New development on implantation

Report from ESHRE2006

• Dr Clement Ho• MB BS (HK), FRCS (G), FRCOG, FH

KCOG, FHKAM (Obstetrics & Gynaecology)

• Dr Alexander K. Doo• MB ChB (G), MRCOG,• FHKCOG, FHKAM (Obstetrics & Gynae

cology)

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 cleav

age) • 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 ass

ociated 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 formation4000 embryos – looked at nuclear alignment, nucl

eolar 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 d

ay 3• Number of multinucleated blastomer

es• 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 e

mbryos• 106 embryos, 4 cell stage analyzed• FISH performed (13,15,16,18,21,22,X and Y)• Embryos in 3 group

All mononucleated blastomereAt least one multinuclear blastomereAll 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 Euploid Mosaic AneuploidGroup A (%) 45 42 13Only mononuclear cells sgr (%) a vs b

86 vs 14Group B (%) 0 56 44Mono/Multinuclear cells sgr (%) a vs b

38 vs 62Group C (%) 0 0 100Only 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 for selection

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 sho

wed 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 bifring

ence.• 7 embryos HZB/HZB, 9 embryos HZB/LZB, and 14 e

mbryos 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 frag

mentation 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 associ

ated with nucleoprotein filaments• 65 sperm sample : 8 N, 50 OAT, 7 TESE49 fresh 16 thawed oocytes (ICSI birefringent sperm) vs 47 fresh

16 thawed (control)

Morphology - sperm Normal OAT TESE

% Bi 88.1+/-11.3 55.5+/-28.6 14.3+/-9.1Range 65-97 8-98 4-32

OAT sample :Progressive Motility No progressive motility65.9+/-24.8% range 8-98 28.8+/-19.1% range 12-71

Fertilization rate Study Control p value80% 66% 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 clea

vage 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

• Uterine environment• Embryonic genome• Selction criteria• Embryos for transfer• Monozygotic twinning• Sex ratio• Cryopeservation

Morphology Computer assisted multilevel analysis

• Lausanne/Linz group2PN zygotes computer program quantitative analy

sis of digital images• 40 features were measured• Main features are zygote and PN size, PN posi

tion, cytoplasmic halo area, number and distribution of nucleolar precusor bodies (NPBs)

Morphology Computer assisted multilevel analysis• 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 be

tween 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+/-1.4; NS)

Morphology Computer assisted multilevel analysis• NPB were less dispersed in center B for PN2

(12.5+/.3.9m vs. 13.5+/-3.8m; p<0.005) but not for PN1 (16.9+/-4.2m vs. 16.4+/-3.9m;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 B1) Higher NPB number in PN1 compared to PN22) Higher NPB dispersion in PN1 compared to PN23) 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 recently- inherited 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 recurren

t 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 - mosiacismProblem of mosiacism – ? 1 cell ? 2 cell analysis, a nu

mber 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 meta-analysis• 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 eligiblePGS for repeated miscarriage- no eligibleOnly 6-8 pairs of chromosome analyzed –

cost and time

PGD

• McArthur et al, Sydney• Experience with day 3 biopsy vs day 5or 6 bl

astocyst biopsy of the trophoblast cells for PCR and FISH.• Higher implantation rate per embryo transferre

d (29% vs 41%)• Higher clinical implantation rate (28%vs39%)• Lower miscarriage rate• Higher take home baby rate at normal birth wei

ght.

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 resultsPolar bodies analysisComplete genomic hybridization – more accu

rate 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 a

cids 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, >5m)

Pre-implantaion molecular screening• Blastocyst development - cultured for 5-7 d

ays• 5.6% (1/18) when lipofuscin bodies >5m• 24.2% (8/33) when lipofusin bodies 3-5m• 32.2% (82/267) when lipofusin bodies <3mTotal amino acid concentration is significantly red

uced,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 ISCIResults 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 = 78.0-99.3)

Pre-implantation molecular screening

• HLA-G and implantation• HLA-G is a non-classical HLA class I gene with restri

cted 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 recep

tors 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 fetal-derived alloantigen expressed by trophoblast cells.

In vivo selection

• Maybe of benefit• Hohmann et al. JCEM 2003• RCT Mild stimulation protocol (late fol

licular 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 con

ventional (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