Maternal Cadmium Exposure During Pregnancy Is Associated With Fetal Growth Restriction And Its Mechanism

The association between maternal exposure to cadmium during pregnancy and fetal growth restriction(FGR) remains controversial. The current study will analyze the relative risk(RR)and 95% confidential interval CI)of low birth weight(LBW) and small for gestational age(SGA) among subjects with high cadmium level during pregnancy when compared to the subjects with low cadmium level in Hefei Birth Cohort, which based on a exsisting research platform for China Anhui Birth Cohort Study(C-ABCS). Based on the established mouse model of growth restriction in fetuses whose mothers were exposed to cadmium during pregnancy, we will observeon cadmium distribution in maternal mice, placentas and fetal mice, and the adverse effects of maternal cadmium exposure on placental structure and function. In vivo study, we will observe the regulatory effects of maternal cadmium during pregnancy on placental endoplasmic reticulum stress in mice. In vitro study, we will further explore the modulatory effects of cadmium treatment on endoplasmic reticulum stress in human trophoblasts. To clarify the role of reactive oxygen species(ROS) in cadmium-induced endoplasmic reticulum stress in placental trophoblasts, the present study investigate the antagonistic effects of N-tert-butyl-α-phenylnitrone(PBN) on cadmium-induced placental endoplasmic reticulum stress. To clarify the role of IRE1 signaling pathway in cadmium-impaired placental trophoblast function, we will explore the antagonistic effects of IRE1 si RNAs transfection on cadmium-downregulated nutrient transporters and growth factors in human trophoblasts. The present study will provide a scientific basis for clarifying the mechanism in cadmium-induced FGR.Part 1: Maternal exposure to cadmium during pregnancy is associated with fetalgrowth restriction(FGR)Objective The present study will analyze the RR and 95% CI of LBW and SGA among subjects with high cadmium level during pregnancy when compared to the subjects with low cadmium level.Methods A subsample of the China-Anhui Birth Cohort Study(C-ABCS) The present study analyzed. Total 3352 mother-and-singleton-offspring pairs were eligible for this study. Maternal data, birth weight and maternal serum were collected. Maternal serum cadmium content during pregnancy was detected by graphite furnace atomic absorption spectrometry(GFAAS).Results Average maternal serum cadmium concentration was 0.89 mg/L in the birth cohort study. According to maternal serum cadmium level, the subjects were divided into high cadmium(≥P75) and low cadmium(<P75). The incidence and RR of LBW and SGA infants based on maternal serum cadmium level was analyzed. Results on maternal data of three trimesters showed 4.3% newborns were with LBW among subjects with high cadmium level(RR: 2.25; 95%CI: 1.45, 3.49; P<0.001). Adjusted RR for LBW was 2.21(95%CI: 1.43, 3.42; P<0.001) among subjects with high cadmium level using multiple logistic regression model. Ten point five percents of newborns were with SGA among subjects with high cadmium level(RR: 1.43; 95%CI: 1.09, 1.86; P=0.009). Adjusted RR for SGA was 1.41(95%CI: 1.08, 1.85; P=0.011) among subjects with high cadmium level. Results on maternal data of the first trimester showed 2.5% newborns were with LBW among subjects with high cadmium level(RR: 1.26; 95%CI: 0.52, 3.08; P=0.608). Adjusted RR for LBW was 1.26(95%CI: 0.52, 3.08; P=0.606) among subjects with high cadmium level. Nine point four percents of newborns were with SGA among subjects with high cadmium level(RR: 1.20; 95%CI: 0.75, 1.93; P=0.446). Adjusted RR for SGA was 1.18(95%CI: 0.73, 1.90; P=0.498) among subjects with high cadmium level. Results on maternal data of the second and third trimester showed 5.2%newborns were with LBW among subjects with high cadmium level(RR: 2.70; 95%CI: 1.63, 4.49; P<0.001). Adjusted RR for LBW was 2.79(95%CI: 1.68, 4.66; P<0.001) among subjects with high cadmium level. Eleven point one percents of newborns were with SGA among subjects with high cadmium level(RR: 1.55; 95%CI: 1.12, 2.13; P=0.008). Adjusted RR for SGA was 1.54(95%CI: 1.11, 2.12; P=0.009) among subjects with high cadmium level.Conclusion Maternal exposure to high cadmium in the second and third trimesters elevated the risk of LBW and SGA infants.Part 2: Adverse effects of maternal exposure to cadmium during pregnancy on fetal development, placental structure and function in miceObjective The present study will explore the adverse effects of maternal exposure to cadmium during pregnancy on fetal development and placental development in mice.Methods This study consisted of three experiments. Experiment 1, the present study established two mouse models of growth restriction in fetuses whose mothers were exposed to cadmium during pregnancy. One was maternal cadmium chloride(4.5 mg/kg, i.p.) exposure at GD 9; the other was maternal cadmium chloride(0.5 mg/kg, i.p.) exposure from GD 13 to GD 17. All pregnant mice were killed at GD 18. Fetal crown-rump length and fetal weight were measured. Experiment 2, to explore the immediate and long-term effcts of maternal exposure to cadmium during pregnancy on cadmium distributions in maternal mice, placentas and fetal mice, the pregnant mice were injected with cadmium chloride(4.5 mg/kg, i.p.) at GD 9, and killed at GD 10 and GD 18. Maternal sera, maternal livers and kidneys, placentas and fetal samples were collected. Cadmium content in the samples was detected using GFAAS. Experiment 3, to investigate the effects of maternal exposure to cadmium during pregnancy on placental structure and function in mice, The pregnant mice were injected withcadmium chloride(4.5 mg/kg, i.p.) at GD 9. Mouse placentas were collected, weighted and measured at GD 10 and GD 18. Placenta was analyzed by histopathological method and quantitative RT-PCR technique.Results Experiment 1 indicated maternal cadmium exposure in the second or third trimester markedly decreased crown-rump length and fetal weight, whereas cadmium in the doses didn’t lead to maternal signs of poisoning. These results suggested that maternal exposure to cadmium during pregnancy induced FGR. Experiment 2 showed that cadmium level in maternal serum,maternal liver and kidney from GD 10 mice was increased by 2.8, 80.5, and 13.4 times, respectively; Cadmium level in placenta and embryo at GD 10 was increased by 60.2 and 11.5 times; Cadmium content in maternal serum,maternal liver and kidney from GD 18 mice was increased by 2.2, 300.9, and 34.3 times, respectively; Cadmium level in placenta and embryo at GD 18 was increased by 47.7 and 4.9 times. The above results suggested that maternal cadmium exposure during pregnancy markedly elevated cadmium level in mouse placentas, whereas a small part of cadmium was transported to fetal mice via placenta. Experiment 3 showed that maternal exposure to cadmium during pregnancy significantly reduced placenta weight and diameter. HE staining showed average blood sinusoid area of the labyrinth layer was obviously decreased in cadmium-treated placenta. TUNEL and immunostaining for proliferating cell nuclear antigen(PCNA) showed that maternal exposure to cadmium during pregnancy markedly induced cellular apoptosis, and inhibited cellular proliferation in the labyrinth layer of mouse placenta. Results of q RT-PCR showed that m RNA expression of nutrient transporters(Glut1, Znt1, Zip1 and Pcft), growth factors and their receptors(Pgf, Igf2, Vegfr1 and Igf2r) was significantly decreased in cadmium-treated placenta at GD 10.Conclusion These results suggested that maternal cadmium exposure during pregnancy markedly increased cadmium content in mouse placenta, impaired placental structure and function, and induced fetal growth restriction.Part 3: Role of IRE1 signaling pathway in cadmium-impaired placental trophoblast function and its mechanismObjective The current study will investigate the role of reactive oxygen species(ROS) in cadmium-induced placental ER stress and the role of IRE1 signaling pathway in cadmium-impaired placental trophoblast function.Methods This study consisted of three experiments. Experiment 1, to explore the effects of cadmium treatment on placental endoplasmic reticulum(ER) stress, the pregnant mice were injected with cadmium chloride(4.5 mg/kg, i.p.) at GD 9. Mouse placentas were collected at 0, 2, 8 and 24 h after cadmium treatments. Expression of ER stress-related m RNA and proteins was detected in mouse placenta. Experiment 2, to determine the role of reactive oxygen species(ROS) in cadmium-induced placental ER stress, GD 9 mice were administered with two doses of PBN, one(100 mg/kg, i.p.) injected 0.5 h before cadmium chloride(4.5 mg/kg, i.p.) treatment, and the second(100 mg/kg, i.p.) injected 4 h after cadmium chloride(4.5 mg/kg, i.p.) treatment. Mouse placentas were collected at 8 and 24 h after cadmium treatments. Oxidative stress and ER stress-related indicators were analyzed in mouse placentas. Experiment 3, to explore the role of IRE1 signaling pathway in cadmium-impaired placental trophoblast function, human JEG-3 cells were transfected with IRE1 si RNAs before cadmium treatments. Cells were collected at 12 h after cadmium treatments. The m RNA expression of nutrient transporters and growth factors was analyzed using q RT-PCR technique.Results Experiment 1 showed that maternal exposure to cadmium during pregnancy significantly up-regulated the expression of ER stress-related m RNA(Grp78 和 Atf4) and proteins(GRP78, pe IF2? and CHOP) in placenta. Cell research showed that different concentrations of cadmium obviously upregulated ER stress-related proteins(p IRE1?, pe IF2?, CHOP, p JNK and GRP78) in human trophoblasts in time-dependent relationships. Protein expression of GRP78 in human trophoblasts was significantlyupregulated at 2 h, whereas protein expression of GRP78 in human trophoblasts was markedly downregulated at 12 and 24 h after cadmiun treatment. Additionally, protein expression of p IRE1? in human trophoblasts was significantly upregulated at 6 h, whereas protein expression of p IRE1? in human trophoblasts was markedly downregulated at 24 h after cadmium treatment. However, protein expression of pe IF2?, CHOP and p JNK was increased in human trophoblasts with time. These results suggested that cadmium obviously induced ER stress and unfolded protein response(UPR) in placental trophoblasts. Experiment 2 showed that PBN pretreatments markedly attenuated cadmium-upregulated placental ER stress-related proteins(GRP78, pe IF2? and CHOP), alleviated cadmium-induced placental glutathione(GSH) depletion, and antagonised cadmium-upregulated the protein expression of 3-nitrotyrosine(3-NT) and heme oxygenase 1(HO-1) in mouse placentas. Experiment 3 showed that cadmium obviously downregulated the expression of nutrient transporters(GLUT1, FATP4, PCFT) and growth factors(PGF, IGF2) m RNA. IRE1 si RNAs pretreatment markedly antagonised cadmium-downregulated the m RNA expression of growth factors(PGF, IGF2).Conclusion The above results suggested that ROS partially contributed to cadmium-induced placental ER stress, whereas IRE1 signaling pathway played a key role in cadmium-impaired endocrine function in placental trophoblasts.Taken together, the forementioned results allowed us to draw three conclusions. At first, maternal exposure to cadmium during pregnancy increased the risk of FGR. Next, maternal cadmium exposure during pregnancy markedly increased cadmium content in mouse placenta, impaired placental structure and function, and induced fetal growth restriction. Finally, maternal cadmium exposure during pregnancy induced placental ER stress and UPR in mice. ROS played a key role in cadmium-triggered placental ER stress, whereas IRE1 signaling pathway exerted a mediate effect on cadmium-impaired endocrine function in placental trophoblasts.