Progesterone Receptor To Pathogenesis Of Endometriosis

ObjectiveEndometriosis (EMS) is a common gynecological condition in which the endometrium—like tissue is found outside the uterine cavity. It is highly important to understand its etiology ,because it has been linked to pelvic pain ,multiple operations, and infertility and difficult to cure of. Nowadays, the exact mechanism for the development of endometriosis is unclear.The most widely accepted theory is implantation of viable endometrium on peritoneal surfaces after retrograde menstruation .However, it is well established that menstrual debris is present in the peritoneal cavity of 90% of menstruating women,but only 10—15% women are capable of establishing ectopic endometrial implants. There are several possible explanations for such susceptibility,including differencesin genetic predisposition,increased exposure to menstrual debris,abnormal eutopic endometrium,altered peritoneal environment,reduced immune surveillance,and increased angiogenic capacity. Familial and epidemiologic studies support that this disease is a genetic disorder of polygenic/multifactorial inheritance. Molecular and clinical observations support that endometriosis has the characters of steroid-dependence and progesterone resistance. This study lays stress on progesterone receptor ( PR ) to study its isoform ( PRA and PRB ) gene expression ,polymorphism and promoter methylation status. The aim is to find the action of PR in the development of endometriosis. On second thoughts ,because inactivation of 17_β-estradiol to estrone is catalyzed by 17_βhydroxy steroid dehydrogenase type2 (17_βHSD₂) in the human endometrium , progesterone stimulates to produce 17_βHSD₂ by the action of PR .Study on 17_βHSD₂ will be beneficial to understand the machanism that endometriosis is an estrogen-dependent disease.Material and MethodsMaterial1,sample:(1) peripheral blood sample;(2) endometrium sample:three portionsa.stored at -70℃b.10% formalin and embedded in paraffinc. EMs Nude mice medel: endometrial fragments was transplanted into the perotoneal cavity of nude mice .Seven days later,implanted endometrial lesions were obtained and stored at -70℃and 10% formalin.(3) ovary endometriotic tissue sample.2,DNA and RNA extraction related agents.3,Western-blot related agents.4,RT-PCR related agents.5,Methylation-specific PCR(MSP) related agents.6,Immunohistochemical staining related agents.Methodes1,PCRDNA was isolated by phenol/chloroform extraction from peripheral blood samples from EMs and control. Polymorphism of the progesterone receptor gene PROGINS was examined by PCR.2,Western-blotWestern blot analysis was performed after immunoprecipitation to detect bands specific for PRA and PRB in eutopic endometrium from endometriosis and control (patients with cervical intraepithelial neoplasia,CIN), endometriotic tissue from nude mice grafts, and ovary endometriotic tissue.3,RT-PCRTotal RNA was extracted from eutopic endometrium and ovary endometriotic tissue using Trizol,the cDNA synthesized from the mRNA was followed,then amplified PCR products of 17_βHSD2,asssyed mRNA .4,MSPDNA was isolated by phenol/chloroform extraction from eutopic endometrium and ovary endometriotic tissue paraffin-embedded sections and modified by sodium bisulfite. Methylation and unmethylation alleles were examined by PCR.5,Immunohistochemical stainingTwo anti-PR antibodies,one specific to PRB and the other to PRA+B were used to identify PRB and PRA protein expression in eutopic endometrium and ovary endometriotic tissue by SP method.6,Statistical analysisStatistical analysis was performed using SPSS for windows 11.0 software. Statistical significance was calculated with t-test and chi-squared independence test.The difference was considered significant when the p value was less than 0.05.Results1,Western-blot for PRA and PRBPRA was detected positively in all eutopic endometrium of EMS(n=28) and control (n=15).The level was lower in EMS than in control,but the difference was not significant (p>0.05);PRB was expressed negatively in eutopic endometrium of endometriosis, but positively in that of control. The level was lower significantly in endometriosis than in control(p<0.01); PRA was detected positively in all transplanted endometrium samples of EMS(n=20) and control (n=10) in mice.The difference of two groups was not significant (p>0.05); PRB were expressed negatively in transplanted endometrium of mice, but positively in that of control. There was a significant difference in level in two groups(p<0.01);PRA was expressed positively in ovarian endometriotic tissue and paired eutopic endometrium of endometriosis ,and PRB was negatively(n=28).The level is no significant difference beteewn them(p>0.05).2,RT-PCR17_βHSD2 were expressed positively in eutopic endometrium of endometriosis(n=28) and control(n=15). There was no significant difference in level beteewn them(p>0.05). 17_βHSD2 were expressed negatively in ovarian endometriotic tissue and there was significant difference in level compared with its eutopic endometrium (p<0.05).3,PCRIn both patients with endometriosis (n=66)and control(n=56) ,the genotype frequency pattern showed dominance of the allele T1 homozygosity.However,the frequency of the mutant allele T2 was significantly different between the study and control groups. Frequencies of the mutant allele T2 was 0.14 in endometriosis and 0.04 in control ((T1/ T2 and T2/T1 vs T1/T1),odds ratio : 4.54 [95%CI: 1.50-13.78] )P = 0.004. Homozygosity for allele T2 was present in 3.0% of women with endometriosis. 4,MSP and Immunohistochemical stainingAlleles of PRA was methylated in 39 /62endometriosis samples (n=62)and 17/33 controls(n=33) ,(p>0.05); Alleles of PRB was methylated in 61/62 endometriosis samples and 22/ 33 controls (p<0.01). Most of all in endometriosis and controls were positive expression for PRA alleles (p>0.05) in immunohistochemical staining; The negative expression of PRB alleles in 20/62 endometriosis cases was significantly higher than that in 2/33 controls(p<0.01).The loss of unmethylation alleles was well correlated with negativity in immunohistochemical staining for PRB of endometrial samples (p<0.01).Conclusion1,The absence of PRB in eutopic endometrium and endometriotic tissue of endometriosis may be associated with endometriosis..2,The absence of 17βHSD2 in endometriotic tissue may be associated with endometriosis. 3,PROGINS may be associated with an increased risk of endometriosis in Chinese women of Han .4,CpG methylation in eutopic endometrium may be associated with the inactivation of progesterone receptor B gene of endometriosis.