Factors Influencing The Live Birth Rate Following Fresh Embryo Transfer Cycles In Infertile Women After Endometrioma Cystectomy And NBDHEX Inhibits GSTM4 In Endometriosis

Part I Factors influencing the live birth rate following fresh embryo transfer cycles in infertile women after endometrioma cystectomyBackground:Endometriosis is defined as the appearance of endometrial glands and stroma in ectopic locations,including ectopic pelvic peritoneum,ovary,and rectovaginal septum.It affects nearly 10%of women of childbearing age and 30%-50%of women with chronic pelvic pain and infertility.Ovarian endometriotic cysts,also known as endometriomas,are so-called "chocolate cysts",which are usually associated with infertility and increase the risk of endometriosis-related ovarian cancer.Until now,whether to perform surgery before assisted reproductive technology(ART)has been controversial.Some researchers believe that laparoscopic surgery is the gold standard for the diagnosis of endometriosis,and that it can improve fertility in these patients by removing lesions,separating adhesions,assessing fallopian tubes and ovaries function,and restoring normal pelvic anatomy.However,some researchers believe that surgery before ART may lead to impaired ovarian reserve,poor ovarian response,reduced oocytes abtaine,and even adverse pregnancy outcomes.In fact,a significant proportion of women with endometriosis-related infertility choose to have surgery not to increase their chances of pregnancy,but to improve their quality of life.The question what we focused on is not whether pre-ART surgery is better than direct ART,but what are their chances of having a live birth and finding ways to increase them for patients after endometrioma cystectomy?Therefore,the purpose of this study was to identify the predictors affecting the clinical outcomes of in fresh IVF/ICSI-ET cycles among infertile women who had undergone ovarian cystectomy for endometriomas.And to investigate whether different stimulation protocols influence the live birth rate.This may help fertility specialists and their patients to determine the expectations of appropriate treatment strategies for ovarian endometrioma before starting assisted reproductive treatment.Methods:We recruited 513 infertile women with a history of ovarian cystectomy for endometriomas who underwent their first fresh ET with different stimulation protocols following IVF/ICSI cycles at the Reproductive Hospital Affiliated to Shandong University from January 2014 to December 2018.One or two embryo are implanted.Firstly,we investigated the differences in demographic and main treatment IVF/ICSI cyles characteristics between live birth and non-live birth groups.Clinical and laboratory parameters potentially affecting the live birth rate following fresh ET cycles were analyzed.Univariable and multivariable analyses were performed to assess the relationship between predictive factors and live birth rate.Results:The overall live birth rate was 240/513(46.8%).Multivariable modified Poisson regression models showed that two factors were significantly lowers the probability of live birth:female age ≥ 35 years(aOR 0.603;95%CI 0.389-0.933;P=0.023);BMI range 21-24.99 kg/m2 compared with BMI<21 kg/m2(aOR 0.572;95%CI 0.372-0.881,P=0.011).And two factors significantly increased the probability of live birth:AFC>7(aOR 1.591;95%CI 1.075-2.353;P=0.020);two embryos transferred(aOR 1.607;95%CI 1.089-2.372;P=0.017).Conclusions:For these infertile women who had undergone ovarian cystectomy for endometriosis,female age<35 years,AFC>7,and two embryos transferred might achieve better clinical fresh IVF/ICSI-ET outcomes.BMI<21 kg/m2 or≥25 kg/m2 might also have positive effects on the live birth rate,but different ovarian stimulation protocols had no significant effects.However,a larger sample size may be needed for further study.Part II NBDHEX ameliorates the progression of endometriosis by regulating GSTM4 expression to inhibit proliferation and induce apoptosisBackground:Endometriosis is a chronic disease caused by the presence of endometrial glands and stroma in ectopic locations,including ectopic pelvic peritoneum,ovary,fallopian tubes,broad ligament,abdomen,rectovaginal septum and some time even to lungs.It is usually associated with dysmenorrhea,dyspareunia,chronic pelvic pain,and infertility.Although endometriosis is a benign disease,it has the characteristics properties similar to malignant cancer,such as ectopic implantation,growth,migration and invasion.The source of ectopic endometrium has not yet been elucidated.At present,the widely accepted "retrograde menstruation and ectopic implantation theory" believes that the endometrial glandular epithelium and stromal cells can enter the pelvic cavity with menstrual blood reflux through the fallopian tube during menstruation,but most women of reproductive period have menstrual blood reflux,only 10%-15%of them have the disease,this doubt may be explained by the "Eutopic endometriosis determinism theory".During menstruation,endometrial cells in the uterus flow back to the pelvic cavity with menstrual blood.Cells with strong adhesion,invasion and angiogenesis ability are more likely to be planted in the ovary and the adjacent pelvic peritoneum,where they continue to grow and spread,forming internal lesions.They believe that the biological characteristics of the present endometrium are the decisive factors in the development of endometriosis,and the local microenvironment in which it is located is an important factor in the development of endometriosis.We screened the proteomic profiles of endometrial tissues from endometriosis patients and healthy women.Acquired data revealed that GSTM4 was highly expressed in endometriosis patients.Drug therapy is appropriate for most patients with endometriosis.The first-line therapy for drug treatment include oral contraceptive pills or nonsteroidal anti-inflammatory drugs.However,with these therapeutic approaches,many patients have had little efficacy,or only short-term remission,and some patients even get worse.The mechanism underling endometriosis progression is not fully understood,but several studies demonstrated that it is associated with disorder of the oxidative balance.Oxidative stress is resulted from unbalanced production of reactive oxygen species(ROS)and the cell’s own antioxidant defense.Cancer stem cells proliferate spontaneously takeing the advantage of the aberrant redox system.In addition,the imbalance of immune system resulting in the accumulation of inflammatory factors is also one of the pathogenesis of endometriosis.In order to find a better therapy for endometriosis,we urgently need to find new and effective drugs to treat this disease.GSTM4 gene was first isolated from cervical carcinoma cell line HeLa and the protein forms functional dimers was comprised of 218 amino acids with a Mr of approximately 26.4kDa.We screened the proteomic profiles of endometrial tissues from endometriosis patients and healthy women.Acquired data revealed that GSTM4 was highly expressed in endometriosis patients.A study specifically targeting GSTM4 has found that NBDHEX was used to inhibit the pharmacological activity of GSTM4 and significantly limited cellular proliferation and oncogenic transformation in Ewing sarcoma cells.A large amount of evidence emphasizes that NBDHEX can inhibit GSTs catalytic activity and induces apoptosis of tumor cells,which can be used as a new potential anticancer drug.The inhibition of GSTM4 expression by NBDHEX in endometriosis has not been reported.This study aims to explore the role of GSTM4 in endometriosis and to detect whether NBDHEX can affect cell proliferation,migration,invasion and apoptosis by regulating the expression of GSTM4,so as to provide a research basis for clinical treatment.Methods:This study recruited nonpregnant women of reproductive age.15 cases of human endometriosis eutopic endometrium for immunohistochemical was collected as the experimental group and 15 cases of normal endometrial tissues were collected as control group.The primary endometrial stromal cells(euESC)were isolated and cultured from 9 pairs of fresh endometrium from endometriosis patients and corresponding healthy women for follow-up experiments.The expression level of GSTM4 in endometrial stromal cells of endometriosis patients and healthy women was investigated by western blot analysis(WB).NBDHEX is an inhibitor of GSTM4,the effects of different concentrations of NBDHEX(0.25μM,0.5 μM,0.75 μM,1 μM,1.5 μM,2 μM,2.5 μM,3 μM,3.5 μM,4 μM)on the proliferation of primary endometrial stromal cells in patients with endometriosis were detected by CCK8 assay.And to find the half maximal inhibitory concentration for endometriosis eutopic endometrium.Tanswell assay was used to study the effects of different concentrations of NBDHEX on cell migration and invasion ability.WB assay was used to detect the effects of NBDHEX on the expression levels of PCNA and MMP-9 in primary endometrial stromal cells of endometriosis patients.The effect of NBDHEX on apoptotic cell rate was detected by flow cytometry,and the expression levels of Survivin,Bcl-XL and Bax were detected by WB assay.WB was used to detect the effects of NBDHEX on the expression levels of oxidative stress-related pathways Keapl and Nrf2 in primary endometrial stromal cells of patients with endometriosis,and to explore the mechanism of NBDHEX.Because in addition to inhibiting the expression of GSTM4,NBDHEX may also inhibit the expression of other GSTs family proteins.In order to further elucidate the role of GSTM4 in the progression of endometriosis,we transfected an siRNA containing an GSTM4 targeting sequence(si-GSTM4)and negative control siRNA(si-NC)in eutopic endometrial stroma cells with endometriosis and control endometrial stroma cells.The effects of knockdown GSTM4 on cell functions such as proliferation,migration,invasion and apoptosis both in eutopic endometrial stroma cells with endometriosis and control endometrial stroma cells were detected,and the oxidative stress signaling pathway of inhibiting GSTM4 protein expression by NBDHEX was investigated.Results:IHC showed that GSTM4 protein was expressed in the cytoplasm of both stromal cells and glandular epithelial cells in endometrial tissue,but not in the nucleus.IHC and WB results showed that GSTM4 was significantly overexpressed in endometriosis patients compared with controls.CCK8 results showed that the proliferation of endometriosis cells decreased with the increase of NBDHEX concentration.The IC50 value of primary endometrial stromal cells with endometriosis was about 1.44 μM.After treating the cells with GSTM4 inhibitor NBDHEX,the migration and invasion functions of the endometrial stromal cells from endometriosis patients were significantly decreased,while the apoptotic function was significantly enhanced.Treating cells with NBDHEX did not affect the expression of Keapl,but reduced the expression of Nrf2.After transfection with si-GSTM4,the protein level of GSTM4 was down-regulated by nearly 70%.The silencing of GSTM4 inhibited the proliferation,migration and invasion of endometrial stromal cells and the gene expression of its signature proteins in endometriosis patients and controls.GSTM4 protein may interact with Nrf2 through a series of reactions to reduce the expression levels of apoptotic protective proteins such as Survivin and Bcl-XL,and increase the expression level of pro-apoptotic protein Bax to induce apoptosis,but it does not affect the expression of the upstream protein Keapl of oxidative stress.Conclusions:This study found that GSTM4 was highly expressed in endometriosis,and inhibition of GSTM4 by NBDHEX could suppresses cell growth,migration,invasion and interact with Nrf2 to induce apoptosis,but has no effect on the expression of Keap1 in endometriosis.The use of siRNA to knockdown GSTM4 more accurately confirmed its ability to ameliorate the progression of endometriosis.NBDHEX may have therapeutic potential in the treatment of endometriosis.