| Backgrounds and ObjectivesPreterm birth?PTB?is defined as birth before 37 completed weeks of gestation.It could cause great harm to the maternal and the offspring and cause mental stress and a huge economic burden to the families of preterm infants.The average incidence of PTB worldwide is 9.6%-11.1%,while the average incidence of PTB in China is 6.7%-11.0%.However,the incidence varies considerably in different regions.The related factors of PTB include PTB history,pregnancy complications,and nutritional status during pregnancy.In recent years,the association between vitamin D?Vit D?deficiency in pregnancy and the risk of PTB has been widely concerned.However,the current research conclusions are not consistent,and they mainly focus on Vit D during a certain trimester.Furthermore,the timing of detection is also inconsistent.There is still a lack of research on the association between the Vit D in the three trimesters and PTB.Besides,the single nucleotide polymorphism?SNP?in Vit D metabolic pathway genes are related to the level of 25?OH?D?25-hydroxyvitamin D?in plasma.However,previous studies have not considered that the variation of the Vit D metabolic pathway genes may affect the association between Vit D and the risk of PTB.Although the etiology of PTB has been extensively studied,it has not been elucidated.Most studies suggest that PTB is mainly related to infection and inflammation.Vit D has an anti-inflammatory effect,the levels of inflammatory cytokines are altered in pregnant women with Vit D deficiency.Pyroptosis is involved in the inflammatory process of many diseases and pyroptosis-related proteins are expressed in pregnancy-related tissues.There were no previous studies that investigated the possible mechanisms by which Vit D deficiency mediates pyroptosis in PTB.This study was carried out in Zhoushan Maternal and Child Health Hospital of Zhejiang Province.A cohort study design was used to explore the association of plasma 25?OH?D levels in the first,second and third trimesters with the risk of PTB,and to explore the association of Vit D and its metabolic pathway-related genes?GC,CYP24A1,CYP27B1,LRP2,VDR?with the risk of PTB.Besides,a 1:2 nested case-control study was designed to explore the association between the levels of IL-1?,IL-10,IL-18,TNF-?,Eotaxin,G-CSF in the third trimester with the risk of PTB.Besides,a 1:1 nested case-control study was designed to explore the difference of m RNA expression between NLRP3,Pro-IL-1?,Pro-Caspase-1,and ASC in the PTB and control group.This study aimed to provide scientific evidence for Vit D supplementation during pregnancy and PTB prevention strategies.Materials and MethodsThis study was conducted in Zhoushan Maternal and Child Health Hospital,Zhejiang,from August 2011 to May 2018.Participants'social-demographic characteristics,lifestyle factors,and other information were obtained from epidemiology questionnaires.Fasting blood samples were collected at three time points after obtaining informed consent.From the electronic medical record system?EMRs?of the hospital,we extracted medical history,gravidity,parity,pregnancy complications,and follow-up information of pregnant women from antenatal EMRs,and extracted the time of labor,gestational weeks,labor complications and other information from delivery EMRs.Genotyping for the selected genetic polymorphisms was performed using the Mass ARRAY system method.A high-performance liquid chromatography-tandem mass spectrometry was used to detect plasma 25?OH?D levels.Multiple statistical methods including linear regression,logistic regression,crossover analysis,and stratified analysis were applied to explore the association of Vit D in three trimesters and Vit D metabolic pathway-related genes with gestational week and the risk of PTB.Based on the above cohort study,a 1:2 nested case-control study matched by age was conducted.25 cases of PTB and 50 controls of full-term birth with blood samples in the third trimester were selected.A magnetic Luminex assay was used to test the plasma levels of inflammatory factors?IL-1?,IL-10,IL-18,TNF-?,Eotaxin,G-CSF?.Furthermore,a 1:1 matched nested case-control study was also conducted in 10 cases of PTB and 10 controls of full-term birth.5 m L peripheral blood sample at the third trimester was collected,and human peripheral blood mononuclear cells were isolated by density gradient centrifugation.The m RNA expression levels of NLRP3,Pro-IL-1?,Pro-Caspase-1,and ASC were detected by real-time PCR.Multiple linear regression models,conditional logistic regression model,and stratified analysis were applied to explore the association of Vit D in the third trimester,the inflammatory factors with gestational week and the risk of PTB.Differences between NLRP3,Pro-IL-1?,Pro-Caspase-1,and ASC m RNA expression in two groups were compared using the paired t test.ResultsA total of 3465 pregnant women,of which 202 were PTB,accounting for 5.8%,were included in the cohort study.3004,1849,and 1248 participants had detected25?OH?D in the first,second,and third trimester,respectively.The average 25?OH?D levels in three trimesters were 18.48±8.55 ng/m L,26.92±12.55 ng/m L,and 27.06±12.20 ng/m L,respectively.After adjusting for potential confounders,Vit D insufficiency??=0.28,P=0.004?and sufficiency??=0.25,P=0.015?in the second trimester was associated with higher gestational week compared with Vit D deficiency.Besides,Vit D sufficiency in the third trimester was associated with higher gestational week compared with Vit D deficiency??=0.22,P=0.013?.However,no significant association was found between Vit D and the risk of PTB.A total of 2245 pregnant women were included in the further analysis of Vit D metabolic pathway-related genes,of which 173 women were PTB.After adjusting for confounders,the gestational week of individuals with rs7041 CC genotype was lower,in comparison with that of the individuals with AA genotype??=-0.44,P=0.010?.Compared with rs10210408 CC genotype,the gestational week of individuals with TT genotype was higher??=0.30,P=0.018?,and had a decreased risk of PTB?OR=0.60,95%CI:0.37-0.97?.The gestational week of individuals with rs2228171 CC genotype was higher??=0.61,P=0.026?and had a decreased risk of PTB?OR=0.46,95%CI:0.23-0.94?compared with individuals with TT genotype.According to the results of crossover analysis,the gestational week of individuals with rs10210408 CC genotype and Vit D deficiency was significantly lower?second trimester:?=-0.63,P<0.001;third trimester:?=-0.38,P=0.030?compared with individuals with TT genotype and Vit D non-deficiency.In the second and third trimesters,the gestational week of individuals with rs2209314 CC genotype and Vit D deficiency was lower,in comparison with individuals with TT genotype and Vit D non-deficiency?second trimester:?=-0.58,P=0.005;third trimester:?=-0.50,P=0.022?.In the second and third trimester,compared with individual with rs2544381 GG genotype and Vit D non-deficiency,the gestational week of individual with CG genotype and Vit D deficiency was lower?second trimester:?=-0.88,P=0.007;third trimester:?=-0.99,P=0.001?.We also found that rs7041 and Vit D might exert interaction on gestational week and the risk of and Vit D might exert interaction on gestational week in the second trimester(P inter=0.036);rs4334089 and Vit D might exert interaction on gestational week in the third trimester(P inter=0.024).Similar results were found in pregnant women tested plasma25?OH?D in the first and second trimester.For spontaneous deliveries,there were significant associations of Vit D and rs7041 with the gestational week at first,second,and third trimesters.There were significant associations of Vit D and rs10210408 with the gestational week at first and second trimesters.Furthermore,significant associations of Vit D and rs1155563,rs10877012,rs2209314,rs11568820,rs4334089 with gestational week were found at second and third trimesters.We did not find significant associations between TNF-?,IL-10,IL-1?,IL-18,G-CSF,Eotaxin,and IL-10/IL-1?ratio with the risk of PTB.Compared with IL-18levels below 294.41 pg/m L,IL-18 levels above 294.41 pg/m L was associated with higher gestational week when Vit D deficiency??=2.98,P=0.040?.Similar results were found in the spontaneous delivery group.The m RNA expression of NLRP3 was higher in the PTB group than that in the control group?P=0.031?,while we did not find the significant difference in ASC,Pro-IL-1?and Pro-Caspase-1 m RNA expression levels.ConclusionsWomen with Vit D deficiency are associated with shorter gestational weeks.However,no significant association was found between maternal Vit D levels and the risk of PTB.SNPs in Vit D metabolic pathway genes were significantly associated with gestation week and the risk of PTB,mainly in GC and LRP2 genes.Besides,maternal Vit D and GC,VDR genes might exert interaction on the gestational week and the risk of PTB.Vit D deficiency in the third trimester might mediate PTB through inflammation induced by the NLRP3 inflammasome.Further studies with a large sample size are recommended for validation. |
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