| Research backgroundIntrauterine adhesions (IUA) is the atresia of uterine cavity and (or) cervical tube which is caused by many reasons such as dilatation and curettage, infection, electrocautery that can damage the basal layer of the endometrium. After the damage, uterine start scar healing, and lost its physiological function. This kind of disease was first report in the year of 1894, in 1984, Asherman report intrauterine adhesions caused by uterine curettage after abortion and delivery in details. Asherman named this kind of disease "traumatical amenorrhea". The symptom of IUA are mainly amenorrhea, dysmenorrhea, infertility, although some of the patients can get pregnant after treatment by themselves, they are also prone to face many serious obstetric complications like miscarriage, premature birth, adherent placenta, postpartum hemorrhage, fetal intrauterine growth retardation, fetal malformation and so on. So IUA not only raise up the economic and psychological burden of patients, it also challenge the obstetrics and gyneacology doctors around the world. The cause of IUA are mainly artificial abortion, early pregnancy and late pregnancy curettage, hysteroscopy surgery etc. The pathogenesis of intrauterine adhesions is not clear until now, but there are many research hypothesis such as fibrosis hyperplasia theory, neural reflex theory, abnormal differentiation of stem cells, the most widely studied is fibrosis hyperplasia theory. Pathological changes of IUA contains endometrial tissue fibrosis, decreased endometrial glands, and this is the same with fibrosis diseases, so reserch IUA as fibrosis disease may be a breakthough to find the method to treat this disease. Diagnosis of IUA mainly rely on hysteroscopy, a comprehensive assessment of uterine endometrial morphology, distribution and degree of damage are observed under direct vision. Hysteroscopy is an accurate and preffered method for the diagnosis of IUA. There are many other methods to diagnose IUA, such as ultrasonography, MRI, hysterosalpingography, however, all these methods except hysteroscopy are not able diagnose IUA correctly.Because there is no method can cure IUA by itself, so we are using comprehensive treatment to treat this disease. The most advanced methods to treat IUA are mainly:transcervical resection of adhesions(TCRA), during the operation, intrauterine device, supporting balloon of the uterine cavity and the application of the biological glue material are arranged. After TCRA, estrogen are supplement to patients aiming to promote endometrial repairment and proliferation, so as to restore the physiological activity of endometrial.In the numerous adjuvant treatment of IUA, estrogen supplement is widely used by domestic and foreign scholars as the empirical treatment. Except the special physiological effect on endometrium, estrogen plays an important role in the treatment of many fibrosis disease such as liver fibrosis, renal fibrosis and myocardial fibrosis. This anti fibrosis effect of estrogen make it more promising in the treatment of IUA. In the treatment of IUA, the use of estrogen to prevent adhesion recurrence, promote endometrial repair after TCRA has become a consensus. However, although the widely clinical application of estrogen in the treatment of IUA, the treatment effect is different and the basical research of the therapeutic effect of estrogen in IUA is not deep enough. There are many controversy on the application of estrogen in the treatment of IUA from domestic and foreign scholars, the disputes are mainly concentrated on the dose of estrogen supplement to the patient post TCRA. Many domestic and foreign clinical trials showed that supplement high dose of estragen after TCRA has significant effect on the prevention of IUA recurrence, postoperative menstrual recovery and later pregnancy rates. Some studies showed that high dose of estrogen can make worse of IUA condition. However, due to the less of these clinical cases, geographical restrictions, and the prospective cohort study is difficult to carry out, so the supplementary dose of estrogen after TCRA remains to be further researched.Estradiol valerate(E2V) is a synthesis ester of natural estradiol, in 1950s, E2V was put into clinical application, until now, E2V has become the most common estradiol ester in clinical practice. The main clinical use of E2V is the treatment of estrogen deficiency diseases, menstrual disorders and endometrial lesions such as premature ovarian failure, perimenopausal syndrome, irregular menstruation, IUA and so on. E2V is the most commonly used drug in the adjuvant therapy of IUA. In this paper, the effect of E2V on the prevention and treatment of IUA was studied by animal experiments.Estrogen receptors (ERs) are a group of proteins widely expressed intracellular and extracellular, which can specificly bind estrogen, ERs is activated after the combination of estrogen and ERs, when activated by estrogen, ERs can transfer to the nucleus and correspond to DNA to play biological effects. The latest research also confirms that ERs can play biological effect without bind to DNA. ERs can be divided into two types:the nuclear estrogen receptor (belonging to the nuclear receptor family) and the membrane estrogen receptor (belonging to the G protein coupled receptor family). Nuclear estrogen receptors contains ER-a and ER-β, the human encoding ER-a gene is located in chromosome 6q24-q27, because ER-a commonly expression in endometrial tissue, and ER-β is more extensivly expression in the ovary, brain and bone tissue, so this experiment mainly discusses the effect of ER-a in IUA. Latest research shows that in liver fibrosis, myocardial fibrosis and other fibrotic diseases ER-a have negative correlation with the fibrosis degree, namely that high expression of ER-a correlated with reduced fibrosis, on the contrary low expression of ER-a correlated with aggravated fibrosis, so ER-a has protective effects on organ fibrosis. In uterine tissue, estrogen mediated through ER-a, promoting the proliferation and repair of endometrial glands and stroma, the physiological role of ER-α in endometrial makes it become a potential therapeutic target for the treatment of IUA. However, the role of ER-α in the development of IUA is not researched in-depth, there is few reports in foreign literature, a few domestic research results also have a great deal of controversy. Research results showed that a negative correlation between the expression level of endometrial ER-α and the severity of IUA, patients with high level of ER-α have a better prognosis. There are also research showed the opposite, that IUA degree are positively related to the expression level of ER-α, abnormal high expression level of ER-α can increase the degree of IUA.Transforming growth factor(TGF-β) is a muti-effect growth factor whose physiological effect pathway is through Smad protein family. The latest study results demonstrated that structure related molecules TGF-β1,2,3 is common in mammals, The encoding gene of this cytokine in human is in chromosome 19q13. TGF-β1 is widely secreted in many cells including fibroblasts, fibroblast cells, monocytes, epithelial cells, endothelial cells, and so on. Gene polymorphism makes TGF-β1 showing a variety of biological functions, such as promoting organ fibrosis, protecting the nerve, regulating the immune response, regulating cell cycle and other biological effects. The theoretical study of TGF-β1 in organ fibrosis is one of the basic of this research, a large number of domestic and foreign research displayed that in the regulating of extracellular matrix, TGF-β1 increase collagen, glycoprotein synthesis and expression, at the same time reduce the synthesis of matrix degrading protease, which makes TGF-β1 become a potent profibrotic factor, and play an important role in organ fibrosis. TGF-β1 plays an important role in liver fibrosis, pancreatic fibrosis, pulmonary fibrosis, renal fibrosis and other fibrosis diseases. The pathology of IUA is similar with fibrosis disease and numerous clinical retrospective study showed that in patients with IUA TGF-β1 expression is over expressed, consistent with the fibrosis disease. The results showed that endometrial TGF-β1 expression level was positively correlated with the degree of IUA, severe IUA patients endometrial TGF-β1 expression level was significantly higher than that in patients with mild to moderate IUA.Matrix metalloproteinases (MMPs) family is a group of protease which need catalytic of metal elements, most of MMPs need zinc binding, some others can bind with cobalt, this protease are widely distributed, it can be synthesized by many cells like neutrophils, mononuclear cells, smooth muscle cells, stellate glial cells, vascular endothelial cells and so on. MMPs is synthesized in inactive condition, after activated by extracellular protease, MMPs degrade extracellular matrix components such as collagen protein IV, collagen protein V and reduce the accumulation of extracellular matrix. Therefore, the physiological role of MMPs is contrary with TGF-β1, it is one kind of protease which can decompose extracellular matrix. MMPs regulate many physiological processes such as embryonic development, bone development, angiogenesis, wound healing and so on. The targets of MMPs include gelatin, elastin, type IV collagen, type V collagen and laminin, all these targets can be degraded by MMPs, so that MMPs can degrade the fibrotic tissue [10]. In organ fibrosis diseases. MMPs is the most widely researched fibrosis degradation factors, in liver fibrosis, pulmonary fibrosis, pancreatic fibrosis diseases MMPs showed protective role in these organ fibrosis disease. MMP9 is one member of the family of MMPs, the encoding gene of this cytokine in human is located on chromosome 20:46.01-46.02Mb. At present, there are some studies indicate that the expression level of MMP9 in endometrial tissue of patients with IUA was significantly lower than that in the normal control group, and the expression level was negatively correlated with the degree of IUA. Conversely, up regulation of MMP9 can promote the degradation of extracellular matrix, which is expected to reduce or even reverse the organ fibrosis. In IUA, the use of estrogen to prevent adhesion recurrence and promote endometrial repair has become a consensus. Studies showed that estrogen can significantly increase the expression level of MMP9 in endometrial tissue while decreasing endometrial fibrosis in patients with IUA. Our team’s previous study also found that estrogen can increase the expression level of MMP9 and inhibit the formation of IUA after endometrial injury in rats.Therefore, this study adopts mechanical and infectious injury method to build IUA in SD rats, and supplement different doses of E2V in different periods to rat IUA model. Evaluate the prevention and treatment effect of estrogen to IUA by detecting serum E2 concentration, and observing endometrial fibrosis area ratio, uterine endometrial glands, endometrial expression level of TGF-β1, MMP9 and ER-a, in order to further guide clinical trials and clinical application.Experimental methods1. experimental groupsForty-eight 8-10 weeks old female SD rats were randomly divided into 8 groups, 6 rats in each group (Rat uterus is "Y" font double uterus, so each group consists 12 uterus). The experimental groups were as follows:(1) blank control group:Rats without IUA modeling, and without E2V treatment by gavage, only gavage saline.(2) Experimental control group:construct rats IUA model, the modeling method using mechanical and infectious injury, after modeling gavage saline instead of E2V.(3) Low dose estrogen group A (low estrogen A group), medium dose estrogen group A (medium estrogen A group), high dose estrogen A group (high estrogen A group):these three groups are prophylactic application of E2V groups, after IUA modeling, gavaging defferent doses of E2V 0.206mg/(kg·d),0.514mg/(kg·d), 1.028mg/(kg d) to each group.(4) Low dose estrogen group B (low estrogen B group), medium dose estrogen group B (medium estrogen B group), high dose estrogen B group (high estrogen B group):these three groups are treatment application of E2V groups, after IUA modeling, the 9th d gavaging defferent doses of E2V 0.206mg/(kg·d),0.514mg/(kg·d), 1.028mg/(kg d) to each group.2. Methods of construction rat model of IUAThe 10-0 medical sterile sutures were soaked in the 6mg/L of the lipopolysaccharide,4℃ placed 24h standby. The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate 0.35ml/100g each rat, strictly aseptic operation, choose lower abdominal incision in rats, expose the uterus, make a longitudinal incision above cervix about 5mm, length is about 4mm. use self-made rat uterine cervical spatula doing rat uterine curettage, stop curettage when feeling the uterine wall is rough. Put the lipopolysaccharide sutures into uterine, leave suture tails a certain length in the abdominal wall so that the next step we can get out easily. Use saline peritoneal lavage, suture the abdominal wall of rats.24h later removed lipopolysaccharide sutures and after 9d IUA model is established.3. Dose conversion of estrogenThe dose of E2V using animal experiment equivalent dose conversion algorithm [5]. The dose gavage to each group 0.206mg/(kg·d),0.514mg/(kg·d),1.028mg/(kg·d) equivalent to the oral dose of 60kg adults 2mg/d、5mg/d、lOmg/d. After TCRA in human, estrogen supplementation duration is usually 3-6 physiological cycles, because the physiological cycle of rat is about 4-6 days, so the preventive and therapeutic application of E2V duration is 24 days, about 4-6 physiological cycles of rats.4. Experimental procedureThe blank control group, experimental control group and the three prophylactic use of estrogen groups were cut the tail to get blood again on the 24thd of the experiment, and the experimental animal were collected bilateral uterine tissue; three therapeutic application estrogen groups began gavaging estrogen at the 9thd of modling, the duration is also 24 days, at 33thd of the experiment rats were cut the tail to get blood, and collected bilateral uterine tissue. The blood samples were collected to detect serum concentration of E2 by ELISA, the rat uterus collected to count numbers of endometrial glands by HE staining, and detect endometrial fibrosis area ratio by Masson staining, TGF-β1, MMP9 and ER-a expression level was detected in endometrial by Immunohistochemistry.Statistical analysisStatistical analyses were performed using SPSS version 20.0, 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. The mean between the two groups were compared using independent samples t test.Experimental results1. Rats serum estradiol (E2) concentration(1) Serum E2 concentration in each group before modlingBefore modeling the peripheral blood serum E2 concentration of rats in each group showed no significant difference between the 8 groups of rats (F=0.752, P=0.643), this shows that the rats were in the similar endocrine status before modling.(2) Rats second time blood serum concentration of E2 compare to its first blood concentrationExperimental control group and blank control group were gavaged saline,24d later serum E2 concentration compare to E2 concentration before modeling showed no significant difference (P>0.05); the 3 group A began intragastric administration of E2V from the modling day and the duration is 24d, by measuring the concentration of serum E2 in each group, the concentration is much higher than before modeling(P<0.05); 3 group B rats intragastric administration of estrogen at 9th d, the duration is 24d, serum level of E2 had significant difference with the E2 concentration before model construction(P<0.05); P=0.002-0.000.(3) comparation of rats second times serum E2 concentrationThe blank control group and experimental group, have no significant difference in serum E2 concentration (P=0.240); 6 estrogen groups compared with 2 control groups, the serum E2 concentration was significantly increased, there were significant differences (P<0.05), P values were: P=0.000.3 A groups were compared, with the gavaging E2V dose increased, the concentration of serum E2 increased, the result have significant difference (P<0.05). medium group A is higher than low group A (P=0.000), high group A is higher than medium group A and low group A (P=0.000). Three groups B were compared, with gavaging E2V dose increased, serum E2 concentration also increased. The result have significant difference (P<0.05), estrogen group B is higher than that of low estrogen group B (P=0.000), high estrogen group B is higher than medium group B and low estrogen group B (P=0.000). Gavage the same dose of E2V rats group compared, low group A and low group B, medium group A and medium group B; high group A and high group B showed no significant difference, P values were:0.656,0.189,0.721 respectively.(4) Summary of the results of E2 detection in serumComparison of serum E2 concentrations in rats showed that the serum E2 levels were positively correlated with the dose of E2V administrate to rats.2. The fibrosis area ratio of endometrium in rats of each group(1)Comparison of blank control group with the other groupsCompared with the blank control group, after IUA molding,7 groups of rats uterine endometrial fibrosis area were significantly higher than blank control group (P<0.05), P values were:P experimental control group=0.001, P low group A=0.000, P medium group A=0.043, P high group A=0.006, P low group B=0.000, P medium group B=0.039, P high group B =0.025.(2)Comparison of estrogen groups and the experimental control groupCompared with the experimental control group, fibrosis area ratio in 3 group A was higher than in the experimental control group, there is significant difference (P<0.05),the P value were Plow group A=0.013,Pmedium groupA=0.021,Phigh group A= 0.003 respectively. There was no significant difference in the fibrosis area ratio between the 3 group B and the experimental control group, the P values were P=1.000.(3) Comparison of the fibrosis area ratio in the endometrium of the rats in each estrogen groupComparison between 3 group A, medium estrogen group A and high estrogen group A is significantly lower than the low estrogen group A (P<0.05), the P values were:0.022 and 0.082; comparison of medium estrogen group A and high estrogen group A have no significant difference(P=0.988). In group B, medium estrogen group B and high estrogen group B have no significant difference with the low estrogen group B, the P values were:P=1.000; comparison of medium estrogen group B and high estrogen group B have no significant difference(P=1.000).(4)Summary of the results of the endometrial fibrosis area ratio in rats3 estrogen group A endometrial fibrosis area ratio decreased, the medium estrogen A group and the high estrogen A group higher than the low estrogen A group, while the medium estrogen A group and the high estrogen A group have no significant difference.3 estrogen group B endometrial fibrosis area ratio have no improvement.3. The endometrial glands of rats in each group were counted(1) Comparison of endometrial glands in modeling groups and blank control groupCompared with the blank control group, the other 7 groups of rats have significantly decreased endometrial glands count, P values were P=0.000.(2) Comparison of endometrial glands in estrogen groups and experimental control groupCompared with the experimental control group, three group A rats endometrial glands increased significantly(P<0.05), the P value were P=0.000; three group B rats endometrial glands had no significant difference with experimental control group (P>0.05), P values were:Plow group B=0.536, Pmedium group B=0.536, Phigh group B=1.000.(3) comparision of endometrial glands in estrogen groupsComparison between 3 group A, medium estrogen group A and high estrogen group A is significantly higher than the low estrogen group A (P<0.05), the P values were:0.002 and 0.022; comparison of medium estrogen group A and high estrogen group A have no significant difference(P=0.224). In group B, medium estrogen group B and high estrogen group B have no significant difference with the low estrogen group B, the P values were:P=0.224 and 0.536; comparison of medium estrogen group B and high estrogen group B have no significant difference(P=0.536). (4)Summary of the endometrial glands in rats3 estrogen group A endometrial glands increased, the medium estrogen A group and the high estrogen A group higher than the low estrogen A group, while the medium estrogen A group and the high estrogen A group have no difference.3 estrogen group B endometrial glands have no improvement.4. The expression of TGF-β1 in the endometrium of rats in each group(1) Comparison of the expression levels of TGF-β1 in endometrium of rats with the blank control groupCompared with the blank control group, the expression levels of TGF-β1 were significantly increased in the 7 modeled rats, and the difference was significant (P<0.05), the P value were 0.000.(2) Comparison of expression levels of TGF-β1 in the endometrium of rats with the experimental control groupThe expression levels of TGF-β1 in the endometrium of 6 estrogen groups rats were significantly lower than that in the experimental control group, the difference was significant (P<0.05), the P value were 0.000.(3) Comparison of the levels of TGF-β1 expression in the endometrium of rats in each group of estrogen groupsComparison between 3 group A, medium estrogen group A and high estrogen group A is significantly lower than the low estrogen group A (P<0.05), the P values were:0.003 and 0.002; comparison of medium estrogen group A and high estrogen group A have no significant difference(P=0.842). In group B, medium estrogen group B and high estrogen group B is significantly lower than the low estrogen group B (P<0.05), the P values were:P=0.001 and 0.002; comparison of medium estrogen group B and high estrogen group B have no significant difference(P=0.918). Comparison between groups of the same dose of E2V groups, low estrogen group A and low estrogen group B, medium estrogen group A and medium estrogen group B, high estrogen group A and high estrogen group B endometrial TGF-β1 expression results have no significant difference, P values were respectively:0.747,0.984,0.778.(4) Summary of the expression of TGF-β1 in endometrium of ratsTGF-β1 expression level in 6 estrogen groups was much lower than experimental control group, medium dose and high dose estrogen group was lower than that of low dose group, high dose group and medium dose group have no significant difference.5. Endometrial expression of MMP9 in rats(1) modeling rats endometrial MMP9 expression levels compared with the blank control groupCompared Blank control group and experimental control group, endometrial MMP-9 expression level have no significant difference (P=0.672). The remaining six estrogen groups rats endometrium MMP9 expression level were significantly increased compared with the blank control group (P<0.05), the P values were 0.000.(2) MMP9 expression levels in estrogen groups compared with experimental control groupCompared with experimental control group, MMP9 expression levels in 6 estrogen groups were significantly increased(P<0.05), the P values were 0.000.(3) The comparison of endometrial MMP-9 expression level in estrogen groups3 group A were compared, in medium estrogen group A and high estrogen group A endometrial MMP9 expression is higher than low estrogen group A significantly(P=0.000); medium estrogen group A and high estrogen group A have no significant difference (P=0.379).3 group B were compared, in medium estrogen group B and high estrogen group B endometrium MMP9 expression is higher than low estrogen group B significantly(P=0.000); medium estrogen B group and high estrogen group B have no significant difference(P=0.550). Comparison between groups of the same dose of E2V groups, low estrogen group A and low estrogen group B, medium estrogen group A and medium estrogen group B, high estrogen group A and high estrogen group B endometrial MMP9 expression results:there were no significant differences,the P value were 0.893,0.421 and 0.279 respectively.(4) Summary of endometrial MMP9 expression in each groupMMP9 expression level in 6 estrogen groups was much higher than experimental control group, medium dose and high dose estrogen group was higher than that of low dose group, high dose group and medium dose group have no significant difference.6. The ER-a expression of rat endometrial in each group(1) Modeling rats endometrial ER-a expression levels compared with the blank control groupCompared with the control group, ER-a expression level of modeled rats was significantly decreased, P=0.000.(2) The ER-a level of estrogen groups compared with experimental control group6 intragastric administration of E2V groups rats endometrial ER-a expression level was significantly higher-than the control group (P<0.05),P=0.000.(3) The intragastric administration of E2V groups rats endometrial ER-a expression level3 groups A were compared, endometrial ER-a expression of medium group A and high group A were significantly higher than low group A (P=0.000); medium estrogen groups A and high estrogen groups A have no significant difference (P=0.513); 3 groups B were compared, endometrial ER-a expression of medium group B and high group B were significantly higher than low group B, P=0.000; medium estrogen groups B and high estrogen groups B have no significant difference (P=0.536). comparison between groups of the same dose of E2V groups, low estrogen group A and low estrogen group B, medium estrogen group A and medium estrogen group B, high estrogen group A and high estrogen group B endometrial ER-a expression results have no significant differences, P values were respectively:0.359, 0.378 and 0.359.(4) Summary of rats endometrial ER-a expression levelER-a expression level in 6 estrogen groups was much higher than experimental control group, medium dose and high dose estrogen group was higher than that of low dose group, high dose group and medium dose group have no significant difference.Conclusions1. After treatment with E2, compared with experimental control group, three estrogen group A present with decreased fibrosis area and increased endometium glands, we consider that administrate E2 immediately after endometrial damage may have protective effect to avoid IUA formation. However in three group B, the fibrosis area and endometium glands have no improve compared with experimental control group, we consider that after the formation of IUA treatment effect of E2 is poor, may need comprehensive treatment.2. In estrogen group, TGF-β1 expression level of medium and high dose group is decreased copared with low dose group, MMP9 expression levels is increased, and the medium and high groups have no significant difference. This indicate that after TCRA,application of medium dose of estrogen is the lowest effective dose.3. ER-a expression of medium and high doses of estrogen group is higher than low dose estrogen group, suggesting that E2 can induce the synthesis of ER-a, high dose groups and medium dose groups have no significant difference, indicating that the synthesis of ER-a is not entirely concerned with the dose of estrogen, further research needs to be investigated the mechanism of expression of ER-a. |
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