Cleavage vs Blastocyst Transfer in the Time Lapse Era.

Cleavage vs Blastgthe Time L

Barry Behr, y ,Prof

Director, IVFDirector, IVFCo-Director IVF

Stanford UniversiStanford Universi

tocyst Transfer in yLapse Era.

Ph.D., HCLD,fessorF LaboratoryF LaboratoryF/ART Programity Medical Centerity Medical Center

Disclo

I am a founder of AuxI am a founder of IviGI am a founder of IviG

osure

xogynGen/BlastogenGen/Blastogen

Out

Lets make this interac

tline

ctive

Human Embryoo Development

Why go to B

Embryo Selection

HAVING to go to blastbenefit of the TLM techbenefit of the TLM tech

Blastocyst?

t removes some of the hnologyhnology

What does a cembryo ne

Be euploidCleaveCleaveCompactF i bl bl tForm a viable blastocy

cleavage stage g geed to do?

tyst

What does a Blado

Be euploidDifferentiate into ICMDifferentiate into ICM HatchAApposeAttachInvade

astocyst need to yo?

ad TEad TE

$$64,000 q

Are you doing fresh vsAre you doing PGD/S?Are you doing PGD/S?When is the endometr

questions

s frozen ET???rium most receptive?

Cleavage vs B

Simpler to measure eaFaster ( ie can ET earFaster ( ie can ET earMay be safer

Blastocyst TLM

arly eventslier)lier)

What are we meaWhat are we mea

May determine which – Genetic (ie when is besGenetic (ie when is bes– Metabolic– (both?)(both?)

asuring with TLC?asuring with TLC?

day we ETst to do a bx)st to do a bx)

Parameters mestagestage

easure cleavage eventsevents

Human Embryo TiPaper Published Samples

Payne et al. Hum Reproduction 1997 50

2PN • ObsHum Reproduction 2PN

Lemmen et al.RBM Online 2008 102

2PN oocytes

• Rep• Cor

preg

Mio et al. Am J Obstet Gyn 2008 286

oocytes • Obs• Rep

Wong et al 242

• Idenby DWong et al.

Nature Biotechnology 2010 2422PN

y• Dem

exp• Dev

Pribenszky et al. RBM Online 2010 5

2PN • RepRBM Online 2010 2PN p

Meseguer et al. Hum Reprod 2011 247

2PN • Eva

Hashimoto et al. 2012 80 EHashimoto et al.Fert Steril 2012 80

2PN • Eva

Swann et. al.Fertil steril 2012 10 oocytes

or zygotes • Cor

ime-lapse StudiesConclusions

served details of the fertilization process to 20 hrs

ported PN appearance & disappearancerelated synchrony in nuclei appearance after 1st cleavage with gnancy success

served details of the fertilization processported two ICMs - monozygotic twins

ntified cell cycle parameters that predict blastocyst formationDay 2ymonstrated that parameters correlate to embryo gene pression dataveloped cell tracking software

ported a live birthp

aluated cell cycle parameters to implantation

l t d ll l t f bl t t litaluated cell cycle parameters for blastocyst quality

related cytoplasmic movements with Ca2+ oscillations.

Oct 2010

Imaging and MoleEmbryonEmbryon

ecular Analysis of nic Cellsnic Cells

Nature Biotech ePub Oct 3, 2010

Custom MicroIndividual culture within t

o‐Well Dishesthe same media drop

Comparison to a Morphologic

SenSpep

1. F. Guerif et al., Limited value of morphological assessment at dA prospective study based on 4042 embryos. Hum Reprod; 20

Studyof Common cal Predictors1

4 morphological criteria:

Pronuclear morphology on day 1; early cleavage, # cells, and fragmentation on day 2

sitivity ~ 0.65cificity ~ 0.65

and fragmentation on day 2

y

days 1 and 2 to predict blastocyst development potential: 007

Imaging Does Not PParam

Alter Fundamental tmeters


Arrested EmbryosyCytokinesis Duri

DiviNormal

Divi

Mild

Severe

phenotype

phenotype

s Exhibit Abnormal ing 1st Cleavage sionsion

4 ESSPs of Early Ger

Wong et al, submitted

rm Cell Development

Maternal inherited (1)EGA (2)EGA (2)Blastocyst (3)Ubiquitous (4)


Embryo Arrest and GIndividual BIndividual B

Gene Expression in BlastomeresBlastomeres


MoleculaCorrelated gene expression profilin

1 Embryos with aberrant cytokinesis1. Embryos with aberrant cytokinesis demonstrate underlying defects in molecular programs:

ar Analysisng revealed:

2. Using our predictors, embryo transfer can2. Using our predictors, embryo transfer can be performed prior to embryonic gene activation, minimizing risk of adverse outcomes:

7

8Gene Transcription Activation

ProposedCurrent Embryo Transfer

Point

3

4

5

6p

Transfer Point

0

1

2

3

1-cell 2-cell 3-cell 4-cell 6-cell 8-cell 9-cell Morula Blastocyst

DiscriminationBlastocyst Receiver Operating

SenSpep

23

n Potential of Predictorsg Characteristic (ROC)

sitivity = 0.94 cificity = 0.93y

Example Tracking on Whole Dish

Eeva AnalysFully automated, state-of-the-art computer vis

the end o

sis Software

Eeva Timing

sion software predicts blastocyst formation by of Day 2

Eeva Parameter Eeva Prediction

Timing

20 minPASS

12 hrsPASS

PASS1 hrs

20 iPASS

15 hrs

20 min

FAIL

2 hrs

PASS

Refinement of cell cyc

Parameter Measurements Wong/Lo

Refinement of cell cyc

Duration of first cytokinesis 14.4+

Time between first and second mitosis

11.1+2

Time between second and third mitosis

1+1.6

ParameterMeasurements

Normal CGH

Duration of first cytokinesis

14.4+4.2 Min.

Time between first and second mitosis

11.8+0.71 Hourssecond mitosis

Time between second and third mitosis

0.96+0.84 Hours

cle imaging parameters

oewke et al. Present Study

cle imaging parameters

+6 Min. 14.4+4.2 Min.

2.2 Hours 11.8+1.2 Hours

6 Hours 0.85+0.79 Hours

Meiotic Error Mitotic Error

117.2+166.5 Min. 36.0+66.9Min.

4.0+5.2 Hours 6.4+6.6 Hours

2.0+4.3 Hours 2.0+3.9 Hours

Detection of chromosomal dusimple and comp

plications/deletions and both plex mosaicism

Calculation of Embryonic ERi k U i M h lRisk Using Morpholog

Asses

A table showing the calculation (number of euploid embryos/total number expressed as a percentage for each morphological and/or parameter assehighest percentage of embryonic euploidy over aneuploidy was obtained wand cellular fragmentation analysis.

Euploidy Versus Aneuploidyi l d/ P tgical and/or Parameter

ssment

of embryos) and the resulting probability of embryonic euploidy essment. (B) Graphic representation of the table demonstrating that the with the combination of cell cycle parameters that predict normal A-CGH

Wong et al., 2010, Mesg , ,and Hashimoto

Table courtesy of Michael Tucker

seguer et al., 2011 g ,et al., 2012

Putting it aall together


Summary

Summ

Any TLM approach is betteUterine receptivity will play– Which will influence which sy

Practical matters like best tinfluence the decision

mary

er than a static system a role in when we ETystem will dominatetime to bx or vit may also

Cleavage vs Blastocyst Transfer in the Time Lapse Era.

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