Current technical and medical developments in embryo testing and the possible impact on the extent of the application.

Transcription

1 Current technical and medical developments in embryo testing and the possible impact on the extent of the application Luca Gianaroli

2 PREIMPLANTATION GENETC DIAGNOSIS PGD oocyte PGD embryo

3 BIOPSY PB Cleavage Advantages - No embryo mass reduction - Several days for analysis - No mosaicism - Analysis of male and female contribution - Possibility of second biopsy in case of no result Blastocyst -Several cells available for analysis - No embryo mass reduction (trophectoderm biopsy) Disadvantages - No analysis of paternal contribution - Need for embryo biopsy in case of no result - Need for embryo biopsy if the oocyte carries a recessive mutation - Embryo mass reduction - Mosaicism - A few hours for analysis

4 ESHRE PGD CONSORTIUM Data BIOPSY METHOD 2870 PB Of which 759 sent by SISMER PB1 only Cleavage 49 PB + Cleavage 99 Blastocyst Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

5 PGD on embryo - Legal Status in Europe Allowed Forbidden Not Regulated

6 PGD INDICATIONS AND DATA STATE OF THE ART

7 ESHRE PGD CONSORTIUM List of 22 countries contributing data to the last data collection (2007) Argentina Australia Belgium Brazil Czech Republic Denmark Finland France Germany Greece Israel Italy Japan Portugal Spain Sweden Taiwan The Netherlands Turkey UK Ukraine USA Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

8 ESHRE PGD CONSORTIUM No. CENTRES Data collection I II III IV V VI VII VIII IX X 1/97-9/98 to 5/2000 to 5/ / Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

9 ASSISTED REPRODUCTIVE TECHNOLOGY IN EUROPE: EIM REGISTERS PGD CONSORTIUM EIM PGD cons Year No. EIM cycles Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

10 ESHRE PGD CONSORTIUM No. CYCLES Data collection I II III IV V VI VII VIII IX X 1/97-9/98 to 5/2000 to 5/ / Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

11 MEAN NUMBER OF CYCLES REPORTED BY THE CONTRIBUTING CENTERS - TEN DATA COLLECTIONS Data collection I II III IV V VI VII VIII IX X 1/97-9/98 to 5/2000 to 5/ / No. of centers No. of cycles Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

12 CLINICAL OUTCOME THROUGHOUT THE YEARS a b % abc def d e c f Pregnancy rate / ET Pregnancy rate / OR Implantation rate 5 0 Data collection V VI VII VIII IX X abcdef P<0.001 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

13 ESHRE PGD CONSORTIUM To December 2007: cycles, 5135 children born Aneuploidy (61%) Sex-selection (2.5%) Translocations (15.5%) Monogenic disorders (17%) X-linked disorders (4%) Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

14 PGD FOR GENETIC DISORDERS Present Clinical Applications PGD can be carried out for any disorder in which the gene responsible for the disease has been identified

15 PGD FOR GENETIC DISORDERS 2 µl of the PCR reaction product are used for a second PCR reaction (internal primers) for each 1 locus st PCR 2 nd PCR Cell Lysis External Multiplex PCR Nested PCR Mutation Analysis External primers for the amplification of: the gene regions with the mutations linked STR markers for ADO detection STR markers for aneuploidy (optional)

16 BETA THALASSEMIA COD.39 C T Sequencing Analysis Minisequencing Normal Allele Mutated Allele Normal Allele Mutated Allele

17 PGD - Present Clinical Applications Estimated prevalence in Europe Nov 2010 Autosomal Recessive Autosomal Dominant X-linked Triplet repeats expansion Cystic Fibrosis 12/ Beta Thalassemia 0.5/ Sickle Cell Anemia 11/ Spinal Muscular Atrophy (SMA) 7.8/ (It) Retinoblastoma 5.4/ Neurofibromatosis NFI 25/ ; NF2 0.5/ Li Fraumeni syndrome reported 400 families Duchenne / Becker Muscular Dystrophy 5/ Hemophilia A 11/ Hemophilia B 2/ Fragile X 14.25/ Myotonic Dystrophy 4.5/ Huntington 5.9/ Adrogenital Syndrome (SAG) 1/ births Familial Adenomatous polyposis 5.25/ Congenital Adrenal Hyperplasia (CAH) 10/ Fanconi Anemia 1/ Medium chain Acyl-CoA dehydrogenase deficiency (MCAD) 1/68 UK, 1/101 DK, 1/133 It Achondroplasia 4.5/ Hypochondroplasia 3.3/ Multiple Endocrine Neoplasy 2 3.5/ Marfan Syndrome 20/ Tay Sachs disease 0.3/ births Osteogenesis Imperfecta 6.5/ Alpha-1-Antitripsin 25/ Tuberous Sclerosis 8.8/ Epidermolysis Bullosa 2.5/ Retinitis Pigmentosa 27.5/

18 THALASSEMIA 1.5% carriers in the world affected 12% carriers 700 affected 12.5% carriers affected 7% carriers affected

19 Autosomal recessive. Data Cystic Fibrosis Beta thalassemia Sickle cell (+/-HLA) Spinal muscular atrophy + retinitis pigmentosa HLA compatibility HLA compatibility + specific disease Cycles to OR Clinical pregnancy rate (%) per OR per ET Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

20 Autosomal dominant. Data Huntington Myotonic dystrophy 1 Neurofibromatosis 1 Charcot-Marie- Tooth Cycles to OR Clinical pregnancy rate (%) per OR per ET Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

21 Other diseases. Data Other diseases Cycles to OR 1292 Clinical pregnancy rate (%) per OR per ET Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

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23 Specific X-linked. Data Duchenne Becker muscular dystrophy Haemophilia A - B FRAXA Cycles to OR Clinical pregnancy rate (%) per OR per ET Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

24 X-LINKED. Data X-Linked Data X-Linked Data Cycles to OR 1167 Cycles to OR 110 Cycles to transfer 880 Cycles to transfer 86 Clinical pregnancies (% per OR) (% per ET) 228 (19) (26) Clinical pregnancies (% per OR) (% per ET) 22 (20) (26) Miscarriages (% per pregnancies with follow-up) 2 (10)

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26 PGD FOR CHROMOSOME ABNORMALITIES Present Clinical Applications Structural: Translocations Deletions Inversions Numerical: Altered karyotype sex chromosomes

27 PGD FOR TRANSLOCATIONS - METHODS -Robertsonian translocations Acrocentric 13, 14, 15, 21, 22 Enumeration probes - Reciprocal translocations Chromosome painting in 1 st PB Breakpoint spanning probes Cell conversion Centromeric and telomeric probes Normal Derivatives Combination of probes proximal and distal (subtelomeric) to the breakpoints

28 PGD FOR TRANSLOCATIONS - METHODS Normal Balanced Unbalanced

29 Chromosome abnormalities. Data Robertsonian translocations Recirpocal translocations Sex chromosome aneuploidy Others Cycles to OR Clinical pregnancy rate (%) per OR per ET Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

30 Chromosome abnormalities. Data 2007 Robertsonian translocations Recirpocal translocations Sex chromosome aneuploidy Others Cycles to OR Delivery rate (%) per OR per ET Miscarriage rate (%) Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

31 PGD FOR ANEUPLOIDY Present Clinical Applications Chromosome numerical abnormalities in patients with a normal karyotype Aneuploidy screening (PGS)

32 ANEUPLOIDY Aneuploidy Data Aneuploidy Data Cycles to OR Cycles to OR 3753 Cycles to transfer Cycles to transfer 2638 Clinical pregnancies (% per OR) (% per ET) 3210 (19) (27) Clinical pregnancies (% per OR) (% per ET) 781 (21) (30) Miscarriages (% per pregnancies with follow-up) 93 (14) Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

33 Aneuploidy. Data PGS cycles Advanced maternal age Advanced maternal age + other poor prognosis indications Cycles to OR Delivery rate (%) per OR per ET a a a P<0.001 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

34 Aneuploidy. Data PGS cycles CLINICAL OUTCOME THROUGHOUT THE YEARS c Pregnancy rate / ET % ab a c bd e d e Pregnancy rate / OR Implantation rate 5 ab P<0.01 cde P< Data collection I+III IV V VI VII VIII IX X Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

35 PGD PREGNANCIES

36 Evolution of pregnancy No. clinical pregnancies 5187 Lost to follow-up 193 Pregnancy loss (miscarriage, TOP, ectopic) No. deliveries (% over pregnancies with follow-up) (83) Singletons 3182 Twins 921 Triplets 37 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

37 Data on live born children Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

38 Congenital malformations at birth No. data available 4021 No. babies with malformations 154* (3.8%) Major 84 (2.08%) (37 in twins, 1 in a triplet) Minor 74 (1.8%) (23 in twins) *some babies had more than one malformation

39 Neonatal complications at birth No. data available 3917 No. deliveries with complications 301* Singletons 51 Twins 270 Triplets 30 *some babies had more than one complication

40 PGD Misdiagnoses

41 No. transferred embryos No. implanted embryos Misdiagnoses Monogenics (1%) Sexing for X- linked diseases Translocatio ns (1.7%) (0.5%) PGS (0.3%) Sex selection (0.7%) No misdiagnoses reported in 2007 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October Hum Reprod 2010;25:

42 PGD RECENT ADVANCES

43 RECENT ADVANCES - BIOPSY

44 BIOPSY Combination of PB + blastomere biopsy FISH analysis on PB with the probes specific for chromosomes 13 (red), 16 (aqua), 18 (pink), 21 (green) and 22 (yellow). PB1 is euploid No calling for PB2 due to pulverization of the signals Oocyte unknown The same probes are tested on a blastomere biopsied from the corresponding day 3 embryo that is called as chromosomally normal

45 BIOPSY Combination of PB + blastomere biopsy The combination of polar body and embryo biopsy does not affect embryo viability Magli et al. The combination of polar body and embryo biopsy does not affect embryo viability. Hum Reprod 2004;19:

46 BIOPSY Cleavage stage: How many cells to biopsy?

47 BIOPSY The biopsy of one cell does not affect embryo viability De Vos A, Staessen C, De Rycke M, Verpoest W, Haentjens P, Devroey P, Liebaers I, Van de Velde H. Impact of cleavage-stage embryo biopsy in view of PGD on human blastocyst implantation: a prospective cohort of single embryo transfers. Hum Reprod 2009;

48 BIOPSY Blastocyst stage: Trophectoderm biopsy Zona opening on day 3 (day 5) Biopsy on day 5

49 BIOPSY Viability of biopsied blastocysts 149 biopsied blastocysts transferred to 48 patients (no genetic or chromosomal analysis) 52 implanted with Fetal Heart Beat 34.9 Implantation rate Jones G, Cram DS, Song B, Kokkali G, Pantos K, Trounson AO. Novel strategy with potential to identify developmentally competent IVF blastocysts. Hum Reprod 2008;23:

50 BIOPSY Viability of biopsied blastocysts 219 biopsied blastocysts transferred to 208 patients (PGD for monogenic diseases) 97 implanted with Fetal Heart Beat 44.3 Implantation rate The biopsy of trophectoderm cells does not affect embryo viability Leigh d, mcarthur s, DE Boer K, Marshall J, Traversa M, Jansen RPS.PGD by blastocyst biopsy. Reprod Biomed Online 2009;18:3-S6..

51 RECENT ADVANCES - BIOPSY - CRYOPRESERVATION

52 PGD - CRYOPRESERVATION There are several situations when embryos may be frozen in cases of PGD: - prior to the biopsy (for example in cases of ovarian hyperstimulation syndrome) - after the biopsy to give more time to perform the diagnosis - after the biopsy and diagnosis where fresh embryos have been transferred but unaffected surplus embryos remain.

53 PGD - CRYOPRESERVATION Slow freezing Cryopreserved biopsied cleavage stage embryos show a lower survival rate than cryopreserved intact embryos (Magli et al., 1999; Stachecki et al., 2005). Embryos biopsied at Day 3 and cryopreserved at blastocyst stage show a higher survival rate than if cryopreserved on Day 3 (Zhang et al., 2009). Blastocysts having been biopsied on Day 3 have similar survival and implantation rates when compared with intact blastocysts (Magli et al., 2006; El-Thoukhy et al., 2009)

54 CRYOPRESERVATION Vitrification Vitrified biopsied blastocysts show a higher survival rate than slow freezing. Slow freezing Vitrification P No. cycles Survival 71% 95% <0.05 Mean age 37.8± ±3.4 Mean no. blastocysts transferred 1.9± ±0.4 Births + ongoing PR (%) 20 (23) 28 (37) <0.05 IR 38/146 (26) 36/99 (36) <0.05 Miscarriages 4% 5% Keskintepe L, Sher G, Machnicka A, Tortoriello D, Bayrak A, Fisch J, Agca Y. Vitrification of human embryos subjected to blastomere biopsy for pre-implantation genetic screening produces higher survival and pregnancy rates than slow freezing. J Assist Reprod Genet 2009;26:

55 RECENT ADVANCES - BIOPSY - CRYOPRESERVATION - MICROARRAYS

56 ARRAY CGH DNA TO BE TESTED + NORMAL CONTROL DNA DNA EXTRACTION Cy3 Cy5 DNA AMPLIFICATION LABELLING

57 ARRAY CGH HYBRIDIZATION Patient ID

58 ARRAY CGH

59 ARRAY CGH No variation Loss of DNA Patient ID Gain of DNA

60 ARRAY CGH Euploid, XX

61 ARRAY CGH Aneuploid, XY +6, 22-5, 14, 19, 21

62 ARRAY CGH Is this a reliable technique?

63 ARRAY CGH ESHRE Proof of principle study -To perform the analysis within a time period that is compatible with fresh transfer -To test the reliability of the technique by evaluating the concordance between biopsied cell and corresponding oocyte / embryo /blastocyts 12 hrs 226 oocytes for reanalysis in 3 months Geraedts J, Collins J, Gianaroli L, Goossens V, Handyside A, Harper J, Montag M, Repping S, Schmutzler A. What next for preimplantation genetic screening? A polar body approach! Hum Reprod. 2010;25:

64 ARRAY CGH ESHRE Proof of principle study Number of patients 41 Number of cycles 42 Average age 40.0 Average number of zygotes 5.5 Total number of zygotes 226 Geraedts et al. Submitted.

65 PB1 PB2 2 pn OOCYTE

66 ARRAY CGH ESHRE Proof of principle study Number PB biopsied 452 Number amplified % Number diagnosed % Euploid % Aneuploid % Concordance PB1+PB2+oocyte = 94% Geraedts et al. Submitted.

67 Microarrays CGH Whole Genomic Amplification SNP BAC probes Labelling Hybridization Single Nucleotide Polymorphism Labelling Scanning / Analysis

68 SNP Microarrays Loss of heterozygosity (LOH) probability plots for (A) a single cell derived from a female cell line with monosomy 21, and (B) a single cell derived from a male cell line with trisomy 13. Treff et al. Accurate single cell 24 chromosome aneuploidy screening using whole genome amplification and single nucleotide polymorphism microarrays. Fertil Steril 2010;94:

69 Karyomapping PGD + PGS. PGD Translocations + PGS - Genome wide genotyping of parents and appropriate family member(s) for ~340,000 biallelic single nucleotide polymorphisms (SNPs) - Whole genome amplification of single or small numbers of cells biopsied from each embryo and SNP genotyping - Mendelian analysis of parental and grandparental haplotypes - Construction of karyomaps identifying the parental and grandparental origin of each chromosome or chromosome segment in each embryo Handyside et al. Karyomapping: a Universal Method for GenomeWide Analysis of Genetic Disease based on Mapping Crossovers between Parental Haplotypes. J Med Genet 2010;47:

70 Karyomapping PGD Translocations In translocation cases: discriminates between normal vs. balanced

71 RECENT ADVANCES - BIOPSY - CRYOPRESERVATION - MICROARRAYS Can also be applied for: Simultaneous PGD + PGS PGD for translocations Simultaneous PGD for translocations + PGS

72 SNP Microarrays PGD + PGS Carriers for GM1 gangliosidosis, an autosomal recessive lysosomal storage disorder, after the diagnosis of the disease in their child (female carrier for the R201C mutation; male carrier for the R59C mutation, both housed within the GLB1 gene). The couple also recently had had a 1 st trimester spontaneous abortion. Brezina et al. Single-gene testing combined with single nucleotide polymorphism microarray preimplantation genetic diagnosis for aneuploidy: a novel approach in optimizing pregnancy outcome. Fertil Steril Advanced online access.

73 Array CGH PGD for Translocations Patient with a reciprocal translocation: 46XX,t(11p;19q) Gain 11q Loss 19q

74 Array CGH PGD for Translocations + PGS Patient with a reciprocal translocation: 46XX,t(5q;8p) Gain 8p Loss 11q Gain 19p Loss 5q Loss 16

75 Arrays CLINICAL RESULTS Blastocyst biopsy + Vitrification

76 Arrays Trophectoderm biopsy Blastocyst Vitrification Comprehensive Chromosome Screening (CCS) Frozen Blastocyst Transfer based on CCS results To date, 269 blastocyst CCS cycles including an FET have been completed for the following indications: 1.Advanced maternal age 2.Recurrent miscarriage 3.Repeated IVF failure Schoolcraft et al. Fertil Steril 2010

77 Arrays Results and Outcome (n=269): Mean maternal age = 37.4 years Survival following vitrification = 97.4% (486/499) Mean # Blastocysts Transferred = 1.8 Probability a euploid blastocyst will implant = 61.2% Miscarriage rate = 4.1% Schoolcraft et al. Fertil Steril 2010

78 CGH in blastocysts vs. polar bodies Results and Outcome Blastocyst biopsy (16 patients): Mean maternal age = 38.4 years (32-43) Previous IVF failures = 4.4 (3-10) Diagnosed following devitrification = 94% (73/78) Mean # Blastocysts Transferred = 1.9 Implantation rate = 58.3% Clinical pregnancy rate = 69.2% Results and Outcome PB biopsy (16 patients): Mean maternal age = 39.9 years (35-44) Previous IVF failures = 3.8 (3-7) PB diagnosed following zygote devitrification = 87% (203/234) Mean # day 2 embryo transferred = 1.9 Implantation rate = 11.5% Clinical pregnancy rate = 21.% Fragouli E, Katz-Jaffe M, Alfarawati S, Stevens J, Colls P, N-neka Goodall BA, Tormasi S, Gutierrez-Mateo C, Prates R, Schoolcraft WB, Munne S, Wells D. Comprehensive chromosome screening of polar bodies and blastocysts from couples experiencing repeated implantation failure. Fertil Steril 2010;94:

79 RECENT ADVANCES - BIOPSY - CRYOPRESERVATION - MICROARRAYS - PCR-based PGD protocol for translocations

80 PCR-based PGD protocol for translocations Monosomy Trisomy Euploidy Robertsonian 13;14 Test both partners for informative STR (short tandem repeats) markers that flank each breakpoint in Reciprocal translocaions. For Robertsonian translocations, STR markers located at any point along the chromosomes involved allows for differentiation between aneuploid embryos and normal / balanced embryos by simply enumerating peak signals. Fiorentino F, Kokkali G, Biricik A, Stavrou D, Ismailoglu B, De Palma R, Arizzi L, Harton G, Sessa M, Pantos K. Polymerase chain reaction-based detection of chromosomal imbalances on embryos: the evolution of preimplantation genetic diagnosis for chromosomal translocations. Fertil Steril 2010;94:

81 PCR-based PGD protocol for translocations Embryo with UPD14 Female Male Fiorentino F, Kokkali G, Biricik A, Stavrou D, Ismailoglu B, De Palma R, Arizzi L, Harton G, Sessa M, Pantos K. Polymerase chain reaction-based detection of chromosomal imbalances on embryos: the evolution of preimplantation genetic diagnosis for chromosomal translocations. Fertil Steril 2010;94:

82 RECENT ADVANCES - BIOPSY - CRYOPRESERVATION - MICROARRAYS - QUALITY MANAGEMENT: GUIDELINES FOR PGD

83 ESHRE PGD GUIDELINES - Harton GL. Braude P, Lashwood A, Schmutzler A, Traeger-Synodinos J, Wilton L, Harper JC. ESHRE PGD consortium best practice guidelines for organization of a PGD centre for PGD/preimplantation genetic screening. Hum Reprod Advanced online access. - Harton GL, Magli MC, Lundin K, Montag M, Lemmen J, Harper JC. ESHRE PGD Consortium/Embryology Special Interest Group Best practice guidelines for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS). Hum Reprod Advanced online access. - Harton GL, Harper JC, Coonen E, Pehlivan T, Vesela K, Wilton L. ESHRE PGD consortium best practice guidelines for fluorescence in situ hybridization-based PGD. Hum Reprod Advanced online access. - Harton GL, De Rycke M, Fiorentino F, Moutou C, SenGupta S, Traeger- Synodinos J, Harper JC. ESHRE PGD consortium best practice guidelines for amplification-based PGD. Hum Reprod Advanced online access.

84 PGD PREVENTIVE ART Reproductive history of 202 couples SISMER Dec infertile 81 not proven fertility 105 fertile 1 dead at the age of 2 years - Wiskott-Aldrich 1 dead at the age of 3 years - SSCP 1 dead at the age of 14 months spinal-muscolar atrophy 1 dead 6 hrs after birth type II glicogenosis 1 dead at the age of 7 months - spinal-muscolar atrophy 1 dead 5 hrs after birth - Potter 1 1 dead a the age of 6 years - Wiskott-Aldrich 1 dead after 12 hrs - 21 hydroxylase deficiency 1 dead at the age of 2 yrs Menkes 1 dead at the age of 2 yrs type II glycogenosis 1 dead at the age of 9 months spinal-musclar atrophy 78 TOP affected fetus 57 affected children born