Proteomic Analysis On The Materno-Fetal Interface Of Early Pregnancy Loss And Follicular Developmental Potential

IntroductionProteomics is the large-scale study of proteins, modifications, complexes, polymorphism and interactions from a given cell line or organism. Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients combined with protein identification by mass spectrometry is currently the workhorse for proteomics. In spite of promising alternative or complementary technologies that have emerged recently, 2-DE is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. Matrix-assisted laser desorption/ioniza tion-time of flight mass spectrometry is a high throughput and highly sensitive technique, which is technically possible to have more detailed information on proteins with differences in protein expression and modification.A major focus of biomedical research is identification of the molecular bases of germinal cell development and maturation, implantation, embryo development and fetal growth, so as to improve the knowledge about reproduction; further, improve the diagnostic capabilities and therapeutic options to those relative diseases. However, we have still known little about the the complex interactions between numerous biological molecules during these processes. With Proteomics-based approaches support, it is able to offer great potential in unraveling these complex biological problems or pathways in reproduction related disease pathogenesis.Early pregnancy loss is the most common complication of human reproduction. Given the complexities of early development, it is likely that many mechanisms are involved. Thus identification of putative proteins affected in early pregnancy loss is indispensable to our understanding of the complex biomolecular background associated with this multifactonal event. The proteomic tools enable a systematic analysis of the proteins involved on a larger scale.One the other hand, human follicular fluid has been suggested to influence oocyte development potential, and some of human follicular fluid proteins may be potential markers for oocyte maturation during follicular development. The complexity of human follicular fluid, however, hinders the efforts to identify such proteins. Mass spectrometry techniques have revolutionized the proteomic study of biology and biochemistry by permitting sensitive, rapid, high throughput, and specific analysis of peptides and proteins at molecular level. Accurately measured molecular masses can be directly used for the assignment of novel antigens or markers for diagnostic, prognostic, or therapeutic use.Thus proteomics, in conjunction with high throughput polymorphism analysis, may enable us to unravel particular molecular complexes or pathways in the physiology and pathogenesis during early embryo developmental and explore additional potential markers for oocyte maturation during follicular development.Part One Establishment and Optimization of the Two-dimensional Electrophoresis of Chorionic Villi of Early Pregnancy LossObjective: To establish and optimize the two-dimensional electrophoresis (2-DE) of villous tissues of early pregnancy loss for proteomic analysis, and study the difference of global patterns between early prenangcy loss (EPL) and normal pregnancy. Methods: Running immobilized PH gradient-isoelectric focusing electrophoresis as the first dimension, then vertical sodium dodecylsulfate- polyacrylamide gel electophoresis (SDS-PAGE) as the second dimension. A series of important steps, such as sample solubility, volume of loading, electrophoresis paraments and protocol for staining wereoptimized. We performed 2-DE on six placental villous tissues from patients with EPL and six from normal pregnant women, followed by comparison of the silver-stained 2-DE profiles.Results: The 2-DE patterns of EPL and normal control with good quality have been obtained. Using PH 4-7 IPG strips in the first dimension, 1735±121 protein spots were obtained in EPL and 1552±153 protein spots in normal control group, respectively. When compared EPL and normal control with PDQuest software, it showed that 18 protein spots displayed quantitative changes in expression, of which 5 spots increased and 13 'spots decreased in abundance.Conclusion: Integrating protein spots extracted by enhanced solubilising solutions is beneficial for achieving intact 2-DE map of villous tissues. The modified silver staining protocol compatible with mass spectrometry does not decrease resolution of protein spots. The immobilized pH gradient strips with narrow pH range get better resolution than those with wide pH range do. The differentially expressed proteins are useful for studying the pathophysiological mechanisms underlying EPL. Part Two Proteomic Analysis on the Alteration of Protein Expression in the Villous Tissues of Early Pregnancy LossObjective: Early pregnancy loss is the most common complication of human reproduction. Given the complexities of early development, it is likely that many mechanisms are involved. Knowledge of differences in protein expression in parallel profiling is essential to understand the comprehensive pathophysiological mechanism underlying early pregnancy loss.Methods: To identify proteins with different expression profiles related to early pregnancy loss, we applied a proteomic approach and performed MALDI-TOF MS on the protein spots with different expression which had been decteted on the gels from EPL group compared with those from normal control. Results: It was found that 13 proteins were down-regulated and 5 proteins were up-regulated significantly (P<0.05) in early pregnancy loss as determined by spotvolume. Among them, 10 down-regulated and 2 up-regulated spots were identified by MALDI-TOF MS. Anomalies of these proteins, including three principle antioxidant enzymes (copper/zinc-superoxide dismutase, peroxiredoxin 3 and thioredoxin-like 1 protein), S100 calcium binding protein A11, galectin-1, chorionic somatomammotropin hormone 1, transthyretin, fas inhibitory molecule, eukaryotic translation elongation factor, RNA-binding protein, ubiquitin-conjuating enzyme E2N and proteasome beta-subunit, indicate widespread failure in cell regulations and processes such as antioxidative defense, differentiation, cell proliferation, metabolism, apoptosis, transcription and proteolysis in early pregnancy loss.Conclusion: This study has identified several proteins that are associated with placentation and early development, shedding a new insight into the proteins which may be potentially involved in the pathophysiological mechanisms underlying early pregnancy loss. Part Three Proteomic Analysis on the Alteration of Protein Expression in the Decidual Tissues of Early Pregnancy LossObjective: To study the knowledge of differences in protein expression of decidual tissues of EPL in parallel profiling is essential to understand the comprehensive pathophysiological features and mechanism underlying EPL.Methods: To identify proteins with different expression profiles related to early pregnancy loss, we applied a proteomic approach and performed two-dimensional gel electrophoresis (2-DE) on five decidual tissues from patients with EPL and five from normal pregnant women, followed by comparison of the silver-stained 2-DE profiles.Results: The 2-DE patterns of EPL and normal control with good quality have been obtained. Using pH 4-7 IPG strips in the first dimension, 1213±97 protein spots were obtained in EPL and 1277±82 protein spots in normal control group, respectively. When compared EPL and normal control with PDQuest software, it showed that 9 proteins were down-regulated and 13 proteins were up-regulated significantly (P＜0.05) in earlypregnancy loss as determined by spot volume. Among them, 7 down-regulated and 3up-regulated spots were identified by MALDI-TOF MS. Anomalies of several proteinssuch as glusoce-regulated protein 78, valosin- containing protein, actin-related protein 2,myosin regulatory light chain 3, F-actin capping protein, proteasome subunit,ubiquitin carboxyl- terminal esterase L3 etc., had been identified.Conclusion: This study has indicated 10 proteins that are associated with widespreadfailure in cell regulations and processes such as cellular stress response, cell structure,cell growth, metabolism and proteolysis in decidual tissues of EPL, shedding a newinsight into the proteins which may be potentially involved in the pathophysiologicalmechanisms underlying EPL.Part Four Specific Peptide Patterns of Follicular Fluids at Different GrowthStages Analyzed by Matrix-assisted Laser Desorption/Ionization Time-of-flightMass SpectrometryObjective: Human follicular fluid (HFF) has been suggested to influence oocytedevelopment potential, and some of HFF proteins may be potential markers for oocytematuration during follicular development.Methods: Using MALDI-TOF MS, the presence of specific peptide peaks in HFFwhich could represent the follicle development potential was evaluated. HFF fromdifferent developmental stages were first digested and the resultant peptide mixtureswere directly analyzed by MALDI-TOF MS. It was shown that the frequencies ofspecific peaks demonstrated higher reproducibility than peak intensities after multiplemeasurements (= 6 times) per sample. Using this approach, a reliable peak list for eachdifferent sample could be generated by combining the information from multiplemeasurements.Results: By comparing the peak lists from different samples at different growth stages,we found that 5 specific peaks appeared in the 100% frequency category of 6 replicatesin all the HFF samples containing mature oocyte. Similarly, such 25 peptide peaks werealso identified for HFF containing immature oocyte. These specific peaks could be usedto distinguish HFF from different stages as biomarkers related to follicle developmentand maturation. After searching the protein database, some proteins that are known tobe involved in the development and maturation of oocyte were identified, such asapolipoprotein A-I, collagen type IV, integrin, et al.Conclusion: Identification of such proteins in our experiment further proved that thedirect analysis of tryptic digests could be of practical value.