Current technical and medical developments in embryo testing and the possible impact on the extent of the application Luca Gianaroli
PREIMPLANTATION GENETC DIAGNOSIS PGD oocyte PGD embryo
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
ESHRE PGD CONSORTIUM Data 1997-2007 BIOPSY METHOD 2870 PB Of which 759 sent by SISMER PB1 only 23541 Cleavage 49 PB + Cleavage 99 Blastocyst 0 5000 10000 15000 20000 25000 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Hum Reprod 2010;25:2685-2707.
PGD on embryo - Legal Status in Europe Allowed Forbidden Not Regulated
PGD INDICATIONS AND DATA STATE OF THE ART
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 2008. Hum Reprod 2010;25:2685-2707.
ESHRE PGD CONSORTIUM No. CENTRES 60 50 40 30 20 10 0 Data collection I II III IV V VI VII VIII IX X 1/97-9/98 to 5/2000 to 5/01 5-12/01 2002 2003 2004 2005 2006 2007 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Hum Reprod 2010;25:2685-2707.
500000 ASSISTED REPRODUCTIVE TECHNOLOGY IN EUROPE: EIM REGISTERS PGD CONSORTIUM 450000 400000 350000 300000 EIM PGD cons 250000 200000 150000 100000 50000 0 Year 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 No. EIM cycles 203893 232443 258460 279267 289690 257682 295081 367066 419037 459170 479288 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Hum Reprod 2010;25:2685-2707.
ESHRE PGD CONSORTIUM No. CYCLES 6000 5000 4000 3000 2000 1000 0 Data collection I II III IV V VI VII VIII IX X 1/97-9/98 to 5/2000 to 5/01 5-12/01 2002 2003 2004 2005 2006 2007 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Hum Reprod 2010;25:2685-2707.
MEAN NUMBER OF CYCLES REPORTED BY THE CONTRIBUTING CENTERS - TEN DATA COLLECTIONS 120 100 80 60 40 20 0 Data collection I II III IV V VI VII VIII IX X 1/97-9/98 to 5/2000 to 5/01 5-12/01 2002 2003 2004 2005 2006 2007 No. of centers 16 25 24 36 43 50 45 39 57 57 No. of cycles 392 926 782 1890 2219 2984 3358 3488 5858 5887 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Hum Reprod 2010;25:2685-2707.
CLINICAL OUTCOME THROUGHOUT THE YEARS 40 35 a b % 30 25 20 abc def d e c f Pregnancy rate / ET Pregnancy rate / OR 15 10 Implantation rate 5 0 Data collection V VI VII VIII IX X 2002 2003 2004 2005 2006 2007 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 2008. Hum Reprod 2010;25:2685-2707.
ESHRE PGD CONSORTIUM To December 2007: 27630 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 2008. Hum Reprod 2010;25:2685-2707.
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
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)
BETA THALASSEMIA COD.39 C T Sequencing Analysis Minisequencing Normal Allele Mutated Allele Normal Allele Mutated Allele
PGD - Present Clinical Applications Estimated prevalence in Europe www.orpha.net Nov 2010 Autosomal Recessive Autosomal Dominant X-linked Triplet repeats expansion Cystic Fibrosis 12/100 000 Beta Thalassemia 0.5/100 000 Sickle Cell Anemia 11/100 000 Spinal Muscular Atrophy (SMA) 7.8/100 000 (It) Retinoblastoma 5.4/100 000 Neurofibromatosis NFI 25/100 000; NF2 0.5/100 000 Li Fraumeni syndrome reported 400 families Duchenne / Becker Muscular Dystrophy 5/100 000 Hemophilia A 11/100 000 Hemophilia B 2/100 000 Fragile X 14.25/100 000 Myotonic Dystrophy 4.5/100 000 Huntington 5.9/100 000 Adrogenital Syndrome (SAG) 1/10 000 births Familial Adenomatous polyposis 5.25/100 000 Congenital Adrenal Hyperplasia (CAH) 10/100 000 Fanconi Anemia 1/100 000 Medium chain Acyl-CoA dehydrogenase deficiency (MCAD) 1/68 UK, 1/101 DK, 1/133 It Achondroplasia 4.5/100 000 Hypochondroplasia 3.3/100 000 Multiple Endocrine Neoplasy 2 3.5/100 000 Marfan Syndrome 20/100 000 Tay Sachs disease 0.3/100 000 births Osteogenesis Imperfecta 6.5/100 000 Alpha-1-Antitripsin 25/100 000 Tuberous Sclerosis 8.8/100 000 Epidermolysis Bullosa 2.5/100 000 Retinitis Pigmentosa 27.5/100 000 http://www.orpha.net/orphacom/cahiers/docs/gb/prevalence_of_rare_diseases_by_alphabetical_list.pdf
THALASSEMIA 1.5% carriers in the world 400.000 affected 12% carriers 700 affected 12.5% carriers 1.600 affected 7% carriers 1.300 affected
Autosomal recessive. Data 1997-2007 Cystic Fibrosis Beta thalassemia Sickle cell (+/-HLA) Spinal muscular atrophy + retinitis pigmentosa HLA compatibility HLA compatibility + specific disease Cycles to OR 643 700 285 269 Clinical pregnancy rate (%) per OR per ET 35 30 25 20 15 10 5 0 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Hum Reprod 2010;25:2685-2707.
Autosomal dominant. Data 1997-2007 Huntington Myotonic dystrophy 1 Neurofibromatosis 1 Charcot-Marie- Tooth Cycles to OR 530 586 25 17 Clinical pregnancy rate (%) per OR per ET 35 30 25 20 15 10 5 0 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Hum Reprod 2010;25:2685-2707.
Other diseases. Data 1997-2007 Other diseases Cycles to OR 1292 Clinical pregnancy rate (%) per OR per ET 30 25 20 15 10 5 0 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Hum Reprod 2010;25:2685-2707.
Specific X-linked. Data 1997-2007 Duchenne Becker muscular dystrophy Haemophilia A - B FRAXA Cycles to OR 157 75 311 Clinical pregnancy rate (%) per OR per ET 30 25 20 15 10 5 0 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Hum Reprod 2010;25:2685-2707.
X-LINKED. Data 1997-2007 X-Linked Data 1997-2007 X-Linked Data - 2007 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)
PGD FOR CHROMOSOME ABNORMALITIES Present Clinical Applications Structural: Translocations Deletions Inversions Numerical: Altered karyotype sex chromosomes
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
PGD FOR TRANSLOCATIONS - METHODS Normal Balanced Unbalanced
Chromosome abnormalities. Data 1997-2007 Robertsonian translocations Recirpocal translocations Sex chromosome aneuploidy Others Cycles to OR 1213 2413 337 290 Clinical pregnancy rate (%) per OR per ET 35 30 25 20 15 10 5 0 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Hum Reprod 2010;25:2685-2707.
Chromosome abnormalities. Data 2007 Robertsonian translocations Recirpocal translocations Sex chromosome aneuploidy Others Cycles to OR 204 440 45 40 Delivery rate (%) per OR per ET Miscarriage rate (%) 35 30 25 20 15 10 5 0 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Hum Reprod 2010;25:2685-2707.
PGD FOR ANEUPLOIDY Present Clinical Applications Chromosome numerical abnormalities in patients with a normal karyotype Aneuploidy screening (PGS)
ANEUPLOIDY Aneuploidy Data 1997-2007 Aneuploidy Data - 2007 Cycles to OR 16806 Cycles to OR 3753 Cycles to transfer 12071 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 2008. Hum Reprod 2010;25:2685-2707.
Aneuploidy. Data 1997-2007 16806 PGS cycles Advanced maternal age Advanced maternal age + other poor prognosis indications Cycles to OR 5400 2058 Delivery rate (%) per OR per ET 25 20 15 a a 10 5 0 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 2008. Hum Reprod 2010;25:2685-2707.
Aneuploidy. Data 1997-2007 16806 PGS cycles CLINICAL OUTCOME THROUGHOUT THE YEARS 35 30 c Pregnancy rate / ET % 25 20 15 10 ab a c bd e d e Pregnancy rate / OR Implantation rate 5 ab P<0.01 cde P<0.001 0 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 2008. Hum Reprod 2010;25:2685-2707.
PGD PREGNANCIES
Evolution of pregnancy No. clinical pregnancies 5187 Lost to follow-up 193 Pregnancy loss (miscarriage, TOP, ectopic) No. deliveries (% over pregnancies with follow-up) 854 4140 (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 2008. Hum Reprod 2010;25:2685-2707.
Data on live born children 1997-2006 2007 Harper et al. (2010) ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Hum Reprod 2010;25:2685-2707.
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
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
PGD Misdiagnoses
No. transferred embryos No. implanted embryos Misdiagnoses Monogenics 6260 957 10 (1%) Sexing for X- linked diseases Translocatio ns 1598 228 4 (1.7%) 3934 609 3 (0.5%) PGS 21543 3120 10 (0.3%) Sex selection 993 143 1 (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 2008. Hum Reprod 2010;25:2685-2707.
PGD RECENT ADVANCES
RECENT ADVANCES - BIOPSY
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
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;19:1163-1169.
BIOPSY Cleavage stage: How many cells to biopsy?
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;2988-2996.
BIOPSY Blastocyst stage: Trophectoderm biopsy Zona opening on day 3 (day 5) Biopsy on day 5
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;23:1748-59.
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..
RECENT ADVANCES - BIOPSY - CRYOPRESERVATION
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.
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)
CRYOPRESERVATION Vitrification Vitrified biopsied blastocysts show a higher survival rate than slow freezing. Slow freezing Vitrification P No. cycles 85 75 Survival 71% 95% <0.05 Mean age 37.8±2.4 38.2±3.4 Mean no. blastocysts transferred 1.9±0.4 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;26:629 635.
RECENT ADVANCES - BIOPSY - CRYOPRESERVATION - MICROARRAYS
ARRAY CGH DNA TO BE TESTED + NORMAL CONTROL DNA DNA EXTRACTION Cy3 Cy5 DNA AMPLIFICATION LABELLING
ARRAY CGH HYBRIDIZATION Patient ID
ARRAY CGH
ARRAY CGH No variation Loss of DNA Patient ID Gain of DNA
ARRAY CGH Euploid, XX
ARRAY CGH Aneuploid, XY +6, 22-5, 14, 19, 21
ARRAY CGH Is this a reliable technique?
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;25:575 577.
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.
PB1 PB2 2 pn OOCYTE
ARRAY CGH ESHRE Proof of principle study Number PB biopsied 452 Number amplified 428 95% Number diagnosed 419 93% Euploid 155 34% Aneuploid 264 58% Concordance PB1+PB2+oocyte = 94% Geraedts et al. Submitted.
Microarrays CGH Whole Genomic Amplification SNP BAC probes Labelling Hybridization Single Nucleotide Polymorphism Labelling Scanning / Analysis
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;94:2017-2021.
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;47:651-658.
Karyomapping PGD Translocations In translocation cases: discriminates between normal vs. balanced
RECENT ADVANCES - BIOPSY - CRYOPRESERVATION - MICROARRAYS Can also be applied for: Simultaneous PGD + PGS PGD for translocations Simultaneous PGD for translocations + PGS
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 2010. Advanced online access.
Array CGH PGD for Translocations Patient with a reciprocal translocation: 46XX,t(11p;19q) Gain 11q Loss 19q
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
Arrays CLINICAL RESULTS Blastocyst biopsy + Vitrification
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
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
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;94:875-877.
RECENT ADVANCES - BIOPSY - CRYOPRESERVATION - MICROARRAYS - PCR-based PGD protocol for translocations
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;94:2001-2011.
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;94:2001-2011.
RECENT ADVANCES - BIOPSY - CRYOPRESERVATION - MICROARRAYS - QUALITY MANAGEMENT: GUIDELINES FOR PGD
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 2010. 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 2010. 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 2010. 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 2010. Advanced online access.
PGD PREVENTIVE ART Reproductive history of 202 couples SISMER 1997-7 Dec 2010 16 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