| Background:Intrauterine adhesions (IUA) is partial or complete obliteration in the uterine cavity and (or) the cervical canal because of the basal layer of endometrium severely damaged that due to endometrial repair disorders and scar repair. IUA is common happened after aitificial and curettage. The manifestations of IUA include the decrease in menstrual flow, amenorrhea infertility, periodic abdominal pain, secondary infertility, obstetric complications (abortion, premature delivery, placenta accreta, placental abruption, placental implantation, postpartum hemorrhage, etc), which significantly influence the reproductive health.IUA is common disease in the world, it has now become the second largest cause of female secondary infertility. Hysteroscopy examination and treatment is currently the gold standard for clinical diagnosis and treatment, but postoperative adhesion regenerate easily. Endometrial need some time to repair, easy to occur adhesion during this period after transcervical resection of adhesions. There are many methods for prevention the postoperative adhesion regeneration, and most scholars tend to the comprehensive treatment mainly to barrier treatment, such as placing intrauterine device combined with estrogen, placing children balloon catheter, uterine cavity injection of hyaluronic acid gel, amnion grafting and stem cell therapy, etc. At present the theatment universally adopted is the the comprehensive treatment of transcervical resection of adhesions combined with intrauterine device and estrogen, But there is no one ideal method. Therefore, it has become a problem for clinical doctors pay close attention to preventing adhesion regeneration after operative. Menstrual symptoms has improved, but the pregnancy and livebirth rate are still not ideal by taking some anti adhesion material application to treat IUA. So to evaluate IUA therapeutic effect of the point is not only the improving clinical menstruation symptom, but more important is to improve the clinical pregnancy rates and live birth rates.Oxidized regenerated cellulose (Interceed) is a sterile absorbable off-white knitted fabric prepared by the controlled oxidation of regenerated cellulose. Interceed has advantages such as good biocompatibility, biodegradable, non-toxic and safe and effective, etc. It has been widely used in general surgery and abdominal surgery of obstetrics and gynecology, the antiadhesion effect is very clear, but it is very few that use Interceed to the uterus. Some scholars has adopted the (Interceed) surrounding the intrauterine device combination with large dose of estrogen for treating intrauterine adhesions after surgery successfully, obtained certain antiadhesion effect, but the pregnancy rates that observed were no significant difference.The key factor for embryo implantation successfully are the embryo quality and endometrial receptivity. With the development of assisted reproductive technology, the embryo quality has got improved significantly,but it is still the bottleneck that the low embryo grow rate of the development of human assisted reproductive technology. Endometrial receptiblity reflect the endometrial accept ability of embryos. Endometrial receptiblity is a consequence that the allowance is available of apposition, adhesion, invasion and embryo implantation by the alteration in endometrial mesenchymal. Currently the expression of pinocytosis and integrin avP3 is recognized as the markers of endometrial receptivity. Endometrial receptiblity has the strict specificity of space and time, Pinocytosis appears in 6-8d after ovulation, continuous less 48 hours. Pinocytosis of rabbits express in 7d-8d after mating, This period mach with blastocysts endometrial adhesion. Integrin avβ3 and pinocytosis synchronization appear.The research of the endometrium receptivity for IUA is very little, and the research is none that whether affect the endometrial receptivity by using Interceed for uterine cavity. Therefore, this study adopts the methed of mechanical and infection double damage to build IUA animal models on New Zealand white rabbit, To evaluate the treatment efficacy of IUA by observing a series of changes of such as endometrial morphology, histology,etc after Interceed combined with estrogen therapy. To assess the influence on the endometrial receptiblity by observing the expression of pinocytosis and interin avβ3 in the endometrial on implantation window, as to provide theory basis for the clinical treatment of IUA.Chapter 1 The treatment efficacy of Interceed combined with estrogen on rabbit intrauterines adhesionsObjective:To explore the treatment efficacy, adopting the methed of mechanical and infection double damage to build IUA animal models, giving placement Interceed in the uterus combined with injection estrogen therapy. As to provide theory basis for the clinical treatment of IUA.Methods:1. Ninety-six healthy unfertilized female New Zealand white rabbits were divided into 6 groups(16 rabbits in each group).(1)Normal control group:Model was not made in normal control group, which was performed sham operation.(2)Interceed control group:the Interceed was placed into the normal rabbit’s uterus.(3)Model group:the rabbits were not treated after the modeling.(4)Interceed treatment group:the rabbits were placed Interceed into the uterus at 1 w after the modeling.(5)Estrogen treatment group:the rabbits were injected estrogen at 1 w after the modeling.(6)Combined treatment group:the rabbits were placed Interceed into the uterus and injected estrogen at 1 w after the modeling.The intrauterine adhesions mode of rabbits were established by the double damage method of mechanical injury and infection in the last four groups.2. Four rabbits were sacrificed in each group before treatment(1w after the modeling), after treatment 1w,2 w and 4 w respectively. Draw blood from the ear marginal vein, serum estradiol levels were detected by the methed of radioimmunoassay. The uterine tissuss were collected. HE was performed to observe endometrial morphology and calculate glands. Masson staining was performed to detect the degree of endometrial fibrosis. The expression of TGF-β1 protein was detectd by immunohistochemistry.3. Statistical Analysis. Statistical analyses were performed using SPSS version 19.0, all data are presented as "mean values±SD". Homogeneity of variance were detected, One-Way ANOVA was utilized to analyze the multiple groups, the definition of significant level a=0.05, P<0.05 was considered statistically significant, If conform to the homogeneity of variance, two samples test were adopt LSD test. If do not conform to the homogeneity of variance, two samples test were adopt Tamhane’s test.Results:1. The uterus anatomical observation in each groupThe appearance of normal rabbit’s uterus is pink, the endometrial area are pale, and the endometrial surface has no congestion or haemorrhage, the opening glands were visible. There were no obvious changes about uterus anatomy structure in Interceed control group. Interceed has been completely absorbed in 2 weeks after placement in uterine cavity in Interceed control group. The degree of uterus edema and uterine cavity effusion increase gradually with the extension of modeling time. The number of glands were few, Thickness of endometrial were uneven. In Interceed treatment group, estrogen treatment group, combined treatment group, the edema degree of uterus and uterine cavity effusion reduce gradually, the glands repair with the extension of treatment time. Anatomy of the uterus form basic returned to normal after treatment of 4 weeks in combined treatment group. Other organs such as experimental groups of ovarian, fallopian tube had no obvious change.2. The comparison of serum E2 in each groupThere are significant differences in six group about the concentration of serum E2 (F=6.585, P=0.000), and there are significant differences at different time after treatment (F=9.501, P=0.000).(1)Before treatment, there have no statistical significance in six group about athe concentration of serum E2 (F=0.564, P=0.726).(2)One week after treatment, there was significant difference among all groups(F=412.198, P<0.000). The concentration of serum E2 in combined treatment group, estrogen treatment group were significantly higher than other groups(P< 0.05), compared with normal control group the differences had no statistical significance in other groups (PInterceed treatment group=0.664, P model group= 0.631, PInterceed treatment group=0.573).(3)Two weeks after treatment, there was significant difference among all groups(F=35.301, P<0.000). The concentration of serum E2 in combined treatment group, estrogen treatment group were significantly higher than other groups(P< 0.05), compared with normal control group the differences had no statistical significance in other groups(PInterceed treatment group=0.804, P model group= 0.764, PInterceed treatment group=0.737).(4)Four weeks after treatment, there have no statistical significance in six group about athe concentration of serum E2 (F=1.107, P=0.391).3. The comparison of the number of endometrial glands in each group by HE stainingNormal rabbit uterus contains endometrium layer, muscular layer and placenta layer, endometrium layer can be divided into the mucosa and submucosa. The mucosa is covered with simple columnar epithelium cells, there were glands, blood vessels, and a small amount of fibroblasts in submucosa. The number of endometrial glands was increased in the three treatment group.(1)Before treatment, the number of glands among six groups was statistically significant difference(F=46.992, P<0.001). Each group (except Interceed control group) compared with the normal control group differences were statistically significant (Pmodel group=0.000, PInerceed treatment group=0.000, Pestrogen treatment group=0.000, Pcombined treatment group=0.000). Interceed control group compared with normal control group, tehere was no statistical difference (P= 0.674). The combined treatment group compared with Interceed treatment group and estrogen treatment group respectively, differences were no statistical difference (P= 0.766, P= 0.766).(2)One week after treatment, the number of glands among six groups was statistically significant difference (F=34.428, P<0.001). Groups (except Interceed control group) compared with normal control group, the differences were statistically significant(Pmodel group=0.000, PInterceed treatment group=0.000, Pestrogen treatment group=0.000, Pcombined treatment group=0.002). Compared with normal control group, there was no statistically significant difference in Interceed control group (P=0.774). Combined treatment group compared with Interceed treatment group and estrogen treatment group, the differences were statistically significant (P=0.000, P=0.000).(3)Two weeks after treatment, difference was statistically difference between six groups (F=17.758, P<0.001). There was significant difference in model group, Interceed treatment group and estrogen treatment group compared with group normal control group respectively (Pmodel group=0.000, P interceed treatment group=0.040, Pestrogen treatment group=0.004). There was no significant difference in Interceed control group, combined treatment group compared with normal control group respectively (P=0.907, P=0.907). There was significant difference in combined treatment group compared with Interceed treatment group, estrogen treatment group respectively (P= 0.017, P=0.017).(4)Four weeks after treatment, there was significant difference among all groups (F=18.832, P<0.001).There was statistically difference in model group compared with normal control group (P=0.000), There was no statistical significance in another groups compared with normal control group (P Interceed control group=0.517, Pinterceed treatment group 0.104,Pestrogen treatment group 0.108, Pcombined treatment group =0.186). There was statistical difference in combined treatment group compared with Interceed treatment group and estrogen treatment group respectively (P= 0.004, P=0.004).4. Comparison of endometrial fibrosis area ratio in Masson stainingNormal rabbit intrauterine membrane in Masson staining can be observed from interstitial collagen fiber contained a small amount of neat rows of blue. As the duration of treatment, the degree of fibrosis reduce gradually.(1)Before treatment, there was significant difference among all groups about fibrosis area ratio (F=79.978, P<0.001). Between groups (except Interceed control group) compared with normal control group, the differences were statistically significant (Pmodel group=0.000, PInterceed treatment group=0.000, Pestrogen treatment group=0.000, P combined treatment group=0.000). There was no significant difference in Interceed control group compared with normal controls (P= 0.836). The differences had no statistical significance in combined treatment group compared with Interceed treatment group and estrogen treatment group respectively (P= 0.605, P= 0.605).(2)One week after treatment, there was significant difference among all groups about fibrosis area ratio (F=60.685, P<0.001). There was significant difference in model group, Interceed treatment group, estrogen treatment group compared with normal control group respectively (Pmodel group=0.000, PInterceed treatment group=0.000, Pestrogen treatment group=0.000). There was no statistically significant difference in Interceed control group and combined group compared with normal control group respectively(P=0.941, P=0.744). Combined treatment group compared with Interceed treatment group and estrogen treatment group respectively, the differences were statistically significant (P= 0.001, P=0.002).(3)Two weeks after treatment, there was significant difference among all groups about fibrosis area ratio (F=150.764, P<0.001). The difference had statistical significance in Model group and estrogen therapy group compared with normal control group (Pmodel group=0.000, Pestrogen treatment group=0.007). There were no statistically significant differences in Interceed control group, Interceed treatment group and combined group compared with normal control group (PInterceed control group=0.977, PInterceed treatment group=0.641, Pcombined group=0.206). There was no significant difference in combined treatment group compared with Interceed treatment group (P=0.707), but there was significant difference compared with estrogen treatment group (P=0.000).(4)Four weeks after treatment, there was significant difference among all groups about fibrosis area ratio (F=155.768, P<0.001). Model group compared with normal control group, difference was statistically significant (P=0.000). while another groups compared with normal control group, there were no statistical significance (Plnterceed control group=0.998, PInterceed treatment group=0.254, Pestrogen treatment group=0.387, Pcombined group=0.062). There was no significant difference in combined treatment group compared with Interceed treatment group (P=0.996), but there was significant difference compared with estrogen treatment group (P=0.000).5. Analysis of each group expression of endometrial TGF-β1 protein levels.TGF-β1 protein was expressed in endometrial luminal epithelium, glandular epithelium and stroma, brownish yellow granules.(1)Before treatment, there was significant difference among all groups about TGF-β1 protein expression in endometrial (F=55.664, P<0.001). Each group (except Interceed control group) compared with normal control group, the difference was statistically significant (Pmodel group=0.000, Plnterceed treatment group=0.000, Pestrogen treatment group=0.000, Pcombined treatment group=0.000). Thers were no significant differences in combined treatment group compared with Interceed treatment grouo and the estrogen treatment group respectively (P= 0.534, P=0.584).(2)One week after treatment, there was significant difference among all groups about TGF-β1 protein expression in endometrial (F=59.684, P<0.001). While Model group, Interceed treatment group, estrogen therapy group and combined treatment group compared with the normal control group, the difference were statistically significant (Pmodel group=0.000, PInterceed treatment group=0.000, Pestrogen treatment group=0.000, Pcombined treatment group=0.000). The difference was not statistically significant in Interceed control group compared with the normal control group (P=0.066). There was significant difference in combined treatment group compared with Interceed treatment group and estrogen treatment group respectively (P= 0.009, P= 0.043).(3)Two weeks after treatment, there was significant difference among all groups about TGF-β1 protein expression in endometrial (F=102.618, P<0.001). Model group, Interceed treatment group, estrogen treatment group compared with the control group, the differences were statistically significant (Pmodel group=0.000, PInnterceed treatment group=0.000,Pestrogen treatment group=0.007). Interceed control group and the combined treatment group were in the control group showed no significant difference (PInterceed control group=0.971, Pcombined treatment group=0.104). Compared with Interceed treatment group and estrogen treatment group, differences in combined treatment group were statistically significant different (P=0.000, P=0.009).(4)Four weeks after treatment, there was significant difference among all groups about TGF-β1 protein expression in endometrial (F=174.634, P<0.001). The differences were statistically significant in the model group and Interceed treatment group compared with the normal control group (P=0.000, P=0.002). there was no significant difference in other group compared with normal control group (P Interceed control group=0.940, Pestrogen treatment group =0.608,Pcombined treatment group=0.993).here Was significant difference in combined treatment group compared with Interceed treatment group (P=0.001), but there was no significant difference compared with estrogen treatment group (P=0.170).Conclusion:1. The method of mechanical and infection double damage can establish a stable rabbit IUA model,and has good repeatability. We can evaluate the efficacy efficacy of Interceed combined with estrogen on IUA.2. Interceed can not cause inflammation, and it has no obvious adverse effect on the normal endometrium.3. Interceed treatment group, estrogen treatment group and treatment group, three therapy metheds can reverse the number of endometrial glands express a certain level, the degree of endometrial fibrosis and endometrial TGF-β1 protein expression. The combined treatment method works best.Chapter 2 The influence of endometrial receptivity by Interceed combined with estrogen on rabbit intrauterines adhesionsObjective:In order to evaluate the influence of endometrial receptivity, adopting the methed of mechanical and infection double damage to build IUA animal models, giving placement Interceed in the uterus combined with injection estrogen therapy, observed the expression of endometrial pinocytosis and integrin avβ3, As to provide theory basis for the clinical treatment of IUA.Methods:1. One hundred and forty-four healthy unfertilized female New Zealand white rabbits were divided into 6 groups (24 rabbits in each group):normal control group, Interceed control group, model group, Interceed treatment group, estrogen treatment group and combined treatment group as we introduced in the chapter 1. We observed two oestrous cycles in which time we scrape the vaginals of these rabbits with sterile stick to obligate ovulation and pseudopregnancy in the second week of treatment.2. We executed 8 rabbits from 6th to 8th day of the pseudopregnancy and removed the uterines immediately. We divided the uterines into two parts averagely. The first part were washed with PBS liquid before vertically cut and then fixed in the glutaraldehyde solution to observe the expression of endometrial pinocytosis under scanning electron microscopy. The second part were fixed in 4% paraformaldehyde for 24 hours to observe the expression of avβ3 integrin under mmunohistochemical dying.3. Statistical Analysis. Statistical analyses were performed using SPSS version 19.0, all data are presented as "mean values±SD". Homogeneity of variance were detected, One-Way ANOVA was utilized to analyze the multiple groups, the definition of significant level α=0.05, P<0.05 was considered statistically significant, If conform to the homogeneity of variance, two samples test were adopt LSD test. If do not conform to the homogeneity of variance, two samples test were adopt Tamhane’s test.Results1. The expression of endometrial pinocytosis during implantation window period non-ciliated cells with microvilli covered the surface of the endometriun before the growth of pinopodes under SEM,The pinopodes protrude as mushroom-like ecptomas on the surface of the endometriun.(1)Normal control group:A great number of cells with shortened or vanished microvilli protruded to the uterine cavity and the grown pinopodes came into being on the 6th day of pseudopregnancy. Bare surface, non-microvilli, unanimous size, clear boundary and mushroom-like ecptomas presented in the total mature pinopodes which covered the surface of the endometrium on the 7th day of pregnancy. Degenerated pinopodes with Shrinking surface presented on the 8th day of pseudopregnancy.(2)Interceed control group:A great number of cells with shorten microvilli began to protrude to the uterine cavity and the developmental pinopodes presented on the 6th day of pseudopregnancy. Grown pinopodes shown as membrane-like ecptomas with regular morphology as well as occasional short and thin microvilli presented on the 7th day of pseudopregnancy. Degenerated pinopodes with microvilli and increasing ruga on the surface appeared again on the 8th day of pseudopregnancy.(3)Model group:A few cells with different maturation, some developmental pinopodes along with grown pinopodes as well as a large number of microvilli distributed in some regions of the endometrium on the 6th day of pseudopregnancy. The pinopodes on the endometrial surface were inhibited and shown decreased distribution and out of sync in maturation on the 7th day of pseudopregnancy. Microvilli appeared again and pinopodes disappeared on the 8th day of pseudopregnancy.(4)Interceed treatment group:a large number of cells with various sizes protruded to the endometrial cavity and developmental pinopodes with shorten microvilli presented on the 6th day of pseudopregnancy. Abundant pinopodes with various sizes and maturation appeared on the 7th day of pseudopregnancy. Pinopodes with different maturation deceased or shrunk on the 8th day of pseudopregnancy.(5)Estrogen treatment group:a large number of cells with various sizes protruded to the endometrial cavity and developmental pinopodes with shorten microvilli presented on the 6th day of pseudopregnancy. Abundant pinopodes with various sizes and maturation appeared on the 7th day of pseudopregnancy. part is fully developed pinocytosis, part for began to degenerate pinocytosis,Degenerated pinopodes with microvilli and increasing ruga on the surface appeared again on the 8th day of pseudopregnancy.(6)Combined treatment group:A large number of cells with consistent sizes and obviously shorten microvilli as well as developmental pinopodes appeared on the 6th day of pseudopregnancy. Abundant pinopodes with consistent sizes, shapes, clear boundary and total maturation appeared on the 7th day of pseudopregnancy. A great number of degenerated pinopodes shrunk on the 8th day pseudopregnancy.2. The expression of integrin αvβ3 of the endometrium in six groups during implantation window period(1)On the 6th day of pseudopregnancy:the discrepancy of expression of integrin αvβ3 on endometrium in 6 groups are statistically significant. Model group, Interceed treatment group and estrogen treatment group is statistically significant compared to the normal control group respectively(Pmodel group=0.000, P Interceed treatment group=0.005, Pestrogen treatment group=0.002). Combined treatment group, Interceed control group is without statistical significance compared to normal control group respectively (P=0.100,P=0.891). Combined treatment group and Interceed treatment compared to estrogen treatment group is statistically significant(P=0.001, P=0.006).(2)on the 7th day of pseudopregnancy:the discrepancy of expression of integrin avβ3 on endometrium in 6 groups in statistically significant(F=22.761, P<0.001). Model group, Intercede treatment group and estrogen treatment group are statistically significant compared to the normal control group respectively.(Pmodel group=0.000, P Interceed treatment group=0.006, P estrogen treatment group=0.003). Combined treatment group, Interceed treatment group are without statistical significance compared to normal control group(P=0.110, P=0.465). Combined treatment group and Interceed treatment group are statistically significant compared to estrogen treatment group(P=0.032, P=0.023).(3)on the 8th day of pseudopregnancy:the discrepancy of expression of integrin αvβ3 on endometrium in 6 groups in statistically significant(F=25.546, P<0.001). Model group, Interceed treatment group and estrogen treatment group are statistically significant compared to the normal control group respectively(Pmodel group=0.006, PInterceed treatment group=0.006,Pestrogen treatment group=0.008,Pcombined treatment group=0.018). Interceed control group is without statistical significance compared to the normal control group (P=0.460). Combined treatment group is statistically significant compared to Interceed treatment group and estrogen treatment group respectively (P=0.007, P=0.048).Conclusion:1. As the endometrial pinopodes expressd regionally and integrin avβ3 decreased dramatically in modle group, it indicates that the endometrial receptivity decreases after the mechanical and infectious damage towards the endometrium.2. It is Similar that expression of integrin αvβ3 and development of pinocytosis in Interceed control group with that in the normal control group. Interceed has no significant adverse effects to control endometrial receptivity.3. Interceed treatment group, estrogen treatment group and combined treatment group can improve the expression and development of emdometrial αvβ3 integrin and pinopodes in the adhesive uterine in some way.4. Combined treatment group improve in the expression of pinopodes and avP3 integrin maximum, which indicates that the combined treatment may improve the endometrial receptivity of intrauterine adhesion. |
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