Transcript What is New on the Horizon PGD/PGS and its application Non

Oocyte and Embryo Selection
using
Sequential Embryo Selection
(SES)
Lynette Scott
Fertility Centers of New England
Reading, MA, USA
Abnormal gametes
generally do
not produce
normal embryos
Gametes
Early embryo parameters may be
a window back to the gametes
Day 1
and
Day 2
Day 3
Day 5
Differentiation
Later development
reflects gene
expression,
differentiation,
developmental
controls
Human Oocyte
Cumulus
Cells
-NO
-gene up/down
regulation
-
Oocyte
-Mt load
-cAMP
-nuclear/
Cytopasmic
maturation
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
Layers
Alignment
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
Note Spindle Alignment
And Zona Alignment
Courtesy of Laura Rienzi
Abnormal Spindle Shapes
Courtesy of David Keefe
Fertilized Oocyte Scoring
Looks 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
Normal = equality between nuclei
Abnormal = any inequality
NPB RATIO
L=5
R=6
L=4
R=4
NPB Ratio vs. Outcome
ALL
ET
Not
Preg
Preg
FHB
100%
>12
Implant 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
Condensed chromatin
13, 14, 15,
21, 22
NPB
RNA
Polymerase
Fibrillar component and center
Dense fibrillar component
Granular component
Pre-RNA
NPB IN FERTILIZED OOCYTES
Looking at chromatin
13, 14, 15, 21, 22
NPB
L=5
R=6
Evidence for epigenetic/ imprinting
errors in these oocytes
Sperm or oocyte?
NPBs associated with imprinting
errors, heterochromatin
34 yr old, GO, PO
Sever male factor, Sperm problem not an egg one
6 IVF attempts, no pregnancy, 2 PGD, no normal’s
Embryo
1
2
3
4
5
6
7
XY
0
X0
X0
0
XXY
XX
XY
13
1
1
1
0
2
1
2
15
0
1
2
2
3
1
2
16
0
1
1
1
2
1
2
17
1
1
2
1
1
2
1
18
1
0
1
1
1
2
2
21
2
2
2
1
1
2
1
22
2
1
1
1
1
1
1
i n terpre tati on
complex
complex
complex
complex
complex
complex
complex
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 Size
Why 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
30%
Small
Rosette
Day 1
Day 2
Day 3
Polyploid
Complex Abnormal
Day 1 and Day 2 Correlations
Deliveries according to early
morphometrics
Scott et al., 2007
Day 3 Cell Number
P<0.05
90
7-8 cell
5-8 cell
80
70
60
50
40
30
20
10
0
All
ET
NEG
POS
FHB
100%
Early Scoring Parameters
Significant for Delivery
Scott et al., 2007
Early Scores and Delivery
Scott et al. 2007
Complex Abnormal
MN
Good Grade 8-cell
XXXY
1x 16
1x 21
3x 22
MN
0x 15
Good Blastocyst (D6)
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 SES
Impact on Delivery Rates
50
45
40
35
30
25
20
15
10
5
0
1.9*
2005
2006
2007
2.1
2.4
2.1
2.5
2.2
5%
2%
1%
<38
* Mean # ET 2.1
38-40
Total
Twin Rate 30-40%