| Background:Intrauterine adhesions(IUA),known as Asherman syndrome since 1948,refer to the partial or total adherence of endometrial surfaces with fibrotic tissue.The basal layer trauma,infection,endometrial repair disorders and radiation contribute to the development of intrauterine scar,which is a great threat for secondary infertility and miscarriage with increasing incidence rate and detection rate in rensent years.Typically,IUA can result in conditions such as menstrual abnormalities,infertility,recurrent pregnancy loss and chronic pelvic pain.However,in some rare cases,the disease was diagnosis only when asymptomatic patients went for a hysteroscopy.As the terminations of aberrant fibrosis after endometrium injury,IUA was an urgent clinical puzzle for seriously threatening female reproductive health.Although the applications of physical barriers combined with hormone therapy after the surgery of intrauterine adhesiolysis has gained the therapeutic effects to some extent to promote endometrial healing,the recurrences of IUA and side effect of high dose systemic estrogen happen from time to time.According to the researches,The postoperative recurrence rates of IUAs are 3.1%-23.5%with a moderate to severe risk in 20%-62.5%of cases.The new therapies targeting the cellular mechanisms that lead to the development of those pathologies remain to be explored.Endometrial fibrosis is the main pathological characteristic of Asherman syndrome with an excessive deposition and reorganization of extracellular matrix(ECM)in place of normal endometrial that shed and regenerated according to the menstrual cycle.Biopsy specimens from patients with intrauterine adhesions compared to those who without intrauterine adhesions contain 50%to 80%of fibrous tissue compared with 13%to 20%in the uterine wall.Meanwhile,the functional glands are replaced by inactive cubocolumnar endometrial epithelium.Transforming growth factor-β1(TGF-β1)that is expressed in most cell types is a secreted protein that regulates numerous cellular responses,such as proliferation,differentiation,migration and apoptosis.Mounting evidence shows that perturbation of TGF-β1 signaling plays a pivotal role in the development of all kinds of fibrosis disease.Fibrosis is a common feature of organ dysfunction,during which,TGF-β is known to upregulate many fibrogenic genes,such as extracellular matrix(ECM)proteins and a-smooth muscle actin(a-SMA),via Smad2 or Smad3.Moreover,the transcriptional factor(Spl)can interact with Smad3 to enhance TGF-β1-induced fibrotic response.Recent study proposes that TGF-β1-mediated epithelial-mesenchymal transition(EMT)might also be involved in fibrosis process.Studies have demonstrated that TGF-β1 is able to induce upregulation of autocrine connective tissue growth factor(CTGF)in mesenchymal type cells,including fibroblasts and those arising from EMT in the kidney during renal fibrosis.Nevertheless,whether these pathways involved and how they are integrated in endometrial fibrosis were seldom reported.MicroRNAs(miRs)are endogenous,small,noncoding RNA molecules that can regulate a wide range of biologic processes by suppressing the translation or inducing the degradation of targeted mRNAs.The microRNA-29(miR-29)families that share the same seed binding sequence are well characterized for their ability to regulate extracellular matrix proteins including collagens,elastin,and fibrillins.Recently,a great quantity of researches has showed that the miR-29 family is also a critical mediator of canonical TGF-β1 pathway in fibrosis disease of vital organs,including renal fibrosis,pulmonary fibrosis,cardiac fibrosis,peritoneal fibrosis and so on.Besides,evidences suggested that miR-29b is able to reverse and inhibit TGF-β1 induced EMT process.In our previous work,the expression of miR-29 decreased while TGF-β1 increased according to diverse degrees of severity of IUA in clinical samples.However,the role of miR-29 in the development of endometrial fibrosis and the possible mechanisms through which miR-29 interacts with TGF-β1 remain unknown.A better understanding of the events driving the pro-fibrotic cascade is indispensable to develop therapeutic strategies for IUA.Athought miR-29b was proved to involve in the fibrotic process of some vital organs,its effects on IUA have barely been reported.In this study,we investigated the development of endometrial fibrosis induced by TGF-β1 using primary human endometrial stromal cells(ESCs).The anti-fibrotic effect of miR-29b mimic and its mechanisms were determined in the established cell model of endometrial fibrosis.Further more,we demonstrated the preventive and therapeutic potential of miR-29b in vivo by transfection with agomir-29b(mimicking miR-29b)in a rat model of IUAs using mechanical and infectious injury[uterine curettage and lipopolysaccharide(LPS)treatment].Chapter I The study on the mechanism of TGF-β1 inducing fibrosis in primary human endometrial stromal cellsObjective:To establish an effective method of separation,purification and culture of the primary human endometrial stromal cells in vitro,and study the effects of diverse concentration of TGF-β1 on the expression of COL1A1、α-SMA、p-Smad2/3 and miR-29b or their expression after different time spans after TGF-β1 treated.The biological characteristics and phenotype of the cultured cells were also to be observed.Taken together,we expect to investigate the specific mechanism.Methods:1.The full-thickness endometrial tissue samples were collected under aseptic conditions.We used collagenase I to digest tissues and nylon strainers to filtrate the cells.The ESCs were cultured in DMEM/F-12 containing 10%fetal bovine serum(FBS).The ESCs were subcultured by 1:2-1:3 after fusion growth to 80-90%.We observed the cellular morphology using an inverted phase-contrast microscope.2.The third passages were used for the experiment of cell identification.Immunocytochemical staining was performed.Cells were cultured on the cover slides in 30 mm culture dishes,and fixed using 4%paraformaldehyde,and primary antibodies of cytokeratin 18 and vimentin were applied.3.The third to sixth passages were used for experiments.Primary ESCs were treated with TGF-β1(0,1,5,and 10 ng/ml)for 48 h or TGF-β1(10 ng/ml)for the indicated time(12,24,48,72 h)in DMEM containing 1%FBS.Real-time PCR and Western blot analysis were performed to detect the expression of miR-29b,COL1A1,a-SMA and p-Smad2/3.4.The cultured ESCs were seeded into 30 mm dishes with coverslip.SEM images further demonstrated morphological changes in primate ESCs in response to continuous TGF-β1 stimulation at the concentration of 0,1,5,and lOng/ml for 4 days.Results:1.The primary cultured ESCs were observed spindle-shaped and mostly adherent 24 hours after inoculation.The cells’s number increased,and the majority of the ESCs formed tightly parallel or braided arrangement and grew to confluence 6-7d later.After subculture,ESCs were observed in fibroblast-like shape with perfect vitality,which can sustain to the 7th passage.Immunocytochemical staining on the third passages showed that the ESCs stained positive for the stromal markers vimentin(brown),and negative for the epithelial cell marker cytokeratin 18(not shown).In addition,the purity of the stromal cells was over 98%.2.Real-time PCR and western blot analysis show that COL1A1,a-SMA and p-Smad2/3 expression at mRNA and\or protein level could be dose-dependently increased by TGF-β1.3.Real-time PCR and western blot analysis show that COL1A1,a-SMA and p-Smad2/3 expression at mRNA and\or protein level could be time-dependently increased by TGF-β1.4.Real-time PCR shows that the expression of miR-29b decreased with the treatment of TGF-β1.Compared with the control group,the expression of miR-29b in both 5 ng/ml group(P<0.001)and 10 ng/ml group(P=0.006)significantly decreased;and the expression of miR-29b in 48h group(P=0.003)and 72h group(P<0.001)were also significantly decreased.5.SEM images further demonstrated morphological changes in primate ESCs to myofibroblast-like cells which were characterised with hypertrophy,increased secretion of ECM and pseudopodia,and decreased junctions between cells in response to continuous TGF-β1 stimulation at the concentration of lOng/ml for 4 days.Conclusion:The method that we used in this study to separation the primary human ESCs was successful for the highly purified cells with perfect vitality.The expression of COL1A1,a-SMA and p-Smad2/3 can be time and dose-dependently increased by TGF-β1.However,the expression of miR-29b decreases with TGF-β1.Promoting myofibroblastic transdifferentiation of ESCs and activating TGF-β1/Smad signaling pathway are the two main mechanisms for TGF-β1 inducing fibrosis in ESCs.Chapter II Overexpression of miRNA-29b has anti-fibrotic effect of on TGF-β1-treated ESCsObjective:In this part,we will test the expression of COL1A1、α-SMA、p-Smad2/3 and miR-29b in ESCs when the miR-29b were over-expressed before or after TGF-β1 treatment.We will also investigate the effect of miR-29b on ESCs proliferation,apoptosis and cell cycle.Futher we explored the preventive and therapeutic potential of miR-29b mimics and its mechanisms were determined.Methods:1.Experiment grouping design(n=3):(1)Normal control group:normal cultured ESCs,no treatment;(2)TGF-β1-treated group:10 ng/ml TGF-β1 treatment on ESCs;(3)miR-29b preventive group:transfection of 50 nM miR-29b mimics into ESCs before TGF-β1 treatment at the concentration of 10 ng/ml.(4)miR-negative control preventive group:transfection of 50 nM miR-negative control mimics into ESCs before TGF-β1 treatment at the concentration of 10 ng/ml.(5)miR-29b therapeutic group:transfection of 50 nM miR-29b mimics into ESCs after TGF-β1 treatment at the concentration of 10 ng/ml.(6)miR-negative control therapeutic group:transfection of 50 nM miR-negative control mimics into ESCs after TGF-β1 treatment at the concentration of 10 ng/ml.2.Transfection:In preventive treatment,cells were incubated overnight to a density of 50%all so and incubated for another 24 h in serum-free culture media to synchronize the cell cycle.Afterwards,miR-29b mimics and a scrambled miRNA control(Ribobio,China)were transfected using Lipofectamine 2000 as instructed.The mixed liquor of mimic-lipo2000 was then added into ESCs with certain Opti-MEM.Six hour post-transfection,the media were refreshed as the experiment was designed.3.Real-time PCR analysis was performed to determine the mRNA expression of COL1A1,a-SMA and miR-29b.4.Western blot analysis and immunocytochemical staining were performed to determine the protein expression of COL1A1,a-SMA and p-Smad2/3.5.Cell counting kit-8(CCK8)assay was preformed for cell proliferation assay.The cultured ESCs were seeded into 96-well plates(3 × 10 3 cells per well).10 μl of CCK-8 solution was added to each well,followed by incubation for 2h.Optical densities(OD)were recorded at 450 nm using a microplate reader.6.Flow cytometric analysis was used to determine for cell apoptosis analysis and cell cycle analysis.Results:1.The expression of miR-29b had significant difference among groups(F=175.630,P<0.001).In the miR-29b therapeutic group and miR-29b preventive group,exogenous miR-29b mimics significantly increased the expression of miR-29b after transfection.2.The expression of COL1A1(F=45.551,P<0.001)and a-SMA(F=28.688,P<0.001)mRNA had significant difference among groups.In the miR-29b therapeutic group and miR-29b preventive group,exogenous miR-29b significantly decreased the mRNA expression of COL1A1 and α-SMA,and increased the expression of MEG3 compared with TGF-β1-treated group.And the difference in the miR-29b preventive group was more remarkable.3.The expression of COL1A1(F=86.104,P<0.001),a-SMA(F=139.874,P<0.001)and p-Smad2/3(F=86.451,P<0.001)at protein level had significant difference among groups.In the miR-29b therapeutic group and miR-29b preventive group,exogenous miR-29b significantly decreased the protein expression of COL1A1,a-SMA and p-Smad2/3 compared with TGF-β1-treated group.And the decrease in the miR-29b preventive group was more remarkable.Moreover,the TGF-β1-treated group showed high expression of intranuclear p-Smad2/3.However,overexpression of miR-29b decreased p-Smad2/3 nuclear translocation and accumulation.4.The role of miR-29b in regulating TGF-β1-treated ESCs proliferation was tested by CCK-8 assay.The difference of proliferation rate among groups was significant(F=456.240,P<0.001).In the miR-29b therapeutic group and miR-29b preventive group,the proliferations of fibrotic ESCs were significantly inhibited,showing 58.35± 0.13%and 68.00±1.81%separately.5.The difference of the percentage in G0/G1 phases among groups was significant(F=34.317,P<0.001).MiR-29b showed significant pro-apoptosis effect on activated ESCs compared to TGF-β1-treated group.The transfection of miR-29b increased the percentage of the activated ESCs in G0/G1 phases compared to the TGF-β1-treated group.Conclusion:The loss of miR-29b in ESCs after TGF-β1 exposure leads to abolishment of COL1A1 and a-SMA repression,via activating TGF-β1/Smad signaling pathway.Conversely,overexpression of miR-29b efficiently overcomes the pro-fibrogenic influence of TGF-β1 on ESCs.In addition,miR-29b increases the expression of MEG3,inhibites the proliferations,promotes the apoptosis of fibrotic ESCs and arrests the cell cycle of them at the G0/G1 phase in vitro.Overexpressing miR-29b may become a promising therapeutic strategy for endometrial fibrosis.ChapterⅢ The study of establishment of female SD rats model of IUAObjective:To assess the new established rat model of IUA using a curettage-LPS double injure method,study the pathological process of endometrial tissue at different time points after the surgery.Through this study,we expected to offer the basic knowledge of this model for further therapeutic exploration.Methods:1.The adult virginal female Sprague-Dawley(SD)rats,weighting 250-280 g,were used.10 mg/L solution of LPS(derived from Escherichia coli 055:B5)in normal saline was prepared beforehand.The 10#medical sterile surgical sutures(10 cm)were soaked in the LPS saline solution for 24 h before use.2.Experiment grouping design:forty-five SD rats were randomly divided into 3 groups.Twenty rats were operated to establish the IUA model groups;another twenty rats were operated as the sham groups.Five rats(n=10 horns)in each groups were killed 48 hours,4 days,9 days and 18 days later respectively.Another 5 rats without treatments were used as normal control.3.Vaginal smears were taken daily and examined microscopically.The model forming surgery was conducted at estrum.4.Method for establishing IUA animal models:All the rats were anesthetized with 10%Chloral hydrate(10 mg/kg)through intraperitoneal injection.A 0.3 cm longitudinal incision was made on the uterus 0.5 cm above its bifurcation.Then a mini endometrial curette was applied to scrape the endometrial lining of the upper uterus.Curettage ceased when the uterine wall became rough.After mechanical injury,a LPS surgical suture was inserted in the uterine cavity through the same uterine incision just made.The tail of the LPS surgical suture was placed through the abdominal wall,and was retained at 1 cm length on the surface of the skin to facilitate removal after 48 h.5.The segments of uterus were collected immediately after the rats were sacrificed.The transverse sections were routinely stained with hematoxyline-eosin and Masson stains.On each hematoxylin-eosin-stained slice,the number of glands per high-power field was counted.In order to quantify the degree of endometrial fibrosis,in masson-stained slice,the ratio between the area of endometrial stromal fibrosis and endometrial area were calculated using a quantitative image-analysis system(Image-Pro Plus software).The ratios of four fields were averaged.Results:1L Forty-eight hours after the creation of the IUA model,the regeneration of the epithelial cells was rarely observed and no gland regeneration was evident.Diffuse hemorrhage,edema,and dense lymphocytic infiltration were observed in the stroma;however there is no obvious fibrous tissue in the uterine cavity.At the end of the first estrous cycle(4d)after the creation of the model,avascular fibrous bands with varying amounts of white blood cell infiltration could be observed in the uterine cavity.A few areas without fibrous bands had sparse regenerated flat epithelial cells in the surface,and gland regeneration was seldom seen.At the end of the second estrous cycle(9d)after the model was created,some highly fibrous adhesions can be observed in the uterine cavity when the uterus was cut vertically.Microscopically,the fibrous adhesions become denser as time passes,and the area without adhesions was lined by flattened or low columnar epithelial cells.In addition,increased extracellular matrixes deposited round scattered glands were present beneath the epithelial layer like scars.At the end of the forth estrous cycle(18d)after the injury,the endothelial morphology had failed to reconstruct and the formed scar is similar to that of 9d after injury2.The ratios of fibrosis in rat s endometrium had significant difference among groups(F=197.526,P<0.001).Ratios of fibrosis on 4th day(42.11±3.57%),9th day(65.32±7.98%)and 18th day(67.82±5.77%)after injury were significantly higher than that of rats in the control group(20.49±3.56%).In addition,the ratio of fibrosis in the experimental group 9th day after injury was significantly higher than that of 4th day after injury(P<0.05).Whereas,there was no significant difference between the 9th day group and the 18th day group after injury(P>0.05).3.The number of endometrial glands had significant difference among groups(F=104.983,P<0.001).Fewer glands were observed in 48h(1.9±1.20),4th day(2.30±1.34),9th day(1.90±1.10)and 18th day(1.50±0.71)after injury than in the control group(9.7±0.95).Glands failed to regenerate in the stroma.4.Curettage-LPS double injure only made an endometrial injure model in rat 48h after the surgery.There is no significant difference in the ratio of fibrosis between the IUA model group and the sham groups 48h after the surgery(p=0.108).The IUA model was first created in the 4th day(p<0.001)after the surgery,and last till the 18th days(p<0.001).Conclusion:The method of curettage-LPS double injure can cause the irreversible injury of endometrial,scarring repairment and fibrosis adhesions,so as to establish a stable rats IUA model.Within 48h after the surgery was performed,acute inflammation is the main response and pathology process.ECM is accumulated to some extent 4d after the surgery,and with the development of fibrosis,endometrial ECM undergoes a peak deposition 9d after the model,and keeps on to the 18d.ChapterⅣ The study of preventive and therapeutic potential of agomir-29b on the rat model of IUAObjective:In the current study,we expect to demonstrate the preventive and therapeutic potential of miR-29b as a regulator of collagen synthesis,EMT and fibrosis development in vivo by transfecting agomir-29b,using the new established rat model of curettage-LPS double injury-induced IUA.Further we explored the possible mechanism.Methods:1.Experiment grouping design:thirty-five SD rats were divided into 7 groups randomly:①Sham operation group;②IUA model of 4d group;③IUA model of 9d group;④agomir-29b preventive group;⑤agomir-negative control preventive group;⑥agomir-29b therapeutic group;⑦agomir-negative control therapeutic group.There are five rats(n =10 uterine horns)in each group.2.The method for establishing IUA animal models was the same as was mentioned in the ChapterⅢ.3.The uteri were collected right after rats were killed.Uterine tissue above the incision from each horn was divided into four parts:(1)The first part was fixed with 4%paraformaldehyde for histology(routinely stained with hematoxyline-eosin and Masson stains).Immunohistochemistry was performed to determine the expression of TGF-β1 and COL1A1,and immunofluorescence was performed to determine the expression of a-SMA and p_Smad2/3.(2)The second part was through into TRIzol liquid for later RNA extraction.Real-time PCR analysis was performed to determine the expression of miR-29b,and the expression of COL1 Al,a-SMA and E-cadherin at the mRNAlevel.(3)The third part was stored at-80 degree centigrade for protein extraction.Later western blot analysis was performed to determine the protein expression of COL1A1,α-SMA,E-cadherin,p-Smad2/3,CTGF and Sp1.(4)The rest was spare in liquid nitrogen.Results:1.We first determine the effect of agomir-29b on miR-29b expression in the uteri by qRT-PCR analysis.The difference among groups is significant(F=403.223,P<0.001).The expression of miR-29b was largely decreased in the fibrotic endometrium 4d(0.36±0.05)and 9d(0.23±0.06)after injury,and there is no significant difference between them.In contrast,IUA rats that were given agomiR-29b exhibited a strong expression of miR-29b in the uterine tissues in both preventive group(1.66±0.16)and therapeutic group(1.60±0.10)compared to the IUA model of 9d group(P<0.05).The results indicated that agomir-29b could be effectively transfected in vivo,and realized the overexpression of miR-29b in the uteri of SD rats.2.Hematoxyline-eosin and Masson stains shows that the ratios of fibrosis(F=134.748,P<0.001)and the count of glands(F=87.250,P<0.001)had significant differences among groups.In the agomir-29b preventive group and the agomir-29b therapeutic group,there are glands regenerated and the epithelial cells were observed at different degrees.The fibrous adhesions become alleviated,too.Compared with the IUA model of 9d group(63.56±5.88%;1.80±1.03),the ratios of fibrosis decreased and the count of glands increased in both the agomir-29b preventive group(25.21 ±2.00%;8.00±1.83)and the therapeutic agomir-29b group(43.57±5.58%;4.60±0.84).However,there are no significant differences between the therapeutic agomir-29b group and the IUA model of 4d group(41.57±4.27%)in the ratios of fibrosis.3.The expression of COL1A1 at both mRNA(F=43.620,P<0.001)and protein level(F=820.970,P<0.001)had significant differences among groups.Compared with the Sham operation group,the expression of COL1A1 at both mRNA and protein level increased in both IUA model of 4d group and IUA model of 9d group,and the expression was higher in IUA model of 9d group than in IUA model of 4d group.Compared with the IUA model of 9d group,the expression of COL1A1 in the agomir-negative control preventive group and the agomir-negative control therapeutic group had no significant differences.However the expression of COL1A1 in the agomir-29b preventive group and the agomir-29b therapeutic group decreased significantly.There was no significant difference in the agomir-29b therapeutic group and IUA model of 4d group.The result in Immunohistochemistry was consistent with that in the western blot analysis.4.The expression of a-SMA at both mRNA(F=526.036,P<0.001)and protein level(F=268.308,P<0.001)had significant differences among groups.Compared with the Sham operation group,the expression of a-SMA at both mRNA and protein level increased in both IUA model of 4d group and IUA model of 9d group.Compared with the IUA model of 9d group,the expression of α-SMA in agomir-negative control preventive group and agomir-negative control therapeutic group had no significant differences.However,the expression of α-SMA in the agomir-29b preventive group and the agomir-29b therapeutic group decreased significantly.There was no significant difference in the agomir-29b preventive group and the Sham operation group.The result in Immunofluorescence was consistent with that in the western blot analysis.5.The expression of E-cadherin at both mRNA(F=286.418,P<0.001)and protein level(F=239.158,P<0.001)had significant differences among groups.Compared to the Sham operation group,the expression of E-cadherin at both mRNA and protein level decreased in both IUA model of 4d group and IUA model of 9d group,and the expression was lower in IUA model of 9d group than in IUA model of 4d group.Compared to the IUA model of 9d group,the expression of E-cadherin in agomir-negative control preventive group and agomir-negative control therapeutic group had no significant differences.The expression of E-cadherin in the agomir-29b preventive group and the agomir-29b therapeutic group increased significantly compared to the IUA model of 9d group.6.The MOD of TGF-β1 in the immunohistochemistry was calculated using a quantitative image-analysis system(Image-Pro Plus software).The difference in the expression of TGF-β1 among groups was significant(F=782.731,P<0.001).Compared with the Sham operation group(0.005±0.002),the expression of TGF-β1 at protein level increased in both IUA model of 4d group and IUA model of 9d group,and the expression was higher in IUA model of 9d group(0.111±0.011)than in IUA model of 4d group(0.0422 ±0.008).Compared with the IUA model of 9d group,the expression of TGF-β1 in agomir-negative control preventive group(0.109 ±0.008)and agomir-negative control therapeutic group(0.116±0.007)had no significant differences.Well the expression of TGF-β1 in the agomir-29b preventive group(0.005 ±0.001)and the agomir-29b therapeutic group(0.050 ±0.007)were decreased significantly.There was no significant difference between the agomir-29b therapeutic group and IUA model of 4d group.7.The difference in the expression of p-Smad2/3(F=410.103,P<0.001),CTGF(F=110.733,P<0.001)and Spl(F=167.661,P<0.001)at protein level among groups were significant.Compared with the Sham operation group,the expression of p-Smad2/3,CTGF and Spl at protein level determined by western blot analysis increased in both IUA model of 4d group and IUA model of 9d group,and the expression was higher in IUA model of 9d group than in IUA model of 4d group.Compared with the IUA model of 9d group,the expression of p-Smad2/3,CTGF and Spl in agomir-negative control preventive group and agomir-negative control therapeutic group had no significant differences.Well the expression of p-Smad2/3,CTGF and Sp1 in the agomir-29b preventive group and the agomir-29b therapeutic group decreased significantly.No significant difference had been seen in the expression of p-Smad2/3,CTGF and Sp1 between the agomir-29b preventive groups and the Sham operation groups.And there were also no significant differences between the agomir-29b therapeutic group and IUA model of 4d group.The result of p-Smad2/3 in Immunofluorescence was consistent with that in the western blot analysis.Conclusion:The present study added novel evidence for a protective role as well as therapeutic potential of miR-29b in endometrial fibrosis in the curettage-LPS double injure induced IUA rat model,which was sustained by the finding that double injury induced endometrial fibrosis,including the accumulation of ECM and enhanced EMT,was associated with a loss of miR-29b in uterine,whereas,restored uterine miR-29b protected endometrium against fibrosis and maintained the regenerative ability of the endometrium.Although therapeutic transfection of agomir-29b failed to reverse the already-formed fibrosis,it could retard the process of endometrial fibrosis in the established model of IUA.Furthermore,we demonstrated that blockade of the TGF-β1/Smad-CTGF-SP1 axis may be the mechanism by which exogenous supplement of miR-29b attenuated endometrial fibrosis. |
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