| Purpose: 1.Spontaneous abortion is a common disease in obstetrics and gynecology,but there are many cases of unexplained reasons for abortion.The relationship between villus telomere length and spontaneous abortion was studied by comparing the length of villus telomere between spontaneous abortion and normal abortion.2.Telomerase is a kind of reverse transcriptase,can synthesize telomere DNA,maintain telomere length and function,with high activity expression in early human embryos,human telomerase reverse transcriptase(hTERT)is the core subunit of telomerase.The correlation between villous hTERT promoter methylation and spontaneous abortion was analyzed by comparing the expression differences in methylation of hTERT promoter between spontaneous abortion and normal human flow villus tissues.3.Telomere length between chromosome aneuploid fetal and diploid fetal amniotic fluid cells was compared to further understand the relationship between telomere length changes and chromosome aneuploidy.Methods:1.Thirty-five cases of spontaneous abortion villous tissue were selected as the experimental group(divided into two subgroups based on villous chromosome results),the cases of 70 villous tissues with induced abortion due to contraceptive failure during the same period were in the control group.and 70 cases of normal induced abortion villous tissue were selected as the control group.About 15 g of villus tissue was collected from each case,and then rinsed with sterile saline to remove the contamination of mother blood.Part of fresh villus tissue was taken for cell culture and chromosomal karyotype analysis with G-banded.DNA was extracted from the remaining villus tissue,and the purity and concentration of 1 ul DNA were detected by UV spectrophotometer to ensure sample quality.About 100 ng DNA was collected to detect telomere length by real-time quantitative PCR.2.Thirty-four cases of spontaneous abortion villous tissue were selected as the experimental group,the cases of 41 villous tissues with induced abortion due to contraceptive failure during the same period were in the control group.Approximately 600 ng of the extracted DNA was selected for bisulfite modification,the modified genomic DNA was detected by uv spectrophotometer with OD260/OD280 ratio of 1.7～1.9,and then by methylation specific polymerase chain reaction Reaction,MSP),the methylation level of hTERT promoter was detected,and agarose gel electrophoresis was performed on the reaction products.The results were observed and photographed by Gel Doc XR+ imager.3.Thirty patients underwent amniocentesis for cytogenetic examination,and the amniotic fluid cells were cultured for about 7～10 days,and the supernatant was moved into the new culture bottle to continue the culture.When chromosome karyotype analysis was completed,this fraction of cells was used for DNA extraction for telomere length detection.Results: 1.The median(quartile range)of telomere length was 0.8(0.4)in the experimental group,and 1.0(1.0)in the control group,which was statistically significant(P =0.022).Among the two subgroups in the experimental group: the villous telomere length was 0.7(0.5)in the chromosomal anomaly group,and 0.8(0.4)in the normal group,and there was no significant difference(P =0.612).There was no correlation between villus telomere length and maternal age,length of menopause and duration of pregnancy(P > 0.05).2.In the experimental group,high methylation rate was 47.1%(16/34),moderate methylation rate was 32.4%(11/34),low methylation rate was 20.6%(7/34).In the control group,the high methylation rate was 9.8 %(4/41),the moderate methylation rate was 48.8%(20/41),and the low methylation rate was41.5%(17/41).The methylation level of the experimental group was higher than that of the control group,the difference was statistically significant(P = 0.002).In the experimental group,the methylation level of hTERT promoter was similar between the chromosome abnormality group and the normal group,and the difference was not statistically significant(P= 0.885).There was no correlation between hTERT promoter methylation level and maternal age(P=0.313),but it was significantly correlated with the number of days of menopause and the number of pregnancies(P < 0.01).3.The mean ± standard deviation of telomere length of the aneuploid group is(1.9 ± 1.2),which is relatively shorter than that of the normal group(3.4 ± 1.7)and was statistically significant(P = 0.013).Conclusion:1.High methylation level of hTERT promoter on villus chromosome and telomere shortening may be one of the factors leading to spontaneous abortion.The high methylation level of hTERT promoter in the villous tissue of spontaneous abortion may lead to the decrease of hTERT expression,telomerase activity and telomere shortening,thus leading to the instability of chromosomes and genomes and inducing spontaneous abortion.The correlation between chromosome karyotype and hTERT promoter methylation level and telomere length needs to be further verified by adding more clinical samples.2.The chromosome telomere length of aneuploid fetal amniotic fluid cells is significantly shorter than that of diploid fetuses,further revealing that telomere length plays an important role in maintaining the stability of genetic material.3.This study is helpful to further reveal the pathogenesis of spontaneous abortion in terms of molecular genetics and epigenetics. |
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