Congreso Nacional del Laboratorio Clínico 2016

Size: px

Start display at page:

Download "Congreso Nacional del Laboratorio Clínico 2016"

Baldric Stephens
5 years ago
Views:

1 Actualización en Screening Genético Preimplantacional Maria Giulia Minasi Center for Reproductive Medicine European Hospital Rome, Italy

2 Aneuploidy rate can reach 60% in human embryos Aneuploidy increases with advancing maternal age, while live birth rate decreases Most of the aneuploidies lead to implantation failure or increased miscarriage rate Fragouli et al., 2011; Grifo et al., 2013; Harton et al., 2013

STANDARD MORFOLOGY EXTENDED CULTURE MORPHOKINETIC NOT INVASIVE EMBRYO SELECTION

of studies reporting very conflicting data aneuploid embryos can reach high

parameters could have an influence on development, ploidy and implantation rates

, 2014; Kaser and Racowsky, 2014; Gardner et al., 2015; Minasi et al.

3 STANDARD MORFOLOGY EXTENDED CULTURE MORPHOKINETIC NOT INVASIVE EMBRYO SELECTION STRATEGIES extremely subjective incomplete and fragmentary information high number of studies reporting very conflicting data aneuploid embryos can reach high morphological grades and viceversa remarkable disagreement concerning which parameters could have an influence on development, ploidy and implantation rates Hardarson et al., 2003; Munnè, 2006; Alfarawati et al., 2011; Fragouli et al., 2014; Kaser and Racowsky, 2014; Gardner et al., 2015; Minasi et al., 2016 The attempt to select an euploid embryo on the basis of these selection strategies remain extremely hazardous!

4 ? First version: PGS 1.0 Preimplantation Genetic Diagnosis (PGD) is an invasive method of embryo selection Initially PGD was used in fertile patients with the risk of transmitting genetically inheritable diseases to their offspring Fluorescence in situ hybridization (FISH) analyzing a panel of 9 chromosomes was previously the most applied method Handyside et al., 1990 Several randomized controlled trials were unable to demonstrate an increase in pregnancy rates with this technique Mastenbroek et al., 2007, 2011

5 PGS: Preimplantation Genetic Screening Different techniques allowing the analysis of all the 24 chromosomes new era: PGS 2.0 or PGD-A (Preimplantatione Genetic Screening for Aneuploidy) SET maintaining high clinical, implantation and live birth rates Reduction of miscarriage rate Reduction of multiple pregnancy rate Reduction of time-to-pregnancy Reduction of live births with genetic defects When? AMA? RIF? RM? Severe Male infertility? Ever? Never? Fragouli et al., 2011; Scott et al., 2012; Fiorentino et al., 2014; Bono et al., 2015; Ubaldi et al., 2015; Sermon et al., 2016

Timing of biopsy biopsy Advantages of blastocyst Smaller proportion of only extra-embryonic cells are removed No impact on developmental and

embryos to be analyzed Trophectoderm is an excellent predictor of inner cell mass karyotype Johnson et al., 2010; Greco et al.

, 2014; Minasi and Greco, 2014 Disadvantages of blastocyst There is the risk that some or even all embryos may not reach the blastocyst stage,

6 Timing of biopsy biopsy Advantages of blastocyst Smaller proportion of only extra-embryonic cells are removed No impact on developmental and implantation potential A large number of cells are removed More accurate diagnosis (particularly important to identify mosaic embryos) Less embryos to be analyzed Trophectoderm is an excellent predictor of inner cell mass karyotype Johnson et al., 2010; Greco et al., 2013; Scott et al., 2013; Adler et al., 2014; Minasi and Greco, 2014 Disadvantages of blastocyst There is the risk that some or even all embryos may not reach the blastocyst stage, especially in some categories of patients (low responders, severe male infertility): informed consent Expensive: not all embryos reach the blastocyst stage at the same time, higher consumption of plastic and culture media, more incubators and embryologists are needed

detection + - - + Mitochondrial disorders detection - - + + Speed (hours) 30-40 h 12-15 h 4-12 h 13-16 h Lab workload high medium low high Costs very high high low very low All these enhanced

7 Pros and cons of different preimplantation aneuploidy testing platforms Adapted from Treff and Scott, 2012; Gardner et al., 2015; Brezina et al., 2016 CGH METHOD SNP acgh qpcr NGS Whole chromosome aneuploidy detection Mosaicism detection + +/- - + Triploidy detection +/- +/- + + Segmental detection + + +/- + Uniparental disomy detection Mitochondrial disorders detection Speed (hours) h h 4-12 h h Lab workload high medium low high Costs very high high low very low All these enhanced methodologies have comparable efficacy in identifying whole chromosome aneuploidy but differ widely in their ability to simultaneously identify other structural chromosome abnormalities or mithocondrion copy number. Furthermore, they differ in speed of analysis, cost and laboratory workload.

8 Prospective double blinded trial AIM: evaluate if NGS can be used for aneuploidy screening of human embryos with the purpose of identifying and selecting chromosomally normal embryos for transfer CPR 63.8% OPR 62.0%

9 NGS acgh Reproductive Medicine, European Hospital, Rome, Italy Monosomy 6 Monosomies 2, 15 and atypical losses on chromosomes 7, 8 and 17 NGS-based 24-aneuploidy screening protocol has a high resolution and allows accurate detection of segmental imbalances as small as 14 Mb in size

7%) with a lower miscarriage rate Scott et al.

10 3 RCTs studies young and good prognosis patients Yang et al., 2012: randomized pilot study; single fresh blastocyst ET with (55 couples, 425 blastocysts) or without (48 couples, 389 blastocysts) PGD-A CPR higher with PGD-A (69.1% vs 41.7%) with a lower miscarriage rate Scott et al., 2013: randomized controlled trial; 134 blastocysts (72 patients) in the PGD-A group and 163 blastocysts (83 patients) in the control group statistically significantly improved IVF outcomes (IR: 79.8% vs 63.2%; CPR: 93.1% vs 80.7%) Benefit in terms of IR and PR in this group Forman et al., 2013: randomized, noninferiority trial; 205 couples, 89 single euploid blastocyst ET vs 86 double untested blastocysts ET sustained IR (63.2% vs 51.7%) and comparable ongoing pregnancy rates (60.7% vs 65.1%) but dramatically reduced risk of twins

(at least 2 miscarriages) (at least 2 implantation failures) (at least 2 miscarriages) (at least 2 implantation failures) (FET ET) (Fresh ET) Reproductive Medicine, European Hospital, Rome, Italy

11 (at least 2 miscarriages) (at least 2 implantation failures) (at least 2 miscarriages) (at least 2 implantation failures) (FET ET) (Fresh ET) Reproductive Medicine, European Hospital, Rome, Italy Advanced Maternal Age studies which included a control group where embryos were selected based on morphology 68.9% vs 44.8% 52.6% vs 19.2% 82.2% vs 84.0% 63.2% vs 36.8% vs 40.1% 55.0% vs 41.8% 27.7% vs 8.9% vs 7.3% 35.2% vs 29.2% vs 19.6% 69.2% vs 43.9% Consistently improved IR in the PGD-A group in all studies 4/5 studies reported significantly improved PR However, due to limitations with observational studies, the validity and generalization of the findings makes it difficult to conclude whether PGD-A is clinically effective in this group of patients

WHY Fertility expert group: general questions (PGS indications) HOW WHEN Molecular biologist group: genetic analysis methods Embryologist group: ideal method (timing of biopsy) AIM: analyze the

12 WHY Fertility expert group: general questions (PGS indications) HOW WHEN Molecular biologist group: genetic analysis methods Embryologist group: ideal method (timing of biopsy) AIM: analyze the current opinion and practices concerning the new version of PGS2.0 WHY MOST CHOSEN INDICATIONS: 1. repeated miscarriage, repeated implantation failure 2. advanced maternal age, PGD+PGS, SET 3. male factor infertility, female infertility Consensus is lacking on which patient groups, if any at all, can benefit from PGS2.0 and especially if it should be offered to all IVF patients HOW MOST METHODS USED: 1. WGA (acgh, SNP) 2. qpcr NGS is promising (not evaluated in this study) WHEN MOST METHODS USED: 1. TE PB 3. BLASTOMERE Blastocoele fluid biopsy is still to new and not validated

13 Future perspectives (NGS) Importance of mosaicism detection Role of mtdna as additional biomarker Feasibility of performing double genetic analysis (PGD and PGS) with a single biopsy

Chromosomal mosaicism is the failure of chromosomes to properly segregate during mitosis, leading to the gain or loss of whole chromosomes Mosaicism Is defined as the presence of two or more

abnormalities Chromosomal mosaicism is a relatively common finding in IVF-derived human embryos and may affect: 15 90% of cleavage stage embryos 30-40% of blastocysts The exact threshold at which

14 Chromosomal mosaicism is the failure of chromosomes to properly segregate during mitosis, leading to the gain or loss of whole chromosomes Mosaicism Is defined as the presence of two or more chromosomally distinct cell lines within an individual: mosaic diploid/aneuploid: mixture of diploid and aneuploid cell lines mosaic aneuploidy: mixture of cell lines with different chromosomal abnormalities Chromosomal mosaicism is a relatively common finding in IVF-derived human embryos and may affect: 15 90% of cleavage stage embryos 30-40% of blastocysts The exact threshold at which mosaicism switches from clinically irrelevant to relevant is unknown and differs depending on the stage and severity at onset The impact of mosaicism on implantation and the developmental potential of mosaic embryos is not known: it is reasonable to assume that mosaicism reduces the likelihood of success of IVF Mosaic embryos are not usually transferred because they are deemed abnormal Baart et al., 2006; Fragouli et al., 2008, 2011; Taylor et al., 2014; Novik et al., 2014

mosaic embryo and written informed consent signed Transfer

15 TRANSFER OF MOSAIC EMBRYOS X 1. When there are no fully euploid embryos available 2. When there is at least one mosaic diploid/aneuploid blastocyst Specific genetic counselling for each single mosaic embryo and written informed consent signed Transfer of 1 or 2 mosaic blastocysts If the pregnancy is obtained, invasive prenatal diagnosis is strongly reccomended

16 3802 blastocysts analyzed (May July-2014) 181 (4.8%) diploid/aneuploid mosaic embryos detected 33.3% of CPR and LBR Delivery Biochemical Negative 33.3% 11.1% 55.6% Greco, Minasi and Fiorentino, NEJM, 2015

17 N. ET Mosaic Blastocysts 49 Transferred mosaic blastocysts 50 Beta positive 24 (49%) Biochemical pregnancies 5 (10.2%) Clinical pregnancies 19 (38.8%) Miscarriages 4 Deliveries 15 Newborn 16 LBR 16/50 (32%) Negative Delivery 15 Biochemical Miscarriage

The transfer of embryos with a lower % of mosaicism is related with a better clinical outcome The transfer of embryos with monosomy is related with a better clinical

18 The transfer of embryos with a lower % of mosaicism is related with a better clinical outcome The transfer of embryos with monosomy is related with a better clinical outcome Updated data from Greco E, Minasi MG and Fiorentino F. N Engl J Med ;373: Courtesy of Francesco Fiorentino and Francesca Spinella, GENOMA GROUP

blastocysts tested by array-cgh (Fiorentino

19 Novik et al., 2014 Aneuploidies in embryos according to the indication Aneuploidies in embryos according to maternal age Data from 7000 blastocysts tested by array-cgh (Fiorentino et al., unpublished data) Courtesy of Francesco Fiorentino and Francesca Spinella, GENOMA GROUP

The importance of mosaicism detection Correct scoring of the results Avoid discarding of potentially viable embryos Choose fully euploid embryos (younger

20 The importance of mosaicism detection Correct scoring of the results Avoid discarding of potentially viable embryos Choose fully euploid embryos (younger patients) Additional chances for IVF patients of achieving a clinical pregnancy Ethical value Courtesy of Francesco Fiorentino and Francesca Spinella, GENOMA GROUP

21 This study investigated the biological and clinical relevance of the quantity of mtdna in 379 embryos mtdna higher in embryos from older women mtdna elevated in aneuploid embryos (independent of age) mtdna lower in implanted blastocysts mtdna content could represents a novel biomarker with potential value for IVF, revealing chromosomally normal blastocysts incapable of producing a viable pregnancy

Quantification of mitocondrial dna (mtdna) from blastocysts as a strategy to predict the implantation potential of chromosomally normal embryos F. Spinella, E. Cotroneo, S. Bono, A. Biricik, E.

22 Quantification of mitocondrial dna (mtdna) from blastocysts as a strategy to predict the implantation potential of chromosomally normal embryos F. Spinella, E. Cotroneo, S. Bono, A. Biricik, E. Greco, M.G. Minasi, A. Ruberti, F. Fiorentino The aim of the present study was to investigate if the measurement of the mitochondrial DNA content in preimplantation embryos can improve the identification of the most likely euploid embryo resulting in a baby

23 High mtdna levels are indicative of compromised status of blastocysts Data from 734 blastocysts tested by NGS (Fiorentino et al., unpublished data) Courtesy of Francesco Fiorentino and Francesca Spinella, GENOMA GROUP

24 Embryos with low mtdna level have increased potential to implant and produce a live birth Retrospective analysis of 107 transferred euploid blastocysts mtdna quantification might represent an additional tool to choice the euploid embryos with higher potential to implant Additional prospective clinical data must be obtained before this approach can be evaluated for routine integration into IVF-PGS programs Courtesy of Francesco Fiorentino and Francesca Spinella, GENOMA GROUP

Genetic diseases and aneuploidies can be detected with a single blastocyst biopsy: a new clinical successful approach E. Cursio, M.G. Minasi, A. Ruberti, A.M. Lobascio, V. Casciani, A. Colasante, F.

25 Genetic diseases and aneuploidies can be detected with a single blastocyst biopsy: a new clinical successful approach E. Cursio, M.G. Minasi, A. Ruberti, A.M. Lobascio, V. Casciani, A. Colasante, F. Scarselli, T. Riccio, K. Litwicka, M.T. Varricchio, A. Biricik, F. Spinella, F. Fiorentino, E. Greco The aim of the present study was to investigate if a single blastocyst biopsy is efficient in detecting both genetic diseases and aneuploidies, limiting negative effect on embryo implantation potential and guaranteeing good clinical outcomes in patients who need PGD for genetic reasons

26 OPU 35h after hcg Denudation 38h after hcg ICSI and individual culture at 37 C, 5%O 2, 6%CO 2 When a blastocyst resulted transferable after PGD analysis, its ploidy status was also evaluated by mean of PGS on the same biopsy sample Biopsy DAY Waiting for the genetic result Frozen ET Vitrification

27 PGD+PGS CYCLE FROM OCTOBER 2011 TO MAY 2016 PGD+PGS cycles 283 Patients 217 Female mean age 35.3±4.1 Male mean age 38.1±5.1 COCs 3257 MII 2476/3257 (76%) Thawed oocytes survived 91 Injected oocytes = 2567 Zygotes 1914/2567 (74.6%) Embryo obtained 1874/1914 (97.9%) Thawed embryos survived 23 Blastocysts obtained 1016/1897 (53.5%) Day 4 blastocysts 5/1016 (0.5%) Day 5 blastocysts 616/1016 (60.6%) Day 6 blastocysts 342/1016 (33.7%) Day 7 blastocysts 53/1016 (5.2%) MONOGENIC DISEASES TREATED Achondroplasia Alpha 1 antitrypsin deficit Leukodystrophies Canavan disease Spinal muscular atrophy Spinocerebellar ataxia Talassemia beta Cardiomyopathy Huntington disease Primary Dystonia Duchenne Dystrophy Myotonic Dystrophy Facioscapulohumeral muscular dystrophy Hemophilia Multiple exostosis Cystic fibrosis Hydrocephalus Hyperkeratosis Krabbe Disease Neurofibromatosis Osteogenesis imperfecta Spastic paraparesis Polycystic Kydney Disease Tuberous sclerosis Alport disease Crisponi disease Currarino disease Lowe syndrome Muenke syndrome Noonan syndrome Treacher- Collins Hereditary deafness

28 PGD+PGS CYCLE FROM OCTOBER 2011 TO MAY 2016 Transferable blastocysts (PGD+PGS) 281/1016 (27.7%) Mosaic blastocysts 35/1016 (3.4%) Not transferable 643/1016 (63.3%) due to PGD due to PGS due to PGD+PGS 248/643 (38.6%) 289/643 (44.9%)* 106/643 (16.5%) No result 57/1016 (5.6%) N Cryopreserved ET 130 Clinical pregnancies 68/130 (52.3%) Implantation rate 71/139 (51.1%) N of cycles with healthy blastocsysts awaiting for transfer 51 (116 blastocysts) Without performing PGS, 289 blastocysts that resulted healthy for the genetic disease could have been transferred leading to implantation failures, miscarriages or, in some cases, to live births affected by different syndromes!!! * 15 trisomies 13 or 18 or 21.. When a blastocyst resulted transferable after PGD analysis, it should be mandatory to evaluate also its ploidy status by mean of PGS on the same biopsy sample

29 Conclusions I: PGS 2.0 issues still open: The value of PGS as a universal screening test for ALL IVF patients has yet to be determined! The studies reported until now on PGS demonstrate high birth rate after PGS and SET but there are still important limitations: Analysis of data per started cycle rather than per ET could more accurately address the applicability of wider use of this technology The ability to expand these techniques to centers with less experience has yet to be established The miscarriage rates reported also after the transfer of only euploid embryos need to be examined The potential embryonic damage due to the biopsy procedure and epigenetics risks related to the extended culture remain unanswered The comparison among the different techinques for assessing ploidy to determine if any platform is superior is lacking

30 Conclusions II: PGS recommendations: CAREFUL COUNSELLING: the possibility of transfer cancellation, embryo degeneration, the expected outcome, the diagnostic limitation, error rates, the opportunity to perform invasive or not invasive prenatal diagnosis, explanation of genetic result, etc. TECHNICAL EVALUATIONS: right number of incubators, low oxygen tension, blastocyst cryopreservation protocol, stage of embryo biopsy, molecular method for genetic analysis, skilled embryologists, connection with a genetic laboratory, timing of embryo transfer, etc. COST EFFECTIVENESS OF PGS: taking into account all the aspects of this procedure (role of cryopreservation, time to pregnancy, cumulative success rate, obstetrical and neonatal outcomes, molecular technique adopted, etc)

Chromosomal Aneuploidy

The Many Advantages of Trophectoderm Biopsy Compared to Day 3 Biopsy for Pre- Implantation Genetic Screening (PGS) Mandy Katz-Jaffe, PhD Chromosomal Aneuploidy Trisomy 21 Fetus Aneuploidy is the most common