scholarly journals Non-invasive, label-free optical analysis to detect aneuploidy within the inner cell mass of the preimplantation embryo

2020 ◽  
Author(s):  
Tiffany C. Y. Tan ◽  
Saabah B. Mahbub ◽  
Carl A. Campugan ◽  
Jared M. Campbell ◽  
Abbas Habibalahi ◽  
...  
Keyword(s):  
Hyperspectral Imaging ◽  
Inner Cell Mass ◽  
Human Fibroblasts ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Fibroblast Cells ◽  
Primary Human Fibroblast ◽  
Non Invasive ◽  
Human Fibroblast Cells ◽  
Chimeric Blastocysts

AbstractStudy questionCan label-free, non-invasive optical imaging by hyperspectral microscopy discern between euploid and aneuploid cells within the inner cell mass of the mouse preimplantation embryo?Summary answerHyperspectral microscopy shows a variance in metabolic activity which enables discrimination between euploid and aneuploid cells.What is known alreadyEuploid/aneuploid mosaicism affects up to 17.3% of human blastocyst embryos with trophectoderm biopsy or spent media currently utilised to diagnose aneuploidy and mosaicism in clinical in vitro fertilisation. Based on their design, these approaches will fail to diagnose the presence or proportion of aneuploid cells within the fetal lineage (inner cell mass (ICM)) of some blastocyst embryos.Study design, size, durationThe impact of aneuploidy on cellular metabolism of primary human fibroblast cells and mouse embryos was assessed by a fluorescence microscope adapted for imaging with multiple spectral channels (hyperspectral imaging). Primary human fibroblast cells with known ploidy were subjected to hyperspectral imaging to record native cell fluorescence (euploid n= 467; aneuploid n= 969). For mouse embryos, 50-70 individual euploid and aneuploid blastomeres (8-cell stage embryo) and chimeric blastocysts (40-50 per group: euploid; aneuploid; or 1:1 and 1:3 ratio of euploid:aneuploid) were utilised for hyperspectral imaging.Participants/materials, setting, methodsTwo models were employed: (i) Primary human fibroblasts with known karyotype and (ii) a mouse model of embryo aneuploidy where mouse embryos were treated with reversine, a reversible spindle assembly checkpoint inhibitor, during the 4-to 8-cell division. Individual blastomeres were dissociated from reversine treated (aneuploid) and control (euploid) 8-cell embryos and either imaged directly or used to generate chimeric blastocysts with differing ratios of euploid:aneuploid cells. Individual blastomeres and embryos were subjected to hyperspectral imaging. Changes in cellular metabolism were determined by quantification of metabolic cofactors (inferred from their autofluorescence signature): reduced nicotinamide adenine dinucleotide (NAD(P)H), flavins with the subsequent calculation of the optical redox ratio (ORR: Flavins/[NAD(P)H + Flavins]). Mathematical algorithms were applied to extract features from the autofluorescence signals of each cell/blastomere/inner cell mass to discriminate between euploid and aneuploid.Main results and the role of chanceAn increase in the relative abundance of NAD(P)H with a decrease in flavins led to a significant reduction in the ORR for aneuploid cells in both primary human fibroblasts and individual mouse blastomeres (P < 0.05). Mathematical algorithms were able to achieve good separation between (i) euploid and aneuploid primary human fibroblast cells, (ii) euploid and aneuploid mouse blastomeres cells and (iii) euploid and aneuploid chimeric blastocysts and (iv) 1:1 and 1:3 chimeric blastocysts. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve of 0.85, 0.99, 0.87 and 0.88, respectively. We believe that the role of chance is low as multiple cellular models (human somatic cells and mouse embryos) demonstrated a consistent shift in cellular metabolism in response to aneuploidy as well as the robust capacity of mathematical features to separate euploid and aneuploid cells in a statistically significant manner.Limitations, reasons for cautionThere would be added value in determining the degree of embryo mosaicism by sequencing the inner cell mass (ICM) of individual blastocysts to correlate with metabolic profile and level of discrimination achieved using the mathematical features approach.Wider implications of the findingsHyperspectral imaging was able to discriminate between euploid and aneuploid human fibroblasts and mouse embryos. This may lead to the development of an accurate and non-invasive optical approach to assess mosaicism within the ICM of human embryos in the absence of fluorescent tags.Study funding/competing interest(s)K.R.D. is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58-2019). This study was funded by the Australian Research Council Centre of Excellence for Nanoscale Biophotonics (CEI40100003). The authors declare that there is no conflict of interest.

O-083 Non-invasive, label-free optical analysis to detect aneuploidy within the inner cell mass of the preimplantation embryo

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
C Y Tan ◽  
S B Mahbub ◽  
C A Campugan ◽  
J Campbell ◽  
A Habibalahi ◽  
...  
Keyword(s):  
Hyperspectral Imaging ◽  
Human Fibroblast ◽  
Preimplantation Embryo ◽  
Mouse Embryos ◽  
Treated Mouse ◽  
Fibroblast Cells ◽  
Label Free ◽  
Primary Human Fibroblast ◽  
Non Invasive ◽  
Human Fibroblast Cells

Abstract Study question Can we separate between control and reversine-treated cells within the inner cell mass (ICM) of the mouse preimplantation embryo by using label-free and non-invasive hyperspectral microscopy? Summary answer Hyperspectral microscopy is able to discern between control and reversine-treated cells using cellular autofluorescence in the complete absence of fluorescence tags. What is known already Embryo mosaicism (containing cells that are euploid (46 chromosomes) and aneuploid (deviation from the expected number of chromosomes)) affects up to 17.3% of human blastocyst embryos. Current diagnosis of aneuploidy in the IVF clinic involves a biopsy of trophectoderm (TE) cells or spent media followed by sequencing. In some blastocyst embryos these approaches will fail to diagnose of the proportion of aneuploid cells within the fetal lineage (ICM). Study design, size, duration The impact of aneuploidy on cellular metabolism was assessed by using cellular autofluoresence and hyperspectral microscopy (broad spectral profile). Two models were employed: (i) Primary human fibroblast cells with known karyotypes (4-6 independent replicates, euploid n = 467; aneuploid n = 969) and reversine induced aneuploidy in mouse embryos (5-8 independent replicates, 30-44 cells per group). Both models were subjected to hyperspectral imaging to quantify native cell fluorescence. Participants/materials, setting, methods The human model is comprised of euploid (male and female) and aneuploid (triploid and trisomies: 13, 18, 21, XXX, and XXY) primary human fibroblast cells. For the mouse model, we treated embryos with reversine, a reversible spindle assembly checkpoint inhibitor, during the 4- to 8-cell division. Individual blastomeres were dissociated from control and reversine treated 8-cell embryos. Blastomeres were either imaged directly or used to generate chimeric blastocysts with differing ratios of control:reversine-treated cells. Main results and the role of chance Following unsupervised linear unmixing, the relative abundance of metabolic cofactors was quantified: reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavins with the subsequent calculation of the optical redox ratio (ORR: Flavins/[NAD(P)H + Flavins]). Primary human fibroblast cells displayed an increase in the relative abundance of NAD(P)H with a decrease in flavins, leading to a significant reduction in the ORR for aneuploid cells (P &lt; 0.05). The mouse embryos displayed an identical trend as the human model between control and reversine-treated embryos. Mathematical algorithms were applied and able to distinguish between (i) euploid and aneuploid primary human fibroblast cells, (ii) control and reversine-treated mouse blastomeres and (iii) chimeric blastocysts with differing ratios of control and reversine-treated cells. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve. We also showed that hyperspectral imaging of the preimplantation embryo does not impact on embryo developmental competence, pregnancy outcome and offspring health in a mouse model. We believe the role of chance is low as both human somatic cells and mouse embryos showed a consistent shift in cellular metabolism in response to human fibroblast cells that are aneuploid and reversine treated mouse embryos. Limitations, reasons for caution Further validation of our approach could include sequencing of the ICM of individual blastocysts to determine the proportion of aneuploid cells in ICM and correlate this with the metabolic profile obtained through hyperspectral imaging. Wider implications of the findings With hyperspectral imaging able to discriminate between (i) euploid and aneuploid human fibroblast cells and (ii) control and reversine-treated mouse embryos, this could be an accurate, non-invasive and label-free optical imaging approach to assess mosaicism within the ICM of mouse embryos, potentially leading to a new diagnostic tool for embryos. Trial registration number Not applicable

scholarly journals Salamander-Derived, Human-Optimized nAG Protein Suppresses Collagen Synthesis and Increases Collagen Degradation in Primary Human Fibroblasts

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Mohammad M. Al-Qattan ◽  
Medhat K. Shier ◽  
Mervat M. Abd-AlWahed ◽  
Ola H. Mawlana ◽  
Mohammed S. El-Wetidy ◽  
...  
Keyword(s):  
Human Fibroblast ◽  
Collagen Synthesis ◽  
Human Fibroblasts ◽  
Inhibitory Effect ◽  
Collagen Degradation ◽  
Fibroblast Cells ◽  
Primary Human Fibroblast ◽  
Dual Effect ◽  
Collagen Production ◽  
Human Fibroblast Cells

Unlike humans, salamanders regrow their amputated limbs. Regeneration depends on the presence of regenerating axons which upregulate the expression of newt anterior gradient (nAG) protein. We had the hypothesis that nAG might have an inhibitory effect on collagen production since excessive collagen production results in scarring, which is a major enemy to regeneration.nAGgene was designed, synthesized, and cloned. The cloned vector was then transfected into primary human fibroblasts. The results showed that the expression of nAG protein in primary human fibroblast cells suppresses the expression of collagen I and III, with or without TGF-β1 stimulation. This suppression is due to a dual effect of nAG both by decreasing collagen synthesis and by increasing collagen degradation. Furthermore, nAG had an inhibitory effect on proliferation of transfected fibroblasts. It was concluded that nAG suppresses collagen through multiple effects.

scholarly journals Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marino Maemura ◽  
Hiroaki Taketsuru ◽  
Yuki Nakajima ◽  
Ruiqi Shao ◽  
Ayaka Kakihara ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Primitive Endoderm ◽  
Cell Stage ◽  
Supportive Environment ◽  
Mouse Zygotes ◽  
Single Blastomeres ◽  
Multicellular Organisms

AbstractIn multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

Changes in the properties of the developing trophoblast of preimplantation mouse embryos as revealed by aggregation studies

Development ◽  
1977 ◽  
Vol 40 (1) ◽  
pp. 143-157
Author(s):  
Paul S. Burgoyne ◽  
Thomas Ducibella
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Supporting Cell ◽  
Mouse Embryos ◽  
Mixed Cell ◽  
Time Period ◽  
Preimplantation Mouse Embryos ◽  
Wide Range ◽  
Preimplantation Mouse

Mouse embryos (8-cell to early blastocyst) were denuded with pronase, and apposed in pairs which represented a wide range of stage combinations. These pairs either formed aggregates which differentiated into double-sized blastocysts, or they failed to aggregate. The 8–16-cell stages would not envelop late morulae/early blastocysts to form layered aggregates. This must mean that as the embryo differentiates into a blastocyst, the outer surface of the trophoblast loses its capacity for supporting cell spreading. The aggregation data also demonstrate that embryos almost completely lose their potential for aggregation at a very discrete stage in development – namely, between 8 and 9 h before blastocoel formation. It is argued that this is the stage at which the zonular tight junctional seal is completed, and that it is this physical barrier which prevents aggregation. It has been argued previously that the zonular tight junctional seal allows the creation of the special microenvironment which is necessary for the determination of the inner cells as inner cell mass. The completion of this seal 8–9 h before it is required for the formation of a blastocoel would provide a suitable time period for this cell determination to occur. The results obtained also relate to the technique of chimera production. Since the aim of this technique is to generate mice with mixed cell populations, it is important that the blastocyst formed following aggregation should have both cell lines present in the inner cell mass. This can best be assured by using relatively late morula stages (75 h post-HCG injection) since these will have already segregated their inner cells, but the incomplete seal will still allow aggregation to take place.

Abnormal development of pre-implantation mouse embryos grown in vitro with [3H]thymidine

Development ◽  
1973 ◽  
Vol 29 (3) ◽  
pp. 601-615
Author(s):  
M. H. L. Snow
Keyword(s):  
Specific Activity ◽  
Inner Cell Mass ◽  
Cell Damage ◽  
Cell Mass ◽  
Cell Number ◽  
Mouse Embryos ◽  
Abnormal Development ◽  
Tritium Concentration ◽  
Cell Stage

Mouse embryos were grown in vitro from the 2-cell stage to blastocysts in the presence of [3H]thymidine. Methyl-T-thymidine and thymidine-6-T(n) were used and both forms found to be lethal at concentrations above 0·1 μCi/ml. Both forms of [3H]Tdr at concentrations between 0·01 and 0·1 μCi/ml caused a highly significant (P &lt; 0·001) reduction in blastocyst cell number. The reduction in cell number, which was positively correlated with specific activity and tritium concentration, was associated with cell damage typical of radiation damage caused by tritium disintegration. Thymidine-6-T(n) also significantly reduced the number of 2-cell embryos forming blastocysts whereas methyl-T-Tdr did not. This difference in effect is assumed to be caused by contamination of one form of [3H]Tdr with a by-product of the tritiation process. A study of the cleavage stages showed that almost all the reduction in cell numbers could be accounted for by selective cell death occurring at the 16-cell stage. Cells which survive that stage cleave at a normal rate. The cells that are most susceptible to [3H]Tdr damage were found to normally contribute to the inner cell mass. The [3H]Tdr-resistant cells form the trophoblast. It is possible to grow blastocysts in [3H]Tdr such that they contain no inner cell mass but are composed entirely of trophoblast. Comparatively short (12 h) incubation with [3H]Tdr at any stage prior to the 16-cell stage will cause this damage. Possible reasons for this differential effect are discussed, and also compared with damage caused by X-irradiation.

Potential of two-cell mouse embryos to develop to term despite partial damage after cryopreservation

1995 ◽  
Vol 29 (3) ◽  
pp. 320-326 ◽  
Author(s):  
Th. Rülicke ◽  
P. Autenried
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Freeze Thaw ◽  
Foster Mothers ◽  
Vital Stains ◽  
Partial Damage

Approximately 18% of cryopreserved 2-cell mouse embryos of 26 different batches showed various degrees of morphological damage after the freeze-thaw process. Normal and damaged morphology were assessed by light microscopy and the ability of an embryo to develop in vitro to a blastocyst, or to develop to term, after transfer to foster mothers. Using vital stains such as Fluorescein-diacetate (FDA) and 4',6-Diamidino-2-Phenylindole (DAPI) it was found that in approximately 82% of the cases, both of the 2 blastomeres of the cryopreserved embryos survived the freeze-thaw process; in 10% only one cell survived the process; and in 8% none survived. Normally, only intact 2-cell embryos are considered for transfer. Here it was shown that over 60% of the partially damaged embryos developed in vitro to the blastocyst stage and, of those, 26% developed to term after transfer to suitable foster mothers. Although the inner cell mass (ICM) appeared to remain smaller during culture after the transfer of partially damaged 2-cell stage embryos, no difference during gestation period was found compared with intact embryos.

Influence of Fe3O4 Nanoparticles in Hydroxyapatite Scaffolds on Proliferation of Primary Human Fibroblast Cells

2016 ◽  
Vol 25 (6) ◽  
pp. 2331-2339 ◽  
Author(s):  
H. Maleki-Ghaleh ◽  
E. Aghaie ◽  
A. Nadernezhad ◽  
M. Zargarzadeh ◽  
A. Khakzad ◽  
...  
Keyword(s):  
Fe3o4 Nanoparticles ◽  
Human Fibroblast ◽  
Fibroblast Cells ◽  
Primary Human Fibroblast ◽  
Human Fibroblast Cells

Developmental pattern of hexaploid mouse embryos produced by blastomere fusion of diploid and tetraploid embryos at the 2-cell stage

Zygote ◽  
2009 ◽  
Vol 17 (2) ◽  
pp. 125-130 ◽  
Author(s):  
Lei Lei ◽  
Na Guan ◽  
Yan-Ning Xu ◽  
Qing-Hua Zhang ◽  
Jing-Ling Shen ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Karyotype Analysis ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Lower Number ◽  
Mouse Embryos ◽  
Developmental Pattern ◽  
Cell Stage ◽  
Positive Expression

SummaryPolyploid mouse embryos are important models for understanding the mechanisms of cleavage and preimplantation development in mammals. In this study, hexaploid (6n) mouse embryos were produced by the electrofusion of blastomeres from diploid (2n) and tetraploid (4n) embryos at the 2-cell stage. Furthermore, the developmental pattern of hexaploid embryos was evaluated by blastocyst rate, cell number, karyotype analysis, cytoskeleton staining and Oct-4 immunofluorescence. The results showed that 72.7% of the hexaploid embryos were able to develop to the blastocyst stage, which is a lower number than that found with normal diploid embryos (98.0%, p < 0.05). The cell number in hexaploid blastocyst was 12.3 ± 2.0, which was less than that found in diploid or tetraploid blastocysts (41.2 ± 7.2; 18.4 ± 3.5). Karyotype analysis confirmed that the number of chromosomes in hexaploid embryos was 120. β-Tubulin and Oct-4 immunofluorescence indicated that the hexaploid blastocysts were nearly lacking inner cell mass (ICM), but some blastomeres did show Oct-4-positive expression.

Sign in / Sign up

Export Citation Format

Share Document