Oocyte and Embryo Selection using Sequential Embryo Selection (SES) Lynette Scott Fertility Centers...
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Transcript of Oocyte and Embryo Selection using Sequential Embryo Selection (SES) Lynette Scott Fertility Centers...
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