Folic Acid In The Prevention Of HOXA10Aberrant Methylation Of Fetal Origin Endometriosis Susceptibility Genes

BackgroundEndometriosis is a hormone-dependent common disease of women, and the incidence is increasing, but so far it’s etiology is unknown. More and more evidence suggesting that endometriosis is an epigenetic disease, and DNA methylation is one of the most important epigenetic regulation in which DNA methyltransferase (DNMT) play a major role. HOXA10gene is an important transcription factor of endometriosis susceptibility genes, and its aberrant methylation patterns is associated with the biological behavior and prognosis of endometriosis. Diet during pregnancy can cause long-term impact on the offspring genome. Folic acid is essential vitamins in the human body and involve the whole process of metabolism in human. Folic acid, is an essential vitamins in human DNA synthesis and involved cell differentiation, doubling and closely related to its function which is significant impact on producing offspring. Folic acid is the ultimate source of the methyl group in human body, playing an important role in maintaining DNA methylation. Clinical studies and animal experiments have confirmed that maternal folate deficiency or metabolic abnormalities are associated with embryonic malformations. Before pregnancy and pregnancy folic acid supplementation can prevent or reduce the incidence of offspring birth defects. Endometriosis as epigenetic disease, whether there is a significant difference between fetus of pregnant with endometriosis and normal pregnant both of them without folate supplement, and whether pregnant women with endometriosis taking folic acid can affect the HOXA10the methylation status of the fetus’s endometriosis susceptibility genes, these hadn’t yet seen the research reported.ObjectiveThis study aim to detect the HOXA10aberrant methylation status of the female fetus endometriosis susceptibility gene between fetus of pregnant with endometriosis and normal both of them without folate supplement, and whether pregnant women with endometriosis taking folic acid can affect the HOXA10the methylation status of the fetus’s endometriosis susceptibility genes, and to understand the role of folic acid in optimizing the intrauterine environment, provide clues for to prevent "adult disease fetal origins".Methods1. specimen collection:Fetal cord blood specimens from2010to2012, Southern Medical University, Zhujiang Hospital and Huizhou Central Hospital, and the female newborn who selected in this study, Endometriosis were confirmed by pathological diagnosis of pre-pregnancy surgery or cesarean section pathological diagnosis. Thirty-six cord blood specimens of pregnant woman with endometriosis, including twenty-one case with0.4mg/d folic acid treatment from Pre-pregnancy to early three month pregnant and fifteen without folic acid supplement, fifty-six cord blood specimens of normal pregnant woman, including thirty cases with folic acid treatment from Pre-pregnancy to early three month pregnant and twenty-six without folic acid supplement.2ml Fetal cord blood collected during childbirth, Saved by Sodium Citrate, and stored at-20℃.2.Experimental methods:(1) Cell DNA bead extraction kit to extract genomic DNA of blood samples; (2) Methylation-specific polymerase chain reaction (MSP) and bisulfite modified sequencing (BSP) were used to detect the methylation status of the HOXA105’CpG promoter region of fetus endometriosis susceptibility genes;3. Statistical analysis:Using SPSS13.0software to analysis data, among groups rate compared with the χ2test and Fisher’s exact probability (the total number of samples is less than40or theoretical frequency less than5), The inspection level a=0.05.Results1. Methylation-specific polymerase chain reaction (MSP):To detect each group fetal endometriosis susceptibility genes HOXA10methylation status, six cases of fetal cord blood of pregnant women with endometriosis without folic acid supplement detected abnormal methylation, methylation rate was40%(6/15); One case of fetal cord blood of normal pregnant women detected abnormal methylation, methylation rate was3.8%(1/26), The difference between two groups was statistically significant (χ2=8.782, P=0.006). The endometriosis group of pregnant women with folic acid supplement was detected one case of fetal cord blood with abnormal methylation, and methylation rate of4.7%(1/21),while The endometriosis group of pregnant women without folic acid supplement was detected six case of fetal cord blood with abnormal methylation, methylation rate was40%(6/15), The difference between two groups was statistically significant (χ2=6.937, P=0.013).2. Bisulfite modified sequencing (BSP):To detect each group fetal endometriosis susceptibility genes HOXA10methylation status, eight cases of fetal cord blood of pregnant women with endometriosis without folic acid supplement detected abnormal methylation, methylation rate was46.6%(7/15); one case of fetal cord blood of normal pregnant women without folic acid supplement detected abnormal methylation, methylation rate was3.8%(1/26), The difference between two groups was statistically significant (χ2=11.106, P=0.002). The endometriosis group of pregnant women with folic acid supplement was detected one case of fetal cord blood with abnormal methylation, and methylation rate of4.7%(1/21),while The endometriosis group of pregnant women without folic acid supplement was detected six case of fetal cord blood with abnormal methylation, methylation rate was46.6%(7/15), The difference between two groups was statistically significant (χ2=8.890, P=0.005).DiscussWith a series of the deepening clinical studies in recent years, the contribution of genetic factors in the pathogenesis of endometriosis was constantly confirmed. First level relatives of endometriosis patients suffering from endometriosis higher risk than non-sick relatives, Present study in chromosomes and genetic analysis of internal endometriosis confirmed that HOXA10of multiple sites gene promoter region5’CpG exist hypermethylation to decline the related gene expression, which may participate and contribute to the occur and progress of endometriosis. HOXA10gene aberrant methylation resulted in gene expression decreased may be one of the pathogenesis of endometriosis.A growing body of evidence in animal models show that many observed factors impacting on the reprogramming of fetal period mediated by epigenetic. Literature reported epigenetic modification can not change the DNA sequence, but can be inherited to offspring, and DNA methylation as one of the most important epigenetic modification,and Epidemiological study confirmed the familial it has the familial aggregation, But its familial aggregation is no animal or human experimental study confirmed. Epigenetic modification of endometriosis as epigenetics disease, whether its abnormal DNA methylation can also affect their offspring yet little research. Which folic acid, we found that female fetuses HOXA10of endometriosis susceptibility genes aberrant methylation rate in endometriosis group is significantly higher than normal group detect methylation specific PCR and bisulfite sequencing assay. In this study, once again prompted endometriosis may be an epigenetic disease, and may have certain hereditary.Folic acid plays an important role in the regulation of genes required for methylation, which must rely on exogenous supply because in the human body is unable to synthesize folic acid. More demand for folic acid during pregnancy, and severe case of insufficient folate. Folic acid in the body The main function is to participate in DNA synthesis and methyl donor, It intracellular methylation reactions and maintain the stability of the genome has an important role in the regulation. Recent studies suggest that DNA methylation is related to human tumorigenesis. DNA methyltransferase (DNMT) as a key enzyme in the methylation plays an important role in the tumorigenesis process. Studies show that folic acid can affect the activity of DNMT to result in tumorigenesis. DNMT showed low expression in normal tissues, while exhibiting high expression in a variety of tumor cells. Studies have found that folic acid supplementation can inhibit DNMT activity of the cells of the tumor lesions, their mRNA and protein expression decreased. Folate status is relate to DNMT activity and expression level, show that Level of folic acid in the body can affect the activity and expression of DNMT, thus affecting the level of DNA methylation. In the experiment of Wang Jintao found that folic acid can inhibit the expression and activity of DNMT and thus participate in the occurrence and development of cervical cancer and precancerous lesions. These are further illustrated folic acid by influencing the the DNMT level, thereby reducing the level of DNA methylation. However, this phenomenon is also confirmed in endometrial DNMTs, Planted in normal pseudopregnant mouse embryonic endometrial DNMTs at normal levels found in the study of Ding YB, The lining of the pseudo-pregnant mice given a low folate diet DNMT1, DNMT3A and DNMT3B were increased, this also shows that folic acid can affect the level of the DNMTs the endometrial. Similarly, Wu et al. detected13cases of ectopic endometrium of patients with endometriosis,10cases of eutopic endometrium and8cases of normal endometrium in DNMT expression levels The results showed that ectopic endometrium methyl transferase enzyme (Dnmt1ANMT3A,, and DNMT3B) genes were overexpressed in patients with endometriosis abnormal DN A methylation.In human subjects found that the placenta is the decisive factor of the transmission to the fetus of maternal plasma folic acid, folic acid through placental folate receptor transmission, and folic acid in fetal blood even several times than maternal folate levels. Endometriosis as an epigenetic disease, whether folic acid supplementation can prevent the occurrence of the female adult disease such as endometriosis? Sie KK experimental found that folic acid supplementation during pregnancy found that pregnant rats, Offspring risk of colon cancer decreased by64%, indicating that folic acid can affect rats intrauterine environment and reduce the risk of their offspring risk of colorectal cancer.Therefore, for this embryonic methylation remodeling period, the experimental study of pregnant women with endometriosis folic acid supplementation between progestation and early pregnancy, to compare fetal HOXA10aberrant methylation of endometriosis susceptibility genes, former aberrant methylation was4.7%, while the latter is40%detected by methylation specific PCR, both methylation rate difference compared was statistically significant (P<0.05), former aberrant methylation was4.7%, while the latter is46.6%detected by Bisulfite sequencing assay, both methylation rate difference compared was statistically significant (P<0.05). Analysis:The analysis may be due to reduced folic acid supplementation by inhibiting DNMT activity and expression, which in turn makes endometriosis susceptibility genes HOXA10methylation level. In early pregnancy correct abnormal methylation status may have far-reaching implications for the prevention of the diseases of women with endometriosis adult.Conclusions1.Both of two groups without folic acid supplement, compared with normal pregnant, female fetal of pregnant women suffering from endometriosis HOXA10methylation rate of endometriosis susceptibility genes significantly increased, further suggesting that endometriosis may be an epigenetic hereditary.2. Pregnant women suffering from endometriosis with folate supplement, female fetal HOXA10methylation rate of endometriosis susceptibility genes significantly reduced than that without folate supplement, suggesting that folic acid may play role in the prevention of fetal origin endometriosis HOXA10aberrant methylation of susceptibility genes.