Expression And Significance Of ACE2-Ang-(1-7)-Mas Axis In The Endometrium Of Patients With Polycystic Ovary Syndrome

Objective:First,by measuring the expression of each component ofrenin-angiotensin system's two axis (ACE-AngⅡ-AT1R/AT2R andACE2-Ang-(1-7)-Mas) in normal human endometrium during menstrualcycle,we wish to confirm local existence of RAS in the endometrium and todiscuss the relationship with ovarian hormones.Second,by observing thedifferrent expression of each component of renin-angiotensin system in theendometrium between that of polycystic ovary syndrome(PCOS) and that ofnormal endometrium,we wish to discuss the changes of endometrial RAS onthe effect of endometrial function and to provide a new way for thepathogenesis and therapeutic approaches in PCOS.Methods:1Objects of study:1.1The collection of paraffin-embedded specimens of endometriumParaffin-embedded specimens were chosed from the clinic patients ofpathology department in the second hospital of Hebei Medical Universityfrom November2009to July2011.The research objects was60cases ofmarried women,age with22～38,mean age29.31±1.07.By pathologicalexamination,all the endometrium was normal,in which30cases withproliferative phase,30cases with secretory phase.1.2The collection of liquid nitrogen specimen of endometriumExperiment group:The research objects were chosed from the clinicpatients of reproductive department and gynecological endocrinologydepartment in the second hospital of Hebei Medical University from January2010to November2011,which include15cases of married women werediagnosed as PCOS,age with24～37years old,mean age23±0.67years old.Control group:15cases of women were diagnosed as infertility in preparation to assisted reproduction treatments of couple infertility due tomale factor or tubal obstruction,age with24～38years old,mean age23±0.67years old.In the experiment group,endometrial specimens were collected from8to14days in the menstrual cycle with oligomenorrhea patients or from any daywith amenorrhoea patients,and about the control group,endometrial specimenswere collected from8to14days in the normal menstrual cycle.All specimens were obtained by cruettage when undergoing diagnostichysteroscopy,one part of samples were fixed in10%buffered formaldehyde,using for clinical diagnosis by hematoxylin-eosin,another part of samples werequickly immersed in liquid nitrogen,using for RQ-PCR quantitative analysis.1.3Inclusion criteria:Referring to the2003Rotterdam diagnostic criteria[8]:①Oligo-and/oranovulation②Clinical and/or biochemical signs of hyperandrogenism③Polycystic ovaries (presence of12or more follicles in each ovary measuring2±9mm in diameter,and/or increased ovarian volume(>10ml)).PCOS wasdiagnosed by①+③and/or②.The control group was satisfied with regularmenstrual, normal level of sex hormones,with no abnormal pelvic ultrasound.All the patients require exclusion of other endocrine diseases fromadrenal or pituitary and without taking hormone medication in the last threemonths,without heart,liver,kidney disease history.2Experimental methods:Routine HE staining was to determine the phases of histology ofendometrial specimens.Immunohistochemistry was used to detect the level ofAngⅡ,AT1R,AT2R,ACE2,Ang-(1-7) and Mas protein in endometria.Endometrial specimens were analyzed by Real-Time Quantitative PCR(RQ-PCR) for detection of AT1R,AT2R,ACE2and MasmRNA.3Statistical analysis:All the experimental data were mean±standard(X±S),processed bySPSS l3.0statistical software.The data conform the normal distribution usingthe t test.Statistical significance was defined as P<0.05. Results:1The results of AngⅡ,AT1R,AT2R,ACE2,Ang-(1-7) and Mas proteinexpression by ImmunohistochemistryAng Ⅱ, AT1R,AT2R,ACE2,Ang-(1-7) and Mas protein expression by apositive tan or brown.They were localized in the cytoplasm and cellmembrane, and both were mostly expressed diffusely.1.1Ang Ⅱ was localized in the endometrium during normal menstrualcycles.In proliferative endometrium,Ang Ⅱwas mainly detected in glandularepithelium.In contrast,in secretory intense immunostaining was seen in theperivascular stromal cells around the endometrial spiral arterioles.(Fig.1)1.2AT1R and AT2R were localized in the endometrium throughout normalmenstrual cycles.The mean levels of AT1R and AT2R specific bindingincreased from the early to the late proliferative endometrium.Both AT1R andAT2R levels were maximal in the early secretory endometrium and thendecreased in the late secretory endometrium.They were mainly localized inendometrial glands epithelial.(Fig.2-3)1.3ACE2was localized in the endometrium throughout normal menstrualcycles,which were more abundant in epithelial cells than in stromal cells,andin the secretory vs. proliferative phase.(Fig.4)1.4Ang-(1-7) was localized in the endometrium during normal menstrualcycles,which were more abundant in epithelial cells than in stromal cells,andin the secretory vs. proliferative phase.(Fig.5)1.5Mas was localized in the endometrium during normal menstrualcycles,which were more abundant in epithelial cells than in stromal cells,andin the secretory vs. proliferative phase.(Fig.6)2The results of AT1R,AT2R,ACE2and MasmRNA by RQ-PCR2.1The expression of AT1RmRNAThe AT1RmRNA was detectable in all endometrial tissue samples.Therelative expression of AT1RmRNA was higher in PCOS endometrium(0.59±0.41) than those in proliferative endometrium (0.23±0.26),thedifference was statistically significant (P<0.01).(Table1) 2.2The expression of AT2RmRNAThe AT2RmRNA was detectable in both the proliferative endometriumofnormal menstrual cycle and proliferative endometrium of PCOS.The resultsof the AT2RmRNA expression were0.51±0.33,1.13±0.68. The level ofAT2RmRNA in proliferative endometrium of PCOS was obviously higherthan those in the proliferative,the difference was significant(P<0.01).(Table2)2.3The expression of ACE2mRNAThe ACE2mRNA was detectable in all endometrial tissue samples.Therelative expression of ACE2mRNA was higher in PCOS endometrium(0.91±0.41) than in proliferative endometrium (0.30±0.24),the differencewas statistically significant (P<0.01).(Table3)2.4The expression of MasmRNAThe MASmRNA was detectable both in the normal proliferativeendometrium and proliferative endometrium of PCOS.The results of theMASmRNA expression were0.31±0.26,0.79±0.26.The level ofMASmRNA in proliferative endometrium of PCOS was obviously higher thanthose in the proliferative, the difference was statistically significant(P<0.01).(Table4)Conclusions:1There is local existence of RAS in the endometrium.2The various components of RAS were expressed in the normalendometrium during menstrual cycle and with cyclical changes,which may berelated to ovarian hormones and endometrial development closely.3The increased expression of AT1RmRNA,AT2RmRNA,ACE2mRNAand MasmRNA in the endometrium of PCOS may influence endometrialdevelopment and participate in the pathological process of PCOS.