Preliminary Study Of HA/PLGA Applied In Prevention Of Intrauterine Adhesion

Asherman’s syndrome(AS), also known as intrauterine adhesion(IUA), is defined as a wide range of partial to complete adhesions within the uterine cavity due to scars usually occurring after curettage and various forms of hysteroscopic surgery, including resection of septa, and hysteroscopic resection of solitary and multiple fibroids. The most serious consequences of IUA is secondary infertility. The methods used to prevent intrauterine adhes ion in c linics nowadays include(1) estrogen which is supposed to increase the proliferation of endometrium,(2) placement of intrauterine device,(3) inserting catheter into uterine cavity,(4)hyaluronic acid infusion into uterine cavity. The last three methods were expected to be applied as a physical barrier to separate the anterior and posterior uterine wall. These methods, however, has obvious limitation. Intrauterine device and catheter require another operation to take them out. Hyaluronic acid gel could not dwell in the uterine cavity for a long period. The aim of this research is to produce a biodegradable film which on the one hand requires no operation to take out, and the other hand can dwell in the uterine cavity for longer time than gel. Meanwhile HA secreted from the film could inhibit the endometrial fibrosis. First of all in this research, endometrial fibrosis was regarded as one of mechanisms in IUA formation. The fibrotic extent of endometrium in patients with IUA and animal model of AS was analyzed. The role of fibrosis in formation of IUA was confirmed. Then HA was used for preventing IUA in animal model. The possible mechanism by which hyaluronic acid inhibited the endometrial fibrosis was also studied. At last, a PLGA film containing hyaluronic acid was produced. The characters of HA/PLGA film inc luding mechanical properties, drug release, and cytotoxicity were inspected. The inhibition of endometrial fibrosis by HA/PLGA film was verified in animal model of AS. In conclusion, HA/PLGA film has a potential application value in preventing IUA.PART ONE THE FIBROTIC EXTENT OF ENDOMETRIUM IN PATIENTS WITH INTRAUTERINE ADHESIONObjective: The objective of this part was to evaluate whether fibrotic markers were abnormal expressed in endometrium in patients with intrauterine adhesion and whether fibrosis was involved in the IUA formation.Method: We revaluated endometrial fibrosis using Masson’s stain. We detected the expression of endometrial fibrosis markers(TGF-Beta 1, CTGF, collagen protein I and collagen protein III) in endometrial tissue with or without intrauterine adhesions using IHC.Results: The ratio of the area with endometrial fibrosis to total endometrial area in intrauterine adhesion significantly increased compared with the normal endometrial tissue(P < 0.05); The expression levels of fibrotic markers were higher in the endometrial tissue with intrauterine adhesions compared to normal endometrial tissue(P < 0.05).Conclusion: Aberrant activation of fibrosis may be involved in the pathology of intrauterine adhesions.PART TWO ESTABLISHMENT OF A MOUSE MODEL OF ASHERMAN’S SYNDROMEObjective: To establish a mouse model of AS.Method:(1)Female Kunming mice were divided into 2 groups: the sham operation group and the endometrial damaged group. In sham operation, the mouse underwent abdominal surgical incis ion without uterine curettage. The endometrial damaged group underwent uterine curettage and LPS intrauterine infusion.(2)The extent of endometrial fibrosis was calculated using Massion’s stain.(3)The fibrotic markers(TGFβ1, CTGF, collagen I, and collagen III) in endometrial tissue were detected using IHC and WB.Resluts: The ratio of the area with endometrial fibrosis to total endometrial area in the endometrial damaged group was significantly increased compared to the sham operation group(P < 0.05). The expression of fibrosis markers(TGFβ1, CTGF, collagenⅠ, and collagen Ⅲ) in the endometrium was increased in the damaged group compared to the sham operation group.Conclusion: Successful establishment of a mouse model of AS.PART THREE MECHANISM OF INHIB ITION OF ENDOMETRIAL FIBROSIS BY HYALURONIC ACIDObjective: To identify the effect of HA at high and low molecular weight on fibrosis of the endometrium in a mouse model of AS. Primary cell of endometrium was used to find out the possible mechanism of anti-fibrotic effect of HA on endometrium.Method: Endometrial fibrosis was compared among the control group, LMW-HA, and HMW-HA group. The extent of endometrial fibrosis was calculated using Masson’s stain. The fibrotic markers(TGFβ1, CTGF, collagen I, and collagen III) in endometrial tissue were detected using IHC and WB. WB and q PCR were used to detect the fibrotic protein expression in primary endometrial stromal cells of control group, TGFβ1group, TGFβ1+HA and TGFβ1+HA+Anti-CD44 Antibody group.Resluts: The ratio of the area with endometrial fibrosis to total endometrial area in the HMW-HA group was significantly decreased compared to the control group(P < 0.05). The expression of fibrotic markers(TGFβ1, CTGF, collagenⅠ, and collagen Ⅲ) in the endometrium was attenuated in the HMW-HA group compared to the control group(P < 0.05). Meanwhile the LMW-HA group had no similar effect. The expression of fibrotic markers(TGFβ1, CTGF, collagen Ⅰ, and collagen Ⅲ) in TGFβ1group was significantly increased compared to the control group(P < 0.05). The expression of fibrotic markers in TGFβ1+HA group was significantly decreased compared to TGFβ1group(P < 0.05). The expression of fibrotic markers in TGFβ1+HA+Anti-CD44 Antibody group was significantly increased compared to TGFβ1+HA group. The expression of smad2 has no difference among the four groups. Phosphorylation of smad2 in TGFβ1group elevated compared to the control group. Phosphorylation of smad2 in TGFβ1+HA group decreased compare to TGFβ1group. Phosphorylation of smad2 in TGFβ1+HA+Anti-CD44 Antibody group elevated again compared to TGFβ1+HA group.Conclusion: HA at high molecular weight may attenuate the degree of endometrial fibrosis after endometrial damage, which may contribute to preventing formation of intrauterine adhesions. Inhibition of TGFβ1/smad signal way by binding of HA at high molecular weight to CD44 may be involved in the inhibition of endometrial fibrosis by HA.PART FOUR THE PRODUCTION OF HA / PLGA FILM AND IDENTIFYING ITS CHARACTERSObjective: To produce the HA/PLGA film and identify its characters inc luding its topograph, in vitro and in vivo degradation, non-deformability, in vitro HA release from the film and in vitro cytotoxicity test.Method:Preparation of PLGA and HA/PLGA film was done according to the reference. Scanning electron microscopy was used to indentify the topograph. The film was immersed into the simulated uterine fluid to test the in vitro degradation. The film was also placed in to uterine cavity of mouse to test the in vivo degradation. The non-deformability was tested by mechanical test and isolated organ test. In vitro HA release was tested by immersing HA/PLGA film to simulate uterine fluid. The concentration of HA was determined by sulfuric acid carbazole colorimetry method. MTT was used to detect the in vitro cytotoxicity of the film.Results: There was no significant difference in the topograph among the PLGA film of different proportion of LA:GA. The degradation time of PLGA film(50:50) was fastest. Most weight of the film was lost during the first 4 weeks.The degradation time of PLGA film(90:10) was the slowest. Most weight of the film was not lost till 12 weeks. The degradation time of PLGA film(75:25) was between PLGA film(50:50) and PLGA film(90:10). In vivo degradation, PLGA film(75:25), PLGA film(90:10), HA/PLGA could dwell in uterine cavity for more than 7 days. Elasticity modulus increased with the grown LA proportion. The film was not torn into pieces when going though the uterine cervix. HA could be released from HA/PLGA film in the simulated uterine fluid. Comulative HA released from HA/PLGA film reached 64.15% on 4 weeks. The in vitro cytotoxicity of the HA/PLGA film was not significantly different from the negative control group and was significantly lower than that of positive control(P<0.05).Conclusion: According to the results of degradation time of PLGA film, PLGA(75:25) was chosen to prepare the HA/PLGA film. The HA/PLGA film could go through the cervix without being torn into pieces. It could even provide a distance between the anterior and posterior wall of uterus. Meanwhile HA/PLGA film could release HA persistently. Of course, the HA/PLGA film has no cytotoxicity to the endometrial stromal cells according to the in vitro cytotoxicity test.PART FIVE ANTI-FIBROTIC EFFECT OF HA/PLGA ON ENDOMETRIUM IN A MOUSE MODEL OF ASHERMAN’S SYNDROMEObjective: To identify the effect of HA/PLGA on fibrosis of the endometrium in a mouse model of AS.Method: Endometrial fibrosis was compared among the control group, PLGA, and HA/PLGA group. The extent of endometrial fibrosis was calculated using Masson’s stain. The fibrosis markers(TGFβ1, CTGF, collagen I, and collagen III) in endometrial tissue were detected using IHC and WB.Results: The ratio of the area with endometrial fibrosis to total endometrial area in the HA/PLGA group was significantly decreased compared to the control group(P<0.05). The expression of fibrotic markers(TGFβ1, CTGF, collagenⅠ, and collagen Ⅲ) in the endometrium was attenuated in the HA/PLGA group compared to the control group. Meanwhile the fibrotic extent of the PLGA group was not different from that of ontrol group.Conclusion: HA/PLGA film may attenuate the degree of endometrial fibrosis after endometrial damage, which may contribute to preventing formation of intrauterine adhesions.