Download Understanding Implantation

New development on
implantation
Report from ESHRE
2006
• Dr Clement Ho
• Dr Alexander K. Doo
• MB BS (HK), FRCS (G),
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FRCOG, FHKCOG, FHKAM
(Obstetrics & Gynaecology)
MB ChB (G), MRCOG,
FHKCOG, FHKAM (Obstetrics &
Gynaecology)
Understanding
Implantation
• The goal – improve implantation
and viable pregnancy.
• Worldwide implantation rates
range from 10-45%
Embryo or endometrium?
• 72% of IVF failure is due to
implantation
• ? Role of the embryo in
implantation
• ? Role of the endometrium in
implantation
Embryos
• 55-90% of transferred embryos do
not implant – current selection
techniques are at best minimally
effective
• Embryo selection –
– Morphology
– PGD
– Pre-implantation molecular level
screening
– In vivo selection
Morphology
• Morpholgical scoring for
embryos at early stages is more
reflective of the gametes
normality
• Early scoring include pronuclear
size, alignment of the polar
bodies, cytoplasm texture,
nucleolar precursor body
numbers, size and the
distribution of the two nuclei
Morphology
• Timing of the first mitotic event
(early cleavage)
• The state of nucleation of each
blastomeres at the completion of the
first and second mitotic division and
the relative equality of the cell size
in the cells from the these initial
mitotic events.
• All these scoring parameters have
been correlated with development in
vitro, embryos grade on D3 or D5,
state of aneuploidy after PGD
screening, development to
blastocyst, and implantations.
Morphology
• Main early embryos scoring
parameters associated with
successful implantation are –
• Nucleolar alignment and equality
• Early cleavage
• D2 nuclear state and blastomeres
symmetery
• D3 cell number and lack of
fragmentation
• D5 blastocyst formation
4000 embryos – looked at nuclear
alignment, nucleolar number, size and
alignment; D2 nucleation and cell no.
and symmetry were significantly
correlated with implantation, the others
were not.
Morphology
• Seven or more blastomeres on
Day 3
• Percentage fragmentation <20%
on day 3
• Number of multinucleated
blastomeres
• Arce et al - ? Morphology
reproducible – study of 4100
cycles local vs central grading –
poor
FISH analysis –
mono/multinuclear status
• Hlinka et al Czech Republic
• Assessment of multinucleation in
cleaving embryos
• 106 embryos, 4 cell stage analyzed
• FISH performed (13,15,16,18,21,22,X and
Y)
• Embryos in 3 group
All mononucleated blastomere
At least one multinuclear
blastomere
All multinucleated blastomere
RESULTS – Euploid, Mosaic (sg/a: 2 or 3
cells were euploid), (sg/b: 1 cell only
was euploid) and Aneuploid
FISH analysis –
mono/multinuclear status
• FISH
Group A (%)
Euploid
45
Mosaic
42
Aneuploid
13
Group B (%)
0
56
44
Group C (%)
0
0
100
Only mononuclear cells sgr (%) a vs b
86 vs 14
Mono/Multinuclear cells sgr (%) a vs b
38 vs 62
Only multinuclear cells
Suggest D2 PGS on mononuclear cells. Some
mono/multinuclear cells which frequently displays
mosiacism maybe be cured in a minority of cases by
performing PGD but this is unpredictable. In the group
with only multinucleated cells, it should be rejected for
transfer completely. D2 nuclear status can be a useful tool
Morphology - Oocytes
• Oocyte morphology polar body shape, size,
integrity and cytoplasmic inclusions
bodies – not proven useful in embryo
selection
• Montag et Al, Germany
• Oocytes zona birefringence intensity is
associated with embryonic implantation
potential
• Polarization light microscopy on living M2
cells showed a 3 layered organization of
the zona pellucida – the inner layer showed
natural birefringence/retardance, which
varied between oocytes.
• 30 cycles with at least 4 oocytes available
for ISCI were screened which resulted in 2
embryo transfer.
Morphology - Oocytes
• Classified into High (HZB) or Low (LZB)
zona bifringence.
• 7 embryos HZB/HZB, 9 embryos HZB/LZB,
and 14 embryos from LZB/LZB transferred
with pregnancies of 5/7(71.4%),4/9(44.4%)
and 3/14(21.4%) respectively.
• Speculate zona birefringence as an
indirect measure of oocytes quality and
selection.
• ? Tool for optimizing stimulation regimes.
Morphology - sperm
• Sperm morphology, motility and the level of
DNA fragmentation have been correlated with
development, but no definite controlled trials
• Crippa et al, Italy
• Sperm selection based on presence of
birefringence in the sperm head
• Sperm quality related to fertilizing capacity
• In mature sperm nucleus strong intrinsic
birefringence associated with nucleoprotein
filaments
• 65 sperm sample : 8 N, 50 OAT, 7 TESE
49 fresh 16 thawed oocytes (ICSI birefringent sperm)
vs 47 fresh 16 thawed (control)
Morphology - sperm
Normal
% Bi
88.1+/-11.3
Range 65-97
OAT
55.5+/-28.6
8-98
OAT sample :
Progressive Motility
65.9+/-24.8% range 8-98
TESE
14.3+/-9.1
4-32
No progressive motility
28.8+/-19.1% range 12-71
Fertilization rate
Study
80%
Control
66%
p value
p<0.025
Implantation rate
16.2%
5.1%
p<0.05
? Tool for sperm selection
Blastocyst transfer in
SET
• Panpanikolaou et al. NJEM 2006
• 351 Women randomised to
single cleavage stage (175) vs
single blastocyst transfer
(n=176)
• Ongoing pregnancy rate per
cycle (22 vs 34)
• Embryos croyerserved+/-SD
(4.2+/-4.1 vs 2.2+/- 2.7)
Blastocyst culture vs
day 3
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Uterine environment
Embryonic genome
Selction criteria
Embryos for transfer
Monozygotic twinning
Sex ratio
Cryopeservation
Morphology
Computer assisted multilevel
analysis
• Lausanne/Linz group
2PN zygotes
computer program quantitative
analysis of digital images
• 40 features were measured
• Main features are zygote and PN size,
PN position, cytoplasmic halo area,
number and distribution of nucleolar
precusor bodies (NPBs)
Morphology
Computer assisted multilevel
analysis
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84 patients, 136 zygote (center A)
90 patients, 154 zygote (center B)
Transfer on day 2-3 or 5.
Center A - 2 zygotes randomly
selected cultured to day 2-3
transferred, rest immediately frozen
• Center B – All zygotes were cultured
to day 3 or 5, 1-3 high quality
embryos transferred
Morphology
Computer assisted multilevel
analysis
• Results - Higher implantation rate in
center B (31.2% vs. 16.2%; p<0.005)
• The mean sizes of zygotes and PN, the
angles between PN and polar bodies
were not different between the two
centers.
• The relative area of the cytoplasmic
halo was lower in center B (0.14+/-0.07
vs.0.21 +/- 0.07; p<0.001).
• The mean number of NPB was higher in
center B for PN1 (7.4+/-2.4 vs.6.3+/-1.7;
p<0.001)but not PN2(4.3+/-1.5 vs.4.1+/-
Morphology
Computer assisted multilevel
analysis
• NPB were less dispersed in center B for PN2
(12.5+/.3.9mm vs. 13.5+/-3.8mm; p<0.005) but
not for PN1 (16.9+/-4.2mm vs. 16.4+/-3.9mm;NS)
• Asymmetry of PN1 and PN2, in terms of size
and NPB number and distribution, was
observed in most zygotes from both centers.
The predominant zygote pattern in Center B
1) Higher NPB number in PN1 compared to
PN2
2) Higher NPB dispersion in PN1 compared to
PN2
3) Larger radius of PN1 was more frequently
observed (55% vs. 41.2%: p<0.02)
PGD
• Initial proposal for severe genetic
disease – 15 years later other
indications late onset disease e.g.
Huntington’s and more recentlyinherited cancer – PND generally not
acceptable ? PGD
• A list of indications offered by
various participating centers are
regularly updated on the ESHRE
website. (Over 80 protocols for
disease using PCR has been
developed)
PGD
• Appears to enhance selection
• Most often offered to patients with
recurrent miscarriage, repeated IVF
failures, Azoospermic patients
treated with TESE-ISCI and women
of advanced age.
• Implantation rate improvement and
decrease in miscarriage rate yet to
be proven when number of embryos
transferred is not limited
PGD - mosiacism
Problem of mosiacism – ? 1 cell ? 2 cell
analysis, a number of misdiagnosis were
reported both for PCR and FISH.
If 2 cells were biopsied for PGS at 8 cell
stage, and that 2 of the cells were
abnormal in the blastomere, there is a 53%
chance it is reported as normal for only the
normal cells are analysed, still leaving the
abnormal moscia cells in the blastomere,
43% chance that one of the biopsied cells
is abnormal, and only 4% chance that both
abnormal cells are biposied .
If 6 cells in the cell blastomere were
abnormal, there is a 4 % chance that only
the normal cells are biposied and hence
reported as normal.
PGS in IVF : a metaanalysis
• Twisk et al. Cochrane library 2006
• PGD in IVF
Control PGDS
Live birth
15%
4-17%
Ongoing pregnancy 20%
8-21%
PGS for repeated IVF failure – no eligible
PGS for repeated miscarriage- no eligible
Only 6-8 pairs of chromosome
analyzed – cost and time
PGD
• McArthur et al, Sydney
• Experience with day 3 biopsy vs day
5or 6 blastocyst biopsy of the
trophoblast cells for PCR and FISH.
• Higher implantation rate per embryo
transferred (29% vs 41%)
• Higher clinical implantation rate
(28%vs39%)
• Lower miscarriage rate
• Higher take home baby rate at normal
birth weight.
PGD -Does day 3 diagnosis
predict day 5 diagnosis?
• Baart et al Hum Reprod 2006
• Biopsy of 2 blastomeres done
on day 3 for FISH analysis, and
the entire embryo reanalysed on
day 5, they found that there is
only a overall confirmation rate
of the correct diagnosis in 54%
of cases.
PGD
? Correlate morphology and PGD
results
Polar bodies analysis
Complete genomic hybridization –
more accurate but labour intensive,
takes about 5 days so all biopsied
embryos needs to be frozen –
microarray platform maybe way
forward.
Pre-implantaion
molecular screening
• Amino acid turnover as prognosis
• Preimplantation embryos can consume and produce
amino acids in a manner dependent upon the stage
of development that may be predictive of
subsequent viability.
• Otsuki et al Japan reported on the consumption of
total and various amino acids by oocytes in
lipofuscinogenesis (which is postulated in normal
aging to be due to proteolytic degradation and/or
oxidative stress) and the rate of blastocysts
development in relation to the refractive bodies size.
• Lipofuscin is a complex lipid and degenerated
material found in the refractile bodies of the
ooplasm, which can be stained by the Schmorl
method.
• They reported on the development competence of
oocytes with various refractive bodies size (<3, 3-5,
Pre-implantaion
molecular screening
• Blastocyst development - cultured
for 5-7 days
• 5.6% (1/18) when lipofuscin bodies >5mm
• 24.2% (8/33) when lipofusin bodies 35mm
• 32.2% (82/267) when lipofusin bodies
<3mm
Total amino acid concentration is
significantly reduced,p<0.05 as well as
several amnio acids (Glutamic acid,
Arginine, Alanine, Citrulline, Cystine,
Ethanolamine, Otrnitine), p<0.01 in the
follicular fluids which contained oocytes
with larger lipofuscin bodies.
Pre-implantaion
molecular screening
• Glucose consumption and lactate
production by preimplantation embryos
Sallam et al Egypt reported on the
relationship between glucose consumption
and lactate production and the clinical
outcome for 51 patients treated with ISCI
Results showed significant increase in
glucose consumption per embryo per hour
compared to those who didn’t and lactate
production was also increased but did not
reach statistical significance.
Suggested that based on their calculations
the optimal cutoff is 125pg/embryo/hr at a
sensitivity of 76.9% (95% CI = 46.2-94.7)
and a specificity of 95.7% (95% CI = 78.099.3)
Pre-implantation
molecular screening
• HLA-G and implantation
• HLA-G is a non-classical HLA class I gene
with restricted tissue distribution and
several isoforms including membrane
bound and soluble.
• Several reports have described presence of
HLA-G molecules present in some embryo
culture supernatants as marker for the
prediction of pregnancy outcome after IVF
(Fuzzi et al 2002, Sher et al 2004), but
others report no soluble HLA-G detection
( van Lierop et al 2002, Noriko et al
2004).This could be due to the
reproducibility and sensitivity of the HLA-G
Elisa method. Le Bouteiller, France
reported an improvement in the sensitivity.
Pre-implantation
molecular screening
• HLA-G and implantation
• Its function depends on the specific
targeted receptors expressed by the
maternal cells present at the fetal
maternal interface – blood of the maternal
intervillous space, decidua basalis at the
site of implantation.
• 5 receptors has been described to date
CD8, IL T2, IL T4, KIR2DL4 and CD160
• Upon engagement of these receptors,
maternal decidual NKcells, CD8+ and CD4+
T cells, macrophages (and maybe others)
can be abrogated or modulated which in
turn contribute to the control of the
maternal immune response against fetalderived alloantigen expressed by
trophoblast cells.
In vivo selection
• Maybe of benefit
• Hohmann et al. JCEM 2003
• RCT Mild stimulation protocol
(late follicular start) vs
Standard long stimulation
• 61% vs 29% with embryos
scoring 1
In Vivo
• Baart et al. Human repro 2005\
• Mild (day 5 start with 150rFSH)
vs conventional (day 2 start
with 225IU)
• Embryo biopsy and FISH
analysis day 3
• Average number
• Oocytes 8 vs 12
• Embryos 6 vs 4
• Normal embryos 2 vs 2
Conclusion
• Seeing is not believing
• Morphology is insufficient
• Pre-implantation genetic
screening is unreliable
• In vivo embryo selection may be
helpful
• Pre-implantation Molecular
Screening may the future