| Background and ObjectiveThe inflammatory lesion of fallopian tube such as adhesions and effusions,are usually treated before in vitro fertilization and embryo transfer(IVF-ET).Because diseased fallopian tube may affect the uterine cavity environment and endometrial receptivity,thus reduce the clinical pregnancy rate and implantation rate.In addition to the low pregnancy rate,such patients will also increase the rate of spontaneous abortions and ectopic pregnancy.Papers reported before showed that the treatment of hydrosalpinx before IVF-ET,such as surgical ligation,resection or aspiration of the accretion fluid,resulted in a much higher rate of continued pregnancy in women than in women without any intervention.The treatments of hydrosalpinx mainly include:laparotomy or laparoscopic tubal resection,proximal tubal ligation of fallopian tube,salpingostomy and vaginal ultrasound-guided hydrosalpinx aspiration.Each of these methods has its advantages and disadvantages.The main drawbacks are tubal ligation or resection may affect the ovarian function,and tubal resection and ultrasound guided aspiration of tubal effusiontends may recur,resulting in failure to treat or repeat surgery.Some patients with laparotomy or laparoscopic surgery are not suitable for reoperation.The purpose of this paper is to explore a method of non-surgical treatment of hydrosalpinx,which does not affect ovarian function and has a low recurrence rate.We intended to block the traffic between fallopian tube and uterus by a new formula medical adhesive and removed the adverse effect of hydrosalpinx on embryo implantation.There were also reports about tube occlusion before IVF-ET,which was not a routine treatment method.Previous study about the blocking model were mostly mechanical blocking such as micro-spring coil plugs under intervention.This method of medical adhesive plugging has not been reported.In order to verify its effectiveness and safety,the New Zealand rabbit uterus,which is similar to the size and structure of human tubal lumen,was selected as the object of study.This study is a preliminary animal experiment.First of all,the rabbit uterine fluid model was established by bacterial injection method.Then the ?-cyanoacrylate medical adhesive was used to occlude the uterus of rabbits.After operation,the blocking effect of medical glue on the rabbit uterus was evaluated by hysterosalpingography and the histomorphological changes of uterus,fallopian tube and ovary,immunohistochemical detection,apoptosis detection and electron microscopic observation of various tissues were observed.The effects of medical glue on the blocking effect of rabbit uterus and other organs and tissues were evaluated in order to provide experimental basis for the treatment of fallopian tubes hydrops before IVF-ET in clinic.MethodsNew Zealand adult female rabbits were selected as experimental animals to establish a rabbit model of uterine effusion,and then medical adhesive embolization was performed on the effusion uterus.In order to eliminate the effects of hormones at different stages of the estrus cycle on the observed indexes,vaginal exfoliative cytology was performed on all experimental rabbits to determine their estrous cycle.The sampling time of the study was set at the early to middle stage of estrus of rabbits.1.Establishment of rabbit uterine effusion modelThe model of uterine effusion in rabbits was established by intubation from vulva to vagina and uterus and then injection of bacteria solution.The injected solution was 1.5×108/ml Staphylococcus aureus solution,1.5×108/ml Escherichia coli solution and 1:1 mixed Staphylococcus aureus solution and Escherichia coli solution,respectively.Postoperative observation time was 1 week,2 weeks and 3 weeks,respectively.After operation,through the gross morphology,histology observation and muscle layer thickness comparison,the appropriate bacterial fluid and time were selected to establish the rabbit uterine fluid model and the rabbits with good bilateral uterine fluid formation were selected for the follow-up experiment.2.Medical glue suppository for blocking the uterus hydrops of rabbitsThirty-five adult New Zealand female rabbits with bilateral uterine fluid were randomly divided into experimental group(n=23)and control group(n=12).Another 12 normal rabbits(without uterine fluid)were selected as blank control group.Through the method of catheterization of the vulva into vagina and uterine cavity and assisted observation by laparotomy,the proximal end of the rabbit hydrops uterus was blocked by medical glue plug.Only intubation and laparotomy were performed in the control group and the blank control group and no medical glue plug was performed in these two groups.Each group was further divided into two groups,which were weighed before operation,1 month and 6 months after operation respectively and the changes of body weight were compared.One and six months after operation,hysterosalpingography was performed in both the experimental group and the control group.Then the contrast condition was recorded and the rate of uterine occlusion was compared.In the experimental group,3 rabbits were sacrificed randomly by intravenous injection of air into the ear margin after anesthesia 1 month and 6 months after operation(all rabbits were successful in bilateral uterine cavity occlusion).2 rabbits were killed with the same method in the control group and the blank control group at 1 month and 6 months after operation.In order to comprehensively evaluate the effect of uterine cavity occlusion and the histological changes,the uterus,fallopian tubes and ovaries of each rabbit were observed and histologically sectioned.The samples were observed by optical microscope and transmission electron microscope.The thickness of myometrium was compared,the estrogen and progesterone receptors in tissues were measured and compared and the apoptosis was detected.Results1.Establishment of rabbit uterine effusion modelThe successful rabbit uterus dilated obviously,the intima and muscular layer became thinner.There was significant statistical difference in myometrium thickness between 1 week after operation and 2 weeks and 3 weeks after operation.However,there was no significant difference in myometrial thickness between 2 and 3 weeks after operation.Therefore the degree of hydrated uterus at 2 weeks after operation can meet the requirements of the experiment.Combined with the condition of bacterial fluid injection,the concentration of mixed bacteria solution(Staphylococcus aureus:Escherichia coli=1:1)was settled as 1.5×108/ml and a propriate model of hydrous uterus was established at 2 weeks after operation.2.Medical glue plug for hydrops uterusSurvival and body weight changes od rabbits Among the 35 rabbits with uterus effusion,31 rabbits successfully underwent medical adhesive occlusive operation and survives normally after operation,including 20 rabbits in the experimental group and 11 rabbits in the control group.No significant difference in mortality were calculated between the two groups.There was no significant difference in body weight between the experimental group and the control group at 1 and 6 months after operation though the rabbits did gain weight than before.There exeisted statistical differences when the weights of rabbits at 1 month and 6months were compared with these of before occlusive operation.(P<0.05).Hysterosalpingography One month after operation,hysterosalpingography was applied to observe the uterine cavities and the blocking rate was 95.00%.Six months after operation,the blockage rate was 85.00%.In the control group,the blocking rate was 0 at 1 month and 6 months after operation.There was significant difference in tubal occlusion rate between the experimental group and the control group(P<0.05),whereas no significant difference in uterine occlusion rate at different time points was calculated in the experimental group.Gross view In the control group,hydrous uterus was thickened,dilated.There were no significant changes in fallopian tubes and ovaries.In the experimental group,the uterine cavity was slightly thickened,smooth.One month after operation,the length of the occlusion site was slightly reduced,the wrinkle was hard,and the adhesive polymer was seen in the uterine cavity.Six months after operation,the uterus was obviously wrinkled.ruddy,the blood vessels were distributed and there was no obvious adhesion around the uterus.There were no significant changes in the uterus at the site of effusion as well as the fallopian tubes and ovaries.Histomorphology The normal uterus,hydrops uterus,proximal hydrops uterus,occlusive uterus and proximal occlusive uterus were compared in this paper.Histological observation of uterus In the control group,1 month and 6 months after operation,the hydrous uterus dilated obviously,the intima became thinner,inflammatory cells infiltrated under the intima.In the proximal uterus at the site of hydrops,the endometrium was obviously thickened,the blood vessels in the lamina propria were dilated.Hyperemia and scattered inflammatory cell infiltration could be seen under the intimal epithelium.In the experimental group,one month after occlusion,the uterine cavity was dilated,a large number of filamentous reticular substances(colloids)could be seen in the cavity and disordered stratified epithelial cells or local epithelial loss could be seen in the mucous epithelium.A small number of inflammatory cell infiltration and fibrosis could be observed.In the proximal uterus,the endometrium was covered with a single layer of cubic epithelium and the lamina propria was scattered with a little of eosinophils.There were no obvious pathological changes.In the experimental group,six months after occlusion,the occlusive site was obviously wrinkled.Microscopically,the structure was disorderly and blurred,the normal structure of endometrium disappeared.There was no obvious epithelial tissue and granulation tissue hyperplasia and fibrosis were seen.The histomorphology of the proximal uterus at 1 month after operation was similar to that at 6 months after operation and there were no obvious pathological changes.The thickness of uterine myometrium became thinner 1 month after operation in the experimental group.There was significant difference in myometrium thickness between the experimental group and the control group at 1 month and 6 months after operation(P<0.05).There was significant difference between the experimental group and the control group at 1 month after operation(P<0.05)where as no significant difference between the experimental group and the control group at 6 months after operation.There were no obvious pathological changes in other tissues.Histological observation of fallopian tubes In the control group and the experimental group,the adventitia of the fallopian tube was dilated,hyperemia and a small number of inflammatory cells were infiltrated in the lumen.There were no obvious pathological changes in other tissues.Histological observation of ovary There were no obvious pathological changes in these tissues of the blank control group,control group and experimental group.Detection of estrogen and progesterone receptors(ER,PR)and apoptosisDetection of ER,PR and apoptosis in uterusCompared with the normal uterus,the expression of ER was decreased,PR and apoptosis increased in the hydrous uterus,the occlusive uterus and the proximal hydrops uterus.There were significant differences in ER,PR and apoptosis between normal uterus and hydrops uterus,normal uterus and thrombus site uterus,normal uterus and proximal hydrops uterus.There were significant differences in ER between proximal hydrops uterus and proximal occlusive uterus.There was no statistical difference between the other groups.Detection of ER,PR and apoptosis in fallopian tubes Compared with the blank control group,the expression of ER of the fallopian tubes in the experiment group was decreased and the average optical density was statistically different.The expression of PR in the fallopian tubes of the control group and the experiment group was significantly higher than that of the blank control group.There was no statistical difference in other comparisons.There was no significant difference in apoptosis among the groups.Detection of ER,PR and apoptosis in ovarian tissues There was no statistical difference between groups.Transmission electron microscopic observation Hydrous uterus:swelling of epithelial cells,loss of microvilli,vacuolization of organelles such as mitochondria.The uterus was disorganized with nuclear chromatin reduction and nuclear dissolution.Besides,phagocytes could be seen after occlusion operation.Fallopian tubes:in the control group and the experiment group,the organelles such as mitochondria and endoplasmic reticulum were slightly vacuolated.Ovary:the structures of all kinds of cells and organelles were clear and normal.Mitochondria damage could be seen occasionally.Conclusion1.A stable model of uterine fluid was built by injecting mixed Staphylococcus aureus and Escherichia coli solution(1.5×108/ml)at 1:1 into the uterine cavity by vaginal intubation and laparotomy.The model of uterine fluid could be established for about 2 weeks.2.Medical adhensive can effectively block the hydrous uterus of rabbits through simple and feasible operations.It had no adverse effect on the survival of rabbits.The main effect of occlusion on rabbit uterus was tissue compression and adhesion,but there was no significant harmful effect on the morphology and function of surrounding tissues.All of the results showed that occlusion has no significant effect on receptor expression.Occlusion can effectively mend the adverse effect of fluid uterine on uterine cavity and improve the uterine environment.It also had no significant effect on ovary.3.The rabbit uterus can be used as a research model of human fallopian tubes because the both are similar in size and structure.The study on the feasibility,effectiveness and adverse effects of medical adhesive in blocking rabbit uterus shows that medical adhesive is feasible for the occlusive fallopian tubes in human pre-IVF-ET. |
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