Yugui Cui

Altered global gene expressions of human placentae subjected to assisted reproductive technology treatments

BACKGROUND Researchers are more and more concerning the safety of fetus or offspring derived from assisted reproductive technology (ART) treatment. As the placenta is a critical organ that sustains and protects the fetus, we hypothesize that altered global gene expression of the placenta subjected to ART manipulation may reflect changes associated with ART procedures and subsequently causal related to offspring health. METHODS Three term placenta samples were obtained from patients undergone in vitro fertilization and embryo transfer due to oviductal factors only. Other three control placentae were from those underwent normal pregnancy. A GeneChip Affymetrix HG-U133 Plus 2.0 Array was utilized to analyze the genes. Using qRT-PCR we certified microarray data from 10 dysregulated genes. Five genes were localized precisely in the placenta as per immunohistochemistry. RESULTS Twenty-six differentially expressed genes were identified in the ART-treated placentae: 17 up-regulated; 9 down-regulated. Eighteen of these were classified into six groups according to critical placental function: immune response; transmembrane transport; metabolism; oxidative stress; cell differentiation; and other functions. Genes involved in immune response, such as ERAP2 and STAT4, and those regulating cell differentiations, such as MUC1, were discerned to be differentially expressed. These gene products were expressed in the placental villus tissues, either in the cytoplasm or in the membrane of syncytiotrophoblastic cells. CONCLUSION To our knowledge, this is the first study in comparing differentially expressed genes in placentae from patients undergone ART treatment vs. those underwent normal pregnancy. Abnormal profiles of critical placental functioning genes, such as ERAP2, STAT4 and MUC1, may be valuable biomarkers to understand how the placenta affects fetal development and ART-derived offsprings health problems.

Differential expression of microRNAs in omental adipose tissue from gestational diabetes mellitus subjects reveals miR-222 as a regulator of ERα expression in estrogen-induced insulin resistance.

Omental adipose tissue plays a central role in insulin resistance in gestational diabetes mellitus (GDM), and the molecular mechanisms leading to GDM remains vague. Evidence demonstrates that maternal hormones, such as estradiol, contribute to insulin resistance in GDM. In this study we determined the differential expression patterns of microRNAs (miRNAs) in omental adipose tissues from GDM patients and pregnant women with normal glucose tolerance using AFFX miRNA expression chips. MiR-222, 1 of 17 identified differentially expressed miRNAs, was found to be significantly up-regulated in GDM by quantitative real-time PCR (P < .01), and its expression was closely related with serum estradiol level (P < .05). Furthermore, miR-222 expression was significantly increased in 3T3-L1 adipocytes with a high concentration of 17β-estradiol stimulation (P < .01), whereas the expressions of estrogen receptor (ER)-α protein and insulin-sensitive membrane transporter glucose transporter 4 (GLUT4) protein (P < .01) were markedly reduced. In addition, ERα was shown to be a direct target of miR-222 in 3T3-L1 adipocytes by using the luciferase assay. Finally, antisense oligonucleotides of miR-222 transfection was used to silence miR-222 in 3T3-L1 adipocytes. The results showed that the expressions of ERα and GLUT4, the insulin-stimulated translocation of GLUT4 from the cytoplasm to the cell membrane and glucose uptake in mature adipocytes were dramatically increased (P < .01). In conclusion, miR-222 is a potential regulator of ERα expression in estrogen-induced insulin resistance in GDM and might be a candidate biomarker and therapeutic target for GDM.

Characterization of serum microRNAs profile of PCOS and identification of novel non-invasive biomarkers.

Background: Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women of reproductive age, is characterized by polycystic ovaries, chronic anovulation, hyperandrogenism and insulin resistance. Despite the high prevalence of hyperandrogenemia, a definitive endocrine marker for PCOS has so far not been identified. Circulating miRNAs have recently been shown to serve as diagnostic/prognostic biomarkers in patients with cancers. Our current study focused on the altered expression of serum miRNAs and their correlation with PCOS. Method and Results: We systematically used the TaqMan Low Density Array followed by individual quantitative reverse transcription polymerase chain reaction assays to identify and validate the expression of serum miRNAs of PCOS patients. The expression levels of three miRNAs (miR-222, miR-146a and miR-30c) were significantly increased in PCOS patients with respect to the controls in our discovery evaluation and followed validation. The area under the receiver operating characteristic (ROC) curve (AUC) is 0.799, 0.706, and 0.688, respectively. The combination of the three miRNAs using multiple logistic regression analysis showed a larger AUC (0.852) that was more efficient for the diagnosis of PCOS. In addition, logistic binary regression analyses show miR-222 is positively associated with serum insulin, while miR-146a is negatively associated with serum testosterone. Furthermore, bioinformatics analysis indicated that the predicted targets function of the three miRNAs mainly involved in the metastasis, cell cycle, apoptosis and endocrine. Conclusion: Serum miRNAs are differentially expressed between PCOS patients and controls. We identified and validated a class of three serum miRNAs that could act as novel non-invasive biomarkers for diagnosis of PCOS. These miRNAs may be involved in the pathogenesis of PCOS.

Prenatal androgen excess programs metabolic derangements in pubertal female rats.

Owing to the heterogeneity in the clinical symptoms of polycystic ovary syndrome (PCOS), the early pathophysiological mechanisms of PCOS remain unclear. Clinical, experimental, and genetic evidence supports an interaction between genetic susceptibility and the influence of maternal environment in the pathogenesis of PCOS. To determine whether prenatal androgen exposure induced PCOS-related metabolic derangements during pubertal development, we administrated 5α-dihydrotestosterone (DHT) in pregnant rats and observed their female offspring from postnatal 4 to 8 weeks. The prenatally androgenized (PNA) rats exhibited more numerous total follicles, cystic follicles, and atretic follicles than the controls. Fasting glucose, insulin, leptin levels, and homeostatic model assessment for insulin resistance were elevated in the PNA rats at the age of 5-8 weeks. Following intraperitoneal glucose tolerance tests, glucose and insulin levels did not differ between two groups; however, the PNA rats showed significantly higher 30- and 60-min glucose levels than the controls after insulin stimulation during 5-8 weeks. In addition, prenatal DHT treatment significantly decreased insulin-stimulated phosphorylation of AKT in the skeletal muscles of 6-week-old PNA rats. The abundance of IR substrate 1 (IRS1) and IRS2 was decreased in the skeletal muscles and liver after stimulation with insulin in the PNA group, whereas phosphorylation of insulin-signaling proteins was unaltered in the adipose tissue. These findings validate the contribution of prenatal androgen excess to metabolic derangements in pubertal female rats, and the impaired insulin signaling through IRS and AKT may result in the peripheral insulin resistance during pubertal development.

Investigation of human testis protein heterogeneity using 2-dimensional electrophoresis.

The testis is the male gonad responsible for spermatogenesis and male hormone secretion. The complicated processes of spermatogenesis and steroidogenesis determine the complexity of protein expression control in the testis. In this study, the heterogeneity of human testis proteins was investigated using 2-dimensional gel electrophoresis. A total of 847 protein spots corresponding to 462 unique proteins were identified successfully by mass spectrometry. Notable heterogeneity was evidenced by the presence of more than 1 spot with different molecular weight and/or Isoelectric point values for each of 180 different proteins. Analysis of the detected peptides of these proteins indicated that this heterogeneity was partly the result of alternative splicing and/or proteolysis. SP_PIR_Keywords analysis suggested that alternative initiation sites and various forms of posttranslational modification may also contribute toward this heterogeneity. Using Pro-Q Diamond phosphostain, 68 spots representing 52 proteins were stained, confirming the presence of phosphorylated forms of these proteins in the human testis. These data were used to establish a proteome reference database, which can be accessed over the Internet (http://reprod.njmu.edu.cn/2d). This database provides an initial reference map of the human testis and serves as a useful resource for comparative proteomics studies of the human testis under normal and pathological states. The abundant protein heterogeneity observed in this study and further investigation of its biological significance will contribute toward understanding protein expression regulation in the human testis and will generate insight into the molecular mechanism of spermatogenesis.

Proteomic analysis of testis biopsies in men treated with transient scrotal hyperthermia reveals the potential targets for contraceptive development.

Mild testicular heating safely and reversibly suppresses spermatogenesis. In this study, we attempted to clarify the underlying molecular mechanism(s) involved in heat‐induced spermatogenesis suppression in human testis. We conducted global proteomic analyses of human testicular biopsies before, and at 2 and 9 wk after heat treatment. Thirty‐one and Twenty‐six known proteins were identified with significant differential expression at 2 and 9 wk after heat treatment, respectively. These were used to characterize the cellular and molecular events in the testes when seminiferous epithelia became damaged (2 wk) and recovered (9 wk). At 2 wk post‐treatment, the changed expression of a series of proteins could promote apoptosis or suppress proliferation and cell survival. At 9 wk post‐treatment, the changed expression of proteins mainly promoted cell proliferation, differentiation and survival, but resisted cell apoptosis. Among those heat‐regulated proteins, HNRNPH1 was selected for the further functional study. We found that HNRNPH1 was an anti‐apoptosis protein that could regulate the expression of other heat‐induced proteins. In conclusion, heat‐induced reversible suppression of spermatogenesis occurred by modulating the expression of proteins related to proliferation, differentiation, apoptosis and cell survival pathways. These differentially expressed proteins were found to be key molecular targets affecting spermatogenesis after heat treatment.

Influence of various tubal surgeries to serum antimullerian hormone level and outcome of the subsequent IVF-ET treatment

Abstract Objective: To compare the influence of various tubal surgeries to ovarian reserve via serum level of antimullerian hormone (AMH) and the subsequent in vitro fertilization and embryo transplantation (IVF-ET) outcome in patients with simple tubal infertility. Study design: A prospective cohort study was conducted on 134 IVF cycles undegone by 26 and 34 cases with bilateral and unilateral salpingectomy, respectively, 23 cases with bilateral oviducts interrupted in the proximal and 51 cases with bilateral oviducts obstruction without intervention as controls. Results: Serum AMH displayed its great superiority to traditional markers of ovarian reserve in correspondence with antral follicles count and decisive effect for the number of oocytes retrieved after stimulation in each group. No significant differences on ovarian reserve and responsiveness or IVF-ET outcome existed among four groups comparable on essential characteristics, except for numerically higher clinical pregnancy rate and live birth rate after various tubal surgeries versus no intervention for bilateral oviducts obstruction. Especially, bilateral salpingectomy precursed the statistically highest implantation rate (51.0% versus 28.0%, 39.1%, 30.4%) and numerically best IVF outcome. Conclusion: Tubal surgical procedures have some beneficial effect for improving IVF outcome without significant impact on ovarian reserve or responsiveness. Bilateral salpingectomy appears to be an appropriate procedure before IVF treatment for bilateral salpingitis, especially hydrosalpinx.

Effect of estradiol on proliferation and differentiation of side population stem/progenitor cells from murine endometrium

BackgroundIn our previous study, endometrium side population cells (SP cells) were isolated from postpartum murine uterus, and characterized by a heterogeneous population of stem/progenitor cells. In this study, we investigated the effect of estrogen on the proliferation and differentiation of SP cells.MethodsSP and non-SP cells of postpartum murine endometrium were isolated by DNA dye Hoechst 33342. The expression of estrogen receptor 1 (ESR1) was measured by reverse transcription polymerase chain reaction (RT-PCR), Real-time PCR, Western blot, immunofluorescence and immunohistochemistry. The proliferation and differentiation of SP cells treated with different concentrations [10(-8) M-10(-6) M] of estradiol (E2) and E2+ ICI182780 (Faslodex, inhibitor of ESR1) were measured by 3-(4, 5-dimethylthiazoly1-2)-2,5-diphenyltetrazolium bromide(MTT) and clonogenic assays.Results(1) SP cells expressed ESR1 at a higher level than non-SP cells. (2) The level of E2 in the serum and the expression of ESR1 in the uterus of postpartum murine changed in the same manner with the ratio of SP cells to total uterus cells at a different postpartum time point. ESR1, as ABCG2 is also predominantly located in the stroma and the glandular epithelium of the uterus. (3) 10(-6) M E2 notably promoted the proliferation of SP cells after treatment for 24 h. This effect could be inhibited by ICI182780. E2 at the concentration of 10(-7) M or 10(-8) M was sent to impair the large cloning efficiency (CE) of SP cells.ConclusionsThe effect of estrogen on the proliferation and differentiation of endometrium SP cells via ESR1 was observed and it was in a concentration dependent fashion. Clearly, more work is needed to understand the in vivo effect of E2 at the physiological concentration on the differentiation of SP cells.

Isolation and Characterization of Side Population Cells in the Postpartum Murine Endometrium

Endometrium is a highly active organ that is periodically remodeled during the life span. Previous studies have indicated the presence of an adult stem or progenitor cell population in this tissue. In this study, side population (SP) cells were isolated from the endometrium of postpartum murine uterus but not from the endometrium of a uterus undergoing a normal estrus cycle. Phenotype analysis showed that SP cells were negative for hematopoietic, endothelial, and mesenchymal stem cell (MSC) markers, but they expressed stem cell antigen 1 (Sca-1) and c-kit at various degrees. Side population cell is a heterogeneous population of endometrial stem/progenitor cells that have colony-forming capacity. They were found to reside in quiescence in the stroma but not in the luminal epithelium. These data suggest that, like other tissues and organs, the murine endometrium also contains SP cells. Their specific role in the regeneration of the endometrium warrants further study.

The association between polycystic ovary syndrome and ectopic pregnancy after in vitro fertilization and embryo transfer

OBJECTIVE We sought to assess the association between polycystic ovary syndrome (PCOS) and ectopic pregnancy after in vitro fertilization-embryo transfer (ET). STUDY DESIGN In this retrospective cohort study, we included 5339 women who had clinical pregnancies after in vitro fertilization treatment (PCOS, 205 women; non-PCOS, 5134 women) at Nanjing Medical University (China) between 2007 and 2011. Fresh and cryo-thawed ET cycles were analyzed respectively. The primary outcome measure was the occurrence of ectopic pregnancy. Multivariate logistic regression analysis was used to adjust for important confounders. RESULTS In fresh ET cycles of women who were undergoing controlled ovarian hyperstimulation (COH; n = 3303), women with PCOS had 3.06 times higher risk of ectopic pregnancy compared with those without PCOS (7.0% vs 2.4%; adjusted odds ratio [aOR], 3.06; 95% confidence interval [CI], 1.34-6.96). In the stratified analysis, for women without PCOS, the high estradiol group (>4085 pg/mL) had higher ectopic pregnancy rates compared with the low estradiol group (≤4085 pg/mL; 3.4% vs 2.0%; aOR, 1.99; 95% CI, 1.19-3.35); however, for women with PCOS, both high and low estradiol groups had high ectopic pregnancy rates (5.6% vs 7.7%; aOR, 0.92; 95% CI, 0.15-5.67). In cryo-thawed ET cycles without COH (n = 2036), the ectopic rates between women with and without PCOS were similar (2.2% vs 2.0%; aOR, 0.94; 95% CI, 0.22-4.07). CONCLUSION PCOS was associated with an increased risk of ectopic pregnancy after COH in fresh ET cycles, but not in cryo-thawed ET cycles. A possible explanation is that, compared with women without PCOS, women with PCOS appear to hold a lower threshold of hyperphysiologic estradiol level that triggers the occurrence of ectopic pregnancy after COH.