PGT-A Experience, Managing the increasing workload, and Implementing PGT-M with AmpliseqMURAT CETINKAYA,MD, PHD,

ART AND REPRODUCTIVE GENETICS CENTER

MEMORIAL HOSPITAL, ISTANBUL, TURKEY

PGT-A Trophectoderm Biopsies

200 204382 296

1119

2203

3510

4028

0

500

1000

1500

2000

2500

3000

3500

4000

4500

2011(4 months)

2012 2013 2014 2015 2016 2017 2018(9 months)

Trophectoderm Biopsies

2011 and 2018 values are projected to annual values

aCGH

NGS

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Changes in ART Clinic’s Approach•Gradually offering PGT-A to a broader spectrum of couples• Initially, • Only to the patients with «good» ovarian reserve, thus high number of blastocysts

• Repeated implantation failures

• Patient’s request

• In 2018, PGT-A patients average 36 years of age• ≥37 years of age patients regardless of ovarian reserve

• Routine aneuploidy testing after PGT-M

•Switching from fresh biopsy & diagnosis & transfer to freeze-all strategy

•Switching to blastocyst culture for all suitable patients

3

PGT-A Trophectoderm Biopsies

200 204382 296

1119

2203

3510

4028

44 49 69 57316

766

11441276

0

1

2

3

4

5

6

0

500

1000

1500

2000

2500

3000

3500

4000

4500

2011(4 months)

2012 2013 2014 2015 2016 2017 2018(9 months)

Trophectoderm Biopsies Number Of PGT-A Cycles Average Number of Trophectoderm Biopsies

2011 and 2018 values are projected to annual values

aCGH NGS

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Timeline of Switching Workflows

aCGH

•Fresh Transfer

•High Workload

•~1 run per week

•High cost

•Manageable logistics

aCGH

•Fresh + Frozen –

Thawed Transfer

•Increased Workload

•~5-6 runs per week

•Increased cost

•Common logistics problems

NGS (PGM)

•Frozen – Thawed Transfer

•Still high Workload

•2 chips per week

•Decreased cost

•Intermittent logictics issues

NGS (S5)

•Frozen – Thawed Transfer

•Stable – Low Wetlab Workload

•1 chip per week

•Stable cost

•Better logistics

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Autumn 2011 Spring 2016 Early 2017 Summer 2018

Early NGS Period – PGM •Isothermal Amplification• Inconsistent performance with IA• Better results during training phase and when new shipment arrives, but decreasing over time

• Laboratory equipment or storage issues?

• 3 months of retries, alternative approaches and additional hands-on trainings

•Emulsion PCR• Stable high quality sequencing

• Decreased hands-on time

• Longer duration from sample to report• Lose the ability to work with fresh samples clinicians already opted-out from fresh transfers

•Stable PGT-A workflow with Emulsion PCR and local IR server

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Necessity to Move To A Higher Capacity Platform•However,• Initial planning was• 2017 2500 tests

• ~200 per month

• 2 chips - 1 run / week

• 3510 tests only during 2017 • Especially on 2nd half of 2017

• ~350 per month

• 6 & 8 chips – 3 & 4 runs / week

• Increased usage • Longer interval time between biopsy and

PGT-A report

• Increased work burden

• On laboratory personnel

• For equipment

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0

50

100

150

200

250

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350

400

450

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

2017

Tests Per Month

Real World Business Problems •Talking with the distributor for a feasible transition plan

•Convincing the hospital management for the necessity of transition

•Cost• Distributor ↑

• Hospital management ↓

•>6 months to install S5 after our first proposal of upgrade• Only after an electronic circuit on PGM gave up

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Current NGS Period – S5 •300 – 400 samples per month

•3 – 4 runs per month with 530 chips

•With IonChef automatization • Decreased hands-on time

• Simplified workflow

•PGT-M with AmpliSeq

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Euploidy Rate & Probability of Finding at Least One Euploid Embryo per Cycle According to Maternal Age Groups

50%

39%

28%

16%

9%4%

82%

66%

53%

30%

17%

8%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

< 35 (n=589) 35-37 (n=464) 38-40 (n=808) 41-42 (n=550) 43-44 (n=323) > 44 (n=84)

Euploidy Rate At Least 1 Euploid Embryo

10p < 0.05, Chi Square Test

Probability of Finding at Least One Euploid Embryo According to the Number of Biopsied Embryo(s) and Maternal Age

11Two-way Probability Table

Number of Biopsied Embryo(s)

1 2 3 4 ≥5

Age

<35 42% 64% 88% 98% 95%

35-37 34% 55% 70% 85% 95%

38-40 29% 51% 57% 70% 88%

41-42 13% 24% 45% 60% 56%

43-44 6% 20% 20% 38% 46%

Ongoing Pregnancy Rate Comparison of PGT-A with Single Embryo Transfers and Non PGT-A Groups

61%

68%

59%

54%

38%

11%

63%

53%

34%

19%14%

0%0%

10%

20%

30%

40%

50%

60%

70%

80%

< 35 35-37 38-40 41-42 43-44 > 44

Ongoing Pregnancy PGT-A Ongoing Pregnancy No PGT-A

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Mean Number of Transferred Blastocyst 1 vs 1.25Multiple Pregnancy Rate: 0.5% vs 16.3%

p < 0.05, Chi Square Test

NGS Improves Pregnancy RatesCompared with aCGH

13Friendenthal et al., 2018

Mean Survival Time(Transfer Counts to Reach an

Ongoing Pregnancy)

Mean (95% CI)

Non-PGT-A Group(Mean Age: 29.8)

2.408 (2.317 to 2.499)

AMA PGT-A Group(Mean Age: 39.9)

1.879 (1.755 to 2.003)

p < 0.0001Cetinkaya et al., O-165, ASRM, 2018 Kaplan-Meier Survival Analysis

Decreasing Number of Transfers to Reach Pregnancy with PGT-A

Decreasing Time to Pregnancy with PGT-A

Mean Survival Time(Days to the FET Cycle that Achieve

an Ongoing Pregnancy)

Mean (95% CI)

Non-PGT-A Group(Mean Age: 29.8)

259 (240 to 279)

AMA PGT-A Group(Mean Age:39.9)

157 (137 to 177)

Hazards Ratio: 1.496 (1.328 to 1.924)AMA PGT-A Group patients reach an ongoing pregnancy 1.49 times faster.

Kaplan-Meier Survival Analysisp < 0.0001Cetinkaya et al., O-165, ASRM, 2018

MosaicismHow much do we want to know? *•Clinicians point-of-view• 100 different IVF centers which utilize a single reference genetics laboratory• 12 % request mosaic embryos to be reported

• 88 % opted out of mosaic reporting

•Patients’ point of view• Survey on FertilityIQ• 82% of women wish to know

if they have mosaic embryos

• 48% of women were not toldif their embryos were mosaic or not

16* Joshi et al, ASRM 2018

Mosaic Embryos

33,5%

58,0%

8,5%

0%

10%

20%

30%

40%

50%

60%

70%

Trophectoderm Biopsies NGS Results

Euploid Aneuploid Mosaic

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NGS Diagnosis and Mosaicism Ratio according to Ovarian Reserve

26,4%33,0% 38,6%

6,3%7,6%

9,4%

67,4%59,4%

52,0%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Poor Responders (Mean Age : 38.9) Normo Responders (Mean Age : 36.2) Hyper Responders (Mean Age : 34.6)

Euploid Mosaic Aneuploid

Chi-square test: Mosaic vs Euploid + Aneuploid p < 0.0001

NGS Diagnosis and Mosaicism Ratio according to Ovarian Reserve

80,8% 81,2% 80,4%

19,2% 18,8% 19,6%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Poor Responders (Mean Age : 38.9) Normo Responders (Mean Age : 36.2) Hyper Responders (Mean Age : 34.6)

Euploid Mosaic

Chi-square test: Mosaic vs Euploid p = 0.88

Clinical Results of Mosaic Embryo Transfers

n %

bHCG (+) / ET 20 / 30 66.6

Biochemical Miscarriage 3 / 20 15.0

Clinical Pregnancy 17 / 30 57.6

Clinical Miscarriage * 1 / 17 5.9

Ongoing Pregnancy 16 / 30 53.3

Live Births ** 6 -

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* 1 Ectopic Pregnancy** 2 Preterms (26 weeks, postpartum exitus 10 days)

(34 weeks, postpartum healthy)

Mosaic Embryo Transfer Results According to Mosaicism Percentages

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Higher Mean Mosaicism Rate for Non-Implanted Embryos

Implementing PGT-M with AmpliSeq

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Aim•To construct a NGS based SNP-haplotyping (SNPh)• To develop an algorithm and a bioinformatics pipeline

•Rare genetic disorder carrier couples• Consanguineous marriages

• More than 70% of our PGT-M cases each year very rare disease increased initial treatment costs

•To maximize the cost-effectiveness of PGT-M• With minimum panel sizemaximum information

• Specifically target only the informative SNPs

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Methods – Bioinformatics•Input data for the pipeline • Exome-trio sequencing of the family (BAM files)

• Genetic diagnosis of the disorder and mutation identification

•Vcftools command-line• Extract SNPs at the region of target gene

•R software• Refine the SNP list to include only the informative SNPs

• Secondary algorithm to further select the target regions• Distribute target SNPs evenly throughout the region

• Prioritize regions where more than 1 SNP can be sequenced in a single amplicon

• Modifiable amplicon size

• Modifiable panel size

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PGT-M with AmpliSeq Initial Study•Couple’s first child was lost due to an undiagnosed disease after birth• Exome-trio analysis did not reveal any possible mutation

•After couple’s consent • HBB chosen as target gene for the study

• AmpliSeq custom panel • 88 amplicons containing 113 SNPs within ± 2 Mb of HBB gene

• Rebiopsy of 3 aneuploid embryos• Already tested with PGT-A, aneuploidies other than chr11

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Results –Initial Haplotype

* ** + + +

* Exome Data+ Embryo Data

Results – «Key-SNPs»

* Exome Data+ Embryo Data

* ** + + +

343 vs 856 reads (71.3%)Preferential Amplification

No reads but alleles determined byGeneHunter Haplotype construction

Results –«NGS – STR Haplotyping Comparison»

PGT-M with AmpliSeq First Case•Couple lost two children • Undiagnosed progressive motor decline with spasticity, ataxia, tremor

• Exome-trio analysis revealed homozygous mutation at POLR3A (10q22.3)

•After searching for informative SNPs at ± 2 Mb of POLR3A gene from exome data• First AmpliSeq design • Returned with missed regions

• Second AmpliSeq design• Inclusion of «non-exome targeted» SNPs with average allele frequency

• 96 amplicons with 106 SNPs

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Distribution of SNPs at POLR3A region

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Key-SNPh for POLR3A

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Thanks…To our hardworking team for all the effort

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Thanks…

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