Cross-species meta-analysis of transcriptome changes during the morula to blastocyst transition: metabolic and physiological changes take center stage

2021 ◽  
Author(s):  
Peter Z. Schall ◽  
Keith E. Latham
Keyword(s):  
Inner Cell Mass ◽  
Cholesterol Biosynthesis ◽  
Meta Analysis ◽  
Cell Mass ◽  
Tca Cycle ◽  
Individual Species ◽  
Sequencing Data ◽  
Model Species ◽  
Ampk Signaling ◽  
Estrogen Receptor Signaling

The morula to blastocyst transition (MBT) culminates with formation of inner cell mass (ICM) and trophectoderm (TE) lineages. Recent studies identified signaling pathways driving lineage specification, but some features of these pathways display significant species divergence. To better understand evolutionary conservation of the MBT, we completed a meta-analysis of RNA sequencing data from five model species and ICM-TE differences from four species. While many genes change in expression during the MBT within any given species, the number of shared DEGs is comparatively small, and the number of shared ICM-TE DEGs is even smaller. DEGs related to known lineage determining pathways (e.g., POU5F1) are seen, but the most prominent pathways and functions associated with shared DEGs or shared across individual species DEG lists impact basic physiological and metabolic activities, such as TCA cycle, unfolded protein response, oxidative phosphorylation, sirtuin signaling, mitotic roles of polo-like kinases, NRF2-mediated oxidative stress, estrogen receptor signaling, apoptosis, necrosis, lipid and fatty acid metabolism, cholesterol biosynthesis, endocytosis, AMPK signaling, homeostasis, transcription, and cell death. We also observed prominent differences in transcriptome regulation between ungulates and non-ungulates, particularly for ICM- and TE-enhanced mRNAs. These results extend our understanding of shared mechanisms of the MBT and ICM/TE formation and should better inform the selection of model species for particular applications.

P–530 The use of wide thresholds for detecting intermediate chromosomal CNV up to 80% doesn’t improve PGT-A ability to discriminate true mosaic from uniformly aneuploid embryos

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
L Girardi ◽  
M Serdaroğulları ◽  
C Patassini ◽  
S Caroselli ◽  
M Costa ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
False Negative ◽  
Cell Mass ◽  
Categorical Variables ◽  
P Value ◽  
Sequencing Data ◽  
Preimplantation Genetic Testing ◽  
Exact Test ◽  
Institutional Review ◽  
Wide Range

Abstract Study question What is the effect of varying diagnostic thresholds on the accuracy of Next Generation Sequencing (NGS)-based preimplantation genetic testing for aneuploidies (PGT-A)? Summary answer When single trophectoderm biopsies are tested, the employment of 80% upper threshold increases mosaic calls and false negative aneuploidy results compared to more stringent thresholds. What is known already Trophectoderm (TE) biopsy coupled with NGS-based PGT-A technologies are able to accurately predict Inner Cell Mass’ (ICM) constitution when uniform whole chromosome aneuploidies are considered. However, minor technical and biological inconsistencies in NGS procedures and biopsy specimens can result in subtle variability in analytical results. In this context, the stringency of thresholds employed for diagnostic calls can lead to incorrect classification of uniformly aneuploid embryos into the mosaic category, ultimately affecting PGT-A accuracy. In this study, we evaluated the diagnostic predictivity of different aneuploidy classification criteria by employing blinded analysis of chromosome copy number values (CNV) in multifocal blastocyst biopsies. Study design, size, duration The accuracy of different aneuploidy diagnostic cut-offs was assessed comparing chromosomal CNV in intra-blastocysts multifocal biopsies. Enrolled embryos were donated for research between June and September 2020. The Institutional Review Board at the Near East University approved the study (project: YDU/20l9/70–849). Embryos diagnosed with uniform chromosomal alterations (single or multiple) in their clinical TE biopsy (n = 27) were disaggregated into 5 portions: the ICM and 4 TE biopsies. Overall, 135 specimens were collected and analysed. Participants/materials, setting, methods Twenty-seven donated blastocysts were warmed and disaggregated in TE biopsies and ICM (n = 135 biopsies). PGT-A analysis was performed using Ion ReproSeq PGS kit and Ion S5 sequencer (ThermoFisher). Sequencing data were blindly analysed with Ion-Reporter software. Intra-blastocyst comparison of raw NGS data was performed employing different thresholds commonly used for aneuploidy classification. CNV for each chromosome were reported as aneuploid according to 70% or 80% thresholds. Categorical variables were compared using Fisher’s exact test. Main results and the role of chance In this study, a total of 50 aneuploid patterns in 27 disaggregated embryos were explored. Single TE biopsy results were considered as true positive when they displayed the same alteration detected in the ICM at levels above the 70% or 80% thresholds. Alternatively, alterations detected in the euploid or mosaic range were considered as false negative aneuploidy results. When the 70% threshold was applied, aneuploidy findings were confirmed in 94.5% of TE biopsies analyzed (n = 189/200; 95%CI=90.37–37.22), while 5.5% showed a mosaic profile (50–70%) but uniformly abnormal ICM. Positive (PPV) and negative predictive value (NPV) per chromosome were 100.0% (n = 189/189; 95%CI=98.07–100.00) and 99.5% (n = 2192/2203; 95%CI=99.11–99.75) respectively. When the upper cut-off was experimentally placed at 80% of abnormal cells, a significant decrease (p-value=0.0097) in the percentage of confirmed aneuploid calls was observed (86.5%; n = 173/200; 95%CI=80.97–90.91), resulting in mosaicism overcalling, especially in the high range (50–80%). Less stringent thresholds led to extremely high PPV (100.0%; n = 173/173; 95%CI=97.89–100.00), while NPV decreased to 98.8% (n = 2192/2219; 95%CI=98.30–99.23). Furthermore, no additional true mosaic patterns were identified with the use of wide range thresholds for aneuploidy classification. Limitations, reasons for caution This approach involved the analysis of aneuploidy CNV thresholds at the embryo level and lacked from genotyping-based confirmation analysis. Moreover, aneuploid embryos with known meiotic partial deletion/duplication were not included. Wider implications of the findings: The use of wide thresholds for detecting intermediate chromosomal CNV up to 80% doesn’t improve PGT-A ability to discriminate true mosaic from uniformly aneuploid embryos, lowering overall diagnostic accuracy. Hence, a proportion of the embryos diagnosed as mosaic using wide calling thresholds may actually be uniformly aneuploid and inadvertently transferred. Trial registration number N/A

scholarly journals Network based meta-analysis prediction of microenvironmental relays involved in stemness of human embryonic stem cells

2014 ◽  
Author(s):  
Virginie Mournetas ◽  
Quentin M. Nunes ◽  
Patricia A. Murray ◽  
Christopher M. Sanderson ◽  
David G. Fernig
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Meta Analysis ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Interaction Network ◽  
Transcriptional Networks

Background. Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of in vitro fertilised blastocysts, which can either be maintained in an undifferentiated state or committed into lineages under determined culture conditions. These cells offer great potential for regenerative medicine, but at present, little is known about the mechanisms that regulate hESC stemness; in particular, the role of cell-cell and cell-extracellular matrix interactions remain relatively unexplored. Methods and results. In this study we have performed an in silico analysis of cell-microenvironment interactions to identify novel proteins that may be responsible for the maintenance of hESC stemness. A hESC transcriptome of 8,934 mRNAs was assembled using a meta-analysis approach combining the analysis of microarrays and the use of databases for annotation. The STRING database was utilised to construct a protein-protein interaction network focused on extracellular and transcription factor components contained within the assembled transcriptome. This interactome was structurally studied and filtered to identify a short list of 92 candidate proteins, which may regulate hESC stemness. Conclusion. We hypothesise that this list of proteins, either connecting extracellular components with transcriptional networks, or with hub or bottleneck properties, may contain proteins likely to be involved in determining stemness.

scholarly journals Network based meta-analysis prediction of microenvironmental relays involved in stemness of human embryonic stem cells

2014 ◽  
Author(s):  
Virginie Mournetas ◽  
Quentin M. Nunes ◽  
Patricia A. Murray ◽  
Christopher M. Sanderson ◽  
David G. Fernig
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Meta Analysis ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Interaction Network ◽  
Transcriptional Networks

Background. Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of in vitro fertilised blastocysts, which can either be maintained in an undifferentiated state or committed into lineages under determined culture conditions. These cells offer great potential for regenerative medicine, but at present, little is known about the mechanisms that regulate hESC stemness; in particular, the role of cell-cell and cell-extracellular matrix interactions remain relatively unexplored. Methods and results. In this study we have performed an in silico analysis of cell-microenvironment interactions to identify novel proteins that may be responsible for the maintenance of hESC stemness. A hESC transcriptome of 8,934 mRNAs was assembled using a meta-analysis approach combining the analysis of microarrays and the use of databases for annotation. The STRING database was utilised to construct a protein-protein interaction network focused on extracellular and transcription factor components contained within the assembled transcriptome. This interactome was structurally studied and filtered to identify a short list of 92 candidate proteins, which may regulate hESC stemness. Conclusion. We hypothesise that this list of proteins, either connecting extracellular components with transcriptional networks, or with hub or bottleneck properties, may contain proteins likely to be involved in determining stemness.

scholarly journals Network based meta-analysis prediction of microenvironmental relays involved in stemness of human embryonic stem cells

2014 ◽  
Author(s):  
Virginie Mournetas ◽  
Quentin M. Nunes ◽  
Patricia A. Murray ◽  
Christopher M. Sanderson ◽  
David G. Fernig
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Meta Analysis ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Interaction Network ◽  
Transcriptional Networks

Background. Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of in vitro fertilised blastocysts, which can either be maintained in an undifferentiated state or committed into lineages under determined culture conditions. These cells offer great potential for regenerative medicine, but at present, little is known about the mechanisms that regulate hESC stemness; in particular, the role of cell-cell and cell-extracellular matrix interactions remain relatively unexplored. Methods and results. In this study we have performed an in silico analysis of cell-microenvironment interactions to identify novel proteins that may be responsible for the maintenance of hESC stemness. A hESC transcriptome of 8,934 mRNAs was assembled using a meta-analysis approach combining the analysis of microarrays and the use of databases for annotation. The STRING database was utilised to construct a protein-protein interaction network focused on extracellular and transcription factor components contained within the assembled transcriptome. This interactome was structurally studied and filtered to identify a short list of 92 candidate proteins, which may regulate hESC stemness. Conclusion. We hypothesise that this list of proteins, either connecting extracellular components with transcriptional networks, or with hub or bottleneck properties, may contain proteins likely to be involved in determining stemness.

scholarly journals Pluripotent stem cells: induction and self-renewal

2018 ◽  
Vol 373 (1750) ◽  
pp. 20170213 ◽  
Author(s):  
R. Abu-Dawud ◽  
N. Graffmann ◽  
S. Ferber ◽  
W. Wruck ◽  
J. Adjaye
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Meta Analysis ◽  
Cell Mass ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Self Renewal

Pluripotent stem cells (PSCs) lie at the heart of modern regenerative medicine due to their properties of unlimited self-renewal in vitro and their ability to differentiate into cell types representative of the three embryonic germ layers—mesoderm, ectoderm and endoderm. The derivation of induced PSCs bypasses ethical concerns associated with the use of human embryonic stem cells and also enables personalized cell-based therapies. To exploit their regenerative potential, it is essential to have a firm understanding of the molecular processes associated with their induction from somatic cells. This understanding serves two purposes: first, to enable efficient, reliable and cost-effective production of excellent quality induced PSCs and, second, to enable the derivation of safe, good manufacturing practice-grade transplantable donor cells. Here, we review the reprogramming process of somatic cells into induced PSCs and associated mechanisms with emphasis on self-renewal, epigenetic control, mitochondrial bioenergetics, sub-states of pluripotency, naive ground state, naive and primed. A meta-analysis identified genes expressed exclusively in the inner cell mass and in the naive but not in the primed pluripotent state. We propose these as additional biomarkers defining naive PSCs. This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.

Use of F9 teratocarcinoma STEM cells to study cell-matrix interactions in the early mouse embryo

1992 ◽  
Vol 50 (1) ◽  
pp. 602-603
Author(s):  
Marc Lenburg ◽  
Rulang Jiang ◽  
Lengya Cheng ◽  
Laura Grabel
Keyword(s):  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Embryoid Bodies ◽  
F9 Cells ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Cell Matrix Interactions ◽  
F9 Teratocarcinoma

We are interested in defining the cell-cell and cell-matrix interactions that help direct the differentiation of extraembryonic endoderm in the peri-implantation mouse embryo. At the blastocyst stage the mouse embryo consists of an outer layer of trophectoderm surrounding the fluid-filled blastocoel cavity and an eccentrically located inner cell mass. On the free surface of the inner cell mass, facing the blastocoel cavity, a layer of primitive endoderm forms. Primitive endoderm then generates two distinct cell types; parietal endoderm (PE) which migrates along the inner surface of the trophectoderm and secretes large amounts of basement membrane components as well as tissue-type plasminogen activator (tPA), and visceral endoderm (VE), a columnar epithelial layer characterized by tight junctions, microvilli, and the synthesis and secretion of α-fetoprotein. As these events occur after implantation, we have turned to the F9 teratocarcinoma system as an in vitro model for examining the differentiation of these cell types. When F9 cells are treated in monolayer with retinoic acid plus cyclic-AMP, they differentiate into PE. In contrast, when F9 cells are treated in suspension with retinoic acid, they form embryoid bodies (EBs) which consist of an outer layer of VE and an inner core of undifferentiated stem cells. In addition, we have established that when VE containing embryoid bodies are plated on a fibronectin coated substrate, PE migrates onto the matrix and this interaction is inhibited by RGDS as well as antibodies directed against the β1 integrin subunit. This transition is accompanied by a significant increase in the level of tPA in the PE cells. Thus, the outgrowth system provides a spatially appropriate model for studying the differentiation and migration of PE from a VE precursor.

Decreased inner cell mass proportion in blastocysts from diabetic rats

Diabetes ◽  
1990 ◽  
Vol 39 (4) ◽  
pp. 471-476 ◽  
Author(s):  
S. Pampfer ◽  
R. de Hertogh ◽  
I. Vanderheyden ◽  
B. Michiels ◽  
M. Vercheval
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Diabetic Rats ◽  
Mass Proportion
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