The Relationship Between Gene Polymorphisms,Expression Of ER Gene Beta In Endometrium And Unknown Aetiological Hypomenorrhea

A common symptom of gynecology, which is total menstrual volume less than 30 ml during a menstrual cycle, is called hypomenorrhea. Its general reasons are following:①endocrine factors,example for abnormal secretion of various hormones. In a lower level of estrogen, endometrial proliferation is not sufficient, which displays endometrial humble.②Endometrial itself factors.We examined some patients by hysteroscopy, and found that some patient's endometrium was smooth and thin. These patients are called unknown aetiological hypomenorrhea whose hormone levels are normal and using hormone replacement therapyt is invalid except medical, surgical or congenital diseases. It can affect the imbedding of spermovium in the endometrium to induce habitual abortion and infertility. The assist reproduction technology successful ratio is extremely low and will be easy to abort even though successful pregnancy. It impacts seriously on the patient's physical and mental health. The treatment of these patients has become a very difficult clinical problem. Estrogen combined with estrogen receptor (ER) plays its biological effect. The effects are following two aspects:①To promote the proliferation and repairation of endometrium;②To promote the periodicity reconstitution of endometrial blood vessel. We want to know whether it is caused by ER combining block while we find the patients'estrogen levels are normal. Our immunohistochemistry results showed the expression of ER endometrium was lower obviously than normal menstrual volume women one.ER contains ERαand ERβsubtypes. Our group has found ERαgene polymorphisms were related to the unknown aetiological hypomenorrhea. There are many polymorphisms sites in ERβgene, ERβgene RsaI and AluI polymorphisms are more researched than other sites, which are located in the 5th exons loci and the 8th exons 3 'non-coding region respectively. There are C (cytosine), A (adenine) repeat sequence polymorphisms (D14S1026)in the 5th intron of ERβgene which belong to gene control region and control the gene expression. The different CA repeat numbers are related to some hormone correlated diseases. Nilsson M thought that ER gene polymorphisms might lead to disproportion in gene transcription and translation, and then ER was abnormal in expression and function.The association between ER gene polymorphisms and gynecology hormonal dependent disease is researched widely presently, for example: ovarian dysfunction,endometrial cancer,postmenopausal osteoporosis,endometriosis, and so on. There is no report about the relationship between unknown aetiology hypomenorrhea and ERβgene polymorphisms until now. We plan to study ERβgene polymorphisms and expression to find out the pathogenesy of unknown aetiological hypomenorrhea, and provide a theoretical basis for further research and treatment.PART ONE THE RELATIONSHIP BETWEEN RsaI,AluI POLYMORPHISMS OF THE ESTROGEN RECEPTOR BETA GENE AND UNKNOWN AETIOLOGY HYPOMENORRHEAObjective:To investigate the relationship between RsaI and AluI polymorphisms of the estrogen receptor(ER) gene beta and unknown aetiology hypomenorrhea in Chongqing area.Methods:100 unknown aetiology hypomenorrhea patients(case group) and 100 normal menstrual volume women(control group) were recruited. They all came from Chongqing district. We observed the ERβgene RsaI and AluI polymorphisms'distribution in the two groups by polymerase chain reaction-restriction restriction fragment length polymorp- hisms(PCR-RFLP). The genotypes,allelotypes,haplotypes characters were analysed and the ERβgene polymorphisms were compared with patients'age and induced abortion numbers respectively.Results:RR,Rr, rr genotypic frequency differences were not significant (χ2=8.376, P=0.068) in two groups. R allelotype frequency in case group was 37.5%, and that in control group was 48.5%, the OR was 0.642(95%CI=0.421～0.973),P=0.026. A allelotype frequency in case group was 18.0%, that in control group was 11.5%, OR was 1.689(95%CI=0.960～2.971),P=0.067. RRaa haplotype's OR was 0.049(95%CI=0.006～0.378),P<0.001. There was not statistically significant different among diversity genotypes'age and induced abortion numbers in two groups.Conclusion:ERβgene polymorphisms is associated with unknown aetiology hypomenorrhea."R"allele may be a protective factor for unknown aetiology hypomenorrhea. The RRaaa haplotype may be the potential protective factor for unknown aetiology hypomenorrhea.The RsaI and AluI genetypes are not related to patients'age and induced abortion numbers in unknown aetiology hypomenorrhea.PART TWO THE RELATIONSHIP BETWEEN CA REPEAT SEQUENCE POLYMORPHISMS IN ER GENE BETA AND UNKNOWN AETIOLOGY HYPOMENORRHEAObjective:To investigate the association of a (CA)n dinucleotide repeat sequence polymorphisms in ERβgene with unknown aetiology hypomenorrhea in Chongqing district.Methods:100 hypomenorrhea patients(case group) and 100 normal menstrual volume women (control group) were recruited. They all came from Chongqing district.The (CA)n dinucleotide repeat sequence in ERβgene the 5th introns loci hypervariable region were purifed, cloned and analysed. We observed the ERβgene(CA)n dinucleotide repeat sequence polymorphisms' distribution in the two groups. In addition, the CA repeat sequence polymorphisms were compared with patients'age and induced abortion numbers respectively.Results:All samples were exhibited 9 different alleles, the (CA)n dinucleotide repeats number were 16～24. There was not difference between distributing frequencies of CA repeat sequence polymorphism in control and case groups (χ2= 7.155,P=0.520). SS genotype was defined as repeat number n≤20, LL for n>20. The SS genotypes rate were 66% in case group and 51% in control group respectively, OR=1.865(95%CI= 1.055～3.298). The SS and LL allele genotypes frequency was compared in two groups and the difference was significant (χ2=4.634, P= 0.031). The linkage disequilibrium of ERβgene CA-STR﹑RsaI and AluI haplotype was not strong. The difference had not statistical significance among diversity allele genotypes'induced abortion numbers and age in unknown aetiology hypomenorrhea.Conclusion:A (CA)n dinucleotide repeat sequence polymorphism is associated with unknown aetiology hypomenorrhea. SS allele genotype may be the risk factor for unknown aetiological hypomenorrhea. The CA allele genetypes are not related to patients'age and induced abortion numbers in unknown aetiology hypomenorrhea.PART THREE THE ER GENE BETA EXPRESSION IN UNKNOWN AETIOLOGY HYPOMENORRHEA PATIENTS NDOMETRIUMObjective:To investigate the role of human estrogen receptorβin the development and growth of unknown aetiological hypomenorrhea by comparing the expression of the human estrogen receptorβsubtype between unknown aetiological hypomenorrhea and normal menstrual volume women in endometrium.Methods:The tissues of 40 unknown aetiological hypomenorrhea specimens and 40 normal menstrual volume women endometrium which were in above-mentioned two groups were selected. RT-PCR and computer image analysis were used to investigate the relative expression of the ERβsubtype at mRNA level. Western blot was employed to investigate the protein expression of estrogen receptorβsubtype. The mRNA and protein expression were compared with patients'age,induced abortion numbers, genotype and allelotype respectively.Results:The ERβ-mRNA expression amount was 0.6457±0.2957 in case group and 0.9637±0.3621 in control group. The mRNA expression of ERβdecreased markedly in unknown aetiological hypomenorrhea compared with normal menstrual volume women endometrium, the difference was statistically significant(t=-6.589,P<0.001).The ERβ-protein expression amount was 347.37±30.35 in case group and 445.21±45.67 in control group,the difference was statistically significant (t=-4.353,P<0.001).The differences among diversity genotypes'age and induced abortion numbers were not statistically significant. ERβexpression differences were not statistically significant among RsaI genotype, AluI genotype, CA repeat sequence genotype.Conclusion:The mRNA and protein expression of ERβdecreases in unknown aetiological hypomenorrhea compared with normal menstrual volume women endometrium, which may be related to the menstrual volume reduction of unknown aetiological hypomenorrhea. The expression of ERβis not related to patients'age and induced abortion numbers in unknown aetiological hypomenorrhea. ERβexpression differences are not statistically significant among RsaI genotype, AluI genotype, CA repeat sequence genotype in two groups.