Fluorescence Lifetime Imaging Microscopy (FLIM): egg and ...cme-utilities.com/mailshotcme/Material...
Transcript of Fluorescence Lifetime Imaging Microscopy (FLIM): egg and ...cme-utilities.com/mailshotcme/Material...
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Fluorescence Lifetime Imaging Microscopy (FLIM): egg and embryo under observation
Denny Sakkas, Ph.D.
Ovarian Club XIV 2019
Scientific Director,Boston IVFWaltham, MA, USA
Associate ProfessorYale University School of Medicine
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LEARNING OBJECTIVES
At the conclusion of this presentation, participants should be able to:
● Understand why we need to identify the best embryo
● Understand the current status of Non-Invasive assessment of embryo viability
● Understand the future for Non-Invasive assessment of embryo viability
Ovarian Club XIV 2019
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What is a success in IVF?
• A pregnancy
• A live birth
• A healthy live birth
• A healthy offspring
• A healthy adult
• Future Generations
Selecting the best embryo will be more than picking a viable embryo
❖ 1970s
❖ 1980s
❖ 1990s
❖ 2000s
❖ 2010s
❖ Next
Ovarian Club XIV 2019
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THE “OMICS” – INVASIVE ASSESSMENT OF THE EMBRYO
Ovarian Club XIV 2019
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THE “OMICS” – NON-INVASIVE ASSESSMENT OF THE EMBRYO
Ovarian Club XIV 2019
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THE OMICS –NON-INVASIVE ASSESSMENT
OF THE EMBRYO
Ovarian Club XIV 2019
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Uptake• Glucose
• Pyruvate
• Amino Acids
• Other Sugars
• Oxygen
Drop (5 - 50µl) of defined
culture medium Production• Lactate
• Ammonium
• Amino Acids
• Enzymes eg. LDH
• HLAG
• PAF
• Ubiquitin
• Interleukins
• HCG
• PIF
• DNA
• microRNA
Morphology and
Cleavage
• Morphokinetic History
• Blastocyst Grading
Discovery
Platforms
Semi Non-Invasive
• Blastocoel Fluid
Sampling
• Fluorescence
Imaging Microscopy
• Hyperspectral
Analysis
• Metabolomics
• Spectroscopy- Raman, NIR, NMR
• HPLC
• Secretomics
• Lab on a Chip
Modified from Sakkas and Gardner, 2017Ovarian Club XIV 2019
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Real Time Morphology
Ovarian Club XIV 2019
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0.93
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Uptake• Glucose
• Pyruvate
• Amino Acids
• Other Sugars
• Oxygen
Drop (5 - 50µl) of defined
culture medium Production• Lactate
• Ammonium
• Amino Acids
• Enzymes eg. LDH
• HLAG
• PAF
• Ubiquitin
• Interleukins
• HCG
• PIF
• DNA
• microRNA
Morphology and
Cleavage
• Morphokinetic History
• Blastocyst Grading
Discovery
Platforms
Semi Non-Invasive
• Blastocoel Fluid
Sampling
• Fluorescence
Imaging Microscopy
• Hyperspectral
Analysis
• Metabolomics
• Spectroscopy- Raman, NIR, NMR
• HPLC
• Secretomics
• Lab on a Chip
Modified from Sakkas and Gardner, 2017Ovarian Club XIV 2019
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The Metabolism of Embryos
• Assessment of specific metabolic pathways, their relative activities and their regulation in relation to embryo viability
• Example: – Glucose, Lactate, Pyruvate
– TCA Cycle functions before 8-cell stage and
– Glycolysis functions after the 8-cell stage
It was first shown in 1980 that Glucose metabolism is linked with the viability of embryos
Ovarian Club XIV 2019
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Glucose consumption of single post-compaction human embryos is predictive of embryo sex and live birth outcome.
(Gardner et al., Human Reproduction 2012)
Positive FCA
Negative FCA
Positive FCA
Negative FCA
Day 4 Day 5
Glu
cose
Up
take
(p
mo
l/e
mb
ryo
/h)
Ovarian Club XIV 2019
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Uptake• Glucose
• Pyruvate
• Amino Acids
• Other Sugars
• Oxygen
Drop (5 - 50µl) of defined
culture medium Production• Lactate
• Ammonium
• Amino Acids
• Enzymes eg. LDH
• HLAG
• PAF
• Ubiquitin
• Interleukins
• HCG
• PIF
• DNA
• microRNA
Morphology and
Cleavage
• Morphokinetic History
• Blastocyst Grading
Discovery
Platforms
Semi Non-Invasive
• Blastocoel Fluid
Sampling
• Fluorescence
Imaging Microscopy
• Hyperspectral
Analysis
• Metabolomics
• Spectroscopy- Raman, NIR, NMR
• HPLC
• Secretomics
• Lab on a Chip
Modified from Sakkas and Gardner, 2017Ovarian Club XIV 2019
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Non-Invasive Fluorescence Lifetime Imaging Microscopy to measure embryo metabolism
Non-Linear Microscopy for ART
▪ Directly measures cellular features linked to egg/embryo quality
▪ Quantitative and objective
▪ Deep imaging, not obstructed by surrounding cells
▪ Non-invasive
▪ long wavelength, low power light
▪ endogenous contrast (no reagents needed)
Mitochondria
Metabolic Activity
Non-Linear Microscopy
Daniel Needleman Lab, Harvard University MA, USA and LUMINOVA
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Why NADH and FAD? Both important intermediates in the electron transport chain
NADH acts as a shuttle for electrons during cellular respiration.
NAD+ picks up an electron from glucose, at which point it becomes NADH.
NADH also contributes to oxidation in cell processes like glycolysis to help with the oxidation of glucose.
FAD is a coenzyme of oxidation-reduction can replace NAD+; FAD accepts two electrons and becomes FADH2
NADH and FADH2 are essential to
cellular respiration and ATP production
NADH and FAD both auto fluoresce
No need to add markers
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Fluorescence Lifetime Imaging Microscopy
(FLIM)
• Excited fluorophores take time to de-excite
• FLIM creates arrival
time histograms of
fluorescence photons
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Quantitative Metabolic
parameters
20μm
Measured parameters
for each embryo, for
both NADH and FAD::
1) Brightness
(~ concentration)
2) Fractions bound to
Enzyme
3) Short fluorescence
lifetime
4) Long fluorescent
lifetime
8 parameters sensitive to
metabolic state
NADH image
(Same process for FAD)
Ovarian Club XIV 2019
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Non-Invasive Fluorescence Lifetime Imaging
Microscopy to measure embryo metabolism
Non Invasive Metabolic Assessment
NADH FAD
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Metabolic Development Curves
NADH and FAD
NADH Irradiance (brightness)
Fraction bound
Short lifetime
Long lifetime
FAD+
Irradiance (brightness)
Fraction bound
Short lifetime
Long lifetime
• NADH abundance peaks around compaction and decreases significantly during blastocyst formation, corresponding with an associated increase in energy demands.
• These patterns were preserved among the embryo cohort
Ovarian Club XIV 2019
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METABOLIC TIMELINESS DISTINGUISES EMBRYO VIABILITY
Ovarian Club XIV 2019
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Non-Invasive Fluorescence Lifetime Imaging
Microscopy to measure embryo metabolism
Metabolism and Spindle Imaging
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Metabolic imaging reports quantitative information about embryo metabolic state
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Oxygen Deprivation
FLIM of Mouse embryos under 0% Oxygen
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Mitochondrial Dysfunction
FLIM of ClpP ((caseinolytic peptidase P)
Knockout mice
Mitochondrial imaging reveals
aberrant mt distribution in many
mutant oocytes
Metabolic imaging quantitative parameters
p=0.042
mtDNA measurements
P-values < 10-27
+/+
-/-
Brightfield NADH FLIM
Comparison of FLIM to mtDNA
Ovarian Club XIV 2019
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Young
Old
Still high significance, but less pronounced than CLPP
Metabolic imaging quantitative parameters
p < 10-5p=0.14
mtDNA measurements
Comparison of FLIM to mtDNA
Differences in intensity
images not apparent
AGE
FLIM of Old versus Young mouse oocytes
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0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
55%
60%
65%
70%
75%
Controls (N=10) Illuminated (N=13)
Live births from FLIM-illuminated
embryos • Effective metabolic measurements were obtained every 2h for 48h, then embryos were transferred
to pseudopregnant female mice.
• No significant difference was observed between the illuminated and control groups.
Ovarian Club XIV
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FLIM characterization of discarded
human blastocysts200 – biopsied PGT-A100- not biopsied100- discarded
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Future
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Future
Ovarian Club XIV 2019
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Improved Metabolic Imaging and
Understanding of Embryos
Viability assessment
Assist in media development
Ovarian Club XIV 2019
Future
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Uptake• Glucose
• Pyruvate
• Amino Acids
• Other Sugars
• Oxygen
Drop (5 - 50µl) of defined
culture medium Production• Lactate
• Ammonium
• Amino Acids
• Enzymes eg. LDH
• HLAG
• PAF
• Ubiquitin
• Interleukins
• HCG
• PIF
• DNA
• microRNA
Morphology and
Cleavage
• Morphokinetic History
• Blastocyst Grading
Discovery
Platforms
Semi Non-Invasive
• Blastocoel Fluid
Sampling
• Fluorescence
Imaging Microscopy
• Hyperspectral
Analysis
• Metabolomics
• Spectroscopy- Raman, NIR, NMR
• HPLC
• Secretomics
• Lab on a Chip
Modified from Sakkas and Gardner, 2017Ovarian Club XIV 2019
![Page 35: Fluorescence Lifetime Imaging Microscopy (FLIM): egg and ...cme-utilities.com/mailshotcme/Material for Websites... · • Fluorescence Imaging Microscopy • Hyperspectral Analysis](https://reader034.fdocuments.in/reader034/viewer/2022042805/5f5ca37be9fdbf38281955db/html5/thumbnails/35.jpg)
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versus
RESULTS DAY 5
TE BIOPSY vs. SBM
(N=27)
INFORMATIVITY
81.8%
CONCORDANCES
63%
RESULTS DAY 6/7
TE BIOPSY vs. SBM
(N=81)
INFORMATIVITY
100%
CONCORDANCES
84%
Rubio & Rienzi et al., Fertil Steril online 2019
Concordance between TE biopsies and embryonic cfDNA
N=108 blastocysts
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Clinical outcome after SET of euploid versus aneuploid SBM
Clinical OutcomeEuploid TE/
Euploid SBM
Euploid TE/
Aneuploid SBMTOTAL
Number of transfers 17 12 29
Mean maternal age (SD) 37.5 (2.5) 37.4 (2.3) 37.5 (2.4)
Positive pregnancy test 11 (64.7) 4 (33.3) 15 (51.7)
Biochemical pregnancy loss 2 (18.2) 0 2 (13.3)
Clinical pregnancy rate (%) 9 (52.9) 4 (33.3) 13 (44.8)
Clinical miscarriage (%) 0 2 (50.0) 2 (15.4)
Ong implantation rate (%) 9 (52.9) 2 (16.7) 11 (37.9)
Analysis of the embryonic cfDNA (niPGT-A)
Rubio & Rienzi et al., Fertil Steril online 2019
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Uptake• Glucose
• Pyruvate
• Amino Acids
• Other Sugars
• Oxygen
Drop (5 - 50µl) of defined
culture medium Production• Lactate
• Ammonium
• Amino Acids
• Enzymes eg. LDH
• HLAG
• PAF
• Ubiquitin
• Interleukins
• HCG
• PIF
• DNA
• microRNA
Morphology and
Cleavage
• Morphokinetic History
• Blastocyst Grading
Discovery
Platforms
Semi Non-Invasive
• Blastocoel Fluid
Sampling
• Fluorescence
Imaging Microscopy
• Hyperspectral
Analysis
• Metabolomics
• Spectroscopy- Raman, NIR, NMR
• HPLC
• Secretomics
• Lab on a Chip
Modified from Sakkas and Gardner, 2017Ovarian Club XIV 2019
![Page 39: Fluorescence Lifetime Imaging Microscopy (FLIM): egg and ...cme-utilities.com/mailshotcme/Material for Websites... · • Fluorescence Imaging Microscopy • Hyperspectral Analysis](https://reader034.fdocuments.in/reader034/viewer/2022042805/5f5ca37be9fdbf38281955db/html5/thumbnails/39.jpg)
Lab On A Chip: Metabolic Analysis of Embryos
Urbanski, Johnson, Gardner et al (2008) Noninvasive metabolic profiling using Microfluidics for analysis of single preimplantation embryos. Anal Chem 80: 6500-6507
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Ovarian Club XIV 2019
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Media, culture conditions and assessmentin the future
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Thank you• Daniel Needleman Lab, Harvard University MA, USA• Emily Seidler MD
• LUMINOVA• Tim Sanchez PhD• Marta Venturas
• Emre Seli MD and Lab
• Boston IVF
• David Gardner PhD
Ovarian Club XIV 2019