Ai-Xia Liu

Proteomic Analysis on the Alteration of Protein Expression in the Placental Villous Tissue of Early Pregnancy Loss

Abstract 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. 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 six placental villous tissues from patients with early pregnancy loss and six from normal pregnant women, followed by comparison of the silver-stained 2-DE profiles. It was found that 13 proteins were downregulated and 5 proteins were upregulated significantly (P < 0.05) in early pregnancy loss as determined by spot volume. Among them, 10 downregulated and 2 upregulated spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anomalies of these proteins, including three principal antioxidant enzymes (copper/zinc-superoxide dismutase, peroxiredoxin 3, and thioredoxin-like 1 protein), S100 calcium binding protein, 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. This study has identified several proteins that are associated with placentation and early development, shedding a new insight into the proteins that may be potentially involved in the pathophysiological mechanisms underlying early pregnancy loss.

Insufficient maintenance DNA methylation is associated with abnormal embryonic development

BackgroundEarly pregnancy loss (EPL) is a frustrating clinical problem, whose mechanisms are not completely understood. DNA methylation, which includes maintenance methylation and de novo methylation directed by DNA methyltransferases (DNMTs), is important for embryo development. Abnormal function of these DNMTs may have serious consequences for embryonic development.MethodsTo evaluate the possible involvement of DNA methylation in human EPL, the expression of DNMT proteins and global methylation of DNA were assessed in villous or decidua from EPL patients. The association of maintenance methylation with embryo implantation and development was also examined.ResultsWe found that DNMT1 and DNMT3A were both expressed in normal human villous and decidua. DNMT1 expression and DNA global methylation levels were significantly down-regulated in villous of EPL. DNMT3A expression was not significantly changed in the EPL group compared to controls in either villous or decidua. We also found that disturbance of maintenance methylation with a DNMT1 inhibitor may result in a decreased global DNA methylation level and impaired embryonic development in the mouse model, and inhibit in vitro embryo attachment to endometrial cells.ConclusionsOur results demonstrate that defects in DNA maintenance methylation in the embryo, not in the mother, are associated with abnormal embryonic implantation and development. The findings of the current study provide new insights into the etiology of EPL.

Sustained endoplasmic reticulum stress as a cofactor of oxidative stress in decidual cells from patients with early pregnancy loss.

BACKGROUND Oxidative stress is a common pathological background for different etiologies of early pregnancy loss (EPL). It has been suggested that elevated reactive oxygen species trigger endoplasmic reticulum (ER) stress by influencing ER function. However, it is unclear whether ER stress is associated with EPL. OBJECTIVES The aim of the study was to determine whether and how ER stress occurs during the development of EPL. APPROACHES Proteomic analysis was performed on decidua from women with EPL, and then ER stress markers, redox status, apoptotic features, and cell viability were analyzed in EPL decidual cells (DCs). RESULTS EPL decidua were characterized by decreased levels of glucose-regulated protein 78 (GPR78) and valosin-containing protein and burdened with ubiquitinated proteins. Evidence of ER stress-induced apoptosis in EPL DCs was demonstrated by extensive dilation of ER, morphological features of apoptosis, and activation of caspase-4 and caspase-12. Furthermore, H(2)O(2) reduced the viabilities in both EPL and control DCs, whereas EPL DCs were more vulnerable to additional OS challenge than the controls as a result of failed induction of GRP78 expression. The cell survival percentages of DCs were dose-dependently reduced by H(2)O(2) and could be reversed in the presence of vitamin E. This effect was partly mediated by reducing the amount of misfolded proteins rather than regulating GRP78 expression. CONCLUSIONS The sum of these observations demonstrate for the first time that sustained ER stress occurs in EPL DCs and the potentially vicious relationship between ER stress and oxidative stress is likely to play an important role in the development of EPL.

Comparative proteomic analysis of human placenta derived from assisted reproductive technology

The aim of this study was to use proteomics‐based approach to examine differences in protein expression in placenta derived from assisted reproductive technology (ART) and normal pregnancy. Using 2‐DE we found that, compared with the control group, 12 spots in standard in vitro fertilization group and 18 spots in intracytoplasmic sperm injection group were identified as significantly differentially expressed proteins. Among them, six spots were differentially expressed in both standard IVF and ICSI groups with the same change tendency. Totally, 20 proteins were successfully identified by MALDI TOF/TOF MS, including proteins involved in the membrane traffic, metabolism, nucleic acid processing, stress response and cytoskeleton. Notably, five proteins detected to be differentially expressed in both ART groups were identified as annexin A3, hnRNP C1/C2, α‐SNAP, FTL and ATP5A. Some of the proteins were confirmed by Western blot and immunohistochemistry analysis. Our study allowed for the initial identification of these proteins related to various functions in placentation with significantly altered abundance in ART groups. The present results reveal that abnormal protein profiles are involved in ART placenta and these differentially expressed proteins may be valuable for the evaluation of potential association between ART treatment and offspring outcome.

Endoplasmic reticulum stress induced by oxidative stress in decidual cells: a possible mechanism of early pregnancy loss

Early pregnancy loss (EPL) is one of the most common complications of human reproduction. Combined with our previous proteomic studies on villous and decidual tissues of EPL, we found that alterations of the proteins involved in oxidative stress (OS), unfolded protein response (UPR) and proteolysis presented a complex and dynamic interaction at the maternal-fetal interface. In the present study, we developed a cell model of OS using normal decidual cells to examine cell viability and expression levels of proteins related to endoplasmic reticulum stress (ER stress) and UPR. We found that glucose regulated protein 78 (GRP 78) and ubiquitinated proteins were significantly up-regulated in hydrogen peroxide (H2O2) treated decidual cells in a dose-dependent manner. Excessive OS could influence proper function of UPR by decreasing VCP in decidual cells, thereby leading to cell damage as well as inhibition of cell growth and activation of apoptosis. Furthermore, when pretreated with MG 132, a pharmacological inhibition of the proteasome, the H2O2 treated decidual cells became less viable and could not up-regulate the expression level of GRP 78 to resolve the protein-folding defects, which indicating that malfunction of UPR in decidual cells might aggravate the inhibitory effect of OS in decidual cells. The present results reveal that abnormal protein profiles associated with OS induced ER stress and malfunction of UPR might be involved in the development of EPL, and OS and ER stress are potential targets for pregnant care and prognosis in normal pregnancy and its disorders.

Identification of Estrogen Response Element in the Aquaporin-2 Gene That Mediates Estrogen-Induced Cell Migration and Invasion in Human Endometrial Carcinoma

BACKGROUND Accumulating evidence suggests that aquaporins (AQP) can facilitate cell migration, invasion, and proliferation in tumor development in addition to water transport. OBJECTIVE The aim of this study was to examine AQP2 expression in the endometrial tissues from patients with endometrial carcinoma (EC) and determine the roles and mechanisms of AQP2 in estrogen-related cell migration, invasion, adhesion, and proliferation of Ishikawa (IK) cells. APPROACH AQP2 expression levels were measured in human endometrial cells and estradiol (E(2))-treated IK cells, and the estrogen-response element was identified. After blocking down and up-regulating the endogenous expression of AQP2 in IK cells, cell morphology, capacity for invasion, migration and adhesion, and expression markers of membrane/cytoskeleton were analyzed. RESULTS AQP2 was expressed in endometrial tissues from patients with EC and endometriosis, both of which are estrogen-dependent diseases. In IK cells, E(2) dose-dependently increased AQP2 expression, which was blocked by the estrogen receptor inhibitor ICI182780. An estrogen-response element was identified in the AQP2 promoter. E(2) significantly increased the migration, invasion, adhesion, and proliferation of IK cells. AQP2 knockdown attenuated E(2)-enhanced migration, invasion, and adhesion. AQP2 knockdown reduced not only the E(2)-enhanced expression of F-actin and annexin-2 but also the E(2)-induced alteration of cell morphology. Moreover, higher expression levels of F-actin and annexin-2 were detected in the endometrial tissues from patients with EC. CONCLUSIONS AQP2 mediates E(2)-enhanced migration, invasion, and adhesion through alteration of F-actin and annexin-2 expression and reorganization of F-actin, and inhibition of AQP may be a potential method for antitumor therapy.

Functional expression of large-conductance calcium-activated potassium channels in human endometrium: a novel mechanism involved in endometrial receptivity and embryo implantation.

BACKGROUND Large-conductance calcium-activated potassium channels (BK(Ca) channels) mediate physiological processes in nonexcitable cells. OBJECTIVE The aim of the study was to determine BK(Ca) channel expression in human endometrium and its role in endometrial receptivity and embryo implantation. METHODS BK(Ca) channel expression in human endometrium is described at different phases of the menstrual cycle using quantitative real time-PCR and Western blot techniques. Their effects on embryo implantation were examined using JAr spheroid attachment assays and in vivo mouse model. We examined their effects on endometrial receptivity factors, nuclear factor-κB (NF-κB) activity using quantitative real time-PCR, Western blot, and EMSA analyses. Changes in electrophysiological properties and cytosolic free Ca(2+) were measured in endometrial cells with or without specific BK(Ca) blocker or transfected with BK(Ca) small interfering RNA using patch-clamp and fluorescence analyses, respectively. RESULTS BK(Ca) channels are expressed in human endometrial cells in a phase-related fashion during the menstrual cycle (proliferative, 0.20 ± 0.02, vs. mid-secretory, 0.72 ± 0.07; P < 0.01). Blocking BK(Ca) channel function or knockdown of endogenous BK(Ca) channel expression not only decreased JAr spheroid attachment rate and embryo implantation rate in mice but also significantly reduced the expression levels of endometrial receptive factors, including leukemia inhibitory factor, integrin β3, claudin-4, and DKK-1, in human endometrial cells. Blocking BK(Ca) channels also reduced BK(Ca)-regulated NF-κB activity, cytosolic Ca(2+) concentrations, and membrane potentials in human endometrial cells. CONCLUSIONS These observations demonstrate that BK(Ca) channels: 1) are expressed in endometrial cells; 2) affect embryo implantation by mediating endometrial receptive factors; and 3) alter the activity of NF-κB and homeostasis of Ca(2+) in the human endometrial cells.

Preliminary proteomic analysis on the alterations in follicular fluid proteins from women undergoing natural cycles or controlled ovarian hyperstimulation.

PurposeTo study the differences in protein expression profiles of follicular fluid (FF) between controlled ovarian hyperstimulation (COH) and natural ovulatory cycles.MethodsTwelve infertile women undergoing in vitro fertilization and embryo transfer (IVF–ET), with matched clinical information, were retrospectively recruited in the IVF center of our university hospital, including six undergoing COH and another six with natural cycles. FF was sampled from dominant follicles with mature oocytes. Protein expression profiles in each FF sample were analyzed respectively using two-dimensional gel electrophoresis. Differentially expressed proteins were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and validated by western blotting. Differentially expressed proteins were further analyzed using Ingenuity Pathway Analysis (IPA) software.ResultsTwo proteins were downregulated and 11 proteins were upregulated (change ≥1.5-fold, P < 0.05) in the COH group. We identified one down-egulated and seven upregulated proteins using MALDI-TOF MS. Four differentially expressed proteins, including transferrin, complement component C3 (C3), haptoglobin and alpha-1-antitrypsin (AAT), were further validated by rate nephelometry and western blotting analyses. The IPA analysis revealed a significant network involved in the humoral immune and inflammatory responses.ConclusionsThe eight differentially expressed proteins were related to immune and inflammatory responses in the ovary. Our results provide new insights into the influence of COH on follicular (spp) development and IVF outcomes.

Physiology of Embryonic Development

Human embryo development is a complex process. The life of an embryo begins when a male’s spermatozoa makes contact with a woman’s egg. A zygote cell, the very first representation of the fetus, is the result of this fertilization process. Contained within this one cell is the DNA of both the male and female, as well as the blueprint from which the fetus will develop. This chapter reviews some of the basic physiology of embryonic development.

Impact of Galectin-1 on Trophoblast Stem Cell Differentiation and Invasion in In Vitro Implantation Model

Trophoblast stem cells (TSCs) differentiate in an orderly manner, which plays an important role in the process of embryo implantation, placentation, and early pregnancy maintenance. At the maternal–fetal interface, the dialogue is crucial between trophoblast cells and endometrial epithelial cells. Previous studies suggested that galectin-1 (Gal-1) may play an important role in placental development. In this study, we used Ishikawa (IK) cells–TSC coculture model to simulate the maternal–fetal interface and induce the differentiation of TSCs by differentiation media. The messenger RNA level of each cell type markers, fusion markers, and Gal-1 was detected by quantitative reverse transcription polymerase chain reaction during the differentiation of TSCs. Wound healing and transwell invasion assays were used to detect the migration and invasion ability in each group. We found that coculture with IK cells or conditioned media from IK cells could promote the differentiation and invasion of TSCs and increase Gal-1 expression in TSCs. Furthermore, recombinant Gal-1 could also promote the differentiation and invasion of TSCs, suggesting that some of IK cells secretion increase the expression of Gal-1 in TSCs during implantation, which then induced trophoblast differentiation and invasion in vitro. These findings provide significant insights into the biology of embryo–maternal interactions with the importance of Gal-1 in TSCs for the successful establishment and maintenance of pregnancy.