| Ovarian endometrial cyst,also known as chocolate cyst,is a benign disease with"malignant behaviors",including pelvic adhesion,ovulation failure,oogenesis disorder,embryo implantation disorder,infertility,etc.Most scholars believed that ovarian endometrial cyst would desensitize the response of ovary to ovulation induction in IVF cycle,which led to increased drug dosage and decreased number of developing follicles and retrieved oocytes.This study recruited patients who were diagnosed with endometriosis and experienced in vitro fertilization and embryo transfer(IVF-ET)from our reproductive center in recent five years.We explored the effects of ovarian endometrial cyst on ovarian reserve function,the number of retrieved oocytes,fertility rate,cleavage rate,embryonic number,embryo transfer rate,clinical pregnancy rate,and abortion rate.Further,the factors in ovarian endometrial cyst that disrupts the therapeutic outcomes of IVF were investigated,and prediction model was established.The main features of ovarian fibrosis are thickened capsule of ovary,increased interstitial connective tissue,and decreased or even disappeared follicles.This study revealed that the degree of fibrosis in cyst wall was deeper in ovarian endometrial cyst than in ovarian teratoma,characterized by higher expression of fibrotic factors,including collagen Ⅰ(Col Ⅰ),alpha-smooth muscle actin(a-SMA),connective tissue growth factor(CTGF),matrix metalloproteinase 2(MMP2),tissue inhibitor of matrix metalloproteinase 1(TIMP1),and TIMP2.This research examined the expression of TGF-β1 in cyst wall of ovarian endometrial cyst.We found that ectopic endometrial cells specifically synthesized TGF-β1.With endometrial stromal cells cultureing in vitro and stimulated with TGF-β1,the effect of TGF-β1 on endometrial stromal cell fibrosis was evaluated.And the key factors involved in TGF-β1/Smad pathway were also detected to elaborate the potential mechanism underlying cystic fibrosis in ovarian endometrial cyst.Additionally,high through-put sequencing of microRNA(miRNA)was performed to detect the expression of miRNAs in follicular fluid from mature follicles of patients with ovarian endometrial cyst.The results showed that the expression of miRNAs of let-7 family were significantly elevated.We further explored the correlation between TGF-β1 and let-7 family members and speculated the possible reasons for the decreased quality of oocytes in ovarian endometrial cyst.PART Ⅰ:Research on the factors that affects the therapeutic outcomes in infertility patients with ovarian endometrial cyst after receiving IVF-ET:A 5-year statistical analysisObjective:This study aimed to compare the ovarian reserve function,fertility rate,oocyte retrieve rate,high-quality embry rate,and pregnancy outcome after IVF-ET between ovarian endometrial cyst and other endometriosis.The study explored the main factors that influence the pregnancy outcome in patients with ovarian endometrial cyst after receiving IVF-ET.Our work provided valuable references for clinical diagnosis and treatment.Materials and Methods:In this study,619 patients who were diagnosed with endometriosis and thus experienced IVF-ET were recruited in our reproductive center between January 2011 and December 2015.Among these patients,398 patients were with ovarian endometrial cyst,while the others were with other pelvic endometriosis.Patients received ovulation induction in every period of IVF-ET.Oocytes received fertilization at 2-6 hours after retrieved.According to the quality of blastomere and symmetry,embryo scoring system was divided into 4 grades.Cryopreservation thawed embryo was conducted using classic TESTART’ s slow freeze and rapid thaw in 2011-2012 and using vitrification freeze and thaw from the year of 2013.The quality of embryos and patient condition were evaluated in 3 days after oocyte retrieval.The number of transferred embryos was no more than 3.Embryos were conventionally implanted in the middle of uterine.The choices for the endometrial preparation programs for frozen-thawed embryo transfer were classified into natural cycle,micro stimulation cycle with low dose of Gn,and hormone replacement cycle.Serum β-HCG was detected in 12 days after embryo transfer to determine the pregnancy.Gestational sac was detected using B ultrasound in 35 days after embryo transfer to determine the clinical pregnancy.Data were analyzed using SPSS 20.0 statistical software.Normal distribution was determined using single sample Kolmogorov-Simirnov test.Measurement data were analyzed using Student’s t test and expressed as mean ± SD.Enumeration data were analyzed using Chi-square test.Fisher’s exact test was used when theoretical frequency was less than 1.The correlation was analyzed using Spearman’s rank correlation test to initially screen the potential parameters that affect the therapeutic outcoriies of patients with chocolate cyst.Whether patients were pregnancy was defined as the dependent variables,and the parameters preliminarily screened were defined as the independent variables.The binomial logistic regression model was established using stepwise regression to screen out the possible factors.Values of P<0.05 were considered statistically different.Results:1.The years of infertility in patients with ovarian endometrial cyst were shorter than the patients with other pelvic endometriosis(P<0.01).The numbers of antral follicle in patients with ovarian endometrial cyst were lesser than that in patients with other pelvic endometriosis(P<0.01).This difference was observed in each age sets.The levels of basal follicle-stimulating hormone(bFSH)were correlated with ages.It was found that bFSH was similar in all patients under 30 years old,while in patients between 30 and 38 years old,the levels of bFSH in patients with endometrial cyst were higher than those in patients with other pelvic endometriosis(P<0.05).2.During IVF-ET treatment,in ovarian endometrial cyst group,the number of retrieved oocytes and the number of 2 pronuclear(2PN)embryos by all age group(P<0.01),and the number of 2PN high-quality embryos in patients under 30 years old were lesser than those in patients with other pelvic endometriosis(P<0.05).The fertility rate,cleavage rate,the rate of 2PN high-quality embryos,embryo implantation rate,clinical pregnancy rate,and abortion rate between the two groups were considered similar.3.After receiving treatment,patients who were with ovarian endometrial cyst and successfully received pregnancy at the first-time transfer were featured by more antral follicles,retrieved oocytes,2PN embryos(P<0.01)and lower bFSH(P<0.05),when compared with patients of treatment failure(including patients who were not pregnant after the first-time transfer or who did not have embryos for transfer).Whether there was a correlation between the parameters and the success of treatment was determined by Pearson’ s correlation analysis.The results showed that the years of infertility,and number of antral follicles,retrieved oocytes,2PN embryos,2PN high-quality embryos were related to the successful therapy.Except for the mere correlation between the years of infertility and 2PN high-quality embryos,every other factor was related each other.The five factors were used to establish the binomial logistic regression model using stepwise regression.The key factors to determine whether IVF-ET was effective in patients with ovarian endometrial cyst were the numbers of antral follicles and 2PN high-quality embryos.The two key factors and their corresponding regression coefficients were used to establish the probabilistic model for predicting the therapeutic outcomes of patients who were with ovarian endometrial cyst and experienced IVF-ET.P=1/1+exp(0.870-0.088x(the number of antral follicles)-0.432x(the number of 2PN high-quality embryos)).Job characteristics(ROC)curve of subjects involved in this model was drawn based and the area under the curve(AUC)was 0.734(P<0.01).The 95%confidence interval was 0.684-0.783.4.In this study,in the group of the patients with ovarian endometrial cyst,383 patients were taken operation,while 15 patients were not.There was no difference of ages in the two groups.However,the volumes of ovarian endometrial cysts at the first time visiting(4.1cm vs.2.2cm,P<0.001)and the number of antral follicles(6.9 vs.5.5,P=0.017)the surgical group were larger and more than that in the non-surgical group.The numbers of retrieved oocytes,embryos,and high-quality embryos in the surgical group were more than that in the non-surgical group(P>0.05),which might be due to the limited cases obtained from the non-surgical group,and the patients in two group had different condition before operation.Conclusion:Endometriosis would damage the clinical therapeutic outcomes of IVF-ET.Besides,ovarian endometrial cyst would significantly impair the ovarian reserve function and ultimately affect the therapeutic efficacy of IVF-ET.It remains an unclear issue that whether ovarian endometrial cyst itself or the surgery itself is more harmful to the ovarian function.Thus,in patients with ovarian endometrial cyst,it would be wise to develop a strategy to preserve the ovarian function at the earliest convenience based on ages,ovarian function,fertility or not,and the volume of ovarian endometrial cyst.PART II:Study on the ovarian fibrosis and its Mechanism in ovarian endometriosisObjective:This work was to study the histological characteristics,molecular features,and fibrotic stages of surrounding tissues in cyst wall of ovarian endometrial cyst and mature teratoma.It is suggested that cyst wall of ovarian endometrial cyst may be the formation of ovarian fibrosis.We also explored the expression and profibrotic effects of TGF-β1 in ovarian endometrial cyst.Materials and methods:1.Seventy-one patients were recruited during November 2014 and December 2015,among which forty-two patients were with ovarian endometrial cyst and twenty-nine patients were with mature teratoma.All the patients experienced laparoscopic oophorocystectomy.Specimens of 1×1 cm2 were collected from cyst wall for HE and Masson staining to evaluate the fibrotic degrees.2.RT-qPCR was performed to determine the gene expression of fibrotic factors,such as Col I,a-SMA,CTGF,TIMP1,TIMP2,and MMP2.IHC and western blotting were performed to compare TGF-β1 expression in cyst wall of ovarian endometrial cyst and mature teratoma.3.FISH was performed to determine the site where TGF-β1 was synthesized in ovarian endometrial cyst.4.RT-qPCR was performed to detect the expression of TGF-β1 and related fibrotic genes in ovarian tissue near and away from in ovarian endometrial cyst.Results:1.Results from HE and Masson staining showed that in spite of the similar clinical symptoms,patients with ovarian endometrial cyst were characteristic of more severe cyst fibrosis than those with mature teratoma.RT-qPCR analysis showed that the expression of fibrotic genes,including Col Ⅰ,α-SMA,CTGF,TIMP1,TIMP2,and MMP2,was higher in cyst wall of ovarian endometrial cyst than mature teratoma.2.TGF-β1 was specifically expressed in ectopic endometrial cells from cyst wall of ovarian endometrial cyst but neither in cyst wall of mature teratoma nor epithelial cells near the lumen.The protein expression of TGF-β1 was significantly higher in cyst wall of ovarian endometrial cyst than mature teratoma.FISH results showed that TGF-β1 was specifically expressed in ectopic endometrial cells.3.The expression of TGF-β1 was higher in ovarian tissues near ovarian endometrial cyst than lesions away from.The expression of Col I,a-SMA,and CTGF was also increased(P<0.01)and the fibrosis scored higher.These findings suggested that ovarian fibrosis negatively correlated with the distance from normal tissues to endometriosis lesions.Conclusion:Ectopic endometrial cells from cyst wall of ovarian endometrial cyst synthesized and secreted TGF-β1.Abundant TGF-β1 was accumulated in the surrounding ovarian tissues,which facilitated the reconstruction of extra cellular matrix,triggered fibrosis and tissue adhesion.Due to the adhesion formation,excessive resection of cyst wall would remove partial normal ovarian tissues,caused damaging on ovarian structure,and decreased ovarian reserve.Therefore,how to protect the ovarian function in patients with ovarian endometrial cyst is one of hot debates.It could be anticipated that effective inhibition on abnormal expression of TGF-β1 would suppress the formation of fibrosis in surrounding ovarian endometriosis.PART Ⅲ:Research on the mechanisms underlying ovarian endometriosis-induced ovarian fibrosis and damage on the follicular microenvironmentObjective:This work was aimed to investigate the mechanisms underlying the action of TGF-β1 in inducing ovarian endometriosis-induced ovarian fibrosis and explore the function of activated TGF-β1/Smad pathway during fibrosis.In addition,combined with the phenomenon of increased apoptosis of cumulus cells in endometriosis,we explored the possible correlation between high TGF-β1 and follicular microenvironment from the molecular and gene level,in order to provide experimental basis and ideas for the protection of ovarian function in ovarian endometriosis patients.Materials and methods:1.HE,Masson,and immunochemical staining were performed on cyst wall samples from patients with ovarian endometriosis to testify the expression of TGF-β1 and key factors involved in TGF-β1/Smad pathway.2.In this study,endometrial specimens were obtained from patients who experienced hysterectomy or endometrial biopsy in reproductive center&obstetrics and gynecology department in our hospital.Stem cells were isolated,cultured and authenticated from endometrium.Stem cells were incubated with 10 ng/mL of TGF-β1.Then the expression of fibrotic markers and critical factors involved in TGF-β1/Smad pathway in stem cells were examined.3.Cumulus granulosa cells were obtained from patients who were diagnosed with ovarian endometrial cyst plus infertility or experienced IVF-ET merely owing to tube factors,and the apoptosis index of these cells were measured and compared.4.Follicular fluid were collected from 6 patients experiencing IVF,among which 3 patients were diagnosed with ovarian endometrial cyst and the others suffered from salpingemphraxis.The high-throughput sequencing of miRNAs was performed in total RNA extracted from follicular fluid.5.Based on the results of high-throughput sequencing of miRNAs,293T cells were selected and incubated with SB-431542(an antagonist of TGF-β1).RT-qPCR were performed to determine the expression of miRNAs belonging to let-7 family.Results:1.Results from HE,Masson,and immunochemical staining showed that areas positive for TGF-β1,TGF-βR1,α-SMA,Col Ⅰ,and CTGF were overlapped with fibrotic areas in Masson staining.Meanwhile,areas positive for p-Smad2,p-Smad3,Smad2/3,Smad2,Smad3,and Smad4 were overlapped with areas positive for TGF-β1 and TGF-βR1 and fibrotic areas.2.Endometrial cells-derived stem cells were cultured in vitro and the incubated with TGF-β1.Experimental results showed that the gene expression of fibrotic genes,such as Col I,a-SMA,CTGF,and FN,were increased in a time-dependent manner.Also,the protein expression of Col I,CTGF,a-SMA,and MMP2 were increased in a time-dependent way.Besides,several key factors mediating TGF-β1/Smad pathway,i.e.Smad2,p-Smad2,Smad3,and p-Smad3,were activated immediately after TGF-β1 stimulation.3.Results from high-throughput sequencing of miRNAs suggested that seven miRNAs belonging to let-7 family,that is hsa-let-7f-5p,hsa-miR-98-5p,hsa-let-7e-5p,hsa-let-7a-5p,hsa-let-7d-5p,hsa-let-7g-5p,and hsa-let-7c-5p,were expressed in follicular fluid of patients with ovarian endometrial cyst at a higher level than that of patients who experienced IVF-ET solely owing to male factors.4.There were few significant differences in ages between patients with ovarian endometrial cyst and patients who experienced IVF-ET owing to tube factors.The number of retrieved oocytes was obviously less in patients with ovarian endometrial cyst than that in patients who experienced IVF-ET owing to tube factors,while the percentage of apoptotic cumulus granulosa cells was more than that in patients who experienced IVF-ET owing to tube factors(37.2%vs.9.2%,P<0.01).5.Results showed that TGF-β1 significantly induced the expression of let7a,let-7c,let-7d,let-7e,let-7f,let-7g,and miRNA-98 in 293T cells,while no obvious changes in the expression of these factors in SB-431542-treated cells when ’compared with vehicle-treated cells.These results suggested that TGF-β1 may be involved in regulating let-7 family members.Conclusion:TGF-β1/Smad signaling pathway played a central role in the formation of ovarian endometriosis-induced ovarian fibrosis.Through an analysis on the results obtained from high-throughput sequencing of miRNAs,we found that the expression of seven miRNAs from let-7 family were abnormally increased.Thus,we postulated that TGF-β1/Smad signaling pathway was activated during ovarian fibrosis,which thus upregulates the abundance of let-7 family members.This study elucidates a possible mechanism that TGF-β1 synthesized by ectopic endometrial cells triggered fibrosis in ovary,upregulated let-7 family members,increased the percentage of apoptotic cumulus granulosa cells,and consequently disrupted the follicle microenvironmen,which provides a potential target for the improvement of oocyte quality. |
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