The Association Reseach On LRH-1 And Imprinted Gene P57^kip2 For Patients With Unexplained Recurrent Spontaneous Abortion

BeckgroundRecurrent spontaneous abortion(RSA)refers to spontaneous abortion more than three times(including three times)in the first trimester of pregnancy[1],and its incidence is about 2%-5%in the female-aged female population,accounting for all women naturally.15%-20%of abortion is one of the most common abnormalities in the first 3 months of pregiancy[2].The cause of this abortion is very complicated,except for the bost common genetic factors(chromosomal abnormalities),endocrine abnormalities such as thyroid function problems,immune factors(anti-cardiolipin syndrome or autoimmune disease),infectious diseases,and vascular eibolism diseases[3-6]etc,about half of the patients have unknown causes of abortion,and three(including three)consecutive unexplained recurrent spontaneous abortion(URSA).The etiology and pathogenesis of URSA are complex and diverse.At present,there is no clear indication of repeated abortion and effective methods to improve the success rate of treatment and prevent recurrence of spontaneous abortion.Therefore,the possible pathogenesis of recurrent spontaneous abortion is sought in new research fields.It is a demand for medical development and one of the research hotspots in the field of reproduction.At present,there are many studies on the mechanism of unexplained recurrent spontaneous abortion.In a considerable part of the research,the role of transcription factors is gradually highlighted.Hepatic receptor homolog-1(LRH-1;NR5A2)is one of the transcription factors that plays an important role in animals and humans,and participates in many physiological processes including lipid metabolism[7].Cancer development[8],anti-inflammatory activities[9],etc.Studies have shown that the expression level of LRH-1 in the ovary is significantly higher than other tissues[10,11],indicating the important function of LRH-1 in this organ.It has also been found that LRH-1 regulates ovarian cholesterol intake and promotes steroidogenesis[12].Using the LRH-1 knockout mouse model,it was found that LRH-1 can regulate ovulation[13].Other studies suggest that LRH-1 regulates the cell cycle,coordinates endoderm,and participates in early embryo development in animals.There is a mouse experimental model that knocks out one copy of the LRH-1 allele and knocks out one copy of the sf-1 allele,which can lead to infertility or spontaneous abortion in the first trimester.All of this evidence suggests that LRH-1 may play an important and positive role in pregnancy.The secretion of progesterone in placenta during pregnancy plays an important role in the continuation of pregnancy and the preservation of the fetus.The function of cytochrome cholesterol side chain cleavage is to catalyze the synthesis of steroid hormones including placenta progesterone.Cholesterol is converted to pregnenolone[14,15].LRH-1 is an important transcription factor upstream of CYP19,and CYP19 is a key gene of the key rate-limiting enzyme P450 of estrogen synthesis,which regulates estrogen formation and plays an important role in placental development[16,17].At present,there is a correlation between the occurrence of LRH-1 and human URSA,which is rarely reported at home and abroad.In the process of expression,the imprinted gene mainly transmits genetic information through a single parent.The related discipline is epigenetics.At present,this subject has begun to receive widespread attention.Related studies have found that the imprinting status of imprinted genes changes,the factors affecting the loss of heterozygosity are complex,and closely related to transcriptional regulation,and the effect of methylation abnormalities in the promoter region is also obvious.The imprinted gene p57Kip2 is a member of the Cip/Kip family of cyclin dependent kinases inhibitor(CDKI).On mouse chromosome 7,its characteristic is the paternal imprint and maternal expression.But in human tissue,p57Kip2 is only partially imprinted.Animal experiments have shown that the lack of p57Kip2 expression in mouse placenta can cause massive proliferation of placental trophoblast cells[18],and this factor in human placental tissue is abundantly expressed in the cytotrophoblast of villus tissue.Related studies have found that approximately one in ten human BWS sporadic cases have a certain correlation with this maternal allele[19]Another study found that overexpression of p57Kip2 can lead to early fetal growth retardation and even premature intrauterine death.It has been reported that the expression level of this gene in the placenta of the offspring born using Assisted reproductive technology(ART)is high,which affects the proliferation of placental trophoblast cells,leading to abnormal development of the placenta and the fetus.The study of the imprinted gene p57Kip2 in the offspring of hydatidiform mole and human assisted reproductive technology is reported,but studies related to unexplained recurrent spontaneous abortion still need to be improved.In this study,we investigated the changes in the expression of LRH-1,p57Kip2 and their related genes in the villus and decidua of URSA patients,from single nucleotide polymorphism(SNP)and promoter methylation status.The relationship between LRH-1 and p57Kip2 and URSA is helpful to reveal the relationship between LRH-I and p57Kip2 and human reproduction,and provide a reference for the early diagnosis and treatment of unexplained recurrent spontaneous abortion.PARTI Expression of LRH-1 in villi and decidua of patients with unexplained recurrent spontaneous abortionObjectiveIn view of the important function of nuclear receptor liver receptor homolog 1(LRH 1)in various biological procedures and the physiological changes come along the unexplained recurrent spontaneous abortion(URSA),our study was carried out to investigate the potential roles of LRH-1 in URSA.Methods30 patients with URSA who were treated in the Affiliated Weihai Second Hospital of Qingdao University from August 2015 to September 2016 were selected for re-embryo embryonic villus and decidual tissue,and 30 cases of unplanned pregnancy were selected for abortion.The villus and decidua tissues were compared and analyzed,and the experimental group and the control group were set respectively.The expression levels of LRH-1 gene in the villus and decidual tissues of the two groups were detected by immunohistochemical technique,RT-PCR and Western-Blot.The mRNA and protein expressions of LRH-1,CYP19 and P450 scc genes were detected by the method.Results1.Immunohistochemistry showed that LRH-1 gene protein was expressed in both villus and decidua of both groups.The expression level in the URSA group was lower than that in the control group(p<0.05),but there was no statistical difference in the expression of the decidua between the two groups(p>0.05).2.RT-PCR showed that the LRH-1 mRNA in the URSA group was significantly lower in the villus(p<0.05).In the decidua,the LRH-1 content in the URSA group was not statistically different from the control group(p>0.05).CYP19 and P450scc mRNA expression levels were consistent with LRH-1 expression in the villus and decidua.3.Western-blot analysis showed that the expression of LRH-1 protein in URSA group was significantly lower in villus(p<0.05).There was no statistical difference in LRH-1 protein expression between the two groups in decidua(p>0.05).CYP19,P450scc protein expression levels were consistent with LRH-1 expression in the villus and decidua.Conclusions1.The expression level of LRH-1 in the URSA group was significantly lower than that in the control group(p<0.05).There was no significant difference in the expression of the two groups in the decidual tissue(p>0.05).It is suggested that the decreased expression level of LRH-1 in villus tissue may be related to URSA.2.The expression levels of CYP19 and P450scc in villus were different between the two groups,and the URSA group was significantly lower than the control group(p<0.05).There was no significant difference in the expression of the two groups in the decidual tissue(p>0.05).It is suggested that the decreased expression levels of C YP19 and P450scc in villus tissue may be related to URSA.PART2 Association between LRH-1 gene single nucleotide polymorphism(SNP)and unexplained recurrent spontaneous abortionObjectiveTo explore the genetic relationships between LRH-1(rs2816948),CYP19(rs727479 and rs700518),and P450scc(rs4077582)as a potential mechanism behind unexplained recurrent spontaneous abortions.MethodsA case-control study was used and featured two groups:Patients with unexplained recurrent miscarriage(n=82,abortion group)and those who had normal pregnancy(n=97,control group).Abotion samples were obtained from all patients.Genomic DNA was later extracted and sequenced,after which statistical analyses performed to assess the relationship between single nucleotide polymorphisms and unexplained recurrent spontaneous abortions.ResultsThe distribution frequency of GG genotype and G allele in rs727479 locus of CYP19 gene in URSA group was higher than that in normal control group,the difference was statistically significant(p<0.05).There was no significant difference in genotype and allele distribution between rs700518 locus in CYP19 gene and rs2816948 locus of LRH-1 gene in both groups(p>0.05).The frequency of CC genotype in rs4077582 locus of P450scc gene was lower than that in normal control group,the difference was statistically significant(p<0.05).),the distribution of alleles was not statistically significant between the two groups(p>0.05).Conclusion1.The frequency of GG genotype and G allele of rs727479(single nucleotide polymorphism)in URSA patients was higher than that in normal control group(P<0.05),while the frequency of CC genotype of P450 SCC gene rs4077582 was lower than that in normal control group(p<0.05).2.The genotype and allele distribution of LRH-1 gene rs2816948 and CYP19 gene rs700518 were not significantly different between URSA group and control group.PART3 Expression of imprinted gene p57KiP2 in villi of patients with unexplained recurrent spontaneous abortionObjectiveThe relationship between the expression of p57Kip2 and the cause of unexplained recurrent miscarriage was preliminarily studied by means of the expression of p57Kip2 in the villi of URSA and normal trimester.Methods82 cases of unexplained recurrent spontaneous abortion in early pregnancy(URSA group)and 82 normal pregnancy(control group),taking villi,early spontaneous abortion villi were divided into 7-9 weeks of pregnancy and 10-12 weeks of pregnancy The expression of p57Kip2 in villus tissues was detected by immunohistochemistry.The mRNA and protein expression of p57Kip2 gene were detected by RT-PCR and Western-blotting.Results1.Immunohistochemical detection showed that P57Kip2 in 10-12 weeks samples more strongly expressed in the nap of the spontaneous abortion group,the expression of spontaneous abortion group was obviously higher than in normal pregnancy group(p<0.05),and there was no significant difference in the expression of villus in the URSA group between the 7-9 weeks of pregnancy and the normal pregnancy group(p>0.05).2.RT-PCR test showed that the expression of p57Kip2 in the sample of 10-12 weeks of early spontaneous abortion was higher than that of normal early pregnancy of the same gestational week,and the difference was statistically significant(p<0.05).However,there was no significant difference in the expression of villus in the URSA group between the 7-9 weeks of pregnancy and the normal pregnancy group(p>0.05).3.Western-blot test showed that the expression of p57Kip2 in the sample of 10-12 weeks of early spontaneous abortion was higher than that of normal early pregnancy of the same gestational week(p<0.05).There was no significant difference in the expression of p57Kip2 gene between the URSA group and the normal group of the same gestational age(p>0.05).Conclusion1.In the 10-12 weeks of pregnancy,the expression level of p57Kip2 gene in URSA patients was significantly higher than that in normal pregnancy group(p<0.05).It is suggested that URSA patients have high expression of p57Kin2 in the embryonic suspension of villus tissue in 10-12 weeks of pregnancy.2.In the 7-9 weeks of pregnancy,the expression of p57Kip2 gene in URSA patients was not significantly different from normal pregnancy(p>0.05).PART4 The DNA methylation status research of imprinted gene p57Kip2 promoter in patients with unexplained recurrent spontaneous abortionObjectiveTo investigate the correlation between DNA methylation status in p57Kip2 promoter region and the cause of unexplained recurrent miscarriage.MethodsBy using the method of case-control study of 30 patients with unexplained recurrent miscarriage(URSA group)and 30 normal pregnant women(control group)to collect abortion villus,and extract the villi genomic DNA,DNA methylation sequencing of p57Kip2,NOEY2 and PEG3 gene promoter region was carried out.Based on statistical analysis,the relationship between DNA methylation status of gene promoter region and URSA was studied.ResultsThe methylation level of two sites(site 6,site 7)in the promoter region of p57Kip2 gene in URSA group was lower than that in control group,the difference was statistically significant.(p<0.05).There was no significant difference in DNA methylation status between the other sites of p57Kip2 gene promoter region,no significant difference in DNA methylation status between NOEY2 and PEG3 promoter region(p>0.05).Conclusion1.The methylation level of the two sites in the promoter region of p57Kip2 gene was different between the two groups.The methylation level of these two sites in URSA patients was significantly lower than that of the control group.2.The DNA methylation status of the rest sites of the p57Kip2 gene promoter region were not significantly different between the two groups.DNA methylation status of NOEY2 and PEG3 promoter regions were not significantly different between the two groups.