Oocyte and Embryo Selectionusing

Sequential Embryo Selection (SES)

Lynette Scott

Fertility Centers of New England

Reading, MA, USA

Early embryo parameters may be a window back to the gametes

Abnormal gametes generally do not produce normal embryos

Later development reflects gene expression, differentiation, developmental controls

Day 1and

Day 2

Gametes

Differentiation

Day 3 Day 5

Human Oocyte

CumulusCells-NO-gene up/downregulation-

Oocyte-Mt load-cAMP-nuclear/Cytopasmicmaturation

Zona Pellucida

Laid down by the oocyte so could reflect oocyte quality

Can be visualized and measured using polarized light microscopy

2 systems:A- measures zona density B- measures differential zona layers and

thickness

Porous surface of the zona pellucida

Zona Density, Alignment, Abnormalities

Courtesy of Marcus Montag

LayersAlignment

Spindle• Visualized using polarized light microscopy• Position- should be in the hemisphere

containing the polar body• The shape of the spindle is more important

– Should be bi-polar and ordered

• Correlations with oocyte competence• Draw backs:

– 98% of spindles are in the correct position– The spindle is very dynamic and temperature

sensitive, forming and dismantling in cycles and with temperature drops. The oocyte must also be very carefully positioned for accurate visualization.

– Only true irregularities = abnormal

Metaphase II Spindle

Courtesy of Laura Rienzi

Note Spindle Alignment

And Zona Alignment

Courtesy of David Keefe

Abnormal Spindle Shapes

Fertilized Oocyte ScoringLooks at a part of nucleoli in

the early embryo

Nucleoli Found in all actively dividing cells Sites of rRNA synthesis Develop on the DNA where the genes for ribosomes are

located, rDNA These points on the DNA = NORs

Nucleolar Organizing Regions

Human NORs 5 pairs of NOR-bearing chromosomes

– 13, 14, 15, 21, 22 (acrocentric)

Generally 5-7 NORs in human cells• NOR’s are clustered and this is dependent on heterochromatin

adjacent to rDNA genes Heterochromatin is not inactive and may be involved in

developmental control (Dimitri, 2004)

• Transcription of rDNA results in 3 functional parts:

Nucleolar Precursor Body (NPB) Pattern

15 4 2

Z1

Z2

OA

OB

Z3-1 Z3-2Z3-3 Z3-4

Abnormal = any inequality

Normal = equality between nuclei

L = 5 R = 6 L = 4 R = 4

NPB RATIO

NPB Ratio vs. Outcome

ALL ET Not Preg

Preg FHB 100% Implant

>12 weeks

L/R 4/9 5/8 4/9 5/7 6/7 6/7 6/7

Range 1-15 2-10 2-10 4-9 5-9 5-7 6-7

P<0.05

Fibrillar component and center

Dense fibrillar component

Granular component

Condensed chromatin

Pre-RNA

RNA Polymerase

NPB

13, 14, 15, 21, 22

L = 5 R = 6

NPB IN FERTILIZED OOCYTES

NPB

Looking at chromatin13, 14, 15, 21, 22

Evidence for epigenetic/ imprinting errors in these oocytesSperm or oocyte?NPBs associated with imprinting errors, heterochromatin

Embryo XY 13 15 16 17 18 21 22 interpretation 1 0 1 0 0 1 1 2 2 complex 2 X0 1 1 1 1 0 2 1 complex 3 X0 1 2 1 2 1 2 1 complex 4 0 0 2 1 1 1 1 1 complex 5 XXY 2 3 2 1 1 1 1 complex 6 XX 1 1 1 2 2 2 1 complex 7 XY 2 2 2 1 2 1 1 complex

34 yr old, GO, POSever male factor, Sperm problem not an egg one6 IVF attempts, no pregnancy, 2 PGD, no normal’s

1 DI attempt, Term delivery,3628 gr male

Day 2 Scoring• Cell number

• Blastomere relative size

• Status of nucleation

• Fragmentation

• Timing is important

Day 2 Cell SizeWhy is it important?

• Polarity

• Distribution of cell components

• Embryo axes

• The meiotic and mitotic spindle

MULTI-NUCLEATION and CELL SIZE

30%

4-cell Blastomer Morphology

Small

30%

Rosette

Day 1 Day 3Day 2

Polyploid

Complex Abnormal

Day 1 and Day 2 Correlations

Deliveries according to early morphometrics

Scott et al., 2007

Day 3 Cell Number

0

10

20

30

40

50

60

70

80

90

All ET NEG POS FHB 100%

7-8 cell5-8 cell

P<0.05

Z Score, Z1 and 2 (P<0.01)NPB Ratio, 5-7 per nucleus (P<0.001)*

Day 2 score highly significant (P<0.01)Even cell size (P<0.001)*No multi nucleation (P<0.001)*

– 4 cell best (better than 2-cell, 3 cell reduced FHB, no delivery) (P<0.05)

Day 3 score, 8 cell is best (P<0.05)

Early Scoring ParametersSignificant for Delivery

Scott et al., 2007

Early Scores and Delivery

Scott et al. 2007

Complex Abnormal

MN

MN

Good Grade 8-cell Good Blastocyst (D6)

XXXY1x 161x 213x 220x 15

SEQUENTIAL SELECTION

Accept that “pretty” good

Use biologic criteria and not only morphology In gated scoring, an embryo that does not pass through the

first gate must not be selected at the second level, regardless of morphology

If it does not pass the 2nd gate it should be discarded, as this is the most NB criteria

Introduction and use of SESImpact on Delivery Rates

0

5

10

15

20

25

30

35

40

45

50

<38 38-40 Total

200520062007

5%

2%

1%

2.4

1.9*2.1

2.22.5

2.1

* Mean # ET 2.1 Twin Rate 30-40%

% D

eliv

ery/

>20

wee

ks