group compared with the low VD group (P < 0.01). These results indicate that IL-6 and TNF-α proteins are expressed at low levels when vitamin D levels are higher, while low levels of vitamin D can activate the expression of IL-6 and TNF-α protein, indicating that vitamin D and IL-6 and TNF-α protein may be negatively correlated. However, the specific mechanism requires further research.