| 1. Background and Objection1.1 BackgroundCongenital heart diseases (CHD) was cardiovascular malformations caused by abnormal cardiovascular development during embryo,which characterized by the heart and great vessels in the shape and structure abnormalities or the route should be closed after birth automatically.Common clinical types include ventricular septal defect,atrial septal defects,patent ductus arteriosus,pulmonary stenosis,aortic stenosis,tetralogy of Fallott and complete transposition of the great arteries etc. Some of the patients with CHD will die in infancy, and the survival children with CHD need more medical care and surgery, which will bring heavy mental and economic burden for families and society.Worldwide, CHD had been the most common type of birth defects,the incidence of which was about 4%o to 10%o, more than one million infants born every year.In our country, CHD was the top of birth defects,the incidence of live birth babies was 8%o to 10%o, prenatal morbidity was 10%o to 20%o,1.5 million neonates with CHD had been born every year,and it was about 30% of them would die in infancy,which account for more than 50% of birth defects.Furthermore the surgical treatment for CHD had been up to 120 billion yuan.The U.S. Centers for Disease Control and Prevention reported that about 9% to 18% of CHD resulted from gene or chromosomal abnormalities,the other caused by a combination of genetic and environmental risk factors.Especially in low and middle income countries, the reasons such as poverty and malnutrition during pregnancy were more common.Thus,70% to 80% of CHD could be prevented through improving these adverse factors.Diet is the most easy way to affect the fetus through the placenta,but globally, approximately two billion people, the majority of which were women and young children, were suffering from micronutrient deficiencies, even higher during pregnancy.Many domestic and foreign surveys had found that unreasonable intake of nutrients during pregnancy were very common around the world, and kinds of nutriment shortage were also not completely the same.A large cohort study recruited 2019 pregnant women in London showed that despite higher socioeconomic status,the intake of iron, zinc and folic acid was still below the recommended dietary intake ratio,1/5 pregnant women did not add any of these nutrients supplements.In 2010 the maternal nutritional status of nine provinces (including municipalities) in China indicated that the occurrence of nutrients deficiency during pregnancy was about 15%.A diet investigation of pregnant women in Szechwan found that the dietary energy source of pregnant women in the region:excess fat (39%), low carbohydrate (49.6%), protein only met minimum requirement (12.1%),compared with the rural area,pregnant women in urban area had significantly higher reference nutrient intake(RNI) of maternal malnutrition deficiency in developing countries.A large number of evidence supports the concept that human pregnancy outcome was significantly influenced by the nutritional status of the mother,if pregnant women suffered the lack of nutrients during pregnancy,it would lead to fetal growth restriction, even miscarriage, premature birth, and deformity.There had a large number of studies shown that the lack of certain nutrients associated with CHD,in the sensitive period of rat embryonic development, iron deficiency could cause cardiac enlargement,maternal B vitamins deficiency during pregnancy might increase the risk of fetal aorta transposition,maternal zinc deficiency during pregnancy might cause cardiac anomalies of embryo.As a special economic zone city with a dense population.lt was necessary to understand the incidence and epidemiological characteristics of CHD in nearly five years of which,and analyze the relationship between the maternal intake of nutrients among periconception and fetus CHD.We wanted to find the potential influencing factors of CHD and put forward prevention measures for the primary prevention of CHD.Zinc, as one of the trace elements in human body, played an important role in human health and development,its biological function included:taking participate in synthesizing more than 300 kinds of zinc-containing metal-loenzyme,certain proteins and cell membrane,regulating the DNA replication, repair, and transcription; promoting the growth and development,maintaining normal appetite, taste and the normal immune function,maintaining the stability of cell membrane,taking participate in the metabolite of Vitamin A, etc.Zinc deficiency was one of the ten biggest factors contributing to burden of diseases in developing countries,populations in South Asia, South East Asia, and sub-Saharan Africa were at greatest risk of zinc deficiency. A study conducted in rural Bangladesh showed that about 55% pregnant women were zinc deficient in first trimester[161.Literature around the world had shown 12% to 40% pregnant women suffering zinc deficiency.Scholars had studied 211 cases of pregnant women and women of childbearing age in Xiangyang,whose 15 elements content of hair,and found that there was a significant lack of zinc in both women of reproductive age and pregnant women in the region,and it was more obvious in pregnant women(respectively the constituent ratio of zinc deficiency was78.82%and 82.89%).Zinc deficiency could result in adverse pregnancy outcome.Animal experiments had reported that the incidence of teras was about 47% if zinc deficiency during pregnancy, and found that zinc deficiency during pregnancy would cause intrauterine growth retardation even affected the development of important organs in the embryonic period,which would lead to the related structural abnormalities, including heart, lungs, brain and genitourinary malformations.The cardiovascular abnormalities mainly covered aortic stenosis, ventricular septal defect, ventricular defect cardiac outflow tract malformation and innominate artery dysplasia, etc. Scholars found the whole blood zinc concentration of pregnant women whose fetal with CHD was lower than pregnant women with normal fetus,which suggested that zinc deficiency might be one of the reasons for CHD.Vitamin A was fat-soluble vitamin,including three biologically active forms such as retinol, retinal, retinoic acid(RA) and retinoic ester,and was the required element in the various life activities,mainly involved in the expression of genes,cell proliferation maturation and differentiation,maintaining mucosal surface,the intestinal absorption of iron, reproduction and so on.Vitamin A multiple functions mainly relied on its specific nuclear receptors:retinoic acid receptors (RARs) and retinol retinoid X receptors (RXRs).These receptor gene expression in the early stages of embryonic development were regulated by RA level.And RA was the most important active metabolite of vitamin A.However,at present A lack of vitamin A was quite common, the WHO and United Nations Children’s Fund had determined that iron, vitamin A and iodine deficiency were three big micro-nutrient deficiencies,there were about 19 million pregnant women suffering low serum retinol concentration, and about 15.3% of pregnant women were at risk of vitamin A deficiency in nation.Domestic studies found that levels of serum vitamin A were low in early pregnancy[28].Many animal experiments,such as rat, poultry,cattle,etc,had proved that prenatal maternal vitamin A deficiency or excess could lead to series of fetal congenital defects,including heart, vision, lungs and the central nervous system abnormalities,etc.The cardiac abnormalities caused by abnormal levels of vitamin A, including ventricular septal defect, persistent truncus arteriosus, etc.Zinc status influenced several aspects of vitamin A metabolism, including its absorption, transport, and utilization.Two common mechanisms postulated to explain this relationship:vitamin A transport mediated through protein synthesis that relied on zinc, and the oxidative conversion of retinol to retinal that required the action of a zinc-dependent retinol dehydrogenase enzyme.There was also evidence that vitamin A affected zinc absorption and utilization by changing the zinc dependent binding protein levels. Although animal experiments had found that there existed interactions between vitamin A and zinc, but the experiment cannot isolate the effects of zinc deficiency from effects of the accompanying generalized malnutrition and protein deficiency,which might effect the results.And their possible relationship in the body was still not clear.Because the degree of influence over the status of maternal nutrition for fetus CHD were inconsistent and it was necessary to understand the correlation between fetus CHD and maternal serum zinc,vitamin A levels in Shenzhen city,which would benefit for prevention and risk screening of fetus CHD.And the relationship between levels of vitamin a and zinc of pregnancy women with fetus CHD was still inconclusive.Heart development need the expression of many genes according to the order of time and space to regulate the development. We known from the study of animal models that more than 400 genes were required for normal heart development,Even though mutations in each individual gene was quite rare, the large number of potential causal mutations might make heart defects not rare[35].Although many genes mainly genes had been found to be important for cardiac development in animal studies, so far only a minority of mutations in these genes had been proved in human, e.g.GATA 4, TBX5, NOTCH1 and NKX2.5,etc.In mammals,Notch family members and Notch ligands were expressed throughout the early development of vasculature,which took part in an evolutionary conserved signaling pathway of which functions was to modulate cell-fate decisions of a variety of cell types originating from all three germ layers.Jaggedl gene transcribed into Jagged1 protein,which combined with Notch receptor.Jagged1 protein was a kind of cell surface proteins,regulated the angiogenesis.Mutations in Jaggedl, Notchl, Notchl/Notch4 or components of the Notch signaling cascade in mice resulted in severe vascular defects,for example the heart outflow tracts malformation.In the development of mice heart, Jaggedl gene mainly expressed in atrioventricular channel, outflow endocardium and atrial muscle[39].Alagille syndrome (ALGS) was caused by heterozygous mutations in Jaggedl,a polymalformative disorder affecting the liver, eyes and skeleton, characterized by a peculiar facies and heart, of which the cardiovascular malformations mainly included peripheral pulmonary stenosis, pulmonary artery stenosis and tetralogy of Fallot, etc.To date, over 440 different Jaggedl gene mutations had been identified in ALGS, which revealed Jagged1 gene not only had a high mutation rate, but various mutational forms, such as frameshift mutations, nonsense mutations, splice site mutations, missense mutations, insertion mutations etc. Nonsense mutation meant a stop codon replaced a certain amino acid codon because of a change in the basic group,resulting in the synthesis of the peptide chain termination in advance. Missense mutation meant another amino acid codon replaced a certain amino acid codon because of a change in the basic group, resulting in changing the amino acid sequence of the polypeptide chain. People focused on the Jaggedl mutations in the gene coding region,especially,the mutations relatively concentrated in exons 2-6,23-25and exon 9 . Three polymorphic loci of Jaggedl gene in exon 6 region played an important role in Jagged lgene, including a nonsense mutation c.7650 A (p.Tyr255X), two missense mutations c.814G>T(p.Val 272 Phe), c.834G>T (p.Glu278Asn).For the current studies,the research of Jagged 1 gene mainly focused on the digestive tract, breast and other cancer, nervous system, skeletal system, ALGS, certain types of congenital heart disease (such as pulmonary stenosis, tetralogy of Fallot and so on). And most of Jaggedl gene research was aimed at the European and American population, the study of East Asian populations was not sufficient. Whether maternal-fetal Jaggedl gene polymorphisms were associated with fetus CHD,it remained unknown. Through detecting the fetal-maternal Jaggedl gene polymorphism,to investigate the relationship between the fetus CHD and Jaggedl gene polymorphism.lt was excepted to assess the risk for fetus CHD.1.2 ObjectiveThrough a retrospective analysis of data collected the monitoring data on CHD in Shenzhen city from 2009 to 2013,we understood the occurrence of birth defects and CHD in nearly five years of Shenzhen City and evaluated the epidemiological characteristics of CHD in Shenzhen City.Comparing the difference of the intake of periconceptional maternal nutrients intake between the women whose offspring were diagnosed with CHD and the women whose offspring were normal,we would analyze the relationship between the intake of periconceptional maternal nutrients and fetus CHD.We wanted to find the potential influencing factors of CHD and put forward prevention measures.By detecting maternal serum levels of vitamin A,the whole blood concentration of zinc and Jaggedl genetic polymorphisms,to analyze the correlation of maternal zinc, vitamin A levels, maternal-fetal Jagged 1 gene polymorphism and CHD.The results of the study was expected to provide theoretical basis for risk prediction of fetus CHD2. Materials and Methods2.1 MaterlsAll of the material sources were retrospectively analyzed from the perinatal birth defects in monitoring data of Shenzhen City from 2009 to 2013, and from them we randomly selected the women whose offspring were diagnosed with CHD and the women whose offspring were normal to take an investigation on the intake of periconceptional nutrition. All the selected women lived in Shenzhen more than one year,who delivered from January to December in 2013.According to 1:1 matching, matching conditions:the age difference within 2 year-old, difference of gestational weeks within 2 weeks,live in the same area of shenzhen, the same nation(Han).There was no statistically significant difference (P> 0.05) of maternal age, gestational age in the groups.Altogether 324 questionnaires were collected.Forty-four fetus with CHD and their mothers were recruited into the maternal case group and the fetal case group respectively, while eighty-eight normal fetus and their mothers were divided into the maternal control group and the fetal control group severally.According to 1:2 matching, matching conditions followed the former. There was no statistically significant difference (P> 0.05) of maternal age, gestational age in the groups.All subjects were from the cases of induced labor or delivery pregnant women and fetus from the Maternal and Child Health Hospital of Shenzhen between November 2013 and November 2014.All subjects of the investigation and experience were included in the informed consent.2.2 Methods2.21 We analyzed the incidence and basic epidemiological characteristics of CHD, including maternal residency type,degree of culture,age distribution and the fetal birth season, outcomes, gender,etc,in Shenzhen City from 2009 to 2013. CHD was divided into simple CHD and complex CHD (CHD with other system malformations),of which the mainly epidemiological characteristics were analyzed.2.22 We took an investigation on nutrition by phone call:we investigated the maternal intake of nutrients from the case group and control group during periconception,which mainly covered the intake of dairy products, eggs, beans, fruits, nuts, seafood, multi-vitamins, folic acid, calcium, iron, cod liver oil and protein powder in different stages(that was three months before conception, the first trimester,the second and three trimester).Besides,We also inquired into the intake frequency (including daily,four to six times a week,one to three times a week and less than one time a week)of dairy products, eggs, beans, fruits, nuts, seafood in different stages.2.23 In induced labor or childbirth,gathering the maternal venous blood 8ml from two groups,3ml of blood into the the heparin anti-coagulation tubes then kept in-80℃ refrigerator, to be used for detecting of the whole blood concentration of zinc,3ml of blood into the drying tube,to 3000 r/heart 10 minutes after the separation serum after being and its natural solidification 30 min, then kept in -80℃ refrigerator, to be used for detecting of serum vitamin A level.The other 2ml venous blood was kept using EDTA anticoagulant and 5ml amniotic fluid was collected in induced labor or delivery,together kept in -80℃ refrigerator,to be used for detecting Jaggedl genetic polymorphisms.2.24 Detect the serum level of vitamin A by using ultra-high performance liquid chromatography (UHPLC).Detect the whole blood concentration of zinc by using inductively coupled plasma mass spectrometry system(ICP-MS).2.25 Detect the maternal-fetal Jaggedl gene polymorphism (Ioci765,814,834) by using PCR sequencing technology.2.26 Data were analyzed by the SPASS16.0 statistical software,measurement data, after analysis of normality test, the two samples were compared using independent samples t test for normally distributed data and were expressed as mean ±standard deviation, while using two independent samples of non-parametric test for the non-normally distributed data and were expressed as the median (interquartile range) [M (Q)].Count data were expressed as rate, the two samples were compared using Chi-square/Fisher test,and calculated the OR and 95% CI to show the association strength, only the data P<0.05 can be used for the statistics significance.3. Results3.1 The average incidence of CHD was 7.25‰,94.36% of CHD was checked out by ultrasound, and 33.20% was diagnosed before delivery.The proportions of the age>35 years old,temporary household register and high school and technical secondary school culture level of the pregnant women whose offspring with CHD was 40.72%,44.36% and 41.93% respectively.The majority of CHD was singletons, male, born in the summer and autumn.The most common cardiovascular malformations were patent ductus arteriosus,patent foramen ovale and ventricular septal defect,the incident of which was 24.12‰,18.96%o and 14.81%o respectively.About 14.11% was complex CHD,the mainly other system malformations were musculoskeletal system, facial features,the nervous system.There was about 51.83% of simple CHD complicated with two or more types of cardiac abnormalities.Compared simple CHD and complex CHD,we found that there exited significant differences in the stratification of age, culture degree,prognosis and diagnosis time(P<0.01).3.2 Seven pregnant women in case group did not intake any kind of nutrient supplements during periconception,while five pregnant women in control group.The intake rate of folic acid,multivitamins,calcium and milk powder was higher,and the intake rate of iron agent,cod liver oil and protein powder was much lower.Differences of the intake rate of nutrients including dairy products,nuts,seafood,beans, multivitamins,folic acid, iron and cod liver oil during periconception were statistically significant of two groups(P<0.05),as well as the differences of the dietary habits in first trimester(P<0.05).and all of above nutrients were protective factors(OR<1).Generally,the intake frequencies of dairy products, eggs, fruits, legumes, nuts and seafood in different trimester in the case group were higher(P< 0.05),especially in three months before pregnancy and the first trimester.Multivariate Logistic regression analysis showed that there were six factors associated with fetus CHD,including the intake of multivitamin,folic acid,seafood,dairy products during periconceptional,the intake frequency of dairy products in three months before pregnancy and the early diet habit (P<0.05).3.3 The maternal levels of zinc[5.20 (0.85) mg/L] and serum vitamin A[0.37 (0.16)mg/L] in case group were significantly lower than those in control group[5.67 (1.05) mg/L、0.43 (0.10) mg/L](P<0.05).And we found that the maternal levels of whole blood zinc and serum vitamin A in case group was positively correlated (rp=0.594,P<0.001),but the control group had no obvious correlation (P=0.072)3.4 Among the 132 cases of mother,Jaggedlc.765C>A mutant (including the homozygous and hybrid genotype) frequency was 18.18%, the non-mutant (wild homozygous genotype) was 81.82%.Among the 132 cases of fetus,Jaggedlc.765C>A mutant(including the homozygous and hybrid genotype)frequency was 21.97%, the non-mutant(wild homozygous genotype) was 78.03%.Jaggedlc.834 and c.814 mutations were not found in all samples.Differences of maternal-fetal Jaggedlc.765C>A mutant distribution were statistically significant between the case group and the control group.(P< 0.05).4. Conclusion4.1 The incidence of CHD is more and more higher in Shenzhen city from 2009 to 2013. Simple CHD is most common.The stillbirth rate and prenatal diagnostic rate of complex CHD are significantly higher than simple CHD.When pregnant women are over 35-year-old, the constituent ratio of fetus complex CHD is higher than simple CHD, especially over the age of 40 years old.4.2 A vegetarian diet in first trimester increases the risk of fetal CHD and the risk of fetus CHD can be reduce if women intake the multivitamin, folic acid, nuts and dairy during periconception,and the higher intake frequency of dairy products during three months before pregnancy the risk of fetus CHD will be reduce,too.4.3 The lower maternal levels of whole blood zinc and serum vitamin A during pregnancy are associated with the occurrence of fetal CHD.There existes a positive relationship between the maternal levels of whole blood zinc and serum vitamin A in the pregnant women with fetal CHD,which show a covariant trend.4.4 Maternal-fetal with Jaggedlc.765C>A mutant may associated with the occurrence of fetal CHD. |
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