| IUA refers to the damage and infection of the basilar lining of the endometrium.It may be caused by mechanical trauma,pathogenic microbial infection,inflammatory response which are followed by increased synthesis and extracellular abundance of collagen.These can cause endometrial fibrosis repair.IUA manifested as reduced menstrual flow or amenorrhea,secondary infertility,chronic abdominal pain,severe obstetric complications(such as miscarriage,premature birth,placenta previa,placenta accreta and even implanted),bring patients a great psychological and economic burden.Amnion has great potential in tissue engineering due to its low immunogenicity,bacteriostasis,inhibition of fibrous tissue hyperplasia,secretion of bioactive medium,and stem cell pluripotency.It has been widely used for reconstruction of the ocular surface and skin transplantation.Amnion seems to be promising in promotion of endometrial regeneration and suppression of fibrosis formation.In this study,we will explore the safety,feasibility and the mechanism of amnion in the prevention of intrauterine re-adhesion after TCRA based on in vitro cell experiments and animal experiments.Part I Effect of Amnion on Proliferation and Migration of Endometrial Stromal Cells ObjectiveBy culture of human endometrial stromal cells(ESC),the effect of amnion on the state of cell growth and migration and proliferation of endometrial cells were detected.Material and methods32 cases of endometrial tissue were selected from patients with diagnostic curettage due to unexplained infertility.30 cases of amniotic tissues of full-term caesarean section were collected under sterile conditions.After digestion with trypsin,the epithelial cells are gently scraped off with a scraper.The endometrial stromal cells were seeded on the stromal surface of the amnion(experimental group)or in the DMEM culture medium(control group).The cell scratch test was used to observe the difference of the migration ability.The proliferation rate was determined by CCK-8 method.The expression of proliferating cell antigens(PCNA mRNA)was determined by RT-PCR.Statistical analyses were conducted with SPSS 21.0.Different time points in each group were compared using repeated mesurement design.One-way ANOVA was used to analyze the data in each group.LSD-t test method was used to pairwise comparison.The significance level was defined as α = 0.05.Results1.Compared with those cultured in the control media,cells cultured on the amnion stretched longer and intertwined in a meshwork fashion.Cell migration was significantly increased at different time points in cell with amnion than control(P<0.001).2.The proliferation of the ESCs by detecting the proliferation rate and expression of PCNA mRNA,an increase of which with time was demonstrated.They were significantly higher of cell on amnion than control(all P<0.001).ConclusionsAmniotic membrane promotes migration and proliferation of endometrial stromal cells.Part II Anti-fibrosis Effect on Endometrium of Amnion in a Rabbit Model of Endometrial Injury ObjectivesAdopting the of mechanical and infection double damage to build endometrial injury models in rabbit.Then Observed the effect of amniotic membrane on endometrial morphology,gland count,fibrosis area,TGF-β1 and MMP-2 expression levels.Material and methods48 healthy adult female New Zealand white rabbits(weight 3000-5000 g,age 6-8 weeks)were randomized sheet into 4 groups of 12 rabbits each.Normal control group(group A): Suture the abdominal wall after laparotomy,without hurting its uterus.Amnion control group(group B): Place amnion in uterus,without modeling.Model group(group C): no treatment after modeling;Amnion treatment group(group D): intrauterine placement of amnion one week after modeling.The IUA model was established in rabbits using the double-injury method,mechanical injury and infection.Specifically,a 0.5 cm longitudinal incision was made through the middle and lower thirds of the uterus.The upper endometrium was scratched with a sterile spatula.Cotton soaked in bacterial lipopolysaccharide was placed at the injury site.Endometrial tissues were collected from the animals at the time of before treatment,after 2 and 4 weeks,respectively.The morphological changes of the endometrium were visually observed.HE staining was performed for endometrial morphologic observation and gland count.Masson staining was performed to detect the degree of endometrial fibrosis.The expression of TGF-β1 and MMP-2 was detected by immuno-histochemistry and RT-PCR.Statistical analyses were conducted with SPSS 21.0.Different time points in each group were compared using repeated mesurement design.One-way ANOVA was used to analyze the data in each group.LSD-t test method was used to pairwise comparison.The significance level was defined as α = 0.05.Results 1.The uterus anatomical observationThe appearance of normal rabbit’s uterus is light pink,there was no congestion or haemorrhage in the surface of endometrial.In the amniotic control group,there was no significant change in the endometrium.The amniotic membrane changed from transparent to beige-white shrinkage residue after 1 week,partially decomposed after 2 weeks,was not completely absorbed but the endometrium was almost normal after 4 weeks.With the extension of modeling time,the model group had uneven uterine thickness,surface edema,and mild water accumulation.Endometrial surface uneven,the color becomes dark red,showing a lot of congestive spots.With the prolongation of treatment time,the uterine surface water in the amnion treatment group gradually absorbed,and after 4 weeks,the uterine endometrial morphology can basically return to normal.2.The quantity endometrial glandNo differences were observed between group A and B at different time points(P=0.658,P=0.284,P=0.147).Before treatment,there have no statistical significance in group C and D(P = 0.507).The endometrial glands in groups D and C were significantly less than those in group A and B(P < 0.001).After treatment,the quantity of endometrial glands of group D was larger than that of group C(P < 0.05).The differences were more significant by extension of the treatment(P < 0.001).3.The area of fibrosisThe collagen fibers stained blue-violet after staining.A small amount of collagen fibers were expressed in the endometrial stroma of group A and group B.The collagens in groups C and D were increased and disordered before treatment.The collagen fibers in group D were relatively sparse and arranged more neatly after treatment.No differences were observed between group A and B at different time points(P=1.000,P=0.863,P=0.519).Before treatment,there have no statistical significance in group C and D(P = 0.632).The fibrosis area higher than those in group A and B(P < 0.001).After treatment,the percentage of endometrial fibrosis area was smaller than that of group C(P < 0.05).The differences were more significant by extension of the treatment(P < 0.001).The differences were more significant by extension of the treatment(P < 0.001).4.The expressions of MMP-2We indicated that the expressions of MMP-2 in group A were similar to that of group B(P<0.05).Before treatment,the expression of MMP-2 was significantly lower in groups D and C than in groups A and B(P < 0.001).After treatment,the expression of MMP-2 was higher in group D than in group C(P < 0.01).The differences were more significant by extension of the treatment(P<0.05).5.The expressions of TGF-β1We indicated that the expressions of TGF-β1 in group A were similar to that of group B(P<0.05).Before treatment,the expression of TGF-β1 was significantly higher in groups D and C than in groups A and B(P< 0.001).After treatment,the expression of TGF-β1 was lower in group D than in group C(P< 0.001).The differences were more significant by extension of the treatment(P<0.05).Conclusions1.Amniotic membrane itself has no significant effect on endometrial glands and interstitial fibrosis,suggesting that amniotic membrane can be used safely in the uterine cavity.2.After amniotic membrane transplantation into the uterine cavity,the number of endometrial glands increased and the area ratio of fibrosis decreased,the expression of TGF-β1 decreased,and the expression of MMP-2 increased. |
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