| BackgroundSpontaneous abortion refers to the loss of pregnancy before28weeks of gestation without outside intervention. Although incidence of spontaneous abortion is lower than that of other countries in the world, the increasing tendency of incidence of spontaneous abortion is obvious. The etiology of spontaneous abortion is complicated and not clear.At least50%of spontaneous abortion cases are attributed to heredity factors, immune factors, endocrine dyscrasia and anatomical abnormalities. However, about50%of causes of spontaneous abortion are unexplained, this type of spontaneous abortion is defined as unexplained spontaneous abortion (USA).The consecutive occurrence of two or more pregnancies that end in miscarriage of the fetus, usually before20weeks of gestation, is called as unexplained recurrent spontaneous abortion (URSA).As a kind of pathological pregnancy, USA has an bad effect on body and mental health for pregnant mothers. So it is valuable to explore the causes of USA or URSA and pathogenesis. Several studies showed that high childbearing age, low educational level, occupations exposed to toxic and harmful substances, history of spontaneous abortion, many times of pregnancy and obesity before pregnancy were risk factors for USA. Homocysteine (HCY) is an intermediate product during the metabolic process of methionine or cysteine. Studies showed that metabolic disorder of HCY would lead to high levels of serum HCY which had fetal toxicity and may cause spontaneous abortion. In addition, high levels of serum homocysteine have been linked to imbalance between coagulation and anticoagulation and placenta thrombosis which was related to spontaneous abortion. Many studies have supported that high levels of homocysteine has been linked to URSA. Folate (folic acid) was one of forms of the water-soluble vitamin B and essential for numerous bodily functions. Humans cannot synthesize folate themselves and therefore, folate has to be supplied through the diet to meet their daily requirements. During the metabolism of HCY, folic acid is a kind of key coenzyme. Low levels of serum folate can also lead to homocysteine accumulation which may lead to spontaneous abortion. Methylenetetrahydrofolate reductase (MTHFR) gene regulates folate-dependent remethylation of homocysteine to methionine. The MTHFRC677T gene polymorphismwhich causes the substitution of alanine with valine, causes thermolability and reduces the activity of MTHFR. Then the folate-dependent remethylation of homocysteine to methionine is obstructed which alsoleads to hyperhomocysteinemia. Several studies have found that MTHFRC677T gene polymorphism was associated with high levels of serum HCY, low levels of serum folate and the risk of URSA.However, almost present researches on the risk factors of USA and its relationship with levels of HCY and levels of folate belonged to retrospective methods and detections of levels of serum or plasma HCY and folate were conducted after the diseases of spontaneous abortion had happened. So the recalling bias was inevitable and the power of these studies was limited in the inference of the casual relationship. At present, studies on the interacteraction btween MTHFRC677T gene polymorphism and the risk factors of USA were few. In this study, an observation cohort which recruited2098pregnant women in the first trimmest (<10weeks) were built in twomaternal and child health hospitals in Shandong province fromJan.2011to Feb.2012.At the beginning of study, baseline data and exposure factors information were collected and blood serum and anticoagulation specimen of all objects were stored.And all pregnant women werefollowed up untilpregnancy outcome happened. A1:4matched nested case-control study was conducted which recruited160USA cases, including106URSA cases,with the standard diagnosiscriterion and controls were pregnant women who were selected randomly from the cohort whodelivered healthy live-born term infants.Objectives1. To explore the risk factors of USA and URSA.2. To explore the correlation between the serum levels of HCY,the serum levels of folate and the risk of USA and URSA.3. To explore the correlation betweenMTHFRC677T gene polymorphism and the risk of USA and URSA.4. To explore the interactions between MTHFRC677Tgene polymorphism and the risk factors of USA and URSA.5. To build apredicting model of the risk of USAand find the high-risk population. Methods1. Following up study:A hospital-based cohort was built which included2098pregnant women in the first trimester (≤10weeks) with recruiting criterion and excluding criterion in two maternal and child health hospitals of Shandong province from Jan.2011to Feb.2012.At the beginning of study, baseline data and exposure factors information were collected and blood serum and anticoagulation specimen of all objects were stored. All the members in the cohort were followed up until the pregnancy outcomes happened.2. Matched nested case-contrl study:During the study period, USA cases with uniform diagnosis criterion were recruited into the case group. Controls were pregnant women who were chosen randomly from the cohort who delivered healthy live-born term infants, matched with cases by regionand lastmenstrualperiod±2weeks. Each case was matched with four controls.3. Investigation contents and methods:The data was collected through face-to-face interviews from the beginning of study to pregnancy outcome. The contents of investigation included baseline data and exposure information. Meanwhile, the serum and anticoagulated bloodspecimens of all women in the cohort were collected and stored in the refrigerator (-40℃)4. Detection of serum specimens and classification of MTHFR genotypes:The levels of serum HCY were detected by the method of enzymatic cycling assayand the levels of serum folate were detected by the method of direct chemiluminiscence. MTHFRC677T gene polymorphism was classified by the method of polymerasechain reaction-restrictionfragment length polymorphism (PFLP-PCR).5. Data processing and statistical methods:Database was established using the softwareEpiData3.1. Non-conditional logistic regression model and conditional logistic regression model were used to analyze the effective factors. Multifactor dimensionality reduction (MDR) was used to estimate the gene-environment interaction.Classification tree analysis (CRT) was used to explore the high-risk factors in the predicting model for the risk of USA.Results1. There were2098pregnant women in the first trimester of pregnancy who were included in the observation cohort from the beginning of study to the end of follow-up.The average age of the cohort members was27.08±3.87years old, the maximum age was44years old and the minimum age was20years old.The average gestational weeks of the cohort members was6.81±1.62weeks, the maximum gestational weeks were10weeks and the minimum gestational weeks were1week.There were1240pregnant women (59.10%) in the cohort who were in the first pregnancy. There were31.64%of the cohort members who had one or morn than one time spontaneous abortion.During the study period.160USA cases were recruited in the nested case-control study, the incidence of USA was7.63%and106URSA cases were recruited in thenested case-controlstudy,the incidence of URSA was5.05%.2. Results of multifactor analysis of follow-up study showed that history of spontaneous abortion, history of parity (≥1), underweight before pregnancy, peasants and age were associated with the risk of USA and OR(odds ratio) values were12.521,6.942,2.099,1.765and1.069, respectively (P<0.05). And folic acid supplement before and during pregnancy was a protective factor for the risk of USA, and OR value was0.635(95%CI:0.417,0.967). And peasants, history of parity (≥1). underweight before pregnancy, and age were associated with the risk of URSA and OR values were2.102,1.943,1.809, and1.099,respectively (P<0.05). Folic acid supplement before and during pregnancy was a protective factor for the risk of URSA and OR value was0.569(95%Ci:0.342,0.949).3. The average levels of serum HCY of the USA case group (11.50μmol/L) were significantly higher than those of the control group (9.48μmol/L).P<0.05. Hyperhomocysteinemia in the first trimester of pregnancy was associated with the risk of USA and OR value was2.694(95%CI:1.566,4.635) after adjusting for the potentialconfounding factors. The average levels of serum HCY of the URSA case group(11.02μmol/L) were significantly higher than those of the control group(9.48μmol/L),P<0.05. Hyperhomocysteinemia in the first trimester of pregnancy was associated with the risk of URSA and OR value was2.154(95%CI:1.261,3.678) after adjusting for the potential confounding factors.4. The average levels of serum folate of the USA case group (8.91ng/ml) were significantly lower than those of the control group(10.73ng/ml), P<0.05. Compared to the low levels of serum folate (<6.25ng/ml), high levels of serum folate (>13.78ng/ml) in the first trimester of pregnancy were associated with the risk of USA and OR value was0.215(95%CI:0.102,0.415) after adjusting for the potential confounding factors. The average levels of serum folate of the URSA case group (8.94ng/ml) were significantly lower than those in the control group (10.41ng/ml), P<0.05. Compared to the low levels of serum folate, high levels of serum folate in the first trimester of pregnancy were associated with the risk of URSA and OR value was0.280(95%CI:0.124,0.635) after adjusting for the potential confounding factors.5. The frequency distributions of MTHFRC677T genotypes between the case group and the control group were significantly different for both USA and URSA. For USA, the frequency percents of TT genotype and T allele of the case group were30.6%and53.8%, which were significantly higher than those of control group,17.7%and44.3%, respectively (P<0.01). For URSA, the frequency percents of TT genotype and T allele of the case group were32.1%and52.8%, which were significantly higher than those of control group,16.1%and41.5%, respectively (P<0.01). After adjusting for the potential confounding factors, MTHFRC677TTT genotype was associated with the risk of USA and URSA and OR values were1.891(95%CI:1.172,3.049) and2.488(95%CI:1.505,4.113), respectively.6. The results of MDR method showed that for the risk of USA, underweight before pregnancy and the level of serum folate had a strong antagonism effect and this effect would be weakened when the factor of TT genotype joined into the interaction model. For the risk of URSA. TT genotype and folic acid supplement before and during pregnancy had a strong antagonism effect and this effect would be weakened when the factor of the level of serum folate joined into the interaction model. The results of conditional logistic regression model showed that for the risk of USA, TTgenotype and no folic acid supplement before and during pregnancy, underweight before pregnancy, the low and modest level of serum folate had positive additivity interactions, synergy indexes (S) were3.31,7.69,1.95, respectively, the attributable proportions due to interaction (AP) were52.7%,76.3%,41.3%, respectively and relative excess risks due to interaction (RERI)were2.16,6.19,2.68, respectively. For the risk of URSA, TTgenotype and no folic acid supplement before and during pregnancy, underweight before pregnancy, the low and modest level of serum folate had positive additivity interactions, synergy indexes(S)were2.58,5.81,1.66, respectively, AP were41.2%,72.3%,35.4%, respectivelyand RERIwere1.26,5.71,3.26, respectively.7. The results of Classification Tree analysis showed history of spontaneous abortion, history of parity (≥1), the low level of serum folate and high childbearing age were important effective factors for the risk of USA. History of spontaneous abortion (≥1) was the most important factor for the risk of USA. The correct proportion of predicting was81.5%and theproportion of receiver operating characteristic (ROC) was0.833(95%CI:0.801,0.865) The predictive ability of the classification tree model was relatively good.Conclusions1. History of spontaneous abortion, history of parity (≥1), underweight before pregnancy, peasants and high childbearing age were the risk factors of USA. And peasants, history of parity (≥1), underweight before pregnancyand high childbearing age were the risk factors of URSA.Folic acid supplementation before and during pregnancy may decrease the risk of USA andURSA. Andage, occupation, educational level and history of spontaneous abortion were associated with the status of folic acid supplement before and during pregnancy.2. Hyperhomocysteinemia in the first trimester of pregnancy was associated with the risk of both USA and URSA. High levels of serum folate may decrease the risk of both USA and URSA.3. MTHFRC677T gene polymorphism was associated with the risk of USA and URSA and pregnant women with TT genotype were1.5times higher for the risk of USA and URSA. MTHFRC677TTT genotype and no folic acid supplement before and during pregnancy, underweight before pregnancy, the low and modest level of serum folate had positive additivity interaction for the risk of both USA and URSA.4. Pregnant women who hadhistory of spontaneous abortion, history of parity (≥1), the low level of serum folate and high childbearingage were high-risk population for the risk of USA.Innovations1. A hospital-based cohort was built which included pregnant women in the first trimester (<10weeks) with recruiting criterion and excluding criterion and was followed up until the pregnancy outcome happened. Multifactor analysis was used to find the effective factors of the risk of USA and URSA.2. A matched nested case-control study was used in this study to explore the relationship between the HCY metabolism disorder and the risk of USA and URSA. So the results of serological test could reflect the real levels of serum HCY and serum folate and the time sequenceof cause and outcome was clear. Therefore, the statistic test power was higher and the casual inference between the relationship of the serological marker and the disease were stronger compared to classic case-control study.The results showed hyperhomocysteinemia in the first trimester of pregnancy was associated with the risk of USA and URSA and the high levels of serum folate was the protective factor for the risk of USA and URSA after adjusting for the potential confounding factors.3. MDR method was used to analyze the interactions between MTHFRC677T gene polymorphism and the risk factors for the risk of USA and URSA with conditional logistic regression model used meanwhile.The positive additivity interactions between TT genotype and no folic acid supplement before and during pregnancy, underweight before pregnancy, the low and modest level of serum folate were found. |
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