The Correlation Between Gene Polymorphismss,Expression Of ERa Gene 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 reasons are following :①endometrium damage,②estrogenic hormone level descend,③inborn hypoplasia of uterus, and so on.Now we examine some patients by hysteroscopy, and find that some patient's endometria are smooth and thin, which indicate retarded growth. That doesn't agree with traditional endometrium damage and scar theory. These patients are called unknown aetiological hypomenorrhea, whose estrogen level are normal and invalid through hormone replacement therapy with no medical,surgical or congenital disease. It can affect the imbedding of cytula in the endometrium to induce habitual abortion and aciesis, while the assist reproduction technology achievement ratio is extreme low and will abort even though successful pregnancy.Estrogen plays a important role in menstrual regulation. Estrogen combines with estrogen receptor (ER) to educe its biological effect. The effects embody promote the periodicity reconstitution of endometrial blood vessel, and promote the hyperplasy and reparation of endometrium. And we want to know whether it is caused by ER combining block while we find the patients'estrogen levels are normal.Our former immunohistochemistry showed the expression of ER in unknown aetiological hypomenorrhea endometrium was lower obviously than normal menstrual volume women.ER contains ERa and ERβsubtypes.There are many polymorphisms sites in ERa gene upstream region, PvuII and XbaI restriction fragment length polymorphism enzyme slice sites had been definited and are located in 1 intron which includes enhanser,promotor important sequences. The point mutation may influence gene expression and function. There are T (thymine), A (adenine) repeated sequence in the upstream of ERa gene which belong to gene control region and control the gene expression. Nilsson M thought ER gene polymorphisms might lead to disproportion of gene in transcription and translation, and then ER was abnormal in expression and function.The association between ERa gene polymorphisms and gynecology hormonal dependent disease is widespread presently,for example:habitual abortion;recurrent abortion,Ovarian dysfunction,endometriosis,endometrial cancer,and so on..Up to now,there is not relationship between hypomenorrhea and ERa gene polymorphisms.So we try to study ERa gene polymorphisms and expression to find out the pathogenesy of unknown aetiological hypomenorrhea, and provide some information for further research.PAST ONETHE STUDY ON THE RELATIONSHIP BETWEEN PvuⅡ,XbaⅠPOLYMORPHISMS OF THE ESTROGEN RECEPTOR GENE ALPHA AND UNKNOWN AETIOLOGY HYPOMENORRHEAObjective: To investigate the relationship between polymorphisms of the estrogen receptor(ER) gene alpha PvuⅡ,XbaⅠand unknown aetiology hypomenorrhea in Chongqing districts China.Methods: 100 unknown aetiology hypomenorrhea patients(case group) and 100 normal menstruation women(control group) were recruited. They all came from southen-western China.To observe the ERa gene polymorphisms genotype's distribution in the two groups,PvuII and XbaI restriction fragment length polymorphisms(RFLP)of ERa were analysed, in the meantime,the genotypes,allelotypes,haplotypes characters were analysed and the gene polymorphisms were compared with patients'age and induced abortion numbers respectively.Results: PP, Pp, pp genotypic frequency differences were significant (χ2=14.915, P=0.001). P genotypic frequency in case group is 47.5%, and that in control group is 30.5%, the OR is 2.062(95%CI=1.369～3.105),P=0.001.X genotypic frequency in case group is 20.5%, that in control group is 30.5%, OR is 0.588(95%CI=0.372～0.928),P=0.022. RFLP of PvuII and XbaI in the 2 groups are distributed with polymorphisms. Ppxx genetype constituent ratio was higher in case group(36%) than cotrol group(15%),but ppXx,ppxx genetype constituent ratio was lower in case group(4%,19%)than cotrol group(20%,29%). It was not statistically significant different among diversity genotypes'age but induced abortion numbers in experimental group.Conclusion: ERa gene polymorphisms was associated with unknown aetiology hypomenorrhea."P"allele may be a dangerous factor,"X"allele may be a beneficial factor for unknown aetiology hypomenorrhea women. The Ppxx genetype may be the potential risk factors for unknown aetiology hypomenorrhea. The PvuII and XbaI genetypes are not related to patients'age but induced abortion numbers in experimental group. PART TWO THE ASSOCIATION OF TA REPEAT POLYMORPHISMS IN ERa GENE WITH UNKNOWN AETIOLOGY HYPOMENORRHEAObjective: To investigate the association of a (TA)n dinucleotide repeat polymorphisms with unknown aetiology hypomenorrhea. Methods: 100 normal menstruation person(control group) and 100 hypomenorrhea patients(case group) were recruited. they all came from southen-western China.The (TA)n dinucleotide repeat in ERa gene upstream hypervariable region were purifed,cloned and sequence analysed. we observe the ERa gene(TA)n dinucleotide repeat polymorphisms distribution in the two groups. In addition, the TA repeat polymorphismss were compared with patients'age and induced abortion numbers respectively.Results: All samples exhibted 7 different alleles. The (TA)n dinucleotide repeats were 11～17, The most prevalent allele was TA14 repeat in control group.The distribution of the different alleles of the (TA)n dinucleotide repeat sequence in the case group and control group has significant difference(χ2=27.717,P<0.001).Fisher's exact test was applied to compare the frequency of each allele between two groups.The commonest allele was TA14. The frenquency of the TA13 allele was found to be significantly greater in hypomenorrhea group(33%) than control group(12%)(P=0.001),OR=3.612(1.735～7.519),and the frenquency of the TA15 allele was less in hypomenorrhea group(13%) than control group(31%) (P=0.003),OR=0.333(0.162～0.684). There were not statistically significant different among diversity allele genotypes'induced abortion numbers and age in experimental group.Conclusion: A (TA)n dinucleotide repeat sequence which is in ERa gene upstream hypervariable region was associated with unknown aetiology hypomenorrhea.The TA13 allele was found to be associated with unknown aetiology hypomenorrhea and may be a risk factor; whereas TA15 allele may be a protective factor for patients. The TA allele genetypes are not related to patients'age and induced abortion numbers in experimental group. PART THREE THE ERa GENE EXPRESSION IN UNKNOWN AETIOLOGY HYPOMENORRHEA ENDOMETRIUMObjective: To investigate the significance of human estrogen receptor a in the development and growth of unknown aetiological hypomenorrhea by comparing the expression of the human estrogen receptor a subtype between unknown aetiological hypomenorrhea and normal menstrual volume endometrium.we will comprehend unknown aetiological hypomenorrhea etiopathogenisis and prognostic factors to prophylaxis,diagnose and treat early,which maybe improve it's prognosis.Methods: The tissues of 40 unknown aetiological hypomenorrhea specimens and 40 normal menstrual volume endometrium which were in above-mentioned two groups were selected. All of the specimens were conserved in liquid nitrogen or -70℃refrigerator. RNAs were also extracted. RT-PCR and computer densitometry were used to investigate the relative expression of the ERa subtype at mRNA level. Proteins were extracted and Western blot was employed to investigate the protein expression of estrogen receptor a subtype. Meanwhile, the expression were compared with patients'age,induced abortion numbers,genotype and allelotype respectively. Results: The ERa-mRNA expression amount was 0.8879±0.2741 in case group and 1.2630±0.3532 in control group. The mRNA and protein expression of ERa markedly decreased in unknown aetiological hypomenorrhea compared with normal menstrual volume endometrium,the difference was statistical significant(t=-2.653,P=0.016).The ERa-protein expression amount was 600.8100±212.8439 in case group and 797.2750±85.3860 in control group,the difference was statistical significant(t=-2.709,P=0.014).The difference among diversity genotypes'age was not statistically significant. The difference among diversity genotypes'induced abortion numbers was statistically significant(F=4.985,P=0.019).The difference between induced abortion numbers≥3 and numbers=1,2 was statistically significant(P=0.035,0.007 respective).The PP genotype's mRNA and protein expression were lower than that of pp genotype in experimental group (0.7843±0.1690, 544.0170±118.9401) and than that of control group (0.9857±0.1345, 672.5840±150.1515) (P=0.039,P=0.035).At same time, The xx genotype's mRNA and protein expression were lower than XX genotype in experimental group (0.7629±0.1727,540.0580±123.5944) than control group (0.9671±0.1531, 659.9980±148.8788)(P=0.028, P=0.046). The TA repeated sequence genotype'S expression difference in two groups was not statistically significant(t=-0.5.8,-0.866,P=0.618,0.398,respective).Conclusion: The expression of ERa decreases in unknown aetiological hypomenorrhea compared with normal menstrual volume endometrium. ERa may be related to the menstrual volume of unknown aetiological hypomenorrhea. The expression of ERa is not related to patients'age but induced abortion numbers in experimental group,the more induced abortion numbers patients have,the illness risk is higher. The PP genotype's expression is lower than pp genotype in in unknown aetiological hypomenorrhea, meanwhile the xx genotype's expression is lower than XX genotype in in unknown aetiological hypomenorrhea. The expression of ERa is not related to (TA)n dinucleotide repeat sequence polymorphisms.