| Background:Asherman's syndrome?AS?is disease which is defined as endometrial regeneration disorder as a result of damage in basalis layer of endometrium and mainly present as intrauterine adhesions.Nearly 25-30%of infertility women suffer from this disease and it is the most common cause of uterine infertility.In China,the morbidity of AS increased a lot during recent years due to the high frequency of hysteroscopy operation and painless induced abortion.The main methods for treating AS is transcervical resection of adhesion by hysteroscopy and set intrauterine device or Folly catheter or biomaterial to prevent intrauterine adhesions.However,the re-incident of intrauterine adhesions come out as high as 62.5%,beside,the fibrosis in endometrium impede the embryonic implantation.However,little is known about its molecular features of this aregenerative pathogenesis in AS and how to rebuild the functional endometrium for the patients with AS.Recently,plenty of research aimed at functional effect of UC-MSCs?umbilical cord mesenchymal stem cells?and BMSCs?bone marrow mesenchymal stem cells?in regeneration of different tissues have been established,showing a promising therapeutic option for AS.In the previous study,we have proved that collagen scaffolds loaded with bone marrow mononuclear cells could notably improve regeneration abilities of endometrium as well as pregnancy outcomes in AS patients.To further standardize the cell therapy in AS patients,in the current study,tissue-engineering methods were applied to the regeneration of miniature pig uterine horns and AS patients.We first evaluate the effect of collagen scaffolds loaded with umbilical cord mesenchymal stem cells in the regeneration of uterine endometrium,muscular cells and blood vessels in miniature pig.Next,we use collagen scaffolds combined with umbilical cord mesenchymal stem cells,the first clinical application level UC-MSCs obtained from National Stem Cell Centre transplanted into severe AS patients enrolled in this prospective,experimental,non-controlled study.We hope that this novel treatment could significantly improves the outcome for the patients with severe AS.Chapter one:Transplantation of collagen scaffold with autologous bone marrow mononuclear cells promotes functional endometrium reconstruction via downregulating ?Np63 expression in Asherman's SyndromeObject:to prove ectopically expressed ?Np63 by epithelial cells of the endometrium holds the regenerative key to endometrial reconstruction in patients with AS.Methods:H&E-stained sections were applied to analyze the histological changes in the endometrium during the late proliferation phase.Analyzed the sternness proliferation,differentiation and progenitor properties of the ?Np63+epithelial cells and stromal cells in serial sections by immunochemistry.?Np63 adenovirus?Ad-?Np63?and control adenovirus?Ad-CTL?infected COS-7 cells and primary endometrial epithelial cells to assess endometrial quiescence.BMNCs?bone marrow mononuclear cells?co-cultured with?Np63+endometrial epithelial cells to evaluate the reversal effect of BMNCs.5 patients aged 27-38 years with server AS were recruited.All the clinical inspect were collected during the late proliferating phase of the menstrual cycle by monitoring the dimeter of the follicle by ultrasound;15-17 mm follicle and low progesterone level were defined as the late proliferating phase.Medical history,physical examination,endometrial thickness,intrauterine adhesion score were assessed before and 3 months after cell therapy.Once patients were enrolled,a hysteroscopic evaluation and ultrasound were first performed to gain the endometrial thickness and intrauterine adhesion score.Endometrial biopsies were collected from two points in the most serious area and a nearly normal area of corpora uteri using biopsy seizers and the locations were recorded.On the day of cell therapy,patients received another ultrasound to make sure the menstrual cycle and then 50-80ml bone marrow was extracted from patient and BMNCs were immediately isolated.After that,BMNCs were co-cultures with collagen scaffold?4cm×6cm?and then transplanted into the patients' uterine under the hysteroscopic.10 days before cell therapy,all patients take 6 mg/day × 10 days Progynova?menstrual period day 13?and continuous administration of the same dosage Progynova 30 days after cell therapy.3 month after cell therapy,endometrial regeneration was assessed in terms of restoration of menses,endometrial thickness?by vaginal ultrasound?,adhesion score?by hysteroscopy?,and ongoing pregnancy rate.Results:?1?with IHC technique,?Np63 protein in AS group was dramatically upregulated.?Np63 upregulation is associated with endometrial quiescence in AS,showed as Nanog,and SSEA-1 were downregulated on all ?Np63+cells in the endometria,while KLF4,W5C5 and LGR5 proteins were upregulated.ERa,vimentin,IGF1 and Ki67 proteins were decreased as compared to the control.?2??Np63 overexpression in COS-7 cells and primary endometrial epithelial cells led to reduction in cell numbers and upregulation of cell apoptosis,also a reductions of CCND1 and Ki67 expression.Besides,there exhibited downregulation of Nanog,SSEA-1 and W5C5 and mild upregulation of KLF-4 and LGR5;the cell proliferation markers CCND1 and Ki67 and differentiation markers ERa,IGF1 and vimentin were downregulated.?Np63 induces endometrial quiescence.?3?When ?Np63+epithelial cells of endometrium and BMNCs were co-cultured,expression of ?Np63 mRNA was downregulated,while the expressions of CCND1,Ki67,ERa and IGF1 were significantly upregulated.Autologous bone marrow mononuclear cells?BMNCs?reverse the inhibitory effects of ?Np63 on endometrial epithelial cells.?4?All these 5 patients reported longer and heavier periods,and the ultrasound scan revealed a significant improvement of the endometrial thickness and blood flow.The hysteroscopic images showed a basically normal uterine cavity and a significant reduction of intrauterine adhesion.The BMNC treatment resulted in an increased density of endometrial glands and stromal cells.The Nanog,SSEA-1 and W5C5 proteins were upregulated and the KLF-4 and LGR5 proteins were downregulated CCND1,Ki67,ER?,IGF1 and vimentin were upregulated in vivo at both the mRNA and protein levels.A11 5 patients achieved successful pregnancies and living births,no placental complication were found.Chapter two:The effect of collagen scaffolds loaded with UC-MSCs?umbilical cord mesenchymal stem cells?in the regeneration of the new trauma of miniature pig endometriumObjective:To evaluate the effect of the new trauma of miniature pig uterine horns after a 2.5 cm × 1/2 perimeter endometrial injury treated with collagen scaffolds loaded with UC-MSCs.Methods:Totally 41 female miniature pigs were randomly assigned to four groups,including sham surgery group,spontaneous regeneration group?after endometrial excision the defects were left open without reparation?,collagen/PBS group?after endometrial excision the defects were sutured with collagen membranes?,and collagen/UC-MSCs group?after endometrial excision the defects were sutured with collagen membranes loaded with UC-MSCs?.Each group was divided into pathological group and conception group.In.pathological group,8 weeks and 12 weeks postoperatively,the animals were sacrificed and the uterine horns were prepared for the patency test and gross examination of adhesion,neovascularization and hydrometra.The uterine specimens were fixed with formalin.Sections of the damaged uterus were prepared for HE and immunohistochemistry staining.12 weeks after the injection,conception group animals were mated with male miniature pig to evaluate fertilization ability.In the late trimester of pregnancy?about 100 days?,pregnancy was terminated to compare the growth and development of fetal miniature pig in three group and calculate the rate of placenta implanted in the surgical area.Results:?1?Patency test showed that all groups had 100%patency rate,there is no differences in these groups.8 weeks post-surgery,collagen/UC-MSCs group showed apparent neovascularization,while spontaneous regeneration group and collagen/PBS group showed pale and weak surgical area;12 weeks post-surgery,collagen/UC-MSCs group had normal uterine structure in the surgical area,spontaneous regeneration group and collagen/PBS group still showed pale and come out with scar tissue in the surgical area.?2?HE staining were applied in all the uterine specimens and the calculated the ration of endometrial thickness between surgical area and non-surgical area under low power lens.The results showed that 8 weeks post-surgery,collagen/UC-MSCs group gained higher ratio 0.42±0.13,compared to the other 2 groups,0.16±0.12 and 0.22±0.05 respectively.When it comes to the 12 weeks post-surgery,there is no differences between ratios in the same group,but the differences were still obvious among the 3 groups.?3?Immunohistochemistry staining showed that collagen/UC-MSCs group had higher glands ratio and vascular density ratio.The glands ratio of 8 and 12 weeks post-surgery were:collagen/UC-MSCs group,0.47±0.32 and 0.45±0.39;spontaneous regeneration group,0.13±0.1 and 0.03±0.007;collagen/PBS group,0.05±0.01 and 0.04±0.01.The vascular density ratio of 8 and 12 weeks post-surgery were:collagen/UC-MSCs group,0.58±0.07 and 0.59±0.20;spontaneous regeneration group,0.24±0.15 and 0.30±0.14;collagen/PBS group,0295±0.12 and 0.42±0.21.?4?Pregnancy test showed that the rate of placenta implanted in the surgical area in collagen/UC-MSCs group?55%?was higher than spontaneous regeneration group?10%?and collagen/PBS group?6.25%?.Conclusion:The collagen loaded with UC-MSCs could improve regeneration abilities of uterine endometrium,like capillaries and glands as well as better pregnancy outcomes.Chapter three:Transplantation of collagen scaffold with umbilical cord mesenchymal stem cells promotes functional endometrium reconstruction in server Asherman's syndrome patientsObjective:To evaluate the effect of endometrial regeneration in patients with server AS?Asherman's syndrome?treated with collagen scaffolds loaded with UC-MSCs?umbilical cord mesenchymal stem cells?.Methods:This is a prospective,experimental,non-controlled study with small sample sizes.During November 2014 and June 2015,there were 25 patients aged 27-40 years with moderate or server AS,have the desire for the child,excluded contraindications during pregnancy were recruited.UC-MSCs for transplantation were obtained from National Stem Cell Centre.All the clinical inspect were collected during the late proliferating phase of the menstrual cycle by monitoring the dimeter of the follicle by ultrasound;15-17 mm follicle and low progesterone level were defined as the late proliferating phase.Medical history,physical examination,endometrial thickness,intrauterine adhesion score were assessed before and 3 months after cell therapy.Once patients were enrolled,a hysteroscopic evaluation and ultrasound were first performed to gain the endometrial thickness and intrauterine adhesion score.Endometrial biopsies were collected from two points in the most serious area and a nearly normal area of corpora uteri using biopsy seizers and the locations were recorded.On the day of cell therapy,patients received another ultrasound to make sure the menstrual cycle and then the collagen scaffold?4cm×6cm?loaded with 1×107 UC-MSCs was transplanted into the patients'uterine under the hysteroscopic.10 days before cell therapy,all patients take 6 mg/day × 10 days Progynova?menstrual period day 13?and continuous administration of the same dosage Progynova 30 days after cell therapy.3 month after cell therapy,endometrial regeneration was assessed in terms of restoration of menses,endometrial thickness?by vaginal ultrasound?,adhesion score?by hysteroscopy?,and ongoing pregnancy rateResults:2 of 25 patients were diagnosed with endometrial tuberculosis after the first hysteroscopy inspection and quit the trial to receive antituberculosis therapy,1 patient refused to take the hysteroscopy 3 month after treatment and all the inspection afterwards,these 3 patients were excluded from this clinical trial.All 22 AS patients showed the improved uterine cavity after cell therapy,reduced intrauterine adhesion and heavier menstruate.In all 22 AS patients,2 patients got the 5-6 in AFS,were diagnosed as moderate AS;2 patients were 7,6 patients were 8 and 12 patients were 10 in AFS,these 20 patients were evaluated as severe AS.The intrauterine adhesion score decreased from 8.8± 1.5 to 5.8±3.6?P<0.01?after cell therapy.1 moderated and 3 severe AS patients restored normal uterine cavity,no intrauterine adhesion were observed and the AFS reduced to 0;another 1 moderated and 4 severe AS patients intrauterine adhesion score reduced to 4,evaluated as mild AS;3 patients changed from severe AS to moderate AS;the rest 10 patient were still severe AS.Meanwhile,endometrial thickness increased from 4.4±0.9mm to 5.5±1.1 mm?P<0.01?.Immunohistochemistry staining showed higher ER-?,vimentin and Ki67 expression and increased capillary density in endometrial biopsies after cell therapy.The ER-? positive staining ratio before/after cell therapy was 0.03±0.02/0.28±0.14;similarly,the vimentin positive staining ratio was 0.08±0.05/0.46±0.20 and the Ki67 positive staining ratio was 0.18±0.10/0.40±0.21;the capillary density before/after cell therapy was 7.97 ±2.89/18.36±7.43.7 patients became pregnant after cell therapy and 5 of them have been delivered successfully,no placental complication were found in these 5 patients.The other 2 patients were ongoing third trimester pregnancy.Conclusion:This novel collagen scaffolds loaded with UC-MSCs therapy is a promising therapeutic option for patients with server AS and a wish to conceive. |
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