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Mu

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Mu

  1. 1. PGS with fresh embryo transfer Fresh embryo Santiago Munné, PhD Reprogenetics Laboratories US: Livingston (NJ), Los Angeles (CA), Chicago (IL), Portland (OR), Boca Raton (FL) / Europe: Barcelona (Spain), Oxford (UK), Hamburg (Germany) / Asia: Kobe (Japan), Macao, Abu Dhabi (UAE) / Latin America: Lima (Peru), Buenos Aires (Argentina), Sao Paulo (Brazil), DF (Mexico)
  2. 2. Why PGS?
  3. 3. Most loss of implantation is caused by chromosome abnormalities 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% <35 35-37 38-40 41-42 % euploid no imp % euploid implan % aneuploid Reprogenetics data: 96 centers, >3500 cycles, >19,000 blastocysts analyzed by aCGH to 9/2013
  4. 4. Euploidy decreases with age but not with cohort size # of blastocysts % normal embryos egg donors <35 years 35-37 years 38-40 years 41-42 years N = 4,747 cycles and 29,803 embryos, up to 12/2013. Ata, Munne et al. (2012) Reprod Biomed Online and unpublished data. >42 years 1-3 58% 61% 51% 39% 22% 13% 4-6 62% 60% 52% 38% 23% 17% 7-10 65% 62% 51% 36% 21% 14% >10 68% 63% 55% 37% 25% n/a
  5. 5. The PGS hypothesis - updated • 50% of blastocysts are aneuploid, • Aneuploidy increases with maternal age • Maternal age is inversely proportional to implantation • The error rate of PGS with CCS is low (<2%) • And blastocyst biopsy is non-detrimental Therefore PGS with CCS and blastocyst biopsy: • Improves Implantation rates • Eliminates the Maternal age effect on implantation CCS: comprehensive chromosome analysis, such as array CGH, qPCR, Karyomaping, NGS
  6. 6. WHEN TO BIOPSY
  7. 7. Biopsy stage: before arrays • 30% postmeiotic abnormalities undetected • Triple amount of cells to analyze vs. blastocysts •30%a to 60%b loss of implantation potential • PGD can compensate the damage but does not reach its potential • Centers not proficient in blastocyst culture until recently • Applied clinically in 2005 c • First positive results in 2010 d a Scott et al (2013), b Mastenbroek et al. (2007), c McArthur et al. (2005), d Schoolcraft et al. (2010)
  8. 8. blastocyst biopsy: Advantages Advantages: • More DNA: less no results • Less mosaicism = low error rate • Reduced impact of embryo biopsy • Less embryos to process • Facilitates single embryo transfer • Frozen cycle: Uterine environment optimized after thaw Disadvantages: • Not all embryos reach blastocyst the same day
  9. 9. Evolution of PGS: Reprogenetics data 7000 6000 5000 4000 3000 2000 1000 0 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 Number of Cases Year aCGH day 5 aCGH day 3 CGH FISH 12 FISH 5-9 Reprogenetics Laboratories: 42,000 PGD procedures up to 3/2014
  10. 10. COMPREHENSIVE CHROMOSOME ANALYSIS
  11. 11. aCGH advantages • All 24 chromosome aneuploidies and translocations detected. • Results in <16 hours: allows for day 5 biopsy and 10am day 6 transfer • Parental DNA not required: ad hoc decisions possible. • ICSI not required.
  12. 12. 46,XX+7-10
  13. 13. aCGH validation: reanalysis of blastocysts Reanalysis method Confirmed Euploid Confirmed abnormal Fragouli et al 2011 FISH, aCGH 23/25 27/27 50/52 Capalbo et al. 2013 FISH 19/20 50/50 69/70 Colls et al. 2013 FISH, aCGH 7/7 39/40 46/47 Wells et al. 2013 Next Gen. Sequencing 23/23 67/67 90/90 Total 96% Sensitivity 99.5% Specificity 11% of embryos were mosaic, explaining the 2.4% error rate TOTAL 1.6% Error rate Fragouli et al. (2011) Hum. Reprod. 26: 480-90, Colls et al. (2013) ASRM P-168, Capalbo et al. (2013) Hum Reprod, in press, Wells et al. (2013) ASRM O-435 and unpublished data from Reprogenetics
  14. 14. Speed of different techniques Biopsy Reception Results by qPCR: day 5 day 5, 6pm day 6, am aCGH: day 5 day 5, 6pm day 6, noon NGS: day 5 day 5, 6pm day 6, noon SNPs: day 5 day 5, noon day 6, 6pm
  15. 15. Differences between PGD methods PCR PCR + SNP Next Gen aCGH arrays* Sequencing Detects aneuploidy no yes yes yes Detects gene defects yes yes yes yes Detects mitotic errors no yes no yes >2 month of Preparation yes yes no no Requires affected proband no no yes no # genomes / run 0 0 0 1 * * Karyomapping using BlueGnome ** with NextSeq
  16. 16. NGS: Capacity vs. Depth samples genome depth of / run coverage coverage Genome 1 100% x 30 PGS * 16- 96 ≤ 10% x1 to x3 Wells, Kaur, Rico, Grifo, Anderson, Sherlock, Taylor, Munne (2013) ESHRE, Yin et al (2013) Biol Reprod 88, 69 * Output: MiSeq = PGM << NextSeq
  17. 17. validation of Next Generation Sequencing (NGS) 78 blastocysts previously diagnosed by aCGH were reanalyzed by NGS in a blinded experiment. 21/21 euploid 90/90 aneuploid > 99% concordant Allen Kung et al. (2014) ESHRE and Reprogenetics unpublished data
  18. 18. Overall clinical results with PGD v2 • Blastocyst biopsy • Comprehensive chromosome analysis
  19. 19. Meta analysis of RCTs IMPLANTATION RATES IN RCT STUDIES USING PGS v2: Control PGS Yang et al. 2012 46% 69% Scott et al. 2013 63% 80% Forman et al. 2013 40% 58% TOTAL 53% 73% P<0.001
  20. 20. maternal age effect disappears with full chromosomes analysis
  21. 21. aCGH eliminates the negative effect of maternal age on implantation 60% 50% 40% 30% 20% 10% 0% <35 35-37 39-40 41-42 >42 No PGS * SART bPlGasSto (cayCsGt H) ** Implantation rate Maternal age n/a * SART 2011 ** Harton, Munné et al. (2013) Fertil Steril. And unpublished data to 8/2013. N >800 blast biopsies
  22. 22. Miscarriage rate after blastocyst biopsy Compared to SART: Compared to other studies: 40% 35% 30% 25% 20% 15% 10% 5% 0% No PGS * PGS ** <35 35-37 38-40 41-42 Preg nan cies age SAB This study 307 34.9 7.5% Scott et al. 2013 72 32.2 8.3% *SART, ** Harton et al. (2013) Fertil Steril, and unpublished data
  23. 23. One clear advantage of freezing: EMBRYO BANKING
  24. 24. In embryo banking aneuploidy rates Remain constant 1st cycle Reprogenetics data, unpublished >350 cycles of embryo banking, average age 39.9 2nd cycle 3rd cycle Total Euploidy rate 29% 29% 27% 28% # blastocyst produced 2.4 3.0 2.5 6.3 # euploid blastocysts 0.7 0.9 0.7 2.2
  25. 25. Embryo banking for low responders # of blastocysts or bad prognosis patients % of patients with normal embryos egg donors <35 years 35-37 years 38-40 years 41-42 years N = 3,571 cycles and 19,356 embryos, up to 8/2013. Ata, Munne et al. (2012) Reprod Biomed Online and unpublished data. >42 years 1-3 86% 85% 72% 60% 58% 24% 4-6 95% 97% 95% 88% 69% 54% 7-10 100% 99% 96% 92% 85% 65% >7-10 100% 100% 98% 98% 92% 83%
  26. 26. Success of SET by Euploid Cohort Size 26 No. Euploid Embryos CPR 1 23/55 (41.8%) 2 13/27 (48.1%) 3 9/19 (47.4%) 4 16/21 (76.2%) 5 8/11 (72.7%) 6 6/8 (75.0%) >7 11/15 (78.6%) p < 0.01 S. Morin, K. Melzer, J. Grifo, P. Colls, Z. Zheng, S. Munné (2014) JARG
  27. 27. Cycles needed to accumulate 3 euploid blasts Age Av. blastocysts Av. euploid # cycles to achieve 95% patients w/ 3 euploid 35-39 5.2 2.5 2.7 40-42 4.2 1.1 5.8
  28. 28. Advantages of embryo banking • Less patient “fatigue”: less drop out from cycle to cycle. • Cheaper PGD: One fee per package of IVF cycles • Facilitates “guaranteed baby” plans
  29. 29. Fresh or Frozen?
  30. 30. Does COS affects endometrial receptivity? Roque et al., 2012 RR=1.31 [1.10-1.56] Meta-Analysis Courtesy: Dr. Racowsky Non-PGD Studies
  31. 31. Does COS affects endometrial receptivity? The only effect is on embryos developing to blastocyst on day 6, which in PGS with fresh transfer they are frozen. Non-PGD Studies
  32. 32. Is it worthy to biopsy day 6 blastocysts? The differences between day 5 biopsy and fresh transfer vs. day 5-6 biopsy and vitrification is that the later includes day 6 biopsies: - Day-5 morulas were cultured to day-6 and biopsied if reached blastocyst - SET of blastocysts either biopsied on day 5 or on day 6, thawed transfer Day 5 blastocyst Day 6 blastocyst Implantation 61% 60% N.S. Euploidy 56% 42% P<0.025 Reprogenetics, unpublished
  33. 33. Controlled studies comparing both treatments SET SET SET fresh FET FET Control Control PGS-CCS Cycles 118 272 347 Maternal age 36.1 36.8 37.9 p<0.05 Implantation rate (FHT) 49.2% 52.6% 65.1% p<0.05 SAB 14.3% 14.4% 4.6% p<0.05 Ongoing pregnancy rate 40.7% 43.8% 60.0% p<0.05 Difference between control and PGS: not due to FET Schoolcraft et al. Fertil Steril 2013;100:615–9
  34. 34. Meta-analysis # cycles Ongoing pregnancy rate / transfer FET fresh day 6 FET fresh day 6 Anderson et al. 2013 38 46 55% 66% NS Hill et al. 2013 143 99 56% 45% NS Forman et al. 2013 27 62 55% 65% NS Total 208 207 56% 56% NS Anderson et al. (2013) ASRM O-120, Barrit et al. (2013) ASRM abstract P-167, Forman et al. (2013) Fertil Steril
  35. 35. day 6 transfer and the window of implantation No studies published, but some recommend: • Replace before noon day 6 but… • If last progesterone before trigger is elevated then freeze: the endometrium is advanced and the window of implantation could be missed. • Each center needs to determine their threshold. One center recommends 1.5 or over to freeze.
  36. 36. What IVF centers are doing in the US? 2013 2014 Q1 Day 3 fresh 18.1% 20.9% Day 5 fresh 14.3% 12.4% Day 5 frozen 67.5% 66.6% Total PGD 4359 1460 procedures In Germany is mostly PB biopsy, and in Spain mostly day 3
  37. 37. Fresh: pros and cons Pros - Day 5 blastocysts implant equally well on d6 or FET - Lower cost (no freezing) - Some centers biopsy day 5 morulas - International patients: no need to come back for transfer - INSTANT SATISFACTION Cons - Day 6 blastocysts need to be frozen anyway - Does not allow for embryo banking
  38. 38. Frozen: pros and cons Pros - Day 5 blastocysts implant equally well on d6 or FET - Day 6 blastocysts implant better frozen - Allows for embryo banking for poor prognosis patients - Better uterine receptivity in aggressive stimulations Cons - Higher cost (freezing) - International patients need to come back for transfer - Delayed gratification
  39. 39. Ongoing RCT: Fresh vs. frozen using NGS Procedure: all patients undergo blastocyst biopsy and analysis by Next Gen Sequencing. Group comparison: Patients randomized to day 6 transfer or to FET. Location: Oregon Reproductive Medicine, Portland, OR.
  40. 40. munne@reprogenetics.com www.reprogenetics.com Reprogenetics Scientists Jacques Cohen, PhD (US) Santiago Munne, PhD (US) Dagan Wells, PhD (UK) Renata Prates (US) Samer Alfarawati (UK) Souraya Jaroudi (UAE) Tomas Escudero (US) Mireia Sandalinas, PhD (Spain) Luis Guzman, PhD (Peru) J. Horcajadas, PhD (Latin Am.) M. Konstantinidis, PhD (US) N’Neka Goodall (US) Allen Kung (US) Lia Ribustello (US) Lab & Medical Directors Pere Colls, PhD (US) Carles Gimenez, PhD (Spain) Elpida Fragouli, PhD (UK) Karsten Held, MD (Germany) Tetsuo Otani, MD (Japan) Muriel Roche, PhD (Japan) Braulio Peramo, MD (UAE) Ahmed Yesilyurt, MD (Turkey) Xuezhong Zeng, MD (China) Francisco Rocha (Mexico) Embryologists Kelly Ketterson Catherine Welch Tim Schimmel Genetic Councilors Jill Fischer Amy Jordan Erin Mills G. Manassero, MD

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