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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

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


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


THE “OMICS” – INVASIVE ASSESSMENT OF THE EMBRYO


THE “OMICS” – NON-INVASIVE ASSESSMENT OF THE EMBRYO


THE OMICS –NON-INVASIVE ASSESSMENT

OF THE EMBRYO


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

Real Time Morphology


0.93

Uptake• Glucose

• Pyruvate

• Amino Acids

• Other Sugars

• Oxygen



• Ammonium

• Amino Acids

• Enzymes eg. LDH

• HLAG

• PAF

• Ubiquitin

• Interleukins

• HCG

• PIF

• DNA

• microRNA

Morphology and

Cleavage



Discovery

Platforms

Semi Non-Invasive


Sampling

• Fluorescence

Imaging Microscopy

• Hyperspectral

Analysis

• Metabolomics


• HPLC

• Secretomics

• Lab on a Chip


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


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)


Uptake• Glucose

• Pyruvate

• Amino Acids

• Other Sugars

• Oxygen



• Ammonium

• Amino Acids

• Enzymes eg. LDH

• HLAG

• PAF

• Ubiquitin

• Interleukins

• HCG

• PIF

• DNA

• microRNA

Morphology and

Cleavage



Discovery

Platforms

Semi Non-Invasive


Sampling

• Fluorescence

Imaging Microscopy

• Hyperspectral

Analysis

• Metabolomics


• HPLC

• Secretomics

• Lab on a Chip


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

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

Fluorescence Lifetime Imaging Microscopy

(FLIM)

• Excited fluorophores take time to de-excite

• FLIM creates arrival

time histograms of

fluorescence photons

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)


Non-Invasive Fluorescence Lifetime Imaging

Microscopy to measure embryo metabolism

Non Invasive Metabolic Assessment

NADH FAD

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


METABOLIC TIMELINESS DISTINGUISES EMBRYO VIABILITY


Non-Invasive Fluorescence Lifetime Imaging

Microscopy to measure embryo metabolism

Metabolism and Spindle Imaging

Metabolic imaging reports quantitative information about embryo metabolic state

Oxygen Deprivation

FLIM of Mouse embryos under 0% Oxygen

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


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

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

FLIM characterization of discarded

human blastocysts200 – biopsied PGT-A100- not biopsied100- discarded

Future

Future


Improved Metabolic Imaging and

Understanding of Embryos

Viability assessment

Assist in media development


Future

Uptake• Glucose

• Pyruvate

• Amino Acids

• Other Sugars

• Oxygen



• Ammonium

• Amino Acids

• Enzymes eg. LDH

• HLAG

• PAF

• Ubiquitin

• Interleukins

• HCG

• PIF

• DNA

• microRNA

Morphology and

Cleavage



Discovery

Platforms

Semi Non-Invasive


Sampling

• Fluorescence

Imaging Microscopy

• Hyperspectral

Analysis

• Metabolomics


• HPLC

• Secretomics

• Lab on a Chip


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

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

Uptake• Glucose

• Pyruvate

• Amino Acids

• Other Sugars

• Oxygen



• Ammonium

• Amino Acids

• Enzymes eg. LDH

• HLAG

• PAF

• Ubiquitin

• Interleukins

• HCG

• PIF

• DNA

• microRNA

Morphology and

Cleavage



Discovery

Platforms

Semi Non-Invasive


Sampling

• Fluorescence

Imaging Microscopy

• Hyperspectral

Analysis

• Metabolomics


• HPLC

• Secretomics

• Lab on a Chip


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


Media, culture conditions and assessmentin the future

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


Documents

Fluorescence Lifetime Imaging Microscopy (FLIM): egg and ...cme-utilities.com/mailshotcme/Material for Websites... · • Fluorescence Imaging Microscopy • Hyperspectral Analysis