THE ROLE OF EMBRYO ON IMPLANTATION Başak Balaban, BSc VKV American Hospital Assisted Reproduction Unit, Istanbul, Turkey AMERICAN HOSPITAL

Factors predicting live birth rates Clinical factors: *Women’s age *No.of retrieved oocytes *Aetiology of infertility *No.of embryos transferred....... Embryological factors * QUALITY * Developmental stage Elizur et al.,RBM Online 2005

Developmental Stage?? Day 2 vs. Day 3 ET Day 3 vs. Day 5 BT

Embryo quality&Implantation Transfer of good quality embryos results in higher pregnancy and implantation rates Highest implantation rate with the minimum number of embryos transferred (SET) should be our final goal Therefore selection of the TOP QUALITY EMBRYO/S for transfer is an essential process that should be achieved in IVF lab.s Hsu F&S 1999, Terriou F&S 2001, De Neubourg HR 2004, Schmidt 2005

IN VITRO CULTURE GOOD QUALITY EMBRYO: MOST VIABLE EMBRYO WITH the HIGHEST IMPLANTATION POTENTIAL Sperm IN VITRO CULTURE BLASTOCYST OOCYTE Early Cleavage PN Morphology Day 2 embryo Day 3 embryo Multinucleation 0 16-18 24-25 48 72 96-120

MORPHOLOGICAL ABNORMALITIES OF MII OOCYTE Extracytoplasmic abnormalities **Shape abnormalities (irregular shape of MII oocyte) **ZP abnormalities (dark or thick ZP) **PVS abnormalities (large PVS, PVS granulation) Cytoplasmic abnormalities **Dark cytoplasmic colour (slight granulation) ** Excessive whole or centrally located granulation **Refractile bodies,sERC or vacuoles in the ooplasm

Smooth Endoplasmic Reticulum Clusters (sERC) and vacuolization appearance in MII oocytes 2.1 % IR for sERC(+) cycles Otsuki et al.Hum.Reprod.,2004 EBNER et al. 2008. Higher incidence of obstetric problems, two babies born with imprinting diseases??, abnormal regulation of Ca2+ signaling..

Oocytes without vacuoles (n=1151) Effect of cytoplasmic vacuoles throughout embryo development Oocytes without vacuoles (n=1151) Vacuolar Oocytes (n=47) (3.9%) p value Single vacuole Two vacuoles Multiple vacuoles 31(66) 10 (21.3) 6 (12.7) Fert. (%) 51.6% 43.8% 65.3% 48.9% <.05 Mean diameter of vaculoes with fertilization :9.8m Mean diameter of vaculoes with FF :17.6 m (p<.05) Cut off value :14 m Patients variables similar between vacuole(+) & (-) cycles Ebner et al. F&S, 2005

Both normal and CLCG oocytes Very low OPR% are obtained with the transfer of embryos obtained from CLCG oocytes Both normal and CLCG oocytes Only CLCG oocytes Total No.of cycles 21 18 39 No.of pregnancies 6(28.6) 5(27.8) 11(28.2) Abortion 4(19.0) 2(11.1) 6(15.4) Ongoing pregnancies 2(9.5) 3(16.7) 5(12.8) CLCG-Centrally located granular cytoplasm Fert%+embryo quality: no sig., sig.higher aneoploidy Kahraman&Yakin et al.HR.,2000 Balaban et al., 2007 also reported sig. higher aneoploidy with CLCG

Tesarik,Hum Reprod,1999 Scott,RBM Online,2003 PN MORPHOLOGY

PN score&IR&Deliviries Scott et al.,Hum.Reprod. 2006

Pronuclear patterns& aneuploidy rate Ideal PN pattern Single PN anomaly Double PN anomaly No. Pre-embryos 28 240 41 Cleavage (%) 28 (100) 224 (93.3) 31 (75.6) Grade I + II emb. (%) 21 (75) 126 (56.2) 16 (51.6) Early cleavage (%) 7 (25) 18 (8) ≥8 cell emb. on day 3 (%) 13 (46.6) 56 (25) 3 (9.6) Aneuploidy (%) 7 (25.9) 134 (73.6) 25 (83.3) Blast. from euploid Emb. (%) 90 62.5 33.3 Blast. from aneuploid emb. (%) 28.5 10.4 4 Balaban et al., RBM Online,2004

Relationship between pronuclear morphology, cleavage rate, blastocyst progression, blastocyst grade, and multinucleated blastomeres PN Pattern Equal Sized 2PN Early Cleavage ≥8 cell embryo on D3 Blast BG1+BG2 Hatch MNB* Ideal PN 109 16.1% 27 25.2% 58 54.2% 77 71.9% 61 79.2% 22 28.5% 4 3.7% Single PN Anomaly 477 70.5% 39 8.6% 139 30.9% 160 35.7% 79 49.3% 19 11.8% 59 13.1% Double PN 90 13.4% 3 4.5% 11 16.7% 15 12.7% 7 46.7% 1 6.7% 8 12.1% Balaban, Hum Reprod, 2001

Cleavage rate Blastomere number Slow cleavage Fast cleavage Day 2 embryos that cleave to 4-cell stage result in higher implantation (Giorgetti 1995, Ziebe 1997,Scott 2006) Embryos with at least 4-5 blastomeres on day 2 and at least 7 blastomeres on day 3 yield higher implantation rates (Gerris 1997, Van Royen, 1999,2001) Slow cleavage Lower implantation potential and blastocyst formation should be expected (Alikani 2000) Fast cleavage High levels of mosaicism and polyspermic fertilization (Harper 1994) > 70% chromosomally abnormality (Magli 1998)

Cleavage stage embryo morphology Grade 1 embryo: no fragmentation with equal sized homogenous blastomeres Grade 2 embryo: < 20% fragmentation with equal sized homogenous blastomeres Grade 3 embryo: 20-50% fragmentation with unequal sized blastomeres Grade 4 embryo: >50% fragmentation with unequal sized blastomeres. (Puissant 1987, Staessen 1992, Steer 1992, Ziebe 1997)

Uneven blastomeres Hardarson, Hum Reprod, 2001 Grade I: no fragments, even-sized blastomeres and light, homogeneous cytoplasm Grade IIA: <20% fragments Grade IIB: uneven-sized blastomeres Grade IIC: non-homogeneous cytoplasm Grade III: >20% Grade IV: >50% Hardarson, Hum Reprod, 2001

Cell symmetry&IR&Deliviries Scott et al.,Hum.Reprod.2006

CPR & IR according to the degree of fragmentation Fragmentation rate (%) Total no.of procedures CPR(%) IR(%) 0-5 1062 63.0 31.8 6-15 533 57.8 28.2 16-25 64 51.6 23.4 26-35 41 46.3 >35 27 14.8 6.4** **p<.001 Mean no.of ET:3.2 Alikani et al.,F&S 1999

Embryo fragmentation Patterns Alikani, Fertil Steril, 1999 Type I: minimal in volume and fragments associated typically with only one blastomere Type II: localized and predominantly occupy perivitelline space Type III: small, scattered fragments, these may be in cleavage cavity or peripherally positioned Type IV: large fragments, sometimes resembling whole blastomeres, often distributed randomly and associated with uneven cells Type V: fragments appear necrotic with a characteristic granularity and cytoplasmic contraction within the intact blastomeres Alikani, Fertil Steril, 1999 Sig. lower IR reported with T4

Presence of early cleavage and outcome of ICSI Characteristic No early cleavage Early Cleavage P No of cycles 30 48 Mean age (years) 34.2 33.1 NS # oocytes 11.9 12.8 Fertilization Rate 74.8 77.3 Mean no of ET 3.9 4.0 Implantation Rate 5.1 17.9 < 0.05 Clinical PR/ET 16.7 45.8 Blastocyst progression of excess embryos 25% 56.7% <0.05 Isiklar&Balaban, J Reprod Med, 2002 Strong indicator of embryo quality (Shoukir 1997, Sakkas 1998, Bos-Mikich 2001, Lundin 2001, Lundqvist 2001, Isiklar 2002, Fenwick 2002, Salumets 2003,Ciray 2004,2005,2006,Windt 2004,Montfoort 2004,Fancsovits 2005)

The impact of presence of one or more multinucleated blastomeres on the developmental potential of the embryo until the blastocyst stage Groups n Cleavage on day 3 (%) Blastocyst formation (%) BG1-2 blasts (%) Day 2 embryo without MNB 6414 91,6 51,0 30,7 with 1 MNB 643 56,0 6,4 2,6 with >1 MNB 461 38,8 2,4 0,9 Day 3 embryo 248 - 3,2 216 Yakin&Balaban et al., F&S 2006 Impaired developmental capacity (Alikani 1999, Jackson 1998, Balakier 1997) Correlated with chromosomal abnormalities (Staessen 1998, Kligman 1996, Pickering 1995)

State of nucleation&IR&Deliviries Scott et al.,Hum.Reprod.2006

CUMULATIVE EMBRYO SCORING TO DETERMINE EMBRYO VIABILITY&IMPLANTATION POTENTIAL Sig.higher blast. formation and IR with embryos scored O Rienzi et al.,RBM Online 2005

Cleavage stage embryo charecteristics & IR Sjoblom et al., F&S 2006 Lesourd et al., RBM Online 2006

Embryo quality& Implantation Retr.anal. of 2266 IVF/ICSI double emb day 2ET, ES: Bno+frag.+Bequality+cleavage symmetry+ MNB Holte et al.,Hum.Reprod 2007

Effect of Embryo Charecteristics on Ongoing IR 449 SET in natural IVF cycles, cohort study Pelinck et al.,F&S 2010 in press

ALPHA & ESHRE EMB.SIG CONCENSUS WORKSHOP ON EMBRYO GRADING Concept: 2 day meeting of experts to present, discuss and agree on key aspects required for embryo selection/grading. The experts were asked to join actively with the expectation that all will agree and endorse a final grading scheme at the conclusion of our meeting.   Venue : Istanbul Date : February 26th-27th. 2010 29

CONCLUSIONS Do embryos have any role on implantation and LBR? YES!! The quality of the embryo is directly related with the implantation potential Can morphological observations used in IVF labs. in practical routine help us to determine embryo quality/viability/IMPLANTATION POTENTIAL YES.Irrespective of the stage at which the selection is made,a single observation appear to be insufficient to accurately predict developmental ability. More observations should be conducted at different stages, before and after fertilization, given the fate of the embryo is often decided during oogenesis Is there any parameter to select a top quality embryo with 100% implantation potential ? NO. In addition/without morphological diagnostic tools more predictive markers (molecular, biochemical.....) are still needed