Differences in chromosome abnormalities in eggs and embryos between fertility centers Santi Munné

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Morgan McCarthy
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1 Differences in chromosome abnormalities in eggs and embryos between fertility centers Santi Munné

disclosure None (lol) Except: - Chief Scientific Officer of CooperGenomics - Founder @ Reprogenetics (PGS/PGD company), sold to Cooper - Founder @ Recombine (Carrier Screening

2 disclosure None (lol) Except: - Chief Scientific Officer of CooperGenomics - Reprogenetics (PGS/PGD company), sold to Cooper - Recombine (Carrier Screening company), sold to Cooper - Phosphorus (genomics as a service) - MedAnswers (DTC advise on genetics and infertility) - Board advisors PreVivo (alternative to IVF)

3 Learning objectives 1) Not all PGS techniques are equal. Only hr-ngs can detect mosaics. 2) Aneuploidy increases with advancing maternal age but mosaicism does not 3) PGS v2 increases OPR: evidence from 5 CRTs 4) In addition of maternal age, IVF treatment can also induce chromosome abnormalities 5) PGS can be used as QC for optimizing ART procedures

4 Detection of Aneuploidy, segmentals and Mosaicism By hr-ngs

5 Comparison of current PGS platforms % embryos FISH qpcr acgh Embryo Vu SNP array hr-ngs Total Independent Data Signals* ,700 26,000 32, ,000 Resolution in Mb arm 20M 6M 20M 6M 3M Misdiagnosis aneuploides (a-f) 7% 1% 2% 3% d 2% 0% Unbalanced translocations (g) 2% custom no yes no yes yes Partial aneuploidies 5% no no yes some yes yes Polyploidy 2% yes no no no yes yes Mosaicism (h, i) 20% 20% no 4% no no 20% Miscarriage rate (j, k) 10-20% 20% 13% unk unk 11% a Gutierrez-Mateo et al (2011) Fertil Steril, b Scott et al. (2012), c Treff et al. (2012) Fertil Steril 97:819 24, d unpublished 7 misdiagnoses of 265 samples; e Kung et al. (2015) Reprod Biomed Online,, f Wells et al. (2014) J Med Genet, g Yeobah et al. (2015) ASRM, h Greco et al (2016) NEJM, i Tormasi et al (2015) PGDIS, ASRM. J Rodriguez-Purata et al. (2016) JARG; k Friedenthal et al. (2017) ESHRE * 24M reads per run, 24 samples per run, 30% reads lost = 700,000 reads per sample

6 Mosaicism: Common in day 3 embryos 30% of day-3 embryos were mosaic by FISH. The majority with all cells abnormal: 3[13]1[16]2[18]1[21]3[22] 1[13]1[16]1[18]1[21]1[22] <49% abnormal % abnormal % abnormal 528 1[13]1[16]2[18]2[21]1[22] 3[13]1[16]2[18]1[21]3[22] Munné S, Grifo J, Cohen J, Weier HUG Am J Hum Genet 1994; 55: Munné S, Weier HUG, Grifo J, Cohen J Biol. Reprod. 1994; 51: Colls et al. Fertil Steril 2007;88: [13]1[16]2[18]2[21]2[22] 1[13]1[16]2[18]2[21]1[22] 2[13]1[16]2[18]1[21]1[22] 1[16] 2[13]2[16]2[18]2[21]2[22] 2[13]3[18]1[21]1[22]

7 Higher dynamic range allows NGS to detect mosaics hr NGS Higher dynamic range acgh

8 High resolution NGS vs. acgh: Two center experiences acgh NGS p-value SET transfers Age NS IR (%) 63.9% 71.6% 0.01 OPR (%) 53.1% 61.9% Friedenthal, Maxwell, MD, Munné, Kramer, McCulloh, McCaffrey, Grifo (2017) ESHRE acgh NGS p-value transfers Age NS IR (%) 41.6% 57.8% <0.05 OPR (%) 44.0% 65.7% <0.02 Macer, Barritt, Surrey, Danzer, Ghadir, Wang, Pisarska (2017) PCRS

9 Base line chromosome abnormalities in blastocysts

10 Euploidy decreases with age but not with cohort size (hr-ngs) % euploid blastocysts * # of embryos produced < egg donors years years years years >42 years % 43% 38% 26% 17% 12% % 46% 38% 27% 17% 12% % 47% 39% 30% 16% 11% >10 60% 49% 42% 25% 19% 16% Total 59% 47% 42% 30% 19% 14% N = 42,217 embryos, and 7,425 cycles, Reprogenetics data to 9/2016, * excludes mosaics

11 Prognosis depending on age and ovarian response (hr-ngs) # of embryos produced egg donors % of patients with normal blastocysts <35 years years years years >42 years % 67% 57% 44% 29% 19% % 92% 87% 76% 56% 44% % 98% 96% 91% 73% 50% >10 100% 100% 99% 95% 89% 81% N = 42,217 embryos, and 7,425 cycles, Reprogenetics data to 9/2016, * excludes mosaics

12 N = 103,405 embryos. Reprogenetics and Genesis Genetics data to 1/2017 * Complex: >2 full abnormalities Abnormality rates by high resolution NGS Data from >100,000 embryos Egg donor < >42 Normal 59% 53% 44% 31% 19% 14% Mosaic 16% 18% 17% 13% 10% 8% Aneuploid (± mosaic) 18% 20% 28% 38% 41% 33% Complex (*) 7% 8% 10% 17% 28% 44% Polyploid 1% 1% 1% 1% 1% 1% Mosaics are MITOTIC and therefore do not increase with age Mosaics + Aneuploid and Mosaic show constant rates through age

13 Origin of Aneuploidies by karyomapping analysis Meiotic Errors 370 Maternal Origin Paternal Origin % 63 17% Gain Loss Gain Loss % % 61 Konstantinidis et al. (2016) ASRM 50.8% partial

maternal age and abnormalities detected by karyomapping analysis 100% 80% 60% 40% 20% 15% 36% 52% 24% 24% 26% Meiotic Errors Mitotic Errors 0% < 34

14 maternal age and abnormalities detected by karyomapping analysis 100% 80% 60% 40% 20% 15% 36% 52% 24% 24% 26% Meiotic Errors Mitotic Errors 0% < > 40 Maternal Age Meiotic errors: Difference statistically significant at P<0.05 and P< Mitotic errors: No difference detected (P>0.05)

15 PGS v.2 Overall clinical results

replaced 48 (1) 55 (1) Pregnancy rate 45.8% 70.9% P<0.

16 1 st Randomized trial: acgh + fresh transfer, <35 years old Control PGS patients age <35 <35 replacement Day 6 Day 6 replaced 48 (1) 55 (1) Pregnancy rate 45.8% 70.9% P<0.05 Ongoing preg rate 41.7% 69.1% P<0.05 multiples 0 0 Yang et al. (2012) Molec Reprod

17 2 nd randomized trial: qpcr + fresh transfer Good prognosis patients (average 8 blastocysts) Control replaced on day 5, test biopsied on day 5 and replaced on day 6 PGD Control age N blastocysts Emb replaced 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.

18 3 rd randomized trial: 1 tested vs. 2 untested ongoing pregnancy rate 1 euploid 2 untested blastocyst blastocyst Fresh transfer 65% 70% NS Frozen transfer 55% 52% NS Forman et al. (2013) Fertil Steril Mean maternal age 35 (patients <43)

19 4th randomized trial: acgh but day 3 biopsy, patients y.o PGD-A Non PGD-A p-value No. of cycles performed Implantation rate (IR) 53% 28% < Clinical pregnancy rate/ transfer 54% 43% NS Clinical pregnancy rate/ patient 37% 39% NS Miscarriage rate (%) 3% 39% Ongoing IR 49% 15% < Delivery rate/transfer 53% 24% Delivery rate/patient 36% 22% Rubio et al. (2017) In Vitro Fertilization with Preimplantation Genetic Diagnosis for Aneuploidies (PGD-A) in Advanced Maternal Age: A Randomized Controlled Study. Fertil Steril, in press

PGD/PGS 40% 30% 20% 10% 0% <35 35-37 38-40

20 Life birth rates / transfer SART 2014: Live Birth Rate per Transfer 60% 50% Not Tested PGD/PGS 40% 30% 20% 10% 0% < >42 Analyzed by David McCullough, PhD (NYU, NY)

21 Differences in euploidy rates between centers

22 Objective of the study Objective: To investigate if different fertility centers generate different aneuploidy rates. Problem: Aneuploidy rates could differ due to differences in maternal age and infertility indication. Solution: Differences in aneuploidy rates between Egg Donor cycles is probably ART treatment induced.

23 Aneuploidy rates per center in egg donors Confounders eliminated: - Egg donor age (excluded those >35 years old) - Infertility reason (all fertile) - Day of biopsy (day 5 and day 6 investigated) - Paternal age (using as proxy maternal age) - Other indications (male factor, sperm donation) - Cohort size (from <5 to >20 blastocysts) Confounders still present: - Center Munné et al. (2017) Human Reprod, in press

24 aneuploidy rates are significantly different between centers Cycles of PGS for egg donors: 1645 (13,595 blastocysts) Centers with >10 cycles: 42 Average egg donor (<35 yo): 25.5 Aneuploidy average: 31.0% Aneuploidy range: 18% to 60% p<0.001 Munné et al. (2017) Human Reprod, in press

25 aneuploidy rates are significantly different between centers 6 centers had below average rates and 3 above average rates Munné et al. (2017) Human Reprod, in press

26 Differences in other genetic abnormalities between centers

27 MOSAICISM: Significant difference between centers in egg donor embryos Center ID # N Euploid % Aneuploid % Mosaic % % 37% 16% % 26% 18% % 30% 24% % 25% 29% % 17% 31% % 17% 31% % 27% 33% % 19% 34% % 18% 44% 206 p= Sachdev et al. (2016) ASRM

28 PARTIAL ANEUPLOIDY: Significant Differences between centers Centers: 102 Partial aneuploidy: 0.5% (94/775) to 8% (9/116) (p<0.001) Full aneuploidy in donors: 18% to 43% (p<0.001)

29 MITOCHONDRIAL DNA: Significant difference between centers Embryos generated in 35 different IVF clinics in USA, and over 2000 euploid blastocysts Normal quantity Elevated quantity (low implantation)

30 What is causing these differences?

31 differences between doctors of the same center (study 1) 7 centers had several physicians with >6 egg donor cycles and PGS If doctors used same stimulation (5 centers): There were no differences in aneuploidy rates between physicians If doctors used different stimulation (2 centers): 1 centers with 4 physicians, 86 cycles, had 67% average euploidy rate - but one physician had 55% euploidy rate (p<0.05) 1 centers with 7 physicians, 406 cycles, had 68% average euploidy rate - but one physician had 74% euploidy rate (p<0.05) - another physician had 60% euploidy rate (p<0.05) - another physician had 57% euploidy rate (p<0.05) Munné et al. (2015) PGDIS

32 differences between doctors of the same center (study 2) Population: all patients (not only egg donors) years of age, year 2016, Test: PGS by hr-ngs Physician Total A B C D E Euploid (*) 40% 45% 38% 37% 42% 40% Simple Abnormal 29% 24% 30% 30% 25% 30% Complex Abnormal 11% 9% 13% 14% 9% 11% Mosaic (*) 21% 23% 19% 19% 24% 19% p<0.05 p<0.05 Sundheimer, Akopians, Al-Safi, Surrey, Danzer, Ghadir, Chang, Alexander, Barritt (submitted PCRS)

33 differences between doctors of the same center (study 3) Population: 6 doctors from same center, 423 PGS cycles of egg donation, PGS by acgh Comparison: correlation between euploidy rate per physician and controlled ovarian stimulation parameters such as: MII retrieved (MII), Estradiol on day of trigger (E2), gonadotrophin dose (dose/day), days of stimulation (days), donor age, blastocysts biopsiable (Bx), and number of euploid embryos (#Eup). Results: 1) Significant difference in euploidy rates between physicians (p<0.01) 2) Euploidy rates increased with more MIIs, fewer days, and lower dose/day: Log(#Eup) = x MII x days dose/day McCullogh, alikani, Munné (ASRM 2017) Differences between doctors regarding COH parameters were analyzed by ANOVA, and #Eup was compared to COH parameters using multiple regresion of COH parameters compared to log(#eup)

34 Culture media and chromosome abnormalities (4) KSOM + Sage + p complex protein HSA Fertilization 72% 70% NS #PN rate 5% 5% NS Blastulation 73% 76% NS Implantation,<38 y.o 38% 47% NS Biochem. Preg. loss 24% 7% p<0.025 % euploid embryos 16% 29% p<0.025 ph Values ??? C. Hickman, D. Wells, D. Gwinnett, T. Wilkinson, S. Christiansen, O. Oliana, B. Abramov, A. Carby, S. Lavery (2016) Euploid rate sensitivity to laboratory culture environment: a blind, prospective, randomised, sibling study. Human Reproduction 31; Supp 1: i ; P-203

35 Culture media and mosaicism (5) 27 fertility centers answered a questionaire about culture conditions: Media culture, oxygen in incubators, type of incubator, etc. Mosaicism rates ranged from 6% to 31% Mosaicism rates were correlated with type of media culture: Mosaicism Continuous media: 20% Sequential media: 10% (p<0.001) E. Fragouli, S. Alfarawati, K. M. Simpkins, G. Cutts, K Spath, D. Babariya, N. Kubikova L. Rubistello, S. Munné, D. Wells (2017) ESHRE and unpublished data

36 Summary of potential causes Controlled Ovarian Stimulation (COH): - Live birth rate decreases with increasing Gonadotropin dose (1) - Differences between doctors linked to dosage and duration of COH (2) Size of follicles chosen: - Smaller follicles produce more multinucleated embryos (3) Laboratory conditions: - Culture Media (4,5) - Ph, Temperature (6) (1) Baker et al. (2016) Fertil Steril 104: , (2) McCullogh, Alikani, Munné (2017) submitted ASRM, (3) De Vincentiis (2013) Fertil Steril 99:414 21, (4) Hickmanet al.(2016) Human Reproduction 31; Supp 1: i , (5) Fragouli, Alfarawati, Simpkins, Cutts, Spath, Babariya, Kubikova, Rubistello, Munné, Wells (2017) ESHRE

37 Scientists Santi Munné, PhD, CSO (US) Mark Hughes, MD, PhD (US) Jacques Cohen, PhD (US) Dagan Wells, PhD (UK) Elpida Fragouli, PhD (UK) Joson Horcajadas, PhD (LATAM) M. Konstantinidis, PhD (US) Samer Alfarawati, PhD (UK) Tomas Escudero, MSc (US) Josh Blazek, PhD (US) Mike Large, PhD (US) Katharina Spath, PhD (UK) Ryan Subaran, PhD (US) Sarthak Sawarkar, MSc (US) Dhruti Babariya, PhD (UK) Pere Colls, PhD (US) Tony Gordon, PhD (UK) John Kitchen, PhD (US) Carles Gimenez, PhD (Spain) Mireia Sandalinas, PhD (Spain) Sophia Tormasi, MSc (US) Lia Ribustello, MSc (US) Katie Bauckman, MSc (US) Renata Prates, MSc (US) Luis Guzman, PhD (Peru) Muriel Roche, PhD (Japan) Dr. Araki, PhD (Japan) Bioinformatics, VC scientists Arun Manoharan, PhD (US) Avinash Shanmugan, PhD (US) Ursula Schick, PhD (US) Lauren Hurd, PhD (US) Jim Hayes, PhD (US) Eric Proffitt, PhD (US) Genetic Councilors (R&D) Amy Jordan Erin Mills Rachael Cabey Dina Goldberg Haley Nisson

Mosaicism in human embryos: Etiology and pregnancy outcome Santi Munné

Mosaicism in human embryos: Etiology and pregnancy outcome Santi Munné disclosure - Chief Scientific Officer of CooperGenomics - Founder @ Reprogenetics (PGS/PGD company), sold to Cooper - Founder @ Recombine