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      RNA-Seq-Based Transcriptome Analysis of Changes in Gene Expression Linked to Human Pregnancy Outcome After In Vitro Fertilization–Embryo Transfer

      1 , 2 , 3 , 3 , 3 , 3 , 3 , 1
      Reproductive Sciences
      SAGE Publications

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          Abstract

          To promote the pregnancy rate of in vitro fertilization-embryo transfer (IVF-ET), we proposed to examine the differentially expressed gene during pregnancy success and failure. We used high-sequencing technology to characterize and compare the gene expression profiles of pregnancy success and pregnancy failure patients during 3 different stages: before IVF-ET (stage I), after ovarian stimulation (stage II), and day 15 after embryo transfer (stage III). Selected data from RNA-sequencing experiments were validated by quantitative real-time polymerase chain reaction (qRT-PCR). A total of 282, 208, and 372 genes in stages I, II, and III, respectively, were differentially expressed between pregnancy success and pregnancy failure, respectively. Through confirmation with qRT-PCR, compared to pregnancy failure, we demonstrated much lower level of major histocompatibility complex, human leukocyte antigen class I A, and an much higher level of human leukocyte antigen, class II DQ α1 (HLA-DQA1) in pregnancy success, although the HLA-DQA1 decreased with development duration of pregnancy. Interleukin 1β increased with the development duration of pregnancy in pregnancy failure group and was much higher than that in pregnancy success group. Hemoglobin δ decreased with the development duration of pregnancy in pregnancy failure women and maintained in a lower level in stage I and II but dramatically increased to a much higher level in stage III in pregnancy success women. Minichromosome maintenance complex component 4 significantly increased in stage III in pregnancy failure but not in pregnancy success women. The altered expression of genes implicated in immune response and inflammation, oocyte meiosis, rhythmic process, and so on. Therefore, the current results provide a strong basis for future research to expound the molecular mechanism coping with pregnancy outcome.

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          Most cited references28

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          Next-generation transcriptome assembly.

          Transcriptomics studies often rely on partial reference transcriptomes that fail to capture the full catalogue of transcripts and their variations. Recent advances in sequencing technologies and assembly algorithms have facilitated the reconstruction of the entire transcriptome by deep RNA sequencing (RNA-seq), even without a reference genome. However, transcriptome assembly from billions of RNA-seq reads, which are often very short, poses a significant informatics challenge. This Review summarizes the recent developments in transcriptome assembly approaches - reference-based, de novo and combined strategies - along with some perspectives on transcriptome assembly in the near future.
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            Cytokines, chemokines and growth factors in endometrium related to implantation.

            The complexity of the events of embryo implantation and placentation is exemplified by the number and range of cytokines with demonstrated roles in these processes. Disturbance of the normal expression or action of these cytokines results in complete or partial failure of implantation and abnormal placental formation in mice or humans. Of known importance are members of the gp130 family such as interleukin-11 (IL-11) and leukaemia inhibitory factor (LIF), the transforming growth factor beta (TGFbeta) superfamily including the activins, the colony-stimulating factors (CSF), the IL-1 system and IL-15 system. New data are also emerging for roles for a number of chemokines (chemoattractive cytokines) both in recruiting specific cohorts of leukocytes to implantation sites and in trophoblast differentiation and trafficking. This review focuses on those cytokines and chemokines whose expression pattern in the human endometrium is consistent with a potential role in implantation and placentation and for which some relevant actions are known. It examines what is known of their regulation and action along with alterations in clinically relevant situations.
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              Next-generation Sequencing of Advanced Prostate Cancer Treated with Androgen-deprivation Therapy

              1 Introduction At diagnosis, many men have “incurable” locally advanced or metastatic prostate cancer (PCa), the most common cancer in Europe [1]. PCa progression is initially driven by androgens acting via the cognate androgen receptor (AR) transcription factor. The initial treatment standard for patients with locally advanced or metastatic PCa is androgen-deprivation therapy (ADT), which inactivates the AR for a period of time. After approximately 2–3 yr, these patients can develop castration-resistant PCa (CRPC), for which the prognosis is poor despite newer second-line cytotoxic chemotherapy and endocrine therapies [2–4]. There is an urgent, unmet need for novel therapies for CRPC led by a better understanding of the biology underlying treatment resistance. The mechanisms underlying CRPC are unclear and may be the result of cellular adaptation to or clonal selection by ADT [5]. AR signalling pathways and transcriptional activity may be reactivated [6], or cell growth may be supported by AR-independent outlaw cell signalling pathways [7]. Hence, a greater understanding of ADT-driven molecular changes may yield information on the mechanisms underlying progression to CRPC. Although previously published transcriptome-wide studies have successfully identified ADT-driven transcriptional events [8,9], these analyses have been limited by the inherent bias associated with microarrays [10]. In this study, we undertake quantitative transcriptome profiling of prostate tumours from patients prior to and following ADT using next-generation sequencing (RNA-seq) to identify functionally important novel androgen-regulated pathways and specific gene products that may be reactivated in CRPC as potential targets for therapy. 2 Materials and methods 2.1 Patient samples for RNA sequencing Clinical samples for RNA-seq were prospectively collected as part of the GenTax study [11]. Illumina RNA-seq was performed with complementary DNA sample library normalisation using the Illumina duplex-specific nuclease protocol prior to cluster generation and library sequencing on the HiSeq 2000 sequencer (Illumina, San Diego, CA, USA) with a paired-end sequencing strategy. Further details are given in the Supplement. 2.2 Functional assays All cells were grown at 37 °C in 5% carbon dioxide. LNCaP (CRL-1740, ATCC) cells were maintained in RPMI-1640 medium (Life Technologies, Carlsbad, CA, USA; 31870-025) with 20 mM L-glutamine (Life Technologies, 25030-024) supplemented with 10% foetal bovine serum (PAA Laboratories, Yeovil Somerset, UK; A15-101). LNCaP-AI cells were derived from LNCaP parental cells and maintained as previously described [12]. Proliferation assays were carried out using the WST-1 reagent (Roche Diagnostics, Indianapolis, IN, USA; 05015944001) as per the manufacturer's instructions in medium containing either 10 μm XAV939 (Novartis Pharmaceuticals, Plantation, FL, USA) in 0.1% dimethyl sulfoxide (DMSO) [13] or vehicle. Cell cycle analysis was performed following treatment with 10 μm XAV939 in 0.1% DMSO or vehicle, as previously described [14]. Further details are given in the Supplement. 3 Results 3.1 The transcriptional landscape of androgen-deprivation therapy in clinical prostate cancer RNA-seq was performed on 16 paired pre- and post-ADT samples from eight patients with locally advanced or metastatic PCa (Gleason score >7 [15]; Table 1). The post-ADT sample from patient 8 performed markedly worse on multiple quality control measures, and so both samples from this patient were excluded from further analysis (Supplemental Table 1). Recently, genomic rearrangements rendering ETS-family transcription factors under the control of androgen-responsive or other promoters have been hypothesised as a mechanism driving prostate carcinogenesis [16]. The TMPRSS2/ERG translocation yields the most common PCa-associated gene fusion product, reported in >50% cases [16]. Consistent with this, three of the seven (43%) patients expressed transcripts with sequences corresponding to this fusion event in the pre-ADT samples alone. We observed, on average, a sixfold downregulation of ERG expression following ADT, but expression of TMPRSS2/ERG did not correlate with time to biochemical relapse (data not shown). We also identified 12 additional candidate fusion products, 9 of which were only detectable in pre-ADT samples (Supplemental Table 2). We identified a total of 774 genes upregulated at least twofold (false discovery rate [FDR]  0.05) and only small (approximately 3–5%) differences in the proportion of cells in the S and G2/M phases. However, there was a 17% increase in the proportion of LNCaP-AI cells in the G0/G1 phase (p < 0.05) and a 10% fall in the proportion of cells in the S phase (p < 0.05). These data suggest that, in LNCaP-AI cells, XAV939 treatment specifically causes an accumulation of cells in the G0/1 phase and a reduction of cells in the S and G2/M phases, thereby delaying cell cycle progression. Taken together, these data suggest that the LNCaP-AI subline is particularly dependent on the Wnt/β-catenin signalling pathway for cell growth. 4 Discussion We report the first quantitative transcriptome profiling of clinical PCa from patients prior to and following ADT by using RNA-seq and have substantially enlarged the ADT-regulated gene set, as is expected from a more sensitive sequencing study [29–31]. The substantially better overlap between our and the two previously published microarray gene sets [8,9] contrasts with the poor individual overlap of the gene sets between these two studies, which may be the result of a lack of power of these studies to identify differentially regulated genes. Specifically, we identified several novel pathways perturbed by ADT, including the Wnt/β-catenin signalling pathway. Although activation of Wnt/β-catenin signalling in carcinogenesis is well known in breast and colorectal cancers [32], evidence for this pathway in clinical PCa has been conflicting. Recent exome-sequencing studies have identified mutations with genes encoding components of the Wnt/β-catenin signalling pathway [24,25], but there is no clear consensus on the significance of increased nuclear β-catenin expression in primary PCa based on immunohistochemistry [20]. The current focus of novel therapies for CRPC is the targeting of renewed AR signalling [2,3,19]. In CRPC, an inverse correlation has been observed between β-catenin nuclear localisation and AR expression [33], yet others report no statistically significant difference in expression following progression from hormone-naïve disease to CRPC [34]. We demonstrate overexpression of β-catenin in a subset of CRPC and correlation with expression of AR consistent with AR reactivation. Functional or physical interactions between AR and Wnt/β-catenin signalling have been reported in vitro, with β-catenin functioning as an AR coactivator [20,32] to drive ligand-independent cell growth. Despite conflicting results from immunohistochemistry experiments in clinical PCa, which may be caused by experimental variability [34], the above reports together with our own observations suggest that Wnt/β-catenin signalling may be active in CRPC. In keeping with this hypothesis, expression of putative inhibitors of Wnt/β-catenin signalling and downstream transcription factors were downregulated following progression to CRPC in an LNCaP xenograft model [35]. It is thought that these expression changes may lead to an increase cytoplasmic pool of β-catenin, allowing potential interaction with unliganded AR and other transcription factors. We observed a potent inhibitory effect of XAV939 treatment on LNCaP-AI cell growth, which appeared to be caused by an accumulation of cells in the G0/1 phase of the cell cycle (Fig. 3), suggesting a functional role for Wnt/β-catenin signalling in CRPC. The molecular mechanisms underlying our observations remain unclear, and an exhaustive investigation thereof was outside the scope of this proof-of-principle study. Despite using a well-validated inhibitor of Wnt/β-catenin signalling, a potential limitation of this approach is off-target effects. We identified ADT-driven changes in components of cell signalling pathways, including the Wnt/β-catenin signalling pathway, approximately 22 wk following ADT initiation. Although our patients had not yet developed CRPC at the time of the second biopsy, some did develop early biochemical relapse. A caveat of our approach is the targeted biopsy regime, which, although it yields tumour-rich tissue, could have included a heterogeneous population of epithelial and stromal cells. However, a surprising observation was that our KEGG analysis identified upregulation of expression of cancer gene–related pathways, which would have been expected to be downregulated if stromal expression signatures were overtly expressed. 5 Conclusions Our observations suggest that CRPC may represent repopulation of a tumour with androgen-independent clones reliant on Wnt/β-catenin signalling activity after sustained ADT. In light of recent data suggesting that Wnt ligand expression by the tumour microenvironment may attenuate cytotoxic chemotherapy and confer treatment resistance [36], Wnt/β-catenin signalling may represent a potential target for therapy in PCa. Further mechanistic insights using genetic approaches such as in vitro RNA interference and genetically engineered autochthonous murine cancer models will determine whether this pathway is a potential therapeutic target for CRPC. 

 Author contributions: Hing Y. Leung and Prabhakar Rajan had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

 Study concept and design: Leung, Pedley, Rajan. Acquisition of data: Rajan, Sudbery, Villasevil, Mui, Ahmad, Edwards. Analysis and interpretation of data: Rajan, Sudbery, Edwards, Leung. Drafting of the manuscript: Rajan, Sudbery, Leung. Critical revision of the manuscript for important intellectual content: Sansom, Sims, Ponting, Heger, McMenemin, Pedley. Statistical analysis: Rajan, Sudbery, Edwards, Sims, Heger. Obtaining funding: Rajan, Ponting, Pedley, Leung. Administrative, technical, or material support: Villasevil, Mui, Fleming, Davis, Ahmad, Edwards, Sansom, Sims, Ponting, Heger, McMenemin, Pedley. Supervision: Sims, Ponting, Heger, Leung.

 Financial disclosures: Hing Y. Leung and Prabhakar Rajan certify that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: None.

 Funding/Support and role of the sponsor: The study was supported by grants from the Sanofi-Aventis, NHS Greater Glasgow and Clyde Endowments, Medical Research Council, Cancer Research UK, and Royal College of Surgeons of England, but these bodies did not have any involvement in the analysis, preparation of the manuscript, or decision regarding publication.

 Acknowledgement statement: We are grateful to the patients recruited to GenTax, without whom this work would not have been possible, and staff at the Department of Urology and the Northern Centre for Cancer Care, Newcastle upon Tyne Hospitals NHS Foundation Trust for help with patient recruitment and clinical care. We thank Colin Nixon and David Huels (CR-UK Beatson Institute) for technical assistance and advice, respectively, and Rachana Patel (CR-UK Beatson Institute) and Jacqueline Stockley (University of Glasgow) for critical appraisal of the manuscript.
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                Author and article information

                Journal
                Reproductive Sciences
                Reprod Sci
                SAGE Publications
                1933-7191
                1933-7205
                December 14 2015
                January 2016
                August 06 2015
                January 2016
                : 23
                : 1
                : 134-145
                Affiliations
                [1 ]Department of Obstetrics and Gynecology, Shenzhen Maternal and Child Health Care Hospital, Affiliated to Southern Medical University, Shenzhen, People’s Republic of China
                [2 ]Graduate School, Southern Medical University, Guangzhou, People’s Republic of China
                [3 ]Department of Reproductive Medicine, Affiliated hospital of Dali University, Dali, People’s Republic of China
                Article
                10.1177/1933719115597766
                26249324
                700a6eb6-dfbb-4147-a5f6-f06b661b0d61
                © 2016

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