New methods for embryo selection: NGS and MitoGrade Santiago Munné, PhD US: Livingston, Los Angeles, Chicago, Portland, Miami / 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)
Aneuploidy is the major cause of loss of implantation potential with AMA Aneuploid blastocysts 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 years Reprogenetics data 2012-2015, n= 13,400 PGS cycles by array CGH and blastocyst biopsy
The PGS hypothesis updated PGS v.2: Comprehensive chromosome analysis + blastocyst biopsy Aneuploid increases from 30% to 80% with maternal age Aneuploid embryos miscarry or do not implant The error rate of PGS v.2 is low (<2%) And blastocyst biopsy is non detrimental PGS v.2: Results Improved ongoing Implantation rates Reduces the maternal age effect on implantation
Metanalysis Implantation rates Control PGS Yang et al. 2012 (acgh) 46% 69% Scott et al. 2013 (qpcr) 63% 80% Forman et al. 2013 (qpcr) 40% 58% TOTAL 53% 73% P<0.001
PGS for egg donation PGS donor egg control donor egg Av. donor age 25 25 FET transfers 163 60 Av. emb transferred 1.5 1.5 # positive tests 96% 83% p<0.005 # ongoing pregs 79% 62% p<0.025 Implantation(+sac) 83% 58% P<0.001 P All cycles were FET in both groups. PGS by acgh or NGS. Coates et al. (submitted) Data from Oregon Reprod Medicine and Reprogenetics
acgh eliminates the negative effect of maternal age on implantation 60% Implantation rate 50% 40% 30% 20% 10% 0% <35 35 37 39 40 41 42 >42 Maternal age n/a SART No PGS * blastocyst PGS (acgh) ** * SART 2011 ** Harton, Munné et al. (2013) Fertil Steril. And unpublished data to 8/2013. N >800 blast biopsies
Significant decrease after 42 Total Patients (with follow up) NO MORPHOLOGICAL SELECTION POSIBLE? EGD <35 35 37 38 40 41 42 >42 mtdna? 75 542 359 371 226 203 Av. Euploid embryos 6.4 4.6 3.6 2.5 1.9 1.1 Implantation rate (+sacs) 62% 73% 75% 72% 83% 59% % Pregnant / transfer 69% 66% 64% 63% 65% 54% >18,000 Cycles of acgh, 15,109 of Blastocyst biopsy, but only 1776 with follow up information
Miscarriage rate after blastocyst biopsy and acgh do not increase with age Compared to SART: Compared to other studies: 40% 35% 30% 25% 20% 15% 10% No PGS * PGS ** This study Scott et al. 2013 Preg nan cies age SAB 307 34.9 7.5% 72 32.2 8.3% 5% 0% <35 35 37 38 40 41 42 *SART, ** Harton et al. (2013) Fertil Steril, and unpublished data
Euploidy decreases with age but not with cohort size # of embryos egg donors <35 years % normal blastocysts 35 37 years 38 40 years 41 42 years >42 years 1 3 60% 58% 47% 35% 21% 13% 4 6 64% 55% 47% 34% 19% 14% 7 10 65% 55% 47% 33% 18% 11% >10 66% 56% 43% 33% 21% 11% Total 65% 56% 46% 33% 19% 13% N = 10,852 cycles and 58,798 embryos, up to 5/2015. Ata, Munne et al. (2012) Reprod Biomed Online and Reprogenetics unpublished data.
# of embryos Prognosis depending on age and ovarian response % of patients with normal blastocysts egg donors <35 years 35 37 years 38 40 years 41 42 years >42 years 1 3 83% 80% 71% 57% 36% 22% 4 6 97% 95% 92% 82% 59% 43% 7 10 99% 98% 96% 89% 74% 50% 10 17 100% 99% 99% 97% >17 100% 100% 100% 99% 88% banked 97% banked 64% banked 87% banked N = 10,852 cycles and 58,798 embryos, up to 5/2015. Ata, Munne et al. (2012) Reprod Biomed Online and Reprogenetics unpublished data.
NGS for PGS
Next Generation Sequencing (NGS)
NGS validation: reanalysis of blastocysts Kung et al. 2014 (Reprogenetics) Fiorentino et al. 2014 Wells et al. 2014 (Reprogenetics) Original Analysis method Reanalysis method Sample acgh NGS Same biopsy Karyotype / acgh acgh/ G Band NGS NGS Same biopsy Separate biopsy Confirmed Euploid Confirme d abnormal TOTAL 44/44 108/108 152/152 67/67 141/141 208/208 13/13 28/28 41/41 Total 100% Sensitivity 100% Specificity 0% Error rate Kung, Munne et al. (2014) Hum Reprod. accepted for publication, Fiorentino et al. (2014) F&S, Wang et al. (2014) Biol Reprod, Wells, Kung, Munne et al. (2014) J Med Genetics
First baby born from NGS First NGS baby: David Levy Wells, Kaur, Grifo, Glassner, Taylor, Fragouli, Munne (2014) J Med Genet 51:553 562.
Comparison of current PGS platforms acgh SNPs qpcr t NGS NGS % missed Misdiagnosis full aneuploidy <2% c 2% d <1% e? <1% c 69,XXX w/o aneuploidy no yes no no no 0.2% a UPD w/o other abnormalities no yes no no no 0.01% b Translocations (99% of them) yes no no no yes 2% Partial aneuploidy (>6Mb) yes yes no no yes 5% Mosaicism (>20%) no no no no yes g 12% h Resolution (in Mb) 1 6? >20? 6 19% a Bisignano, Wells, Harton and Munne (2011) RBO. b www.ncbi.nlm.nih.gov/omim, c Kung et al. (2014) ESHRE, d Scott et al. (2012), e Treff et al. (2012) Fertil Steril 97:819 24, f Konstantinides et al (2014) ASRM, g Fiorentino et al (2014) ASRM, h Tormasi et al (2015) PGDIS
NGS calls mosaics better NGS: +7pter-p15.2 and mosaic monosomy 1 and 10 acgh: +7pter-p15.2 and monosomy 1 and 10
Misdiagnosed by acgh Embryo diagnosed as normal by acgh, miscarriage of trisomy 16. Reanalysis by acgh was normal, but reanalysis with NGS of same DNA was mosaic Trisomy 16:
NGS can identify as low as 20% chromosomal mosaicism 3.5 3 2.5 * ** ** ** ** trisomy monosomy Copy number 2 1.5 1 * ** ** ** ** 0.5 0 0 20 40 60 80 100 WGA products (%) * **,p<0,05,p<0,001 Courtesy of Dr. Fiorentino @ Genoma
% of mosaicism detected by NGS at blastocyst stage # % Embryos analyzed 916 97.6 Normal 382 41.7 Complex Abnormal 124 13.5 Full Aneuploid (<=2 chromosomes) 255 27.8 Partial aneuploidy 43 4.7 Mosaic full aneuploidy 78 8.5 Mosaic partial aneuploidy 34 3.7 All mosaic 112 12.2 Tormasi et al. (2015) PGDIS
NGS reanalysis of miscarriages after transfer of euploid by array embryos 39 miscarriages were reported from transfers with euploid embryos (*). 23 miscarriages were undiagnosed and 16 were found trisomic. After NGS analysis of the same biopsies we found: NGS result POC POC Detectable by Total undiagnosed diagnosed acgh, qpcr Euploid 44% 33% 40% Yes Triploid XXX 8% 0% 5% No Mosaic euploid / full aneuploid 12% 50% 28% No Mosaic euploid / partial aneuploid 28% 6% 19% No Complete aneuploid 0% 0% 0% Yes MISCARRIAGES COULD BE FURTHER REDUCED BY NGS (*) miscarriage rate about 7%. Grifo, Munne et al. (2015) submitted
mosaic embryos detected by NGS result in fewer ongoing pregnancies Replaced miscarried ongoing pregnancies NGS result # # % # % p Fiorentino et al* mosaic 16 2 13% 5 31% mosaic 43 5 12% 11 26% Fragouli et al** euploid 51 5 10% 23 51% p<0.001 Mosaics can progress to term but at half the rate of euploid embryos * Fiorentino et al. (2014) ASRM, ** Fragouli et al. (2015) submitted
Is NGS better clinically than acgh? Preliminary results from a single center: acgh NGS Implant 588 56 Not Implant 397 21 Total 985 77 Imp Rate 60% 73% p<0.025 Grifo et al. (unpublished)
NGS mosaics: summary Mosaicism is only detectable by NGS (not by t NGS) 12% of blastocysts are mosaics 60% less miscarriages with NGS than other PGS techniques 50% lower pregnancy than euploid embryos Some mosaics make babies Better implantation rates with NGS than acgh Recommended: First replace euploid embryos and careful discarding mosaics
MitoGrade Selection of the most viable euploid blastocyst by mitochondrial DNA quantification
Blastocyst Mitochondria Mitochondria are cytoplasmic organelles that generate energy for the cell Mitochondria are all maternal in origin, and contain one or more copies of their genome At cleavage stage the mitochondria are still those inherited from the egg The embryo produces its own mitochondria during blastocyst formation Due to a bottleneck mitochondria in the egg and in the blastocyst can be different. Therefore mitochondria competence should be tested in blastocysts, not PBs, or day 3 embryos.
mtdna quantity and blastocyst implantation ability Reprogenetics has discovered that elevated mtdna is associated with failure to implant Above a threshold of mtdna fewer euploid blastocysts implant. 30% of non implanting euploid blastocysts had high levels of mtdna Elevated mtdna levels first appear after first embryonic differentiation Implantation No Implantation Fragouli et al. (2015) PLOS
Mitochondria is an independent biomarker There was no association with blastocyst morphology Only mild association with maternal age and aneuploidy mtdna quantification represents a new independent biomarker of embryo viability No difference in mutations between high and low level mtdna blastocysts Fragouli et al., 2015. PLOS
Mitochondria quantification Blastocyst biopsy WGA PGS by acgh or NGS Quantify mtdna by qpcr (soon by NGS) Targeting multiple mitochondria sites Normalize cell number by comparing to a multi copy nuclear sequence Sequence reads (depth of coverage) 100 50 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Mitochondrial genome (kb) Fragouli et al. (2015) PLOS
PGS + MitoGrade Example patient 35 years old 100% 90% Blastocysts available for transfer 80% 70% 60% 50% 40% 30% 20% 10% 33% 47% 70% 80% aneuploid euploid elevated MitoGrade euploid not implanting (unknown ) euploid implanting 0% no selection PGS PGS + MitoGrade
PGS + Mitograde : Implantation rate after euploid SET 90% 80% 82% implantation 70% 60% 50% 40% 30% 20% P<0.001 10% 0% 4% Euploid + Elevated Mitograde Euploid + Low Mitograde Reprogenetics, to 8/2015
MitoGrade better than Morphology Implantation is higher if the blastocyst is: Euploid, Mitograde normal, morphology BB Than Euploid, Mitograde Elevated, morphology AA
MitoGrade at ASRM O 91 Fragouli E, Cohen J, Munne S, Grifo J, Caroline McCaffrey, Wells D (Reprogenetics): The biological and clinical impact of mitochondrial genome variation in human embryos. Tuesday October 20, 11,15AM SCIENTIFIC PROGRAM PRIZE PAPER
MitoGrade logistics Mitograde is offered jointly with PGS: a single blastocyst biopsy produces Mitograde and PGS results. Mitograde can be offered to any women, of any age, as a booster to obtain even better PGS outcomes. TAT is 2 weeks or less.
THE PGD PORTAL (Jawid)