Differential Proteomic Study On Endometrial Window Of Implantation In Patients With Endometriosis

Background and objectiveEndometriosis (EMs) is classically defined as the presence of functional endometrial glands and stroma outside the uterine cavity. The prevalence of EMs ranges from 10% to 15% of all reproductive age women. There is a rising trend year after year. The occurrence rate of infertility in EMs patients is 40-50%, and is 20 times that of non-EMs population. Therefore, EMs is one of the important reasons for infertility. In 2000, the concept "Endometriosis-associated infertility" was put forward by Buyalos the first time.EMs could result in the infertility by various kinds of mechanisms, such as abnormality of the pelvic anatomy, changes in the peritoneal fluid composition, luteinized unruptured follicle syndrome (LUFS), luteal Phase defect (LPD), and effect on the embryo implantation. But so far, the relationship between the cause of endometriosis-associated infertility and the pathogenesis of EMs is still not clear.With the development of modern assisted reproductive technology (ART) in recent years, in vitro fertilization and embryo transfer (IVF-ET) has become a recognized treatment for refractory endometriosis-associated infertility. However, in patients with EMs undergoing IVF-ET, the embryo transfer rate is up to 85%, but the pregnancy rate was about 20%, which is only half of that of patients with tubal infertility. It indicates that there are many negative factors affecting embryo implantation in the eutopic endometrium of patients with EMs.Endometrial receptivity is the endometrial ability to accept the embryo implantation within a specific period which is known as the "window of implantation" (WOI). WOI is present in the 20th to 24th day of the menstrual cycle. In recent years, studies have indicated that the abnormal regulation of specific gene and protein in the eutopic endometrium of patients with EMs, such as integrins, aromatase, matrix metalloproteinases, angiogenic factors, is associated with the defect in endometrial receptivity. Therefore, decrease of endometrial receptivity of patients with EMs is probably one of the important reasons for infertility.There are many factors that can lower endometrial receptivity of patients with EMs, but so far, It is unclear whether the mechanism and regulatory factors of endometrial receptivity during WOI change and how to change. The morphology and molecular marker indicating change of the eutopic endometrial receptivity has yet to be discovered.As the human genomic analysis gradually completed, exploration for life science has entered the post-genomic era, namely proteomic era. Compared with traditional methods, proteomics technology has such characteristics as high throughput, high efficiency and high accuracy, etc. So currently it has been widely applied in various researches.The change of endometrial receptivity in patients with EMs is a complicated, dynamic process where multiple genes are involved and hundreds of different protein-proteins interact. Proteomics provides an excellent technical platform for the study on the change of endometrial receptivity during WOI. In order to research in the abnormal mechanism of endometrial receptivity, to provide objective and scientific evidence for clinical treatment, and to improve the success rate of ART, we have identified differentially expressed proteins of endometrium during WOI in patients with EMs by the two-dimensional fluorescence differential in-gel electrophoresis (2D-DIGE) and matrix-assist laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS).Chapter 1:Differential Proteomic Study on Endometrial Window of Implantation in Normal Fertile WomenObjectiveFor a better understanding of the molecular mechanism of regulating endometrial receptivity, the goal of this chapter was to establish the methodology of 2D-DIGE and MALDI-TOF-MS for protein analysis of human endometrium during WOI, to acquire the 2D-DIGE protein profiles from normal fertile women, and to identify the differential expressed proteins between pre-receptive stage and receptive endometrium.MethodsTen fertile volunteers were involved in the study. The luteotropic hormone (LH) surge and the ovulatory day (OD) were confirmed by vaginal ultrasound monitoring of follicular development combined with blood and urinary analysis. Endometrial samples were obtained at 1 day after OD (pre-WOI stage, OD+1) and at 6 day after OD (WOI stage, OD+6) respectively, within the same cycle by microbiopsy. A small portion of each specimen was examined histologically. The other portion was stored for proteomics studies. The total proteins were extracted, purified and quantified from two groups, then pre-labeled by Cydye. After IEF and SDS-PAGE, 2D-DIGE gel was scanned and visualized by the multi-channel laser scanner. The 2D-DIGE image was analyzed using Decyder software. Differential protein spots were selected for picking, enzymatic digesting and spotting by Ettan spot workstation. Protein identified by MALDI-TOF-MS was verified in local NCBI and online MASCOT databases.Results1. Endometrial histological biopsies were classified into two groups:early secretory phase (OD+1); mid-secretory phase (OD+6).2. The 2D-DIGE protein profile was acquired and 38 differential expressed proteins were identified with 30 up-regulated and 8 down-regulated on day OD +6 versus OD+1.Summary1. The methodology of 2D-DIGE and MALDI-TOF-MS for protein analysis of human endometrium during WOI was established.2. The 2D-DIGE protein profile from normal fertile women during WOI was acquired.3.38 differential expressed proteins were identified with 30 up-regulated and 8 down-regulated. Chapter 2:Differential Proteomic Study on Endometrial Window of Implantation in Patients with EndometriosisObjective For a better understanding of the molecular mechanism of changingendometrial receptivity in infertile patients with EMs, the goal of this chapter was to acquire the 2D-DIGE protein profiles during WOI from infertile patients with EMs, to identify the differential expressed proteins between pre-receptive stage and receptive endometrium, and to find differential expressed proteins on day OD+6 between EMs group and the control group using DeCyder software.MethodsTen infertile volunteers with EMs were involved in the study. The LH surge and OD were confirmed by vaginal ultrasound monitoring of follicular development combined with blood and urinary analysis. Endometrial samples were obtained at 1 day after OD (pre-WOI stage, OD+1) and at 6 day after OD (WOI stage, OD+6) respectively, within the same cycle by microbiopsy. A small portion of each specimen was examined histologically. The other portion was stored for proteomics studies. The total proteins were extracted, purified and quantified from two groups, then pre-labeled by Cydye. After IEF and SDS-PAGE,2D-DIGE gel was scanned and visualized by the multi-channel laser scanner. The 2D-DIGE image was analyzed using Decyder software. Differential protein spots were selected for picking, enzymatic digesting and spotting by Ettan spot workstation. Protein identified by MALDI-TOF-MS was verified in local NCBI and online MASCOT databases. Differential expressed proteins on day OD+6 between EMs group and the control group were identified using DeCyder software. Results1. Endometrial histological biopsies were classified into two groups:early secretory phase (OD+1); mid-secretory phase (OD+6).2. The 2D-DIGE protein profile was acquired and 42 differential expressed proteins were identified with 38 up-regulated and 4 down-regulated on day OD +6 versus OD+1.3.7 differential expressed proteins between EMs group and the control group on day OD+6 were identified with 4 up-regulated and 3 down-regulated.Summary1. The 2D-DIGE protein profile from infertile patients with EMs during WOI was acquired.2.42 differential expressed proteins were identified with 38 up-regulated and 4 down-regulated.3.7 differential expressed proteins between EMs group and the control group on day OD+6 were identified with 4 up-regulated and 3 down-regulated.Chapter 3:Western Blot Verification of Differential Expressed ProteinsObjectiveWestern blot was performed for semi-quantitative validation of Annexin A4 (ANX A4) and selenium binding protein 1 (SBP1) in the eutopic endometrial tissue during WOI. MethodsPreviously obtained extracted protein samples on day OD+6 of two groups were used for Western blot validation. Differential expressed proteins-ANX A4, SBP1 were selected to carry on the semi-quantitative analysis.Results1. ANX A4:In EMs group and the control group, Western blot analysis revealed protein level of ANX A4 was significantly down-regulated in EMs group on day OD+6 (t=7.654, P=0.002).2. SBP1:In EMs group and the control group, Western blot analysis revealed protein level of SBP1 was significantly up-regulated in EMs group on day OD +6(t=-2.709,P=0.035).Summary1. It is verified that the expressed level of ANX A4 was significantly down-regulated in EMs group.2. It is verified that the expressed level of SBP1 was significantly up-regulated in EMs group.Conclusions1. The methodology of 2D-DIGE and MALDI-TOF-MS for protein analysis of human endometrium during WOI was established. It could provide an excellent technical platform for the study on the change of endometrial receptivity during WOI.2. The 2D-DIGE protein profile from normal fertile women during WOI was acquired and 38 differential expressed proteins were identified.3. The 2D-DIGE protein profile from infertile patients with EMs during WOI was acquired and 42 differential expressed proteins were identified.4. With the help of software,7 differential expressed proteins between EMs group and the control group on day OD+6 were identified with 4 up-regulated and 3 down-regulated.5. By Western blot, It was verified that the expressed level of ANX A4 was significantly down-regulated, but that of SBP1 was significantly up-regulated in EMs group.