Preimplantation Genetic Screening for IVF: Any Benefit for Baby Take Home rate Eray ÇALIŞKAN MD Obstetrics and Gynecology Bahçeşehir University School Of Medicine, Turkey «scientia et amore vitae»

Definition Preimplantation Genetic Diagnosis To search for genetic disease known to be present in one or both of the parents Preimplantation Genetic Screening To screen the embryo for the presence of uneuploidy where the parents are known to be genetically normal

Laser Q PCR aCGH Blastocyst NGS Embryo Biopsy Technique Chemical Mechanical FISH-PCR Q PCR aCGH NGS Polar body Blastomere Blastocyst Staff Experience Laboratory Quality Implantation On going Live birth Single ET Double ET Embryo Transfer Mosaicism Pregnancy Day 5 Day 6 Day 7? Fresh ET Frozen ET -All freeze Per cycle Per transfer

PGS TECHNICAL PROBLEMS Failure to obtain genetic material Nuclear abnormalities Multinucleation Binnucleation Apoptotic Anuclear The single cell may not represent the embryo Mozaicism 20%

Rationale for Preimplantation Genetic screening? The reason for implantation failure is uneuploidy in 50% of the cases 70% of uneuploidies occur during the first meiotic division of the oocytes Blastocysts with good morphology have 30% uneuploidy rate Munne et al., RBM online, 2003

PGS 1.0 FISH based PGS

FISH BASED EXPERIENCE RCT revealed that PGS did not increase baby take home rates Staessen et al 2004, 2008; Mastenbroek 2007; Hardarson 2008; Meyer et al 2008; Debrock 2009) BUT FISH first generation technology 8 chromosome FISH – worst scenario FNR 27% Embryo biopsy experience of the centers ? The timing: polar body, day 3 and blastocyst Johnson et al, Hum Reprod, 2010

FALSE POSITIVE RATE IN FISH TECHNOLOGY When FISH diagnosis was monosomy 65% of embriyos were euploid with array CGH When FISH diagnosis was trisomy 47% of the embryos were found to be euploid by array CGH When FISH diagnosis was complex uneuploidy 63% of the embryos were euploid by array CGH Northrop et al, Hum Reprod, 2010

FISH technology Array CGH Allel drop out per probe in FISH technology 2-3% 6 probe False diagnosis up to 12-18% 8 probe False diagnosis up to 16-24% False diagnosis in all 23 chromosomes 2% Random loss of one allel Over amplification Under amplification Handyside 2004, Rendwick 2006, Spits 2006, Glentis 2009

Spontaneous Abortion Data Bulgu FREKANS 45,X 18 Triploidi 17 Trisomi 16 16.4 Trisomi 22 5.7 Trisomi 21 4.7 Trisomi 15 4.2 Trisomi 13 3.7 Trisomi 18 3 Diğer trizomiler 14.3 Tetraploidi 6 Dengesiz translokasyonlar Diğer 4 51%

PREIMPLANTATION GENETIC SCREENING Was aimed to diagnose most common chromosomal abnormalities found in spontaneous abortions Triploidi Monosomi X Trizomi 16 Trizomi 22 Tetraploidi Trizomi 15 Trizomi 21 Common chromosomes screened with FISH 13,18,21, X, Y – 16, 22 PPV 51% - 16% FNR

VIABILITY AND IMPLANTATION Abortion data and viable uneuploidies do not provide enough information about in vivo embryos, some uneuploidies can implant BUT We were unaware of never implanting chromosomal aberrations? We had the dogma that several chromosomes have a tendency for uneuploidy?

557 embryo test – GENETİKON-Arrayland 8 probe FISH 45% Detection Rate Drop-out %16-24

PB 1 Blastocysr ICM 1

PGS 2.0 Array CGH Next Generation Sequencing

Euploid Trophoectoderm

NGS vs a CGH 100% concordance Similar pregnancy Yang et al, BMC Medical Genomics, 2015

qPCR EUROFERTİL® Flourescent carrying DNA primers Flourescents detected and analyed by computerized systems EUROFERTİL®

384 well plates are used Chromosomal copy numers are calculated Decision for euploid/uneuploid using chromosomal copy numbers

Concordance between ICM and trophectoderm Concordance for euploidy confirmed by CGH or aCGH (97-100% concordance). Fragouli et al., Hum Reprod, 2008 Capalbo et al., Hum Reprod, 2013 - Uneuploid cells are more common in the trophectoderm Gleicer and Orvieto J Ovarian Res 2017 -Uneuploid trophoectoderm and euploid inner cell mass discrepency is up to 50% Gleicher et al RBE 2016

MOSAICISM Sequential trophoectoderm and inner cell mass biopsy 42.3% all cells are euploid 30% all cells are uneuploid 32.4% si mosaic 15.4% mixed uneuploidy from different cell lines, 17% both euploid and uneuploid cells together Mosaic diploid– 6% of the total embryos (20% of uneuploid blastocysts have euploid inner cell mass)

Mosaicism Trophoectoderm biopsy 18 mosaic embryo transfer 6 euploid delivery 5 single uneuploid transfer 3 euploid embryo 6 Uneuploid dx first than, euploid in another lab test with array CGH, ET and 5 euploid baby one biochemical pregnancy All these questions the true diagnosis of uneuploidy Gleicher Fertil Steril 2015, Greco Curr Genom 2012, Gleicher RBE 2016

Abnormalities reaching blastocyst stage Arrested Arrested reached day 3 day 4 blastocyst Euploid: Total 9 (28%) 2 (6%) 21 (66%) Aneuploid: monosomies 14 7 2 (9%) trisomies 15 7 14 (39%) and mosaics (>38% abn.) 19 5 0 (0%) Total 48 (58%) 19 (23%) 16 (19%) Other mosaics: 2n/4n (>38% abnormal) 3 3 3 (33%) 2n/aneuploid or chaotic 54 12 8 (11%) Total 57 (70%) 15 (18%) 11 (13%) Polyploid: 11 (79%) 0 3 (21%) Haploid: 4 (80%) 1 (20%) 0 (0%) Sandainas et al, Hum Reprod, 2001

Advanced Emryo Cultures: More Uneuploids Reach Blastocyst Stage trisomy Monosomy complex mosaic Northrop et al 2010 6 5 1 12 Fragouli et al 2011 7 2 14 Colls et al. 2013 16 10 % of total abnormal 31% 24% 10% 35% Sandalinas et al. 2010 46% 7% 10% 36% Fragouli et al. Hum. Reprod. 2011, Northrop et al, Mol Hum Reprod. 2010, Colls et al. ASRM, 2013, Sandalinas et al., Hum Reprod, 2010

Morphological Evaluation of Embryos and array CGH results Embryo Grade Euploid 111 222 33 1.5 Mosaic Eploid 11111 2222222 333 1.9 Chaotic Euploid 11 222 333 1.7 Uneuploid 1111 2222 333 2.2 Segmental Errors 111111 222 3 1.4 Johnson et al, Hum Reprod, 2010

TÜMGEN® and EUROFERTİL ® Grade I uneuploid ⁺ 11 ⁺ 15 ⁺ 20 Grade I uneuploid ⁺ 11 ⁺ 15 ⁺ 20 Male 47 XYY, female 46 XX TÜMGEN® and EUROFERTİL ®

TÜMGEN® and EUROFERTİL ® + 2, +5, +12, - 13, +18 Euploid Mixed Uneuploid -2, -11p TÜMGEN® and EUROFERTİL ®

TÜMGEN® and EUROFERTİL ® -1 +19+22 Euploid +6 +12+15+19 -1,+5,+6,+12,-13,-14,+18 TÜMGEN® and EUROFERTİL ®

Effect of D 3 vs Trophoectoderm biopsy cleavage stage blastocyst biopsy not biopsy not Implantation rate 31% 53% 52% 54% P<0.05 N.S. Blastocyst biopsy, more DNA-less no results, less mosaicism, reduced impact of embryo biopsy, less embryos to process, facltates single embryo transfer, uterine environment optimized after frozen cycle. Disadvantages: not all embryos reach blastocyst stage Treff et al., Fertil. Steril. 2011

No results day 3 vs Trophoectoderm biopsy Biopsy Embryos Centers* stage undiagnosed range Cleavage 3.2% 1% - 5% Blastocyst 2.3% 0% - 18% Most experienced Untrained Munne S, 2013, ASRM

Euploidy decreased with age but not with cohort size # of blastocysts % normal embryos egg donors <35 years 35-37 years 38-40 years 41-42 years >42 years 1-3 65% 63% 50% 40% 22% 14% 4-6 60% 61% 52% 39% 27% 17% 7-10 64% 62% 53% 25% 16% >7-10 56% 43% 29% N = 3,571 cycles and 19,356 embryos, up to 8/2013. Ata, et al. Reprod Biomed Online, 2012

% of patients with normal embryos Percent of Women with Euploid Embryos: ET possible # of blastocysts % of patients with normal embryos egg donors <35 years 35-37 years 38-40 years 41-42 years >42 years 1-3 86% 85% 72% 60% 58% 24% 4-6 95% 97% 88% 69% 54% 7-10 100% 99% 96% 92% 65% >7-10 98% 83% N = 3,571 cycles and 19,356 embryos, up to 8/2013. Ata, et al. Reprod Biomed Online, 2012

Cycles with no Euploid Embryos Harton, Munne et al., Fertil Steril, 2013

On Going Pregnancy rate / ET Harton, Munne et al., Fertil Steril, 2013

Overall Pregnancy Rate * Harton, Munne et al., Fertil Steril, 2013

CGH and frozen transfer Cycles Mat. Prev. embryos implant. age failed replaced (+ sac) cycles CGH : 45 37.7 2.4 2.0 72% control : 113 37.1 1.2 2.7 46% p=0.0003 Schoolcraft et al., Fertil. Steril., 2010

aCGH + fresh transfer, <35 years old Control PGD (aCGH) Patients 48 55 Maternal age <35 <35 Biopsy on day 5 no yes Transfer on day 6 day 6 Embryos euploid (N) n/a 53.2% (425) Embryos replaced (av.) 48 (1) 55 (1) Pregnancy rate (sac) 45.8% 70.9% p<0.05 Ongoing preg rate 41.7% 69.1% p<0.05 Multiple pregnancies 0 0 Yang et al. Molec Reprod, 2012

qPCR + fresh transfer PGS Control age 32.2 N 72 83 blastocysts 8 7.9 Good prognosis patients (average 8 blastocysts) Control replaced on day 5, test biopsied on day 5 and replaced on day 6 PGS Control age 32.2 N 72 83 blastocysts 8 7.9 Emb replaced 1.9 2.0 implantation 79.8% 63.2% P=0.002 Sustained implant 66.4% 47.9% P=0.03 Delivery rate 84.7% 67.5% P=0.01 Scott et al., 2013 Fertil Steril.

BEST trial: One tested euploid vs. Two untested Delivery Rate   1 euploid blastocyst 2 untested blastocyst Initial Fresh and Frozen transfer 60.6% 65.1% NS Twin 30% Frozen transfer 41.1% 46.1% 6.2% 23% Forman et al., Am J Obstet Gynecol, 2014

PGS for Idiopathic Recurrent Pregnancy Loss PGS decrease miscarriage in women with idiopathic previous recurrent pregnancy loss Werlin et al, Fertil Steril, 2003, Munne et al, Fertil Steril 2005, Garrisi et al, Fertil Steril 2009, Rubio et al, RBM Online 2009, Hodes-Wertz et al, Fertil Steril 2012

Results of PGS by aCGH for RPL: biopsy stage effect day preg. % loss % loss biopsy cycles expected after PGS Day 3 59 36% 9% p<0.001 Day 5 40 42% 3% p<0.001 Total 99 38% 6% p<0.001 Hodes-Wertz et al., Fertil Steril, 2012

What do meta-analysis tell us?

Implantation Chen et al, PLOSone, 2015

RCT Dahdough et al, Fertil Steril, 2015

Observational Studies Dahdough et al, Fertil Steril, 2015

Clinical Pregnancy Rate Chen et al, PLOSone, 2015

Ongoing Pregnancy Rate Chen et al, PLOSone, 2015

Live Birth Rate Chen et al, PLOSone, 2015

Miscarriage Rate Chen et al, PLOSone, 2015

Multiple Pregnancy Rate Chen et al, PLOSone, 2015

Women aged 40-43 Lee et al, J Asist Reprod Genet, 2015

Recently Challenged Ideas Most IVF cycles fail because of aneuploid embryos Their elimination prior to embryo transfer will improve IVF outcomes A single trophectoderm biopsy (TEB) at blastocyst stage is representative of the whole TE TE ploidy reliably represents the inner cell mass (ICM) Ploidy does not change (i.e., self-correct) downstream from blastocyst stage

CONCLUSION Mosaicism may decrease the chance of embryo transfer in women with few embryos after PGS PGS significantly improves ART outomes per embryo transfer not improves per started cycle PGS eliminates the effect of maternal age on implantation Poor Responder Women reported to have no uneuploid embryo for transfer can have second biopsy or transfer