MASARYKOVA UNIVERZITA

Léka řská fakulta

Biologický ústav

Sanatorium REPROMEDA

Dizerta ční práce - přílohy

Preimplanta ční genetická diagnostika chromozomálních abnormit a screening aneuploidií časných embryí v terapii sterilních a infertilních pár ů metodou fertilizace in vitro

Vědní obor: Léka řská biologie 5103V023

Brno, 2009 MUDr. Kate řina Veselá

SEZNAM P ŘÍLOH

Příloha 1 Autosomální dominantní Mendelovsky d ědi čné choroby (4 strany)

Příloha 2 Autosomální recesívní Mendelovsky d ědi čné choroby (8 stran)

Příloha 3 X - vázané Mendelovsky d ědi čné choroby (2 strany)

Sperm and embryo analysis in a carrier of supernumerary inv Příloha 4 (21 stran) dup(15) marker chromosome

Hybridization of the 18 alpha–satellite probe to Příloha 5 (4 strany) revealed in PGD

Příloha 6 What next for preimplantation genetic screening? (3 strany)

ESHRE PGD Consortium data collection VI: cycles from January Příloha 7 (4 strany) to December 2003 with pregnancy follow-up to October 2004

ESHRE PGD Consortium data collection V: Cycles from January Příloha 8 (19 stran) to December 2002 with pregnancy follow-up to October 2003

Příloha 9 Central data collection on PGD and screening (1 strana)

Příloha 1 Autosomální dominantní Mendelovsky dědičné choroby

(odkazuje na www.diseasesdatabase.com)

4-hydroxyphenylpyruvate hydroxylase deficiency Blue Blue rubber bleb nevus syndrome Acro-dermato-ungual-lacrimal-tooth syndrome Branchio-oculo-facial syndrome Acrodysostosis syndrome Brugada syndrome Acrokerato-elastoidosis Bullous ichthyosiform erythroderma Acroreno-ocular syndrome Buschke-Fischer-Brauer syndrome Acute intermittent porphyria C1 esterase inhibitor (C1-INH) deficiency Adams-Oliver syndrome CADASIL Adenylate kinase deficiency Caffey disease Adiposa dolorosa Camptomelic dysplasia Alagille syndrome Camurati-Engelmann disease Albinoidism, Oculocutaneous,autosomal dominant Cardiofaciocutaneous syndrome Alpha-thalassemia/mental retardation syndrome, Cardiomyopathy, familial hypertrophic type Carney myxoma-endocrine complex Alternating hemiplegia of childhood Carotenemia, familial Amyloid polyneuropathy (Portuguese) Cavernous haemangioma Amyloidosis, primary cutaneous Cayler cardiofacial syndrome Amyotrophic lateral sclerosis Central core myopathy Amyotrophy, hereditary neuralgic Char syndrome Andersen cardiodysrhythmic periodic paralysis CHARGE syndrome Anterior segment mesenchymal dysgenesis Cherubism Apert syndrome Chester porphyria Apolipoprotein B deficiency Chondrodysplasia punctata, autosomal dominant Arias oculootoradial syndrome CINCA syndrome Autoimmune polyendocrine syndrome type 2 Cleidocranial dysplasia Autosomal dominant hypophosphatemic rickets Complement factor Hdeficiency Autosomal dominant lateral temporallobe epilepsy Congenital dyserythropoietic anaemiatype 3 Autosomal dominant rhegmatogenous retinal Congenital fiber-type disproportion myopathy detachment Congenital fibrosis of theextraocular muscles type 1 Avellino combined granular-latticecorneal dystrophy Congenital stromal corneal dystrophy Banki syndrome Contractural arachnodactyly Coproporphyria, hereditary Bare lymphocyte syndrome type 2 plana type 1 Bart-Pumphrey syndrome Costello syndrome Benign familial Craniofacialdysmorphism-polysyndactyly syndrome Benign familial infantile convulsions Craniofacial--hand syndrome Benign familial microcytic thrombocytosis Craniometaphyseal dysplasia, Jackson type Benign neonatal epilepsy Crouzon craniofacial dysostosis Benign Rolandic epilepsy Currarino triad Benign symmetrical lipomatosis of Madelung Curry-Hall syndrome Best vitelliform macular dystrophy Cushing proximal symphalangism Cyclical neutropenia Birt-Hogg-Dube syndrome Dentatorubropallidoluysian degeneration Blau syndrome Dentin dysplasia Blepharonasofacial syndrome Dentinogenesis imperfecta syndrome Denys-Drash syndrome

1 Dermatopathia pigmentosa reticularis French type sialuria DiGeorge syndrome Frontotemporal dementia Dihydrofolate reductasedeficiency Gamstorp-Wohlfart syndrome Disseminated superficial actinic porokeratosis Gardner syndrome DOOR syndrome (autosomal dominant) GCK-related permanent neonatal diabetes mellitus Dowling-Degos syndrome Gerstmann-Straussler-Scheinker disease Dowling-Meara epidermolysis bullosa Glaucoma, primary open angle,juvenile-onset type 1 Doyne honeycomb choroiditis Glomerulocystic disease Duane ocular retraction syndrome Glucocorticoid receptor defect Dutch type hereditary cerebral hemorrhage with Glucocorticoid-suppressible hyperaldosteronism amyloidosis Glucose phosphate isomerase deficiency Dysplastic nevus syndrome Glucose transporter type 1 deficiency Dysprothrombinemia, hereditary Goldenhar syndrome Dystonia musculorum deformans type 1 Gordon distal arthrogryposis syndrome Eccrine dermal cylindroma Gorlin basal cell nevus syndrome Ectrodactyly, ectodermal dysplasiaand cleft lip/palate syndrome type 1 Haber syndrome Edstrom myopathy Hailey-Hailey disease Emery-Dreifuss muscular dystrophy, autosomal Hajdu-Cheney syndrome dominant Hand-foot-uterus syndrome Epidermolysis bullosa simplex Haw River syndrome Epidermolysis bullosa simplex, Ognatype Hay-Wells syndrome type 1 Hereditary benign intraepithelial dyskeratosis Episodic ataxia type 2 Hereditary elliptocytosis Episodic ataxia type 5 Hereditary essential myoclonus Epstein macrothrombocytopenia syndrome Hereditary haemorrhagic telangiectasia Erdheim cystic medialnecrosis Hereditary leiomyomatosis and renal cell cancer Erythrokeratoderma variabilis Hereditary liability to pressure palsies Erythrokeratodermia, progressive symmetric Hereditary nonpolyposis colorectal cancer Erythromelalgia, primary Hereditaryonycho-osteodysplasia Essential tremor Hereditary paraganglioma type 1 Exostosis of external auditory canal Hereditary sensorimotor neuropathytype 1 Facio-scapulo-humeral musculardystrophy Hereditary sensorimotor neuropathytype 2 Fahr disease Hereditary sensorimotor neuropathytype 5 Familial adenomatous polyposis Hereditary sensory and autonomic neuropathy type 1 Familial atrial fibrillation type 1 Familial British dementia Hereditary stomatocytosis Familial chondrocalcinosis Hereditary xerocytosis Familial cold urticaria Hibernian fever, familial Familial cutaneous collagenoma Hidrotic ectodermal dysplasia Familial Danish dementia Hischsprungdisease-microcephaly-mental retardation Familial eosinophilia syndrome Familial euthyroid hyperthyroxinemia Holt-Oram syndrome Familial hemiplegic migraine Huntington chorea Familial hypocalciuric hypercalcaemia Huntington disease (Westphalvariant) Familial infiltrative fibromatosis Huntington disease-like 1 Familial juvenile hyperuricemic nephropathy Huntington disease-like 2 Familial partial lipodystrophy type 1 Huntington disease-like 4 Familial partial lipodystrophy type 2 Huriez syndrome Familial visceral amyloidosis,Ostertag type Hutchinson-Gilford progeria syndrome Familial visceral myopathy Hyperexplexia Fatal familial insomnia Hyperferritinemia- syndrome Fechtner syndrome Hyperkalaemic periodic paralysis Hyperlipoproteinemia, familial type 5 Ferguson-Smith epithelioma lanuginosa, congenital Finnish type amyloidosis Frasier syndrome Hypohidrotic ectodermal dysplasia, autosomal Freeman-Sheldon syndrome dominant

2 Hypokalaemic periodic paralysis (primary) Milroy disease Hypotrichosis simplex Mobius syndrome Hystrix-like ichthyosis with deafness Moore-Federman syndrome Icelandic type hereditary cerebralamyloid angiopathy Muckle-Wells syndrome gravior Muenke syndrome Ichthyosis vulgaris Muir-Torre syndrome Idiopathic multicentric osteolysis Multiple cutaneous and mucosal venous Inclusion body myopathy withearly-onset Paget malformations disease and frontotemporal dementia Multiple endocrine neoplasia type 1 INS-related permanent neonatal diabetes mellitus Multiple endocrine neoplasia type 2a Insulin receptor defect with insulin-resistant diabetes Multiple endocrine neoplasia type 2b mellitus Multiple hamartoma syndrome Iridogoniodysgenesis type 1 Multiple hereditary exostoses Isaacs-Mertens syndrome Multiple lentigines syndrome Jackson-Lawler congenital pachyonychia Multiple synostoses syndrome type 1 Jackson-Weiss syndrome Myhre syndrome Jadassohn-Lewandowsky syndrome storage myopathy Jones syndrome Myositis ossificans progressiva Juvenile colonic polyposis Myostatin-related muscle hypertrophy KCNJ11-related permanent neonatal diabetes mellitus Myotonia congenita, autosomal dominant Keratitis-ichthyosis-deafnesssyndrome, autosomal Myotonia fluctuans dominant Keratosis follicularis(congenital) Naegeli-Franceschetti-Jadassohn syndrome Kindler-Weary bullous acrokeratotic poikiloderma Nail dysplasia, isolated congenital Kjer-type optic atrophy , autosomal dominant Kleine-Levin-Critchley syndrome Neuroferritinopathy Neurofibromatosis type 1 Laing distal myopathy Neurofibromatosis type 2 Langer-Giedion syndrome Neuroserpin inclusion bodyencephalopathy, familial Langer-Saldino Neutrophilia, hereditary Lattice corneal dystrophy type 1 Nievergelt-Erb syndrome Laurin-Sandrow syndrome North Carolina macular dystrophy Lenegre-Lev disease Oculocerebrocutaneous syndrome Lenz-Majewski hyperostosis syndrome Oculo-dento-digital syndrome Liddle syndrome Okihiro syndrome Limb-mammary syndrome Lipomatosis, multiple, hereditary Osteoglophonic dysplasia Loeys-Dietz syndrome (malignant) Luder-Sheldon syndrome Lymphoedema praecox Otodental dysplasia Lymphoedema-distichiasis syndrome Machado-Joseph Azorean disease Pallister-Hall syndrome Malignant hyperpyrexia Pancreatitis, hereditary PAPA syndrome Marie Unna hereditary hypotrichosis Paramyotonia congenita of von Eulenburg Maturity onset diabetes of the young Parastremmatic May-Hegglin anomaly Parietal foramina 1 Meesman juvenile epithelial cornealdystrophy Paroxysmal extreme pain disorder Melkersson-Rosenthal-Schuermann syndrome Paroxysmal kinesigenic choreoathetosis Melnick-Fraser syndrome Paroxysmal nonkinesigenic choreoathetosis Melorheostosis Pelger-Huët anomaly Metachondromatosis Peutz-Jeghers syndrome Metaphyseal chondrodysplasia, Jansen type Photic sneeze reflex Metaphyseal chondrodysplasia, Schmid type Piebaldism synthase deficiency Plasminogen deficiency Michelin tire baby syndrome Platelet glycoprotein 4deficiency Micrognathia with peromelia

3 Polycystic kidney disease, adult (autosomal Tietz hypopigmentation-deafness syndrome dominant) Torrance type platyspondylic lethal skeletal dysplasia Polycystic liver disease Townes-Brocks syndrome Polyostotic osteolytic expansile dysplasia Treacher Collins-Franceschetti syndrome Popliteal pterygium syndrome Trichorhinophalangeal syndrome type1 Porphyria cutanea tarda type 2 (familial) Trichorhinophalangeal syndrome type3 Proximal myotonic myopathy Triosephosphate isomerase deficiency Trismus pseudocamptodactyly syndrome type 1, autosomal malabsorption syndrome dominant Tuberous sclerosis Pseudohypoaldosteronism type 2 Twenty nail dystrophy Pseudohypoparathyroidism type 1a Tylosis Pseudohypoparathyroidism type 1b Udd-Markesbery tardive muscular dystrophy Pseudopseudohypoparathyroidism Uhl disease Pseudoxanthoma elasticum dominant type 1 Ulnar-mammary syndrome of Pallister Pseudoxanthoma elasticum dominant type 2 Pulmonary surfactant protein C deficiency Undritz anomaly (congenital) Rapid-onsetdystonia-parkinsonism Variegate porphyria Rapp-Hodgkin ectodermal dysplasia syndrome Velocardiofacial syndrome Reinhardt-Pfeiffer syndrome Vohwinkel mutilating keratoderma Reis-Bucklers corneal dystrophy von Hippel-Lindau syndrome Reticulate acropigmentation of Dohi von Willebrand disease Retinoschisis, autosomal dominant type 1 Riley-Smith syndrome Wagner vitreoretinal degeneration syndrome Robinow-Sorauf syndrome Watson syndrome Romano-Ward syndrome Weber-Cockayne epidermolysis bullosa Rombo syndrome Weismann-Netter-Stuhl syndrome Roussy-Levy syndrome Weissenbacher-Zweymuller syndrome Rubinstein-Taybi syndrome Welander muscular dystrophy Rutherfurd syndrome WHIM syndrome Ruvalcaba-Reichert-Smith syndrome of Cannon Saethre-Chotzen syndrome Witkop syndrome Scalp-ear-nipple syndrome Worster-Drought syndrome Schilbach-Rott syndrome Worth disease Schnyder crystalline corneal dystrophy Xeroderma pigmentosum group B Scimitar syndrome Yellow nail syndrome Sebastian platelet syndrome Zimmermann-Laband syndrome Segawa syndrome, autosomal dominant Severe myoclonic epilepsy in infancy Short QT syndrome type 1 Shprintzen-Goldberg syndrome Sorsby pseudoinflammatory fundus dystrophy Sotos syndrome Southeast Asian ovalocytosis Spheroid body myopathy Spondyloepiphyseal dysplasia congenita Spondyloperipheral dysplasia Steinfeld syndrome Strudwick spondylometaepiphyseal dysplasia Syndactyly type 1 Syndactyly type 4 Tarsal-carpal coalition syndrome Teunissen-Cremers syndrome Thrombocytosis, autosomal dominant

4 Příloha 2 Autosomální recesívní Mendelovsky dědičné choroby

(odkazuje na www.diseasesdatabase.com)

17-beta hydroxysteroid dehydrogenase deficiency of testis Alpers disease 2-methylbutyryl-coenzyme A Alpha-1-antitrypsin deficiency dehydrogenase deficiency Alpha-L-iduronidase deficiency (Hurler 3-alpha-hydroxyacyl- CoA dehydrogenase syndrome) deficiency Alpha-L-iduronidase deficiency (Hurler- 3-beta hydroxylase deficiency Scheie syndrome) 3-Hydroxyisobutyric aciduria Alpha-L-iduronidase deficiency (Scheie 3M syndrome syndrome) 3-Methylcrotonyl-CoA carboxylase Alpha-mannosidase deficiency deficiency Alström syndrome 3-Methylglutaconic aciduria type 1 Aminomethyltransferase deficiency 3-Methylglutaconic aciduria type 3 Amish lethal microcephaly 3-Methylglutaconic aciduria type 4 Amish nemaline myopathy 5-alpha reductase deficiency Andermann syndrome 6-Pyruvoyl tetrahydropterin synthase Anderson disease deficiency Antiplasmin deficiency Aase syndrome Antithrombin 3 deficiency ABCD syndrome Antley-Bixler syndrome Abetalipoproteinaemia Apolipoprotein C-II deficiency Ablepharon-macrostomia syndrome Arginase deficiency Achalasia-addisonian syndrome Arginosuccinate synthetase deficiency Acheiropodia Arginosuccinic aciduria Aromatase deficiency Achondrogenesis, type 1A Arylsulphatase A deficiency Achromatopsia Aspartoacylase deficiency Acrocapitofemoral dysplasia Ataxia telangiectasia Acrocephalopolydactylous dysplasia Ataxia-oculomotor apraxia type 1 Acrocephalopolysyndactyly type 2 Ataxia-oculomotor apraxia type 2 Activated protein C resistance Atransferrinaemia, hereditary Adenine phosphoribosyltransferase Atypical mycobacteriosis, familial deficiency Autoimmune polyendocrine syndrometype 1 Adenosine monophosphate deaminase Autosomal recessive hypercholesterolemia deficiency Autosomal recessive Adenylosuccinate lyase deficiency type 1 Aicardi Goutieres syndrome Baller-Gerold syndrome Aldolase A deficiency Bamforth-Lazarus syndrome Baraitser-Winter syndrome

1 Bartter syndrome Ceroid lipofuscinosis, neuronal 4 Behr syndrome Ceroid lipofuscinosis, neuronal 5 Belgian type mental retardation Ceruloplasmin deficiency Benign familial recurrent cholestasis Chanarin-Dorfman disease Berardinelli lipodystrophy syndrome Charcot-Marie-Tooth disease type 4a Beta thalassaemia (severe/homozygous) Charcot-Marie-Tooth disease type 4c beta-Mannosidosis Charlevoix-Saguenay spastic ataxia Bietti crystalline retinopathy Chediak-Higashi disease Bifunctional peroxisomal enzyme deficiency Cholestanol storage disease Bilateral frontoparietal polymicrogyria Cholestasis-oedema syndrome, Norwegian Bjornstad syndrome type Black locks, oculocutaneous , and Cholesterol ester storage disease deafness of the sensorineural type Chorea-acanthocytosis syndrome Blomstrand chondrodysplasia Christian syndrome 1 Bloom syndrome Chuvash polycythemia Bombay Chylomicron retention disease Bonnet-Dechaume-Blanc syndrome type 2 Bowen-Conradi syndrome COACH syndrome Brown-Vialetto-van Laere syndrome Cockayne syndrome Bruck syndrome Coenzyme Q10 deficiency Burton syndrome Cohen syndrome C11-Hydroxylase deficiency Cold-induced sweating syndrome type 1 C17-hydroxylase deficiency Complement 7 deficiency C21-hydroxylase deficiency Congenital afibrinogenaemia C3b inhibitor deficiency, hereditary Congenital disorder of type 2a Calpainopathy Congenital dyserythropoietic anaemia type 1 CAMFAK syndrome Congenital dyserythropoietic anaemia type 2 Carbamoylphosphate synthetase deficiency Congenital erythropoeitic porphyria Carbohydrate deficient glycoprotein Congenital microvillous atrophy syndrome type 1a Congenital muscular dystrophy, autosomal Carbonic anhydrase type 2 deficiency recessive Carey-Fineman-Ziter syndrome Congenital Carnitine deficiency myopathy Congenital secretory diarrhea, chloride type Carnitine palmitoyltransferase 1 deficiency Congenital secretory diarrhoea, sodium type Carnitine palmitoyltransferase 2 deficiency Cornea plana type 2 Carnosinaemia Corticosterone methyloxidase type Carvajal-Huerta syndrome 1deficiency CD59 antigen deficiency Cortisol 11-beta-ketoreductase deficiency Cenani-Lenz oligodactyly-synostosis Craniomandibulardermatodysostosis syndrome Crigler-Najjar syndrome Centromeric instability of chromosomes 1, 9 Crisponi syndrome and 16 and immunodeficiency Cross-McKusick-Breen syndrome Cerebrohepatorenal syndrome Cumming syndrome Ceroid lipofuscinosis neuronal 2 late Cystathione gamma-lyase deficiency infantile Cystathionine beta-synthase deficiency Ceroid lipofuscinosis neuronal type 8 Cystic fibrosis Ceroid lipofuscinosis, neuronal 1, infantile Ceroid lipofuscinosis, neuronal 3, juvenile

2 Cytochrome P450 oxidoreductase deficiency Folate malabsorption, hereditary D-2-hydroxyglutarate dehydrogenase Fraser-Francois syndrome deficiency Friedreich ataxia Dalmatian hypouricemia Fructokinase deficiency de Barsy syndrome Fructose-1, 6-diphosphatase deficiency De la Chapelle dysplasia Fryns syndrome 3 Desbuquois syndrome Fucosidosis D-glycerate kinase deficiency Fukuyama congenital muscular dystrophy Diastrophic dwarfism Dibasic type 1 Galactokinase deficiency Dibasic aminoaciduria type 2 Galactosamine-6-sulfatase deficiency DIDMOAD syndrome Galactose epimerase deficiency Dihydrolipoamide dehydrogenase deficiency Galactose-1-phosphateuridyl transferase Dihydropyrimidine dehydrogenase deficiency deficiency Galactosialidosis Dimethylglycine dehydrogenase deficiency Galloway-Mowat syndrome Donnai-Barrow syndrome Gamma-aminobutyrate transaminase Donohue syndrome deficiency DOPA decarboxylase deficiency Gangliosidosis GM1, type 1 Dopamine beta-hydroxylase deficiency Gangliosidosis GM1, type 2 Dubin-Johnson syndrome Gangliosidosis GM1, type 3 Dubowitz syndrome Gangliosidosis GM2, type 1 Dyggve-Melchior-Clausen dysplasia Gangliosidosis GM2, type AB Dysequilibrium syndrome Gaucher disease Ellis-van Creveld syndrome Geleophysic dysplasia Emery-Dreifuss muscular dystrophy, Geroderma osteodysplastica autosomal recessive Enlarged vestibular aqueductsyndrome Gilbert disease Epidermolysis bullosa dystrophica,Hallopeau-Siemens type Epidermolysis bullosa letalis Glanzmann thrombasthenia Ethylmalonic encephalopathy Glucose-galactose malabsorption Faciocardiorenal syndrome Glutamate formimino transferase deficiency Factor VII deficiency Factor X deficiency Faisalabad histiocytosis Glutathione peroxidase deficiency Familial alphalipoprotein deficiency (erythrocyte) Familial histiocytic reticulosis Glutathione synthase deficiency Familial hypomagnesemia withsecondary decarboxylase deficiency Glycine N-methyltransferase deficiency Familial paroxysmalrhabdomyolysis Glycogenosis type 1a Fanconi anaemia Glycogenosis type 1b Fanconi-Bickel syndrome Glycogenosis type 2 Fetal akinesia-hypokinesia sequence Glycogenosis type 3 Glycogenosis type 4 Filippi syndrome Glycogenosis type 5 Finnish congenital nephrotic syndrome Glycogenosis type 6 Fleck of Kandori Glycogenosis type 7

3 Goldberg-Shprintzen megacolon syndrome Hyperornithinaemia-hyperammonaemia- Goldmann-Favre syndrome homocitrullinuria syndrome Goldscheider disease Hyperostosis corticalis deformans juvenilis GOMBO syndrome Hyperostosis corticalis generalisata of van Gorlin-Chaudhry-Moss syndrome Buchem GRACILE syndrome Hyperoxaluria, primary type 1 Grebe chondrodysplasia syndrome Hyperoxaluria, primary type 2 Greenberg dysplasia Hyperprolinaemia type 1 Griscelli syndrome type 1 Hyperprolinaemia type 2 Griscelli syndrome type 2 Hypohidrotic ectodermal dysplasia, Guanidinoacetate methyltransferase autosomal recessive type deficiency Hypomyelination and congenital cataract Haemophilia type C Hypophosphatasia Haim-Munk syndrome I-cell disease Hair-brain syndrome Imerslund-Gräsbeck syndrome Hallervorden-Spatz disease Iminodipeptiduria Harboyan syndrome HARP syndrome Immotile cilia syndrome 1 Immunoglobulin A deficiency (selective) Hennekam syndrome Inclusion body myopathy 2 Hepatic venoocclusive disease with Indian familial childhood cirrhosis immunodeficiency Inducible costimulator deficiency Hereditary ACTH resistance Infantile neuroaxonal dystrophy Hereditary orotic aciduria Infantile-onset ascending hereditary spastic Hereditary pentosuria paralysis Hereditary sensory and autonomic Inosine triphosphatepyrophosphohydrolase neuropathy type 2 deficiency Hereditary sensory and autonomic Intestinal enteropeptidase deficiency neuropathy type 3 Intrinsic factor deficiency (congenital) Hereditary sensory and autonomic Isobutyryl-CoA dehydrogenase deficiency neuropathy type 4 Isolated corticotropin deficiency Hereditary sensory and autonomic Isolated lutropin deficiency neuropathy type 5 Isovaleric acidaemia Hermansky-Pudlak syndrome Ivemark syndrome Hexokinase 1 deficiency Jacobs arthropathy-camptodactyly syndrome High molecular weight kininogendeficiency Janz juvenile myoclonicepilepsy Histidinaemia Jarcho-Levin syndrome Holocarboxylase synthasedeficiency Jeune thoracic dystrophysyndrome Homocarnosinase deficiency Johanson-Blizzard syndrome Hunter-Thompson acromesomelicdysplasia Joubert syndrome Huntington disease-like 3 Junctional epidermolysis bullosa,non-Herlitz Hurst microtia-absentpatellae-micrognathia type syndrome Juvenile hyaline fibromatosis Hydrolethalus syndrome Juvenile primary lateral sclerosis Hydroxykynureninuria Kartagener syndrome Hydroxymethylglutaryl-CoA Kaufman-McKusick syndrome lyasedeficiency Kelley-Seegmiller syndrome Hydroxyprolinaemia

4 Keratitis-ichthyosis-deafness syndrome, Marinesco-Sjogren-Garland syndrome autosomal recessive Martsolf syndrome Keutel syndrome Matthew-Wood syndrome Meckel-Gruber syndrome Kohlschutter syndrome Medium chain acyl-CoA dehydrogenase Kostmann disease deficiency Krabbe disease Megacystis, microcolon,hypoperistalsis Krause-van Schooneveld-Kivlin syndrome syndrome Kyphomelic dysplasia Meleda disease L-2-hydroxyglutarate dehydrogenase Metaphyseal chondrodysplasia, McKusick deficiency type Lafora body disease Lamellar ichthyosis Methionine adenosyltransferase deficiency Lange Nielsen syndrome Methionine malabsorption Laron dwarfism Methylenetetrahydrofolate reductase Lathosterolosis deficiency Laurence-Moon syndrome and Leber amaurosis , cblC type Lecithin cholesterol acyltransferase Methylmalonic aciduria type 2 deficiency Mevalonate kinase deficiency Lethal congenital contracture syndrome Michels syndrome Lethal restrictive dermopathy Mirhosseini-Holmes-Walton syndrome -induced hypoglycaemia Mitochondrial acetoacetyl-CoA thiolase Leucocyte adhesion deficiency type1 deficiency Leukocyte-common antigen deficiency Mitochondrial complex III deficiency Leukoencephalopathy with vanishing white Mitochondrial trifunctional protein matter deficiency Leydig cell agenesis Mohr-Claussen syndrome Lipoid congenital adrenal hyperplasia Molybdenum cofactor deficiency Lipoid proteinosis Mounier-Kuhn syndrome Lipoprotein lipase deficiency Mucolipidosis III alpha/beta Lison syndrome Mucolipidosis III gamma Liver glycogen synthase deficiency Mucolipidosis IV Loken Senior syndrome IX Long chain hydroxyacyl-CoAdehydrogenase Mucopolysaccharidosis type IVB deficiency Mucopolysaccharidosis VII Long-chain Acyl-CoA dehydrogenase Mucosulfatidosis deficiency syndrome Lowry-Wood syndrome Muscle-eye-brain disease : alpha-ketoglutarate reductase Muscular dystrophy, late-onset distal deficiency Myeloperoxidase deficiency Majeed syndrome Myoneurogastrointestinal encephalopathy Major histocompatibility complex class I syndrome deficiency Myotonia congenita, autosomal recessive Malonyl-CoA decarboxylase deficiency N-acetylglutamate synthetase deficiency Manitoba oculotrichoanal syndrome NADH-dependent methemoglobin reductase Marden-Walker syndrome deficiency Margarita Island ectodermal dysplasia

5 NADPH-dependent methemoglobin hydroxylase deficiency reductase deficiency Phosphoethanol aminuria Nasu-Hakola disease Phosphogylcerate mutase 2 deficiency Naxos disease Pitt-Rogers-Danks syndrome Nemaline myopathy, autosomal recessive Pituitary dwarfism type 3 Neonatal haemochromatosis Plasma membrane carnitine transporter Neonatal severe primary deficiency hyperparathyroidism Plasminogen activator inhibitor 1 deficiency Nephropathic early-onset cystinosis Platelet factor X receptor deficiency Nesidioblastosis Platelet glycoprotein Ib deficiency Netherton syndrome Polycystic kidney disease, autosomal Neuhauser syndrome recessive Neu-Laxova syndrome Porphobilinogen synthase deficiency Neuroectodermal melanolysosomal disease Postaxial acrofacial dysostosis syndrome Neutral lipid storage disease with myopathy Prekallikrein deficiency Niemann-Pick disease type B Progressiveencephalopathy-edema- Niemann-Pick Disease, Type C hypsarrhythmia-optic atrophy syndrome Nijmegen chromosome breakage syndrome Progressive external ophthalmoplegia, Nonaka myopathy autosomal recessive Non-ketotic hyperglycinemia L protein Progressive familial intrahepatic cholestasis deficiency Progressive pseudorheumatoid dysplasia Non-ketotic hyperglycinemia type3 Propionyl-CoA carboxylase deficiency Norman-Roberts syndrome PCCA type Norrbottnian Gaucher disease Propionyl-CoA carboxylase deficiency Ochoa syndrome PCCB type ( Pseudohypoaldosteronism type 1, autosomal negative) recessive Oculocutaneous albinism (tyrosinase Pseudoinflammatory fundus dystrophy positive) Lavia type Oculocutaneous albinism type3 Pseudoprogeria syndrome Oculocutaneous albinism type4 Pseudotoxoplasmosis syndrome Oguchi congenital night blindness Pseudoxanthoma elasticum recessive type 1 Ohdo blepharophimosis syndrome Pseudoxanthoma elasticum recessive type 2 Omenn syndrome Pulmonary alveolar proteinosis, congenital Opitz trigonocephaly syndrome Pulmonary surfactant protein B deficiency (congenital) Oral-facial-digital syndrome type 3 Purine nucleoside phosphorylase (PNP) Ornithine ketoacid transaminase deficiency deficiency Orofaciodigital syndrome type 4 Pyknodysostosis Osteopetrosis (malignant) Pyridoxine-5'-phosphate oxidase deficiency Osteoporosis-pseudoglioma syndrome Pyridoxine-dependent epilepsy Otospondylomegaepiphyseal dysplasia Pyrimidine 5'-nucleotidase deficiency Owren disease Pyropoikilocytosis, hereditary Papillon Lefevre syndrome Pyruvate carboxylase deficiency Papular atrichia Pyruvate decarboxylase deficiency Pyruvate kinase deficiency Peroxisomal acyl-CoA oxidase deficiency Rabson-Mendenhall syndrome Persistent mullerian duct syndrome type 1 Ramon syndrome

6 RAPADILINO syndrome Sitosterolemia Recessive deafness-onychodystrophy- Sjogren-Larsson syndrome -retardation syndrome Slow acetylator status Recurrent hereditarypolyserositis Smith-Lemli-Opitz syndrome Smith-McCort dysplasia Reticular dysgenesis Sphingomyelinase deficiency Rigid spine muscular dystrophy type 1 Spinal muscular atrophy type 2 Ritscher-Schinzel syndrome Spinal muscular atrophy type 3 Roberts pseudothalidomide syndrome Spinal muscular atrophy type 4 Rolland-Desbuquois dyssegmental dysplasia SPONASTRIME Rosselli-Gulienetti syndrome Stargardt disease Rothmund-Thomson syndrome Stuve-Wiedemann syndrome Rufous oculocutaneous albinism Succinic semialdehyde dehydrogenase Saccharopine dehydrogenase deficiency deficiency S-adenosylhomocysteine hydrolase Succinyl-CoA 3-ketoacidCoA-transferase deficiency deficiency Sucrase-isomaltase deficiency Sandhoff disease Sulphite oxidase deficiency SANDO Systemic infantile hyalinosis Sanjad-Sakati syndrome Taybi-Linder syndrome Sarcosinaemia Tel Hashomer camptodactyly syndrome SC phocomelia syndrome ter Haar syndrome Schimke immunoosseous dysplasia Tetra-amelia, autosomal recessive Schindler disease Thiamine-responsive megaloblasticanemia Schinzel syndrome Schinzel-Giedion midface-retraction Thiopurine methyltransferase deficiency syndrome Thromboxane synthetase deficiency Schopf-Schulz-Passarge syndrome Thyrotropin deficiency Schulman-Upshaw syndrome Tibial hemimelia Schwartz-Jampel-Aberfeld syndrome Torg osteolysis syndrome Sclerosteosis Toriello-Carey syndrome Sea blue histiocytosis Total congenital alopecia Seckel syndrome Transcobalamin II deficiency Segawa syndrome, autosomal recessive Trimethylamine oxidase deficiency Sensenbrenner syndrome Troyer syndrome Sepiapterin reductase deficiency TSH resistance Short chain acyl-CoA dehydrogenase Tumoral calcinosis deficiency Turcot syndrome Short rib-polydactyly syndrome type 1 Tyrosinaemia type 1 Short rib-polydactyly syndrome type 2 Tyrosinaemia type 2 Short rib-polydactyly syndrome type 3 Ullrich congenital muscular dystrophy Short rib-polydactyly syndrome type 4 Unverricht-Lundborg syndrome SHORT syndrome Urocanate hydratase deficiency Shwachman-Diamond syndrome van der Knaap disease Sialidosis type 1 Varadi-Papp syndrome Sialidosis type 2 Very long-chain acyl-CoA dehydrogenase Silverman Handmaker dyssegmental deficiency dysplasia Vitamin D dependent rickets type 1

7 Vitamin D dependent rickets type 2a Vitamin E deficiency, familial isolated Waardenburg syndrome type 3 Waardenburg-Shah syndrome Walker-Warburg syndrome Werdnig-Hoffman disease Werner syndrome Wiedemann-Rautenstrauch syndrome Wilson disease Winchester syndrome Wolcott-Rallison syndrome Wrinkly skin syndrome Xanthinuria type 1 Xanthinuria type 2 Xeroderma pigmentosum group A Xeroderma pigmentosum group C Xeroderma pigmentosum group D Xeroderma pigmentosum group E Xeroderma pigmentosum group F Xeroderma pigmentosum group G Xylosylprotein4-beta-galactosyltransferase deficiency Yemenite deaf-blind hypopigmentation syndrome Young syndrome Yunis-Varon syndrome Zunich neuroectodermal syndrome

8 Příloha 3 X - vázané Mendelovsky dědičné choroby

(odkazuje na www.diseasesdatabase.com)

Aarskog syndrome Adrenal hypoplasia, X-linked congenital Forsius-Eriksson Adrenoleucodystrophy Akesson syndrome Frontometaphyseal dysplasia Allan-Herndon-Dudley syndrome G6PDH deficiency Alpha thalassaemia X-linked mental Gangliosidosis GM3 retardation syndrome Gareis-Mason syndrome Atkin-Flaitz-Patil syndrome GATA1-related cytopenia Glycerol kinase deficiency Bazex-Dupre-Christol syndrome Glycogenosis type 2b Becker muscular dystrophy Glycogenosis type 8 Bertini syndrome Golabi-Ito-Hall syndrome Blue cone monochromatism Gonadal dysgenesis XY femaletype Borjeson-Forssman-Lehmann syndrome Green color blindness Bornholm Haemophilia type A BRESHECK syndrome Haemophilia type B Bruton agammaglobulinemia Hoyeraal-Hreidarsson syndrome Cantu syndrome Hydrocephalus, X-linked Charcot-Marie-Tooth disease, X-linked 1 Hypohidrotic ectodermal dysplasia with Charcot-Marie-Tooth disease, X- immune deficiency linkedrecessive type 5 Hypohidrotic ectodermal dysplasia, X-linked Charcot-Marie-Tooth-Hoffmannsyndrome, Iduronate-2-sulfatasedeficiency X-linked type 2 Immunodysregulation polyendocrinopathy CHILD syndrome and enteropathy, X-linked Chondrodysplasia punctata 1, X-linked Immunoneurologic disorder, X-linked recessive Incomplete androgenin sensitivity Chronic granulomatous disease Jensen syndrome Coffin-Lowry syndrome Juvenile retinoschisis Congenital stationary night blindness Kennedy disease Dent disease Keratosis follicularis spinulosa decalvans Duchenne muscular dystrophy cum ophiasi Lenz syndrome Emery-Dreifuss muscular dystrophy, X- Lesch-Nyhan syndrome linked , X-linked Erythroid 5 aminolevulinatesynthetase Lowe syndrome deficiency Lujan-Fryns syndrome McLeod neuroacanthocytosis syndrome Fitzsimmons syndrome McLeod phenotype

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2 Published-Ahead-of-Print on December 4, 2008 by Journal of Andrology

Sperm and embryo analysis in a carrier of supernumerary inv dup(15) marker chromosome

Running title: Meiotic segregation of inv dup(15)

Eva Oracova1,2, Petra Musilova1,2, Olga Kopecna1, Roman Rybar1,2, Miluse Vozdova1,

Katerina Vesela2 and Jiri Rubes1,2

1Veterinary Research Institute, Brno, Czech Republic; 2Repromeda, Brno, Czech Republic

This work was supported by the Grant Agency of the Ministry of Agriculture (MZE

0002716201).

Correspondence to: Jiri Rubes, Department of Genetics and Reproduction, Veterinary

Research Institute, Hudcova 70, 621 00 Brno, Czech Republic (Tel: +420-533-331401; Fax:

+420-541-211229; E-mail: [email protected])

Copyright 2008 by The American Society of Andrology 2

Abstract

We identified a small, paternally inherited supernumerary marker chromosome inv dup(15), in a phenotypically normal and normozoospermic male from a couple with reproductive problems. Sperm analysis by fluorescence in situ hybridization (FISH) showed that the marker was present in 26% of sperm nuclei. The disomy 15 was ten times higher than in normal control donors. FISH analysis for aneuploidies of the other chromosomes showed an increase in non-disjunction of . We also examined twenty-four embryos by preimplantation genetic diagnosis and ten (41.7%) embryos contained the marker. This report provides information about inheritance of inv dup(15) from a male carrier.

Keywords: chromosome 15, spermatozoa, fluorescence in situ hybridization (FISH), preimplantation genetic diagnosis (PGD)

3

1 Small supernumerary marker chromosomes (sSMC) have been reported in 0.014 - 0.072% of

2 phenotypically normal individuals (for review see Blennow et al, 1994), 0.044% of newborn

3 infants, 0.075% of prenatal cases, 0.288% of mentally retarded patients and 0.125% of

4 subfertile people (Liehr and Weise, 2007). About half of sSMC are represented by

5 heterochromatic markers. These sSMC are usually harmless for their carriers and about 50%

6 of them are of familial origin. About 50% of all sSMC are derived from chromosome 15

7 (Eggermann et al, 2002; Crolla et al, 2005).

8 Chromosomal aberrations play an important role in the aetiology of male infertility, but the

9 influence, if any, of sSMC is uncertain. Older meiotic studies on sperm from sSMC carriers,

10 using human in vitro fertilization (IVF) with the hamster egg bioassay or fluorescence in situ

11 hybridization (FISH), showed 1:1 segregation of the marker chromosome in sperm karyotypes

12 as expected (Martin et al, 1986; Mennicke et al, 1997). More recent FISH analysis of sperm

13 nuclei in sSMC carriers showed a lower proportion of spermatozoa with the marker (Cotter et

14 al, 2000; Paetzold et al, 2006).

15 An influence of sSMC on the occurrence of nondisjunction by the so-called interchromosomal

16 effect (ICE) could also be hypothesized. sSMC might disrupt the normal pairing and

17 disjunction of other chromosomes during meiosis, as is observed in some carriers of

18 chromosomal translocations. Sperm chromosome studies using FISH analysis provide a

19 means of exploring these possibilities in sSMC carriers (Mennicke et al, 1997).

20 Preimplantation genetic diagnosis (PGD) is often used for selection of normal and balanced

21 embryos for embryo transfer during in vitro fertilization (Munne 2002; Simopoulou et al,

22 2003; Munne 2005; Otani et al, 2006). Using appropriate specific probes, PGD detects marker

23 chromosomes in preimplantation embryos from sSMC carriers as well as aneuploidy in the

24 embryos. One or two blastomeres have been examined by the FISH method, using probes for

25 5-9 chromosomes (Munne et al, 1993; Thornhill et al, 2005).

4

26 Here, we report a case study in a phenotypically normal male proband carrying sSMC. A

27 small supernumerary marker chromosome was identified as a familial heterochromatic

28 dicentric derivative of chromosome 15. Investigations of somatic cells combined with sperm

29 analysis informed genetic counseling of the couple. Multi-color FISH analysis of sperm and

30 blastomeres (PGD) was used to determine the segregation ratio the of supernumerary marker

31 chromosome and aneuploidy of chromosomes 13, 15, 16, 18, 21, 22, X and Y. While there are

32 three case studies on sSMC(15) in sperm of carriers (Mennicke et al, 1997; Cotter et al, 2000;

33 Paetzold et al, 2006), this is the first report on the frequency of this sSMC in both sperm and

34 preimplantation embryos.

35

36 Case Report

37 The proband was a 29-year-old male with karyotype 46,XY,+mar. The marker was

38 ascertained during cytogenetic examination performed due to primary sterility of the couple.

39 Cytogenetic analysis of the proband’s parents confirmed the presence of sSMC in all

40 investigated metaphase lymphocytes of his father. The proband reported no health problems,

41 his seminal parameters (volume, concentration, percentages of motile and morphologically

42 normal sperm) were normal according to the World Health Organization guidelines (1999).

43 Sperm chromatin integrity detected by the Sperm Chromatin Structure Assay (Evenson et al,

44 2002) failed to show either increased frequency of sperm DNA breaks or defects in sperm

45 chromatin condensation. His wife, a 30-year-old woman, reported no health problems. Her

46 karyotype was found to be normal. The couple underwent two IVF cycles with PGD analysis

47 for aneuploidy and detection of marker chromosome and FISH analysis of sperm for the same

48 chromosomes as the embryos. The patients gave their informed consent to participate in the

49 study. And this study was approved by the Institutional Review Board of Repromeda centre

50 for reproductive medicine. 5

51

52 Materials and Methods

53 Cytogenetic and Fluorescence In Situ Hybridization Analysis of Lymphocytes–Reverse FISH

54 was performed for identification of the marker. The DNA probe was prepared from the whole

55 marker chromosome using laser microdissection in our laboratory (Kubickova et al, 2002).

56 This probe, labeled with Spectrum Orange, was hybridized with metaphase chromosomes

57 prepared from the cultured lymphocytes of the patient. Furthermore, the origin of the marker

58 chromosome was investigated by FISH analysis using two DNA probes specific for

59 chromosome 15: a satellite III probe D15Z1 (Spectrum Aqua, Abbott, IL, USA) and an α-

60 satellite probe D15Z4 (red, Cytocell, Cambridge, UK). Hybridization was performed

61 according to the manufacturer’s instructions.

62

63 Semen Sample–Semen was collected by masturbation after requested 2–day abstinence. The

64 semen was stored frozen in liquid nitrogen without any cryoprotectives. For the FISH assay,

65 the sample was thawed at room temperature and semen was smeared onto clean microscopic

66 slides and allowed to air-dry. Slides were fixed in methanol/acetic acid (3:1). Sperm nuclei

67 were decondensed by Dithiothreitol (DTT) as described by Robbins et al (1993) and

68 hybridized immediately.

69

70 Fluorescence In Situ Hybridization Analysis of Sperm–We carried out four different FISH

71 experiments. The first FISH experiment was performed for the assessment of the frequency of

72 sperm with the sSMC and disomy for chromosomes 15 and 18. A three-color FISH assay was

73 performed using chromosome 18 α-satellite probe (Spectrum Orange, Abbott), chromosome

74 15 satellite III probe (D15Z1, Spectrum Aqua, Abbott) and a subtelomeric probe for

75 chromosome 15q (green, Q-BIOgene, Illkirch Cedex, France). The next experiment allowed 6

76 determination of the ratio of X- to Y-bearing spermatozoa with sSMC and frequency of sex

77 chromosome disomy. The four-color FISH assay was performed using chromosome X α-

78 satellite probe (a 1:1 mixture of probes labeled with Spectrum Orange and Spectrum Green to

79 obtain a yellow signal, Abbott), chromosome Y satellite III probe (Spectrum Orange, Abbott),

80 chromosome 15 satellite III probe (Spectrum Aqua, Abbott) and a subtelomeric probe for

81 chromosome 15q (green, Q-BIOgene). The two-color FISH was performed using a satellite

82 probe specific for chromosome 16 (green, Q-BIOgene) and a chromosome 21 locus specific

83 probe (Spectrum Orange, Abbott), for the detection of disomy of chromosomes 16 and 21.

84 Finally, the two-color FISH was performed using subtelomeric probes 13qter and 22qter

85 (green and red, Q-BIOgene) for detection of disomy of chromosomes 13 and 22. FISH was

86 performed according to Rubes et al (2005).

87

88 Sperm Scoring after Fluorescence In Situ Hybridization–The slides were examined using an

89 Olympus BX60 fluorescence microscope equipped with FITC/PI dual filter,

90 DAPI/FITC/Texas Red triple-bandpass filter, FITC, Texas Red, aqua single filters (Olympus)

91 and phase-contrast optics. Strict scoring criteria were used for the scoring (Rubes et al, 2005).

92 More than 10,000 sperm nuclei, after hybridization, were scored in each FISH experiment.

93

94 In Vitro Fertilization and Preimplantation Genetic Diagnosis–The couple underwent two IVF

95 cycles which were combined with intracytoplasmic sperm injection (ICSI) and PGD. For ICSI

96 (Palermo et al, 1992), the ejaculate was immediately processed by sperm washing,

97 centrifugation, and swim up techniques. The embryos were biopsied on developmental day 3

98 and one or two blastomeres from each embryo were fixed on a glass slide using the

99 Tween/HCl method (Coonen et al, 1994).

100 7

101 Fluorescence In Situ Hybridization Analysis of Blastomeres–PGD was performed by

102 sequential FISH with probes for eight chromosomes: MultiVysion PB probe (Abbott) specific

103 for chromosomes 13, 16, 18, 21 and 22 was used in the first round; chromosome 15 satellite

104 III probe (D15Z1, Spectrum Aqua, Abbott) and a subtelomeric probe for chromosome 15q

105 (red, Q-BIOgene) were used in the second round; finally, CEP X/Y probe (Spectrum

106 Green/Orange, Abbott) specific for chromosomes X and Y was used in the third round.

107 Hybridizations were performed according to the manufacturer’s protocols. The criteria for

108 signal scoring described previously by Munne et al (1998) were used.

109

110 Statistical Analysis–The data was analyzed by means of the coincidence test for relative

111 frequencies. Statistical analysis was performed by the Statistical Package for Social Sciences

112 version 15.0 for Windows (SPSS, Inc. Chicago, IL) software package. The differences were

113 assumed to be significant when the P-value was lower than 0.05.

114

115 Results

116 Metaphase Chromosomes–The small supernumerary marker chromosomes were found in all

117 metaphase lymphocytes from the investigated proband. The probe prepared by

118 microdissection of the entire marker painted the whole marker chromosome and centromeric

119 region of both acrocentric chromosomes 15 (Figure 1). It was shown that the marker

120 chromosome contains only the centromeric region of chromosome 15, but no sequence from

121 any other chromosome was detected. Hybridization with α- satellite and satellite III probes for

122 chromosome 15 (Figure 2a and b) helped us specify the origin of the marker. Thus, the sSMC

123 was identified as a heterochromatic dicentric marker and the proband’s karyotype was

124 therefore defined as 47,XY,+mar.ish inv dup(15) (D15Z1++,D15Z4+).

125 8

126 Segregation of sSMC(15) in Sperm–In 72.1% of the sperm cells, there was one copy of

127 chromosome 15 without sSMC(15); 26.3% of the sperm cells showed one chromosome 15

128 and sSMC(15); 0.66% of the nuclei had one chromosome 15 and two additional markers;

129 0.25% of the nuclei had disomy 15 and sSMC(15), and finally 0.76% showed only disomy of

130 chromosome 15 (Figure 3a, b and c). Comparison of our data with the published frequencies

131 of disomy for chromosome 15 (Spriggs et al, 1995; Mercier et al, 1998; reviewed by

132 Templado et al, 2005) showed a significantly higher proportion of sperm nuclei with disomy

133 15 in our study (1.01% vs. 0.1%; P<0.01).

134

135 Interchromosomal Effect (ICE)–Further we investigated whether there was any

136 interchromosomal effect leading to an increase in non-disjunction of other chromosomes. The

137 results are summarized in Table 1. The frequencies of aneuploidy and diploidy for studied

138 chromosomes were statistically compared with the previously published results obtained in

139 the control group examined in our laboratory (Rubes et al, 2005) and with the means of

140 disomy published by Templado et al (2005). We found a significant increase in the proportion

141 of sperm nuclei with disomy 21 and a significantly lower number of sperm nuclei with

142 disomy 22. We did not find any distortion of the expected 1:1 ratio of X- and Y-bearing

143 sperm in the carrier. The distribution of the marker was equally frequent in X- and Y-bearing

144 spermatozoa.

145

146 Preimplantation Embryos–In the first cycle, thirteen oocytes were retrieved and eight oocytes

147 were successfully fertilized. In the second cycle, twenty-six oocytes were obtained and

148 twenty-two oocytes were successfully fertilized. Five embryos from the first cycle and

149 nineteen embryos from the second cycle were biopsied; the remaining embryos were

150 unsuitable for PGD analysis (low number of cells and/or fragmentation). A total of twenty- 9

151 four embryos (thirty-eight blastomeres) were evaluated, of which ten (41.7%) carried

152 sSMC(15). The results of screening for sSMC and aneuploidy in the tested chromosomes are

153 summarized in Table 2. In the first cycle, no embryos could be transferred, because all

154 evaluated embryos carried chromosomal aneuploidy and had poor morphology (no embryo

155 developed to blastocyst). A total of eight (42.1%) blastocysts were obtained in the second

156 cycle. In this cycle, all chromosomally normal embryos without sSMC and with good

157 morphology (two hatching blastocysts and one expanded blastocyst) were transferred to the

158 uterus, but no pregnancy was achieved.

159

160 Discussion

161 Isodicentric chromosomes 15 (idic(15) or inv dup(15) chromosomes) are the most common

162 sSMC in humans (Webb, 1994). Several studies reported an increased incidence of sSMC(15)

163 in infertile males with oligo- or azoospermia (Martin-Lucas et al, 1986; Manenti, 1992;

164 Gentile et al, 1993; Morel et al, 2004). Females with sSMC(15) usually show a normal

165 fertility, which could be the reason for prevalence of maternal inheritance of the familial

166 sSMC(15) reported by some authors (Steinbach et al, 1983; Buckton et al, 1985; Webb, 1994;

167 Leana-Cox et al, 1994; Blennow et al, 1995; Liehr et al, 2004a). However, some later studies

168 showed an almost equal inheritance of the marker from both parents (Eggermann et al, 2002;

169 Crolla et al, 2005). Our patient described here is a carrier of a familial heterochromatic

170 dicentric sSMC, which was characterized by FISH analysis as an inv

171 dup(15)(D15Z1++,D15Z4+). He inherited this sSMC from his father. The father of the

172 proband was obviously fertile, and the proband’s sperm fertilized apparently normally with

173 ICSI. The normal phenotype of the proband and his father is consistent with the observation

174 that sSMC(15) contains only heterochromatin.

175 As yet, sSMC segregation in meiosis has been studied in only six males. Based on the human 10

176 in vitro fertilization with the hamster egg bioassay, Martin et al (1986) reported that the

177 frequency of sperm nuclei containing the marker chromosome in two cases of unrelated fertile

178 males (who were carriers of a bi-satellited marker chromosome of unknown origin, but not

179 chromosome 15) was not significantly different from the expected 50%. However, this

180 method allows analysis of only a small number of sperm. Wiland et al (2005) identified

181 sSMC(20) in 4% of lymphocytes and 8.25% of sperm in a phenotypically normal male by

182 FISH. This finding indicated mosaicism among the gametogenic cells, in which the

183 proportion of cells containing the sSMC(20) may reach a minimum of 16%. Concerning

184 sSMC(15), only Mennicke et al (1997) confirmed the expected 1:1 segregation in sperm of a

185 healthy male, with a history of infertility, using FISH analysis. On the other hand, Cotter et al

186 (2000) and Paetzold et al (2006) detected only 6.23% and 17%, respectively, of sperm with

187 the sSMC in the cases they investigated.

188 In our case, the sSMC(15) was observed in 26.55% of sperm nuclei analyzed. This data

189 indicates some form of selection process against the marker during spermatogenesis or the

190 possibility of a tissue-specific mosaicism. Unfortunately, no additional tissue samples from

191 the patient were available for the assessment of potential tissue-specific mosaicism. A similar

192 selection during meiosis was observed in fertile XYY males who predominantly produced

193 normal sperm containing a single-sex chromosome. It was proposed that any germ cell(s)

194 eliminating the extra Y chromosome would have a selective advantage with only 46, XY cells

195 completing meiosis (Faed et al, 1976; Gabriel-Robez et al, 1996). Thus, in those males in

196 whom the extra Y was eliminated, spermatogenesis would proceed normally. A comparable

197 mechanism may exist in some sSMC carriers (Cotter et al, 2000). The association of other

198 chromosomal material with the XY-bivalent was found to be correlated with the impairment

199 of spermatogenesis. When the marker chromosome associates with the XY-bivalent,

200 spermatogenetic failure occurs during prophase of meiosis I, leading to an early disruption of 11

201 spermatogenesis (Jaafar et al, 1994). Similar associations of the trivalent or quadrivalent with

202 the XY-bivalent were observed in infertile translocation carriers with spermatogenetic

203 impairment. In fertile translocation carriers, such associations are less likely to occur (Solari,

204 1999). In two subfertile male carriers of the sSMC, the marker was observed predominantly

205 as univalent (Chandley, 1970). We also tested the hypothesis whether the marker

206 preferentially segregates into gametes with either X or Y chromosome. There was no

207 difference between the frequency of X- and Y- bearing sperm with sSMC(15).

208 Sperm analysis of our patient showed that 1% of sperm was disomic for chromosome 15. This

209 result is significantly higher than the published frequency of chromosome 15 disomy in

210 healthy males (Spriggs et al, 1995; Mercier et al, 1998; reviewed by Templado et al, 2005)

211 and also higher than the 0.3% frequency of disomy 15 ascertained by Paetzold et al (2006) in

212 the sperm of a carrier of similar sSMC(15). Accordingly, it seems that each sSMC(15)

213 individually affects the segregation of normal chromosome 15 during meiosis.

214 Several early reports suggested that the sSMC interfere with the segregation of other

215 chromosomes during meiosis (interchromosomal effect), resulting in an increase of non-

216 disjunction (aneuploidy) associated with the sSMC (Ramos et al, 1979; Bartsch et al, 1980;

217 Anneren et al, 1984). Martin et al (1986) reported an increase in aneuploidy of chromosomes

218 18, 21, 22, X and Y in sperm of the sSMC carrier. However, the authors analyzed only thirty-

219 one sperm cells. Cotter et al (2000) detected that there is no ICE leading to non-disjunction of

220 chromosome 18 due to the presence of a del(15) marker chromosome in their carrier. We

221 examined the frequency of disomy for chromosomes 13, 16, 18, 21, 22, X and Y in sperm of

222 the sSMC(15) carrier. The frequency of sperm nuclei with disomy 21 in our patient was

223 significantly increased compared to the published data (Rubes et al, 2005); this could indicate

224 an ICE. However, it should be taken into consideration that the frequency of sperm disomy

225 increases by only a few tenths of one percent and obviously does not markedly affect 12

226 reproductive functions in its carrier. The risk to the progeny is also very low. Steinbach and

227 Djalali (1983) concluded from a review of the literature that there was no increase in trisomic

228 conceptions or miscarriages in sSMC carriers. We observed a significantly lower occurrence

229 of sperm nuclei with disomy of chromosome 22 in our patient, compared with the mean

230 published by Templado et al (2005). However, they presented the mean frequency of disomy

231 for this chromosome only from two cases, and an extremely high frequency, i.e., 1.21% was

232 described in one of them (Martin and Rademaker, 1999).

233 In our case, sSMC(15) was observed in 41% of preimplantation embryos. A total of 33.3%

234 embryos were euploid for chromosomes 13, 15, 16, 18, 21, 22, X and Y in two IVF cycles.

235 This is in accordance with the previously published frequencies which ranged between 29.7%

236 and 36% of chromosomally normal embryos. These were found by preimplantation genetic

237 diagnosis – aneuploidy screening - in extensive studies performed by Bahce et al (2000),

238 Gianaroli et al (2001), Munne et al (2003) and Baart et al (2006). Our frequency of

239 chromosomally normal embryos in PGD analysis is 40% (3787 tested embryos; unpublished

240 data). It seems that the presence of sSMC(15) does not adversely affect the early development

241 of the embryos’ appearance. The percentage of blastocysts (42.1%) in the second cycle was

242 higher than our clinic’s average at that time. (The average percentage of blastocysts at that

243 time was 31.7%; unpublished data). When the sperm of anonymous donor was used in the

244 third IVF cycle of the couple without PGD analysis, two excellent blastocysts were

245 transferred and pregnancy was established. However, the pregnancy ended with an early

246 miscarriage. Therefore this couple may have a female factor for infertility and the presence of

247 a sSMC may be a coincidental rather than a causative factor.

248 Paetzold et al (2006) suggested genetic risk is low to the offspring of a male carrier of familial

249 heterochromatic sSMC(15). They concluded that potential increase in non-disjunction of

250 chromosome 15 with trisomy rescue mechanism after fertilization (uniparental disomy 15 or 13

251 mosaic trisomy 15) and unequal crossing over of a normal chromosome 15 with the

252 sSMC(15) during male meiosis (duplication or deletion of 15q11.2 in the normal chromosome

253 15 or an insertion of euchromatic material into the marker) can also become risk factors.

254 Generally satellite sSMC carriers are considered to be at low risk of abnormal phenotype as

255 are individuals with inherited markers (Douet-Guilbert et al, 2007).

256 This report provides further information about inheritance of sSMC(15) from a paternal

257 carrier and about variability in segregation of the sSMC. Selection against the sSMC(15)

258 during spermatogenesis is suggested in some patients, but the influence of the sSMC on the

259 fertility of the proband could not be ascertained from the data available. Further studies are

260 necessary to evaluate why and under what circumstances spermatogenesis is prone to failure,

261 selection against, or transmission of the marker chromosome. The significance of sSMC such

262 as reported in this case remains unclear for the purpose of genetic counseling. 14

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Table 1. Comparison of frequencies of sperm aneuploidy and diploidy detected in this study with the previously published results

Proband Control donors

Frequencies Published frequencies (%)

Abnormal categories (%) mean Study

Disomy 13 0.140 0.10 Templado et al, 2005

Disomy 16 0.060 0.07 Templado et al, 2005

Disomy 18 0.060 0.042 Rubes et al, 2005

Disomy 21 0.408* 0.185 Rubes et al, 2005

Disomy 22 0.179* 0.47 Templado et al, 2005

Disomy X 0.040 0.014 Rubes et al, 2005

Disomy Y 0.020 0.071 Rubes et al, 2005

XY sperm 0.060 0.096 Rubes et al, 2005

Diploidy 0.219 0.225 Rubes et al, 2005

* P<0.01

Table 2. The results of PGD analysis of embryos

Embryos without sSMC Embryos with sSMC Total

Euploid embryos 5 (20.8%) 3 (12.5%) 8 (33.3%)

Aneuploid embryos 9 (37.5%) 7 (29.2%) 16 (66.7%)

Total 14 (58.3%) 10 (41.7%) 24 (100%)

23

Figure 1. Hybridization of the probe prepared by microdissection of the entire marker (red) with patient’s metaphase chromosomes. The probe painted the whole marker chromosome

(thin arrow) and pericentromeric region of both acrocentric chromosomes 15 (thick arrows).

Original magnification = 1000x.

Figure 2. Partial metaphase spread showing (a) hybridization of sSMC(15) (thin arrow) and normal chromosomes 15 (thick arrows) with probes D15Z1 (satellite III, green) and D15Z4

(α-satellite, red) and (b) the same chromosomes stained with 4´,6-diamidino-2-phenylindole.

The image was converted to black and white to enhance contrast. Original magnification =

1000x.

Figure 3. Sperm nuclei of the patient hybridized with chromosome 18 α-satellite probe (red), chromosome 15 satellite III probe (D15Z1, blue) and a subtelomeric probe for the chromosome 15q (green): (a) normal sperm, (b) normal sperm with one sSMC(15) and (c) sperm disomy for chromosome 15. Original magnification = 1000x.

RBMOnline - Vol 17 No 5. 2008 695-698 Reproductive BioMedicine Online; www.rbmonline.com/Article/3482 on web 30 September 2008 Article Hybridization of the 18 alpha–satellite probe to chromosome 1 revealed in PGD Petra Musilova obtained her PhD in genetics in 1997 at Masaryk University, Brno, Czech Republic. She is currently working in the Department of Genetics and Reproduction at the Veterinary Research Institute in Brno, where she specializes in the application of fluorescence in situ hybridization (FISH) in various research projects. Since 2004 she has been head of the PGD-FISH section at the Repromeda centre for reproductive medicine. This laboratory has performed almost 600 PGD cycles for aneuploidy and 36 cycles for translocations.

Dr Petra Musilova Petra Musilova1,2,3, Roman Rybar1,2, Eva Oracova1,2, Katerina Veselá2, Jiri Rubes1,2 1Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic; 2Repromeda, Brno, Czech Republic 3Correspondence: e-mail: [email protected]

Abstract

Although the chromosome 18 alpha–satellite probe is considered to have a very low polymorphism rate, the routine use of this probe in prenatal diagnosis revealed rare variants in size and copy number of these sequences. A polymorphic signal was detected in preimplantation genetic diagnosis (PGD) for aneuploidy, in a patient with repeated early miscarriages. A third small signal of chromosome 18 alpha-satellite probe was observed in two of four evaluated embryos. Hybridization to the woman’s metaphasic lymphocytes revealed that the small signal was localized in the pericentromeric region of chromosome 1. Reanalysis of blastomeres with telomeric probes for chromosome 18q confirmed the presence of only two copies of chromosome 18. Options for verifying PGD analysis results, to prevent misdiagnosis in cases of suspected polymorphism, are discussed. Although some authors speculate about a possible role of heterochromatin polymorphism in infertility, this rare polymorphism of 18 alpha-satellite sequences is in itself probably a normal variant. This is the third report of a cross-hybridization of the chromosome 18 alpha-satellite probe and the first report of the localization of the polymorphic 18 alpha-satellite signal to chromosome 1.

Keywords: alpha-satellite, chromosome 1, chromosome 18, FISH, polymorphism, preimplantation genetic diagnosis

Introduction

Preimplantation genetic diagnosis (PGD) for aneuploidy low polymorphism rate may be used without any pre-cycle is carried out to reduce spontaneous abortions, improve work-up. The chromosome 18 alpha–satellite probe (D18Z1) implantation rates and increase the take-home baby rate in IVF is such a probe. Nevertheless, it should be taken into account patients (Munné et al., 2003; Gianaroli et al., 2005; Verlinsky that polymorphism occasionally occurs even in these probes, et al., 2005; Kuliev and Verlinsky, 2008;). In PGD analysis, one and that a failure to reveal the polymorphism can lead to or two blastomeres are usually examined by the fluorescence misdiagnosis. in-situ hybridization (FISH) method, using probes for five to nine chromosomes. The results of FISH can only be verified on Materials and methods discarded embryos, and that is further complicated by the fact that a high proportion of preimplantation embryos are mosaic or even chaotic (Baart et al., 2006). Hence, highly reliable probes Case report are necessary. If using probes that, at an appreciable frequency, cross-hybridize or significantly vary in size, pre-cycle testing The patient was a 36-year-old woman who had experienced on lymphocytes from the partners is recommended (Thornhill two early miscarriages. Karyotype analysis revealed low-level et al., 2005). The probe for the pericentromeric region of mosaicism of chromosome X: 45,X,22ps+[7]/47,XXX,22ps+[1]/ chromosome 15 (D15Z1) or the probe for Yq12 (DYZ1) are 46,XX,22ps+[192] which can be regarded as normal at her age examples. Other probes that are considered to have a very (Russell et al., 2007). The karyotype of her 32-year-old partner 695

© 2008 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK Article - Cross-hybridization of the 18 alpha-satellite probe - P Musilova et al.

was 46,XY,14ps+,16qh+. The patient underwent an IVF cycle all analysed chromosomes. In the embryo that was trisomic for with PGD analysis for aneuploidy. Five embryos were biopsied chromosome 16, the findings in the two blastomeres were not on developmental day 3. Two blastomeres from each embryo identical. One blastomere emitted two telomeric signals; that were fixed using the Tween/HCl method (Coonen et al., 1994). is in accordance with the finding of two regular signals for the centromeric probe. However, the other blastomere showed only FISH analysis on blastomeres one telomeric signal.

PGD for aneuploidy was performed using two rounds of FISH A diploid embryo with two regular chromosome 18 probe with probes for eight chromosomes: MultiVysion PB probe signals was transferred, but pregnancy was not established. (Abbott, IL, USA) specific for chromosomes 13, 16, 18, 21 and Later on, the patient experienced another early miscarriage 22 was used in the first round; CEP X/Y and CEP 15 probes after spontaneous conception. (Abbott) specific for chromosomes X, Y and 15 were used in the second round. The same material was used for the third Discussion hybridization with a telomeric probe for the chromosome 18q (Cytocell, Cambridge, UK). Hybridizations were performed The alpha-satellite probe for chromosome 18 is considered according to the manufacturer’s protocols. to have a low polymorphism rate, and thus its use in PGD without any pre-cycle work-up is acceptable. However, the FISH analysis on lymphocytes routine use of this probe in prenatal diagnosis has revealed rare variants that can lead to misdiagnosis. A reduction in the Metaphase chromosomes, prepared from cultured lymphocytes copy number of alpha-satellite sequences can result in a weak from both partners, were hybridized with the MultiVysion or even missing signal (Skinner et al., 2001; Tepperberg et al., PB probe. Further hybridizations on the woman’s metaphase 2001; Weremowicz et al., 2001). On the other hand, cases have chromosomes were performed with painting probes for been described where the probe hybridized to chromosomes 22 chromosomes 1 and 18 (prepared in the authors’ laboratory). (Thangavelu et al., 1998) and 9 (Wei et al., 2007). An alpha-satellite probe for chromosome 18 (Abbott), which corresponds to the probe included in the MultiVysion PB The occurrence of a small signal of the chromosome 18 alpha- mixture, was hybridized together with the chromosome 1 satellite probe detected in preimplantation embryos could be alpha-satellite and classical satellite probes (Abbott), under caused by either of the above-mentioned polymorphism types. high stringency conditions at 42ºC. However, recognition of the polymorphism type is essential for the correct classification of embryos. Even though a reduction in the copy number of alpha-satellite sequences in one chromosome Results 18 is obviously the more frequently occurring polymorphism type, according to the results obtained by examination of all PGD analysis of five embryos with the MultiVysion PB probe embryos, it seemed more probable, in this case, that a low revealed three signals (two regular and one small) for the number of copies of chromosome 18 alpha-satellite sequences chromosome 18 alpha-satellite probe in all the investigated were present in a non-specific chromosome. This was blastomeres of two embryos (Figure 1a,b). These two embryos unambiguously confirmed when the probe was hybridized to the were normal for all the other analysed chromosomes. Two woman’s metaphasic lymphocytes; a small signal was localized other embryos showed two regular signals for the chromosome to the pericentromeric region of the metacentric chromosome 1. 18 probe, but one of them was trisomic for chromosome 16. It is very useful to have this information on the hybridization of The nuclei of both blastomeres from the last embryo were the probe suspected of polymorphism to parental lymphocytes fragmented and therefore excluded from further evaluation. already available during PGD analysis. Slides with cultured/ uncultured lymphocytes prepared in the pre-cycle work-up can Hybridization of the MultiVysion PB probe to the woman’s be used for this purpose, provided that this test was performed metaphase chromosomes showed a third small signal for the in the couple concerned. Due to the fact that satellite probes do chromosome 18 specific probe, located at the pericentromeric not require a long hybridization time, this investigation could region of chromosome 1 (Figure 2a). FISH with a separate be successfully performed within the time limit for carrying probe for chromosome 18 under high stringency conditions out PGD. Verification of the occurrence of a certain type of excluded non-specific cross-hybridization of the probe to polymorphism in one of the partners helps to explain the results chromosome 1. Chromosome 18 alpha-satellite sequences obtained by examination of embryo blastomeres. were shown to be inserted between the chromosome 1 alpha- satellite and classical satellite sequences (Figure 2b,c). When Colls et al. (2004) detected the polymorphism 16qh− in PGD using the whole chromosome painting probes, no structural analysis. They successfully used telomeric probes in the third rearrangement between chromosomes 1 and 18 was found in round of FISH to confirm the polymorphism and to reclassify the patient. the embryos. This approach was applied to reanalyse embryos from a patient with a polymorphism in chromosome 18 alpha- Reanalysis of the blastomeres with a telomeric probe for the satellite sequences. In the present case, the effectiveness of q arm of chromosome 18 in the third round of FISH confirmed hybridization of the telomeric probe in the third round was the presence of two chromosome 18s in all blastomeres of lowered to about 94% (15 of 16 expected signals were detected the two embryos that emitted a third small signal for the in eight reanalysed blastomeres of four embryos). Hence, this centromeric probe 18 (Figure 1c,d). Two signals were also method can aid in the classification of embryos in cases of 696 detected in both blastomeres of the embryo that was normal for suspected cross-hybridization revealed by PGD, particularly if

RBMOnline® Article - Cross-hybridization of the 18 alpha-satellite probe - P Musilova et al.

Figure 1. Fluorescence in-situ hybridization on blastomeres from an embryo showing a polymorphic signal. (a) Hybridization with MultiVysion PB probe specific for chromosomes 13 (red), 16 (blue), 18 (purple), 21 (green) and 22 (yellow). The polymorphic signal of 18 alpha-satellite probe is indicated by the arrow. (b) Rehybridization of the same blastomeres with telomeric 18q probe (green), showing two signals for chromosome 18.

Figure 2. Analysis of 18 alpha-satellite probe polymorphism in the woman’s lymphocytes. (a) Metaphase spread hybridized with MultiVysion PB probe (the same as in Figure 1). The polymorphic signal of 18 alpha-satellite probe is indicated by the arrow. (b) Partial metaphase spread showing hybridization of polymorphic chromosome 1 with 18 alpha-satellite (green), 1 alpha- satellite (red) and 1 classical satellite (blue) probes. (c) The same chromosome stained with 4',6-diamidino-2-phenylindole. The image was converted to black and white to enhance contrast.

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RBMOnline® Article - Cross-hybridization of the 18 alpha-satellite probe - P Musilova et al.

the analysis is based on 2-cell evaluation. However, a possible for analysis by fluorescence in-situ hybridization.Human false-negative result due to the decreased effectiveness of Reproduction 9, 533–537. hybridization using telomeric probes in the third round of FISH Gianaroli L, Magli MC, Ferraretti AP et al. 2005 The beneficial should be considered. The probability of such a misdiagnosis effects of preimplantation genetic diagnosis for aneuploidy support extensive clinical application. Reproductive BioMedicine Online could be decreased by the use of probes for two different 10, 633–640. sequences of a given chromosome. Kuliev A, Verlinsky Y 2008 Impact of preimplantation genetic diagnosis for chromosomal disorders on reproductive outcome. From the clinical point of view, this rare polymorphism is Reproductive BioMedicine Online 16, 9–10. probably a normal variant, since it was observed in a normal Madon PF, Athalye AS, Parikh FR 2005 Polymorphic variants on adult. Cockwell et al. (2007), who investigated the distribution chromosomes probably play a significant role in infertility. of the common polymorphism in the signal number of Reproductive BioMedicine Online 11, 726–732. Munne S, Sandalinas M, Escudero T et al. 2003 Improved chromosome 15 satellite III sequences (D15Z1), did not uncover implantation after preimplantation genetic diagnosis of aneuploidy. increased frequencies of the polymorphic signal in a group of Reproductive BioMedicine Online 7, 91–97. patients with recurrent miscarriages. However, some authors Russell LM, Strike P, Browne CE et al. 2007 X chromosome loss and highlight the fact that heterochromatin is not an inert part of ageing. Cytogenetic and Genome Research 116, 181–185. the genome and that polymorphism in these regions may play Skinner JL, Govberg IJ, DePalma RT et al. 2001 Heteromorphisms a role in infertility (Buretic-Tomljanovic et al., 1997; Madon of chromosome 18 can obscure detection of fetal aneuploidy by et al., 2005). Chatzimeletiou et al. (2006) detected a 16qh− interphase FISH. Prenatal Diagnosis 21, 702–704. polymorphism in preimplantation embryos of a patient with Tepperberg J, Pettenati MJ, Rao PN et al. 2001 Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH): 2-year repeated IVF failure. Cases of rare polymorphisms caused by multi-center retrospective study and review of the literature. the insertion of a low copy number of alpha-satellite sequences Prenatal Diagnosis 21, 293–301. in unspecific chromosomes, which have been described in the Thangavelu M, Chen PX, Pergament E 1998 Hybridization of literature so far, have been detected in prenatal diagnosis and chromosome 18 alpha-satellite DNA probe to chromosome 22. no effect on fertility has been mentioned (Thangavelu et al., Prenatal Diagnosis 18, 922–925. 1998; Winsor et al., 1999; Wei et al., 2007). In the present Thornhill AR, deDie-Smulders CE, Geraedts JP et al. 2005 couple, however, other heterochromatin variants (22ps+ in the ESHRE PGD Consortium ‘Best practice guidelines for clinical female and 14ps+, 16qh+ in the male partner) could also have preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS)’. Human Reproduction 20, 35–48. an impact on embryo viability. Verlinsky Y, Tur-Kaspa I, Cieslak J et al. 2005 Preimplantation testing for chromosomal disorders improves reproductive outcome of This is the third report describing cross-hybridization of the poor-prognosis patients. Reproductive BioMedicine Online 11, chromosome 18 alpha-satellite probe, and the first report of 219–225. the localization of the polymorphic 18 alpha-satellite signal to Wei S, Siu VM, Decker A et al. 2007 False-positive prenatal diagnosis chromosome 1. In cases of suspected rare polymorphisms in of trisomy 18 by interphase FISH: hybridization of chromosome PGD, besides reanalysis of blastomeres using telomeric probes, 18 alpha-satellite DNA probe (D18Z1) to the heterochromatic the contemporaneous testing of polymorphism type on parents’ region of . Prenatal Diagnosis 27, 1064–1066. lymphocytes is recommended if the slides from the pre-cycle Weremowicz S, Sandstrom DJ, Morton CC et al. 2001 Fluorescence in situ hybridization (FISH) for rapid detection of aneuploidy: work-up are available. experience in 911 prenatal cases. Prenatal Diagnosis 21, 262–269. Winsor EJ, Dyack S, Wood-Burgess EM et al. 1999 Risk of false- Acknowledgements positive prenatal diagnosis using interphase FISH testing: hybridization of alpha-satellite X probe to chromosome 19. This work was supported by the Grant Agency of the Ministry Prenatal Diagnosis 19, 832–836. of Agriculture (MZE 0002716201). Declaration: The authors report no financial or commercial References conflicts of interest.

Baart EB, Martini E, van den Berg I et al. 2006 Preimplantation Received 18 February 2008; refereed 6 March 2008; 13 June 2008. genetic screening reveals a high incidence of aneuploidy and mosaicism in embryos from young women undergoing IVF. Human Reproduction 21, 223–233. Buretic-Tomljanovic A, Rodojcic Badovinac A, Vlastelic I et al. 1997 Quantitative analysis of constitutive heterochromatin in couples with fetal wastage. American Journal of Reproductive Immunology 38, 201–204. Chatzimeletiou K, Taylor J, Marks K et al. 2006 Paternal inheritance of a 16qh-polymorphism in a patient with repeated IVF failure. Reproductive BioMedicine Online 13, 864–867. Cockwell AE, Jacobs PA, Crolla JA 2007 Distribution of the D15Z1 copy number polymorphism. European Journal of Human Genetics 15, 441–445. Colls P, Sandalinas M, Pagidas K et al. 2004 PGD analysis for aneuploidy in a patient heterozygous for a polymorphism of chromosome 16 (16qh–). Prenatal Diagnosis 24, 741–744. Coonen E, Dumoulin JC, Ramaekers FC et al. 1994 Optimal 698 preparation of preimplantation embryo interphase nuclei

RBMOnline® Human Reproduction pp. 1–3,Hum. 2007 Reprod. Advance Access published January 17, 2008 doi:10.1093/humrep/dem424

NEW DEBATE What next for preimplantation genetic screening?

Joyce Harper1,13, Karen Sermon2, Joep Geraedts3, Katerina Vesela4, Gary Harton5, Alan Thornhill6, Tugce Pehlivan7, Francesco Fiorentino8, Sioban SenGupta1, Christine de Die-Smulders9, Cristina Magli10, Celine Moutou11 and Leeanda Wilton12

1UCL Centre for PGD, Institute for Women’s Health, University College London, 86-96 Chenies Mews, WC1E6HX London, UK; 2Department of Embryology and Genetics, Vrije Universiteit Brussel, Laarbeeklaan, 101, 1090 Brussels, Belgium; 3AZ Maastricht, Klinische Genetica, PO Box 5800, 6202 AZ Maastricht, The Netherlands; 4Sanatorium Repromeda, Vinini 235, 615 00 Brno, Czech Republic; 5Genetics and IVF Institute, 3015 Williams Drive, Fairfax, VA 22031, USA; 6The London Bridge Fertility, Gynaecology and Genetics Centre, 1 St Thomas Street, London Bridge, London SE1 9RY, UK; 7Instituto Valenciano de Infertilidad, Plaza de la Policı´a Local, 3, 46015 Valencia, Spain; 8GENOMA, Molecular Genetics Laboratory, Via Po 102, 00198 Rome, Italy; 9PGD Working Group Maastricht, Department of Clinical Genetics, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands; 10SISMER, Via Mazzini 12, 40138 Bologna, Italy; 11Service de la Biologie de la Reproduction, SIHCUS-CMCO, 19, Rue Louis Pasteur, BP120, 67303 Schiltigheim, France; 12Melbourne IVF, 320 Victoria Parade, East Melbourne, 3002 VIC, Australia 13Correspondence address. E-mail: [email protected]

Preimplantation genetic diagnosis for aneuploidy screening (preimplantation genetic screening—PGS) has been used to detect chromosomally normal embryos from subfertile patients. The main indications are advanced maternal age (AMA), repeated implantation failure, repeated miscarriages and severe male factor infertility. Many non-random- ized PGS studies have been published and report an increase in implantation rate, and/or a decrease in miscarriage rate. Recently, two randomized controlled trials have been conducted on patients with AMA as the only indication. Neither study showed a benefit in performing PGS using live birth rate as the measure of success. The debate on the usefulness of PGS is ongoing; the only effective way to resolve the debate is to perform more well-designed and well-executed randomized clinical trials.

Keywords: preimplantation genetic screening; randomized controlled trials; miscarriage; live birth rate

The main goal of aneuploidy screening of embryos in vitro (Harper et al., 2006). In addition, PGS has been used for a derived from subfertile patients undergoing IVF is to increase variety of ‘other’ indications including a previous sporadic their chance of a healthy pregnancy. Such preimplantation genetically abnormal pregnancy, poor embryo quality, pre- genetic screening (PGS) involves the aspiration of a single vious radiotherapy and single embryo transfer (ESHRE PGD cell followed by fluorescence in situ hybridization (FISH) Consortium, unpublished data). using probes for a limited number of chromosomes on the Since the publication of the first papers on PGS on cleavage nucleus of this cell to determine the ploidy status of the stage embryos (Gianaroli et al., 1997) and polar bodies (Munne´ embryo. Subsequently, euploid embryos are selected for trans- et al., 1995; Verlinsky et al., 1995), there have been several fer and aneuploid embryos are discarded, analysed to provide hundred publications on this topic and PGS has been established confirmatory diagnosis or used for research. in many IVF centres worldwide. There has been a steady The main indications suggested for PGS are advanced increase in the number of PGS cycles reported to the Consor- maternal age (AMA; usually defined as maternal age over 37 tium, from 116 cycles in the data collection from 1997–1998 or 38 years), repeated implantation failure (RIF; usually to 2087 cycles in 2004 (Harper et al., 2007). The preliminary defined as three or more failed embryo transfer procedures analysis of data from 2005 follows this trend with 2316 PGS involving high-quality embryos), repeated miscarriage in cycles (ESHRE PGD consortium, unpublished data). patients with normal karyotypes (RM; usually at least three There have been a number of non-randomized comparative previous miscarriages) and severe male factor infertility studies of IVF/ICSI with or without PGS but most report (SMF; usually defined as abnormal semen parameters) only on implantation rate and clinical pregnancy rate per

# The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. Page 1 of 3 All rights reserved. For Permissions, please email: [email protected] Harper et al. embryo transfer procedure whereas the main outcome measure of PGS are that aneuploid embryos which have little or no via- in subfertility studies is suggested to be live birth rate per cycle bility are not transferred or frozen. In this way, high-risk and per patient (Barlow, 2003). For AMA, Obasaju et al. patients potentially avoid miscarriage and a viable abnormal (2001) found no increase in implantation rate or clinical preg- pregnancy. Knowledge of a high frequency of aneuploidy in nancy rate. Munne´ et al. (1999) originally found no increase in their embryos may further help some patients achieve closure implantation rate, but an increase in ongoing pregnancy and or choose other options, such as donor gametes, to achieve live birth rate per embryo transfer procedure. In a later study, pregnancy. Finally fewer trisomic conceptions appear to the same group found a significant increase in implantation result (Munne´ et al., 2005), and fewer embryos are replaced, rate but did not report the live birth rate (Munne´ et al., potentially reducing multiple conception risk. 2003). Gianaroli et al. (1999) reported an increase in implan- The rapid increase in the application of PGS has raised ques- tation rate and clinical pregnancy rate per embryo transfer pro- tions about its efficacy for routine use. The authors of this paper cedure. Recently, other studies have shown a significant believe that the most effective way to resolve the debate about reduction in spontaneous abortions (Munne´ et al., 2006; the usefulness of PGS is to perform well-designed and well- Colls et al., 2007). For RIF patients, Gianaroli et al. (1999) executed randomized clinical trials. The trials should be showed that PGS offered no improvement in the implantation designed to assess the anticipated benefits (a substantial or clinical pregnancy rate per embryo transfer procedure. For improvement in live birth rates and reduction in miscarriage RM patients, Munne´ et al. (2005) showed a significant rates), in relation to the risk and cost for the couple, while reduction in spontaneous abortions. addressing biopsy safety, test accuracy and diagnostic effi- The most obvious criticisms of non-randomized studies are ciency, in order to identify appropriate indications for PGS. their poor experimental design and inadequate control ESHRE is investigating the possibility of setting up a multi- groups. Few of these studies report delivery rate as the end centre randomized controlled trial. The results of this trial point, some involve small numbers of patients, some use should help to clarify whether PGS has any value for IVF two-cell biopsies and some use low numbers of probes. patients, and if so, to specify those subsets of patients for Three randomized controlled trials have been performed for whom PGS is efficacious. AMA (Staessen et al., 2004; Stevens et al., 2004; Mastenbroek et al., 2007). The first two showed no significant difference in References ongoing and live birth rates between the control and PGS group. The latest study showed that the PGS group had a Barlow DH. The design, publication and interpretation of research in Subfertility Medicine: uncomfortable issues and challenges to be faced. significant decrease in the chance of achieving an ongoing Hum Reprod 2003;18:899–901. pregnancy and live birth (Mastenbroek et al., 2007). A random- Cohen J, Wells D, Munne´ S. Removal of 2 cells from cleavage stage embryos is ized controlled trial evaluating the effectiveness of PGS in sub- likely to reduce the efficacy of chromosomal tests that are used to enhance fertile women under the age of 35 undergoing IVF treatment implantation rates. Fertil Steril 2007;87:496–503. Colls P, Escudero T, Cekleniak N, Sadowy S, Cohen J, Munne S. Increased with single embryo transfer (aimed at helping choose the efficiency of preimplantation genetic diagnosis for infertility using “no ‘best’ embryo for single embryo transfer) showed no benefit result rescue”. Fertil Steril 2007;88:53–61. of PGS (Staessen et al., 2007, abstract presented at the Gianaroli L, Magli MC, Munne´ S, Fiorentino A, Montanaro N, Ferraretti AP. Will preimplantation genetic diagnosis assist patients with a poor ESHRE meeting, Lyon). No randomized controlled trials prognosis to achieve pregnancy? Hum Reprod 1997;12:1762–1767. have been reported for other PGS indications. Gianaroli L, Magli MC, Ferraretti AP, Munne´ S. Preimplantation diagnosis for There are also criticisms for each of the three randomized aneuploidies in patients undergoing in vitro fertilization with a poor trials for AMA. One study reported on only 39 patients prognosis: identification of the categories for which it should be proposed. Fertil Steril 1999;72:837–844. (Stevens et al., 2004). Staessen et al. (2004) biopsied two Harper JC, Boelaert K, Geraedts J, Harton G, Kearns WG, Moutou C, cells, as opposed to one as is usual in PGS, which may have Muntjewerff N, Repping S, SenGupta S, Scriven PN et al. ESHRE PGD an adverse affect on embryo viability (Cohen et al., 2007). Consortium data collection V: cycles from January to December 2002 with pregnancy follow-up to October 2003. Hum Reprod 2006;21:3–21. The most recent and comprehensive study by Mastenbroek Harper JC, de Die-Smulders C, Goossens V, Harton G, Moutou C, Repping S, et al. (2007) showed a high percentage of embryos without a Scriven PN, SenGupta S, Traeger-Synodinos J, Van Rij MC et al. ESHRE diagnosis (20%), and did not include probes for chromosomes PGD Consortium data collection VIII: cycles from January to December 15 and 22 (trisomies of which are prevalent among spon- 2005 with pregnancy follow-up to October 2006. Hum Reprod 2007, in press. Mastenbroek S, Twisk M, Van Echten-arends J, Sikkema-Raddatz B, Korevaar taneous abortions). In addition, the pregnancy rate in the JC, Verhoeve HR, Vogel NEA, Arts EGJM, De Vries JWA, Bossuyt PM PGS group was 6% when only undiagnosed embryos were et al. In Vitro Fertilization with Preimplantation Genetic Screening. transferred and this was significantly lower compared to the N Engl J Med 2007;357:9–17. Munne´ S, Dailey T, Sultan KM, Grifo J, Cohen J. The use of first polar bodies non-PGS group suggesting a detrimental effect of the embryo for preimpantation diagnosis of aneuploidy. Hum Reprod 1995;10: biopsy procedure itself. 1015–1021. The debate on the benefit of PGS is ongoing (Twisk et al., Munne´ S, Magli C, Cohen J, Morton P, Sadowy S, Gianaroli L, Tucker M, Ma´rquez C, Sable D, Ferraretti AP et al. Positive outcome after 2006; Cohen et al., 2007). The disadvantages of PGS are that preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod it is labour intensive, invasive to the embryo and expensive. 1999;14:2191–2199. Mosaicism is common in cleavage stage embryos and analysis Munne´ S, Sandalinas M, Escudero T, Velilla E, Walmsley R, Sadowy S, of only one cell means that some embryos, that may have Cohen J, Sable D. Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod Biomed Online 2003;7:91–97. developed normally, would not be considered for transfer if Munne´ S, Chen S, Fischer J, Colls P, Zheng X, Stevens J, Escudero T, Oter M, the single cell tested gives an abnormal result. The advantages Schoolcraft B, Simpson JL et al. Preimplantation genetic diagnosis reduces Page 2 of 3 What next for PGS?

pregnancy loss in women aged 35 years and older with a history of recurrent Stevens J, Wale P, Surrey ES, Schoolcraft WB. Is aneuploidy screening for miscarriages. Fertil Steril Aug 2005;84:331–335. patients aged 35 or over beneficial? A prospective randomized trial. Fertil Munne´ S, Fischer J, Warner A, Chen S, Zouves C, Cohen J And referring Steril 2004;82(Suppl. 2):249. centers PGD group. Preimplantation genetic diagnosis significantly reduces Twisk M, Mastenbroek S, van Wely M, Heineman MJ, van der Veen F, pregnancy loss in infertile couples: a multi-center study. Fertil Steril Repping S. Preimplantation genetic screening for abnormal number of 2006;85:326–332. chromosomes (aneuploidies) in in vitro fertilisation or intracytoplasmic Obasaju M, Kadam A, Biancardi T, Sultan K, Fateh M, Munne´ S. Pregnancies sperm injection. Cochrane Database Syst Rev 2006;1: CD005291. from single normal embryo transfer in women older than 40 years. Reprod Verlinsky Y, Cieslak J, Freidine M, Ivakhnenko V, Wolf G, Kovalinskaya L, Biomed Online 2001;2:98–101. White M, Lifchez A, Kaplan B, Moise J et al. Pregnancies following Staessen C, Platteau P, Assche Van E, Michiels A, Tournaye H, Camus M, pre-conception diagnosis of common aneuploidies by fluorescent in-situ Devroey P, Liebaers I, Steirteghem Van A. Comparison of blastocyst hybridization. Hum Reprod 1995;10:1923–1927. transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective Submitted on October 8, 2007; resubmitted on November 16, 2007; accepted on randomized controlled trial. Hum Reprod 2004;19:2849–2858. December 13, 2007

Page 3 of 3 Human Reproduction Vol.22, No.2 pp. 323–336, 2007 doi:10.1093/humrep/del402 Advance Access publication November 28, 2006.

ESHRE PGD Consortium data collection VI: cycles from January to December 2003 with pregnancy follow-up to October 2004

K.D.Sermon1,11, A.Michiels2, G.Harton3, C.Moutou4, S.Repping5, P.N.Scriven6, S.SenGupta7, J.Traeger-Synodinos8, K.Vesela9, S.Viville4, L.Wilton10 and J.C.Harper7

1Centre for , 2Centre for Reproductive Medicine, University Hospital and Medical School of the Dutch-speaking Brussels Free University (Vrije Universiteit Brussel, VUB), Brussels, Belgium, 3Genetics and IVF Institute, Fairfax, VA, USA, 4Service de la Biologie de la Reproduction, SIHCUS-CMCO, Schiltigheim, France, 5Center for Reproductive Medicine, Academic Medical Center, Fertility Laboratory, Amsterdam, The Netherlands, 6Department of Cytogenetics and Center for Preimplantation Genetic Diagnosis, Guy’s and St. Thomas’ NHS Foundation Trust, Guy’s Hospital, 7UCL Centre for PGD, Department of Obstetrics and Gynecology, University College London, London, UK, 8Laboratory of Medical Genetics, University of Athens, St. Sophia’s Children’s Hospital, Athens, Greece, 9Sanatorium Repromeda, Brno, Czech Republic and 10Melbourne IVF, East Melbourne VIC, Australia 11To whom correspondence should be addressed at: Centre for Medical Genetics, University Hospital and Medical School of the Dutch-speaking Brussels Free University (Vrije Universiteit Brussel, VUB), Laarbeeklaan 101, 1090 Brussels, Belgium. E-mail: [email protected]

The sixth report of the ESHRE PGD Consortium is presented, relating to cycles collected for the calendar year 2003 and follow-up of the pregnancies and babies born up to October 2004. Since the beginning of the data collections, there has been a steady rise in the number of cycles, pregnancies and babies reported. For this report, 50 centres par- ticipated, reporting on 2984 cycles, 501 pregnancies and 373 babies born. Five hundred and twenty-nine cycles were reported for chromosomal abnormalities, 516 cycles were reported for monogenic diseases, 137 cycles were reported for sexing for X-linked diseases, 1722 cycles were reported for preimplantation genetic screening (PGS) and 80 cycles were reported for social sexing. Data VI is compared to the cumulative data for data collections I–V.

Key words: PGD/preimplantation genetic screening/fluorescence in-situ hybridization/PCR/ESHRE PGD Consortium

Introduction wrong time period, or reported after the closure of the collection Since 1999, the ESHRE PGD Consortium has reported on a reg- period, were eliminated from the calculations. ular basis on the current practice of PGD, as reported by their members (ESHRE PGD Consortium Steering Committee, 1999, Results 2000, 2002; Sermon et al., 2005; Harper et al., 2006). The data The membership has increased steadily, and while in the first analysed previously included referral and clinical data on the report data on sixteen centres were included (ESHRE PGD Con- patients consulting for PGD, the cycles they underwent, the sortium Committee, 1999), that figure has now increased to 50. pregnancies that ensued from these cycles and the babies born. This has also led to a steady increase in the number of cycles Because the analysis of the referral and clinical data was reported, from 392 in the first report to 3929 in the current report. biased by several factors, e.g. the underreporting of couples The results are represented in tables according to a now set who did not come through for PGD, this analysis is left out for layout. Accompanying text is limited to the minimum, and the current report. seven tables are only available in an electronic version: Sup- plementary Table IIc with the list of abnormal karyotypes, Materials and methods Supplementary Table IIIc with the list of X-linked diseases for which sexing was carried out, Supplementary Table IVc with Data were collected using a FileMaker Pro 5 database consisting of files for cycle records, frozen cycle records, pregnancies and babies. the list of monogenic diseases for which PGD was carried out, A first general round of correction was carried out, requesting correc- Supplementary Tables VIIIa (data I–V) and VIIIb (data VI) with tions from participating centres, followed by a more in-depth correc- the complications of pregnancy and Supplementary Tables tion by expert co-authors. Cycles with insufficient data, e.g. with no XIIa (data I–V) and XIIb (data VI) with the congenital malfor- cycle or patient identification or no clear indication, cycles from the mations and the neonatal complications.

© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. 323 For Permissions, please email: [email protected] K.D.Sermon et al.

An overview of all cycles collected previously in data of successfully biopsied embryos which gave a diagnosis (91 collections I–V can be found in Table Ia , whereas an over- and 92%), the proportion of successfully biopsied embryos view of the current data collection can be found in Table Ib. with a transferable result (25 and 25%) and the clinical preg- nancy rate per OR (15 and 14%) were similar. PGD cycles for chromosomal abnormalities The relatively low pregnancy rates are likely to reflect that Tables IIa and IIb summarize the 1207 and 529 cycles col- the low proportion of embryos with a transferable result limits lected for data collection I–V and VI, respectively. Overall, the choice of embryo for transfer (24% of embryos biopsied for there was very little difference between the two data collections. all classes of chromosome aberration and only 17% of embryos Reciprocal translocation was the most frequent class of chro- for reciprocal translocation cycles in particular for the data col- mosome aberration; ICSI was the predominant mode of fertili- lection VI). zation; acid Tyrodes with cleavage aspiration was the predominant sampling method. A global average of 14.3 and PGD cycles for sexing for X-linked diseases 14.0 cumulus oocyte complexes (COCs) per oocyte retrieval Table IIIa summarizes data I-V and Table IIIb summarizes the (OR) cycle was collected for the I–V and VI data collections, data from collection VI for sexing only for X-linked disease respectively; the fertilization rate (74 and 70%), the proportion using the PCR or fluorescent in-situ hybridization (FISH).

Table Ia. Overall cycle data collection I–V

Indication PGD PGS PGD-SS Total

Cycles to OR 2868 3078 254 6200 Number infertile 994 2894 41 3929 Female age 33 36 35 34 Cancelled after OR before IVF/ICSI 6 0 0 6 ART method IVF 369 511 113 993 ICSI 2444 2501 128 5073 IVF + ICSI 3 13 0 16 Frozen 9 4 5 18 Frozen and ICSI 14 5 5 24 Frozen and IVF 0 0 1 1 Cancelled ICSI 3 0 0 3 Unknown 18 44 0 62 Unknown and frozen 2 0 2 4 Cancelled after OR 161 128 14 303 Cycles to PGS/PGD 2701 2950 240 5891 FISH 1577 2950 143 4670 PCR 1124 0 97 1221 Zona breaching AT drilling 1703 1808 19 3530 Laser drilling 856 1000 112 1968 Mechanical 128 77 109 314 Unknown 14 65 0 79 Biopsy method Polar body biopsy 251 1781 02031 Cleavage aspiration 25761 24811 130 51871 Cleavage extrusion 85 224 110 419 Cleavage flow displacement 2 16 0 18 Unknown 16 52 0 68 Embryology COC 38955 38561 3291 80807 Inseminated 33353 32839 2772 68964 Fertilized 24239 23323 1891 49453 Biopsied 18368 17549 1589 37506 Successfully biopsied 17998 17232 1505 36735 Diagnosed 15833 15036 1367 32236 Transferable 6085 5353 652 12090 Transferred 4380 4460 449 9289 Frozen 793 687 149 1629 Clinical outcome Cycles to ET 2173 2188 192 4553 hCG positive 656 702 78 1436 Positive heart beat 514 534 59 1107 (clinical pregnancy rate % per OR/% per ET) 18/24 17/24 23/31 18/24 Implantation rate data V only (fetal hearts/embryos transferred %) 15 17 14 16

ART, assisted reproduction techniques; AT, acid Tyrode’s; COC, cumulus oocyte complexes; ET, embyo transfer; FISH, fluorescent in-situ hybridization; OR, oocyte retrieval; PGS, preimplantation genetic screening. PGD column includes PGD for chromosome abnormalities, sexing for X-linked disease and PGD for monogenic disorders. 1Four cycles had polar body biopsy and cleavage stage biopsy. 324 ESHRE PGD Consortium-VI

Table Ib. Overall cycle data collection VI

Indication PGD PGS PGD-SS Total

Total number of cycles 1182 1722 80 2984 Number infertile 501 1473 1 1975 Female age 33 37 38 35 Cycles cancelled before OR 133 9 1 143 Cycles to OR 10471 1713 79 2839 Cycles cancelled before ART 2 0 0 2 ART method IVF 97 124 5 226 ICSI 902 1558 71 2531 IVF + ICSI 17 15 0 32 Frozen + ICSI 261 10 1 37 Frozen + IVF 2 0 0 2 Unknown and frozen 1 0 2 3 Unknown 2 6 0 8 Cancelled post OR 89 45 1 135 Cycles to PGS/PGD 958 1668 78 2704 FISH 537 1668 71 2276 PCR 421 0 7 428 Zona breaching AT drilling 441 716 0 1157 Laser drilling 474 870 1 1345 Mechanical 43 82 77 202 Biopsy method PB biopsy 11 185 0 196 Cleavage aspiration 847 1409 1 2257 Cleavage extrusion 75 74 77 226 Blastocyst 25 0 0 25 Embryology COC 14490 20666 1220 36376 Inseminated 12272 16867 949 30088 Fertilized 8458 11815 644 20917 Biopsied 6254 9496 478 16228 Successfully biopsied 6156 9413 461 16030 Diagnosed 5507 8795 445 14747 Transferable 2065 3193 113 5371 Transferred 1326 2277 92 3695 Frozen 240 454 20 714 Clinical outcome Cycles to ET 709 1283 47 2039 hCG positive 237 409 20 666 Positive heart beat 185 311 15 511 Clinical pregnancy rate (% per OR/% per ET) 18/26 18/24 19/32 18/25 Number of fetal heartbeats 223 384 21 628 Implantation rate (fetal heartbeats/100 embryos transferred %) 17 17 23 17

ART, assisted reproduction technique; AT, acid Tyrode’s; COC, cumulus oocyte complexes; FISH, fluorescent in-situ hybridization; OR, oocyte retrieval; PGS, preimplantation genetic screening. PGD column includes PGD for chromosome abnormalities, sexing for X linked disease and PGD for monogenic disorders 1Includes two cycles with PGD on frozen embryos only. These cycles were not counted in the cycles with OR.

A total number of 137 cycles were collected in this group, with Overall, these data are quite unremarkable and very compa- 134 cycles using FISH and three cycles using PCR. rable to previous data collections (I–V). In contrast to other The biopsy was successful in 98% of embryos biopsied indication groups, the number of cycles for sexing for X-linked and 92% of the embryos successfully biopsied were diag- diseases remains stable. nosed. A transfer was achieved in 76% of the cycles with an average of 1.7 embryos transferred in each cycle. The overall clinical pregnancy rate was 22% per OR and 30% per PGD for monogenic diseases embryo transfer (ET) procedure, with an implantation rate of Amongst PGD cycles initiated for single- disorders 21%. between January and December 2003, the most common auto- Supplementary Table IIIc summarizes the list of 36 indica- somal recessive diseases were cystic fibrosis, β-thalassemia tions. The most common indication was Duchenne muscular (with or without HLA typing), spinal muscular atrophy and dystrophy (n = 33; 25%), haemophilia (n = 25; 19%), retinitis sickle cell anaemia. The most common autosomal dominant pigmentosa (n = 6), adrenoleukodystophy (n = 6), Alport syn- diseases were Huntington disease, myotonic dystrophy, adenom- drome (n = 5) and non-fragile X, X-linked mental retardation atous polyposis coli and Marfans syndrom. The most common (n = 5). specific diagnosis of X-linked diseases was for Duchenne and 325 K.D.Sermon et al.

Table IIa. PGD for chromosomal abnormalities, data collection I–V

Indication Robertsonian Robertsonian Reciprocal, Reciprocal, Sex chromosome Other Total translocations, translocations, male carrier female carrier abnormalities male carrier female carrier

Total cycles 193 167 307 308 144 88 1207 Number infertile 160 70 148 102 127 41 648 Female age 34 32 33 33 34 33 32 Cycles cancelled before OR 8 5 11 12 5 4 45 Cycles to OR 185 162 296 296 139 84 1162 Cancelled after OR before 00105 06 IVF/ICSI ART method IVF 9 25 39 74 14 29 190 ICSI 172 134 252 216 119 55 948 IVF + ICSI 1 1 0 0 0 0 2 Frozen 0 0 1 1 0 0 2 Unknown and frozen 0 0 0 0 1 0 1 ICSI and frozen 3 2 3 5 0 0 13 Cancelled after IVF/ICSI 11 7 19 17 7 4 65 Cycles to PGD 174 155 276 279 127 80 1091 Zona breaching AT drilling 126 109 203 216 68 62 784 Laser drilling 48 41 59 54 33 13 248 Mechanical 0 5 14 9 26 5 59 Biopsy method Polar body biopsy 1 4 0 3 0 0 8 Cleavage aspiration 170 147 264 264 127 79 1051 Cleavage extrusion 3 4 11 11 0 1 30 Cleavage flow displacement 0 0 1 1 0 0 2 Embryology COC 2668 2272 4490 4445 1657 1106 16638 Inseminated 2262 1885 3832 3860 1343 982 14164 Fertilized 1573 1404 2865 2945 965 679 10431 Biopsied 1029 1142 2290 2425 696 533 8115 Successfully biopsied 1003 1120 2248 2387 683 517 7958 Diagnosed 850 1013 2050 2212 614 472 7211 Transferable 328 300 450 470 289 171 2008 Transferred 258 244 385 403 202 128 1620 Frozen 14 22 11 17 19 14 97 Clinical outcome Cycles to ET 135 125 193 204 102 60 819 hCG positive 40 41 50 49 31 18 229 Positive heart beat 34 32 36 37 20 16 175 Clinical pregnancy rate 18/25 20/26 12/19 13/18 14/20 19/27 15/21 (% per OR/% per ET)

ART, assisted reproduction technique; AT, acid Tyrode’s; COC, cumulus oocyte complexes; ET, embryo transfer; OR, oocyte retrieval.

Becker muscular dystrophy, fragile X and haemophilia. Overall, the number of PGD cycles performed for mono- PGD cycles for an additional 47 monogenic diseases were genic disorders between January and December 2003 repre- initiated in 104 cycles, included under ‘other’ in Table IVb, sents the largest number performed in a single year so far. The listed in Supplementary Table IVc. assisted reproduction techniques (ART) and embryology pro- An average of 13.8 oocytes were collected per OR, cedures applied reflect accumulated experience and expertise, biopsy was successful in 98% of embryos, diagnosis was along with technological developments, e.g. laser drilling and achieved in 86% of embryos successfully biopsied, and 75% blastocyst biopsy. of cycles to OR had an ET, with 667 embryos transferred. With respect to the progress and outcome of cycles, includ- Overall, 20% of cycles that reached the stage of OR resulted ing the embryology, rates of diagnosis and clinical outcome in a clinical pregnancy (or 27% clinical pregnancy rate per ET). such as clinical pregnancy and embryo implantation rates, As recommended (Thornhill et al., 2005), 434 cycles had there are no apparent changes compared to previous data col- ICSI and 11 cycles had ICSI and frozen (two of which were lections (Table IVa and Supplementary Table IVc). just frozen embryos). Two cycles were noted as IVF and fro- zen and 3 cycles as IVF and ICSI. Laser drilling was used for zona breaching in 64% of biopsy cycles, with cleavage aspira- Preimplantation genetic screening tion accounting for 85% of biopsy procedures. In addition 17 The cumulative data for preimplantation genetic screening (PGS) cycles (4%) used trophectoderm biopsy in blastocysts; repre- from collections I–V are summarized in Table Va. Table Vb sum- senting the first time, blastocyst biopsy has been reported to marizes the data from collection VI. It is clear that the number of ESHRE for clinical PGD cycles. PGS cycles continues to rise. In last year’s data collection (data

326 ESHRE PGD Consortium-VI

Table IIb. PGD for chromosomal abnormalities, data collection VI

Indication Robertsonian Robertsonian Reciprocal, Reciprocal, Sex chromosome Other Total translocation, translocations, male carrier female carrier aneuploidy male carrier female carrier

Total cycles 91 60 143 159 32 44 529 Number infertile 79 34 103 95 28 24 363 Female age 33 33 34 34 33 33 33 Cycles cancelled before OR 8 9 15 17 0 9 58 Cycles to OR 83 51 128 142 32 35 471 Cancelled before ART 0 0 0 0 0 1 1 ART method IVF 2 4 10 40 4 4 64 ICSI 78 44 106 94 28 28 378 IVF + ICSI 1 1 2 5 0 1 10 ICSI + Frozen 1 2 8 3 0 1 15 Unknown + Frozen 0 0 1 0 0 0 1 Unknown 1 0 1 0 0 0 2 Cancelled after IVF/ICSI 10 6 15 16 3 1 51 Cycles to PGD 73 45 113 126 29 33 419 Zona breaching AT drilling 34 23 74 89 15 11 246 Laser drilling 38 22 34 34 13 19 160 Mechanical 1 0 5 3 1 3 13 Biopsy method Cleavage aspiration 70 42 96 112 29 30 379 Cleavage extrusion 3 3 13 11 0 3 33 Blastocyst 0 0 4 3 0 0 7 Embryology COC 1262 720 1806 1993 420 379 6580 Inseminated 1053 613 1496 1756 359 328 5605 Fertilized 703 423 989 1257 245 224 3841 Biopsied 480 324 746 948 156 179 2833 Successfully biopsied 474 323 738 933 156 177 2801 Diagnosed 432 299 674 868 143 154 2570 Transferable 170 107 117 170 70 55 689 Transferred 100 71 97 141 53 37 499 Frozen 32 6 6 8 5 4 61 Clinical outcome Cycles to ET 55 38 65 76 24 24 282 hCG positive 21 8 21 27 5 3 85 Positive heart beat 18 6 17 21 3 2 67 Clinical pregnancy rate (% per OR/% per ET) 22/33 12/16 13/26 15/28 9/13 6/8 14/24 Number of fetal hearts 23 6 19 23 3 2 76 Implantation rate (fetal hearts/100 embryos transferred) 23 8 20 16 6 5 15

ART, assisted reproduction technique; AT, acid Tyrode’s; COC, cumulus oocyte complexes; ET, embryo transfer; OR, oocyte retrieval.

V), 1211 PGS cycles were performed (Harper et al., 2006), of all biopsied embryos), 2277 were transferred and 454 whereas this year 1722 cycles were reported, which represents a were frozen. In total, 384 embryos implanted (17% implantation 42% increase. The largest increase was seen in cycles for repeated rate). implantation failures (RIF) (275–462; 68% increase) and in cycles The overall clinical pregnancy rate was 18% per OR and for severe male factor (101–188; 86% increase). 24% per ET. These results are similar to the results from col- As for the previous data collections, there was an underre- lection V (16 and 23% clinical pregnancy rate per OR and ET, porting of cycles that were cancelled before OR. Only 9 of respectively). The clinical pregnancy rate per OR ranged 1722 cycles were reported to be cancelled, whereas in general from 9% for advanced maternal age (AMA) + recurrent cancellation rates are in the range of 5–10%. As a result, the miscarriage to 31% for ‘other indications’. These differ- outcome of PGS can only be presented as clinical pregnancy ences in results between various indications reflect at least rate per OR or per ET but not per cycle started. This may partly in part differences in patient and cycle specific data. For be explained by the fact that many reporting centres only instance, in women treated with PGS for AMA (12% receive the blastomeres for analysis, while the IVF cycle is clinical pregnancy rate per OR), the mean age was 41 years done elsewhere, making accurate reporting of cycles more dif- and the average number of oocytes obtained per OR was ficult and as a result incomplete. 9.8. For couples treated with PGS for severe male factor For data collection VI, 9496 embryos were biopsied, and (28% clinical pregnancy rate per OR), the mean age was a result was obtained for 8795 embryos (93% of biopsied only 33 years and the average number of oocytes obtained embryos). Of 3193 embryos diagnosed as transferable (34% was 14.6.

327 K.D.Sermon et al.

Table IIIa. Sexing only for X-linked disease using PCR or FISH, data Table IIIb. Continued collection I–V FISH PCR Total FISH PCR Total Cleavage extrusion 9 0 9 Total cycles 545 68 613 Blastocyst 1 0 1 Number infertile 79 0 79 Embryology Female age 33 29 33 COC 1667 36 1703 Cycles cancelled before OR 31 6 37 Inseminated 1442 27 1469 Cycles to OR 514 62 576 Fertilized 1025 20 1045 ART method Biopsied 753 20 773 IVF 138 10 148 Successfully biopsied 740 20 760 ICSI 374 52 426 Diagnosed 678 20 698 ICSI and frozen 1 0 1 Transferable 235 15 250 Both IVF and ICSI 1 0 1 1 2 1,2 Transferred 150 10 160 Cancelled after OR 28 1 29 Frozen 31 1 32 Cycles to PGD 486 61 547 Clinical outcome Zona breaching Cycles to ET 89 3 92 AT drilling 299 50 349 hCG positive 29 2 31 Laser drilling 163 1 164 Positive heartbeat 27 1 28 Mechanical 24 10 34 Clinical pregnancy rate (%per OR/% per ET) 22/30 33/33 22/301 Biopsy method Number of fetal hearts 32 1 33 Cleavage aspiration 478 56 534 Implantation rate (fetal heart beats/ 21 10 21 Cleavage extrusion 8 5 13 100 embryos transferred) Embryology COC 6787 863 7650 ART, assisted reproduction technique; AT, acid Tyrode’s; COC, cumulus oocyte com- Inseminated 6053 662 6715 plexes; ET, embryo transfer; FISH, fluorescent in-situ hybridization; OR, oocyte retrieval. Fertilized 4322 526 4848 One cycle was combined FISH for sexing and HLA typing with PCR Biopsied 3292 431 3723 1Four clinical pregnancies without data Successfully biopsied 3195 395 3590 Diagnosed 2905 305 3210 PGD cycles for social sexing Transferable 1023 160 1183 Transferred 757 127 884 Table VIb summarizes the data from collection VI for social 3 4 5 Frozen 179 56 235 sexing cases using FISH and PCR. Social sexing for male Clinical outcome Cycles to ET 394 51 445 embryos made up 76% of the cycles (n = 61) while sexing for hCG positive 121 22 143 females made up 24% (n = 19). A total of 80 cycles were col- Positive heart beat 93 16 109 lected in this group, with 79 cycles going to OR and an average Clinical pregnancy rate % per OR/ET 18/246 26/31 19/246 of 15.4 oocytes per retrieval. The embryos in this group were ART, assisted reproduction technique; AT, acid Tyrode’s; COC, cumulus oocyte mainly diagnosed using FISH (91%). The biopsy was suc- complexes; FISH, fluorescent in-situ hybridization; OR, oocyte retrieval. 127 embryos from two cycles frozen before biopsy due to hyperstimulation. cessful in 96% of embryos biopsied and 93% of the embryos 220 embryos frozen before biopsy. biopsied were diagnosed. A transfer was achieved in 60% of 311 cycles with embryos frozen without biopsy or after failed diagnosis included. the cycles which went through PGD, with an average of 2.0 4Thirteen cycles with embryos frozen without biopsy or failed diagnosis included. 5Twenty four cycles with embryos frozen without biopsy or after failed embryos transferred in each cycle. The overall clinical preg- diagnosis included. nancy rate was 19% per OR and 32% per ET, with an 611 embryos transferred removed from calculations dues to lack of information regard- implantation rate [fetal heart beat (FHB)/embryos trans- ing the number of FHB in pregnancies resulting from the transfer of those embryos. ferred] of 23%. These data are very comparable to the I–V set (Table VIa).

Table IIIb. Sexing only for X-linked disease using PCR or FISH, data collection VI Pregnancies and babies The cumulative data for collection I–V is summarized in FISH PCR Total Tables VIIa–XIIa. It is becoming apparent that PGD babies are Total cycles 134 3 137 very comparable to ICSI babies in every respect, e.g. complica- Number infertile 24 0 24 tions of pregnancy, characteristics at birth and major and minor Female age 33 29 33 Cycles cancelled before OR 10 0 10 malformations. Cycles to OR 124 3 127 Of the 666 cycles ending in a positive hCG, no pregnancy Cycles cancelled before ART 0 0 0 was reported in 186 cases: for 89 pregnancies, no information ART method IVF 33 0 33 was given by the centres. For 89 further cycles with a positive ICSI 87 3 90 hCG, no pregnancy was reported because there was no positive IVF + ICSI 4 0 4 heartbeat (these are not the same as the 46 subclinical pregnan- Cancelled post OR 6 0 6 Cycles to PGD 118 3 121 cies that were reported, Table VIIb). A further eight cycles Zona breaching ending in a positive hCG were lost to follow-up. No cycle was AT drilling 64 2 66 reported for 21 pregnancies. This results in the reporting of 501 Laser drilling 46 1 47 Mechanical 8 0 8 pregnancies. Five hundred and one pregnancies ended in 373 Biopsy method reported deliveries, leading to the birth of 453 babies (295 Cleavage aspiration 108 3 111 singletons, 152 twins and 6 triplets). Two pregnancies were 328 ESHRE PGD Consortium-VI

Table IVa. Cycles performed for single gene disorders using PCR, data collection I–V

Indication Autosomal recessive Autosomal dominant Specific sex-linked Other Total

CF3 β-Thal SMA SC6 EB DM1 HD1 AP CMT7 ACH MS DMD8 FRAXA Haem

Total cycles 324 1445 93 27 9 203 146 10 22 7 10 43 51 18 137 1244 Number infertile 107 69 9 6 0 20 14 7 0 0 0 5 14 0 16 267 Female age 33336734313333283334323236293333 Cycles cancelled before OR 36 15 6 2 0 20 16 0 1 0 0 4 3 0 11 114 Cycles to OR 288 129 87 25 9 183 130 10 21 7 10 39 48 18 126 1130 Cycles cancelled before ART 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 3 ART method IVF 150 10 0 1 01 0 0 0 3 2 6 231 ICSI 268 128 81 25 9 176 127 9 21 7 10 36 44 11 118 1070 Frozen 0 1 50 0 0 00 0 0 0 0 0 1 1 8 Unknown 5 0 0 0 0 3 3 0 0 0 0 0 2 0 5 18 Cancelled after OR 14 12 6 1 0 12 5 0 2 1 0 0 4 0 7 64 Cycles to PGD 274 117 81 24 9 168 125 10 19 6 10 39 44 18 119 1063 Zona breaching AT drilling 167 95 43 13 8 85 46 10 2 0 8 22 13 15 43 570 Laser drilling 98 22 28 9 1 78 73 0 17 6 2 17 28 2 63 444 Mechanical 5 0 10 2 0 3 3 0 0 0 0 0 1 1 10 35 Unknown 4 0 0 0 0 2 3 0 0 0 0 0 2 0 3 14 Biopsy method Polar body biopsy 32 002034000001 0 42 172 Cleavage aspiration 2532 114 68 22 9 160 115 10 19 6 10 39 41 17 1082 9912 Cleavage extrusion 14 3 13 0 0 3 3 0 0 0 0 0 0 1 5 42 Unknown 6 0 0 0 0 2 3 0 0 0 0 0 2 0 3 16 Embryology COCs 3738 1768 1112 363 154 2220 1705 111 297 60 112 574 438 208 1807 146674 Inseminated 3275 1479 887 287 125 1961 1445 93 244 49 97 454 380 182 1516 12474 Fertilized 2317 951 621 194 112 1441 1038 82 196 32 79 333 276 134 1154 8960 Biopsied 1778 684 501 137 87 974 738 78 100 24 64 263 193 81 828 6530 Successfully biopsied 1759 673 498 135 87 962 723 78 99 24 63 258 192 80 819 6450 Diagnosed 1492 544 419 116 66 812 608 61 83 20 58 221 163 48 701 5412 Transferable 902 293 264 72 37 355 264 32 35 10 36 152 67 33 342 2894 Transferred 500 230 172 52 23 266 167 25 21 9 25 79 46 30 231 1876 Frozen 173 40 17 33 0 22 37 0 5 0 4 40 10 4 76 461 Clinical outcome Cycles to ET 239 108 77 22 8 138 102 9 11 6 10 33 29 13 104 909 hCG positive 75 42 23 6 3 37 24 3 5 0 2 11 9 6 38 284 Postive heart beat 59 34 19 4 1 29 18 1 5 0 2 10 8 5 35 230 Clinical pregnancy 20/25 26/31 22/25 16/18 11/13 16/21 14/18 10/11 24/45 0/0 20/20 26/30 17/28 28/38 28/34 20/25 rate % per OR/% per ET

ACH, achondroplasia; AP, amyloid polyneuropathy; ART, assisted reproduction technique; AT, acid Tyrode’s; CF, cystic fibrosis (various ); CMT, Charcot–Marie–Tooth disease; COC, cumulus oocyte complex; DM1, myotonic dystrophy type 1; DMD, Duchenne muscular dystrophy (specific); EB, epidermolysis bullosa; FRAXA, fragile-X syndrome; Haem, haemophilia; HD, Huntington’s Disease; MS, Marfan’s syndrome; OR, oocyte retrieval; SC, sickle-cell anaemia; SMA, spinal muscular atrophy; β-thal, β -thalassaemia. 1Includes nine HD with exclusion. 2Three cycles had both polar body biopsy and cleavage stage biopsy. 3Two cycles for two indications: cystic fibrosis and fragile X syndrome; cystic fibrosis and social sexing. 4Eight cycles with missing data. 5Includes three cycles of β -thalassaemia with HLA typing. 6Includes two cycles for sickle cell and β -thalassaemia. 7Includes one cycle for X-linked dominant inheritance. 8Includes one cycle for Beckers muscular dystrophy. terminated because of an encephalocele and spina bifida Two hundred and fifty-seven of the 564 fetal sacs were diagnosed on ultrasound. Sixty-nine of 403 ongoing pregnancies tested prenatally (46%), whereas 203 of 564 fetal sacs were (17%) presented with complications, mainly bleeding and eme- tested post-natally (36%). These figures cannot be added, as sis. One hundred and five of 373 pregnancies were delivered some fetal sacs were tested both prenatally and post-natally. prematurely (28%), 48 singleton pregnancies (16%), 55 twin Abnormalities were found in 13 instances: four prenatally, two pregnancies (72%) and 2 triplet pregnancies (100%). As before, of which ended in a termination of pregnancy (TOP) and nine all parameters e.g. weight, length and head circumference were post-natally. Eight abnormalities were found in miscarriage comparable to those of ICSI babies (Bonduelle et al., 2002) and material, whereas one abnormal karyotype was found at birth. previous PGD Consortium reports (Harper et al., 2006). Of the misdiagnosis: a trisomy 13 was found in miscarriage Seventeen babies were born with malformations (4%), material after PGD for 45,XY,der(13;14)(q10;q10). Although dif- eleven of which were major malformations. ficult to trace, the centre where this misdiagnosis occurred seem to 329 K.D.Sermon et al. 2 2 2 11 449 418 2 2 2 1 cumulus oocyte complexes; cumulus oocyte 100 128 741 3572 (various mutations); COC, (various mutations); Marfan syndrome, FRAXA, Fragile-X syndrome; OR, oocyte retrieval; SC, fic); CF, cystic fibrosis to PGD. 20/33 11/17 0/0 (0/0) 29/36 25/25 13/27 19/25 20/27 dominant Specific sex-linked Others Total 8) 433 45 40 128 (37) , Huntington disease; Marfan, ecker muscular dystrophy (speci ecker muscular dystrophy were counted as cycles going 7356 (8)466 215 (3)111774357 215 (8)466 7356 -thal+HLA) SMA SC HD (HDexcl) DM1 APC Marfan DMD (BMD) Haem FRAXA β pecific); Haem, Haemophilia; HD -thal ( -thal β nique; AT, acid Tyrode’s; BMD, B BMD, acid Tyrode’s; nique; AT, counted as cycles with an OR, but with an OR, cycles counted as -thalassaemia. CF β -thal, β chenne muscular dystrophy (s chenne muscular dystrophy ET) 25/32 (14/33) 20/24 13/14 0/0 (13/13) 26/34 dy screening and PGD for FRAXA. Cycles performed for single gene disorders using PCR, data collection VI IVF+ ICSI 0 0 0 0 (0) 0 1 0 0(1) 0 1 0 0 3 ICSI 6146 (7) 23675 (8)519 815 (3)112388434 815 (8)519 IVF 23675 ICSIIVF + Frozen (7) + FrozenICSI AT drillingLaser drilling 6146 Mechanical bodyPolar Cleavage aspirationCleavage extrusionCOCInseminated 0Fertilized 3 0 0 0 0 (0) 18 38 52 5 16 (0) 26 (7) 6 3 40 (7) 0 0 (0) 0 1 2 (0) 0 0(0) 0 0 15 706 6 0 0 (0) (0) 1 820 1 478 (74) 716 3 3 2 (81) 841 500 (56) 6 35 (8) 27 (0) 0 0 0 0 2 3 275 (0) 12 0 0 41 16 (0) 358 0 195 5 0 60 (0) 1 75 814 (136) 0 37 2 0 0 6 0 (150) 992 556 (9 0 629 0 (0) 0 2 0(0) 2 6 753 0 (0) 0 77 11 (3) 113 0 0 6 (0) 0 67 0 0 119 0 0 0 (0) (60) 187 6 0 (63) 212 0 0 (0) 6 0(0) 0 14 149 176 0 7 0 192 4 0 232 0 1 56 0 1056 0 23 0 1222 2 267 0 5198 6207 129 3 0 6 22 33 11 Cycles to ET 5038 (3) 21658 (8)336 414 (3)121168335 414 (8)336 ET 21658 (3) to 5038 Biopsied biopsiedSuccessfully DiagnosedTransferableTransferredFrozenCycles hCG PositivePositive heartbeatClinical pregnancy rate (% per OR/% per Number fetal heartbeats transferred) embryos hearts/100 rate (fetal Implantation 21 366 (75) 13 (40) 297 367 320 184 (42) 310 103 (39) 258 (4) 152 141 6 (4) 101 22 36 35 16 19 0 141 432 (85) 13 (3) 17 (1) 130 9 (1) 15 (1) 22 (7) 36 82 33 266 49 439 (86) 20 370 (70) 25 42 172 (33) 9 267 3 7 105 (14) 219 3 6 18 30 43 99 0 5 (17) 33 111 0 1 0 56 30 23 (1) 18 20 (7) 20 (1) 22 (3) 27 75 (0) 15 11 (23) 117 7 (17) 102 14 12 85 11 14 6 75 16 (10) 75 69 2 3 (8) 41 572 1 85 3 24 32 70 2595 0 0 0 2 587 25 29 16 482 10 (0) 6 (0) 2648 17 5 (0) 5 (0) 2239 17 209 2 1126 118 4 3 4 667 2 4 3 4 26 19 17 22 147 114 121 90 Two cycles had both aneuploi Two cycles had both not were so they only, embryos frozen-thawed on Two cycles were sickle-cell anaemia; SMA, muscularspinal atrophy; DM1, myotonic dystrophy type 1; DMD, Du type 1; DMD, DM1, myotonic dystrophy Indication recessive Autosomal Autosomal APC, Coli; ART, assisted reproduction Polyposis techAdenomatous Cycles to PGD 6142 (7) 23674 (8)468 817 (3)122182 817 (8)468 23674 PGD (7) to 6142 Cancelled after ARTCycles 3 4 (0) 1 0 2 (0) 8 1 0 0 (1) 0 2 10 32 Table IVb. n cyclesTotal infertileNumber Female age OR before cancelled Cycles to ORCycles Cycles cancelled before ARTART method Zona breaching 5 0 18 69 method Biopsy (1) 7 (0) 22 (8) 53 33 0 64Blastocyst 33 (36)Embryology (7) 46 5 29 6 3 0 33 9 2 (0) 14 24 33 0 90 (8) (1) 22 6 32 (29) 0 13 67 76 (8) 10 33 0 9 1 54 0 28 0 0 8 0 32 9 0 0 (0)1 (4) 17 (0) 0 2 (0) 4 34 (26) 8 0 14 2 (4) 17 32 0 5 27 0 35 4 12 3 0 104 0 23 33 11 (0) 1 20 516 0 33 65 91 114 0 1 2 1 6 2 (0) 0 4 2 17 Clinical outcome 330 ESHRE PGD Consortium-VI

Table Va. Cycles performed for PGS, data collection I–V

Indication AMA AMA + AMA + RIF1 Recurrent Recurrent Severe No Other Total miscarriage1 miscarriage IVF failure male factor2 indication

Total cycles 1043 43 129 483 917 166 67 263 3111 Number infertile 990 36 114 419 893 166 63 213 2894 Female age 40 40 40 36 36 32 33 35 36 Cycles cancelled 32 0 10132 1 2 33 before OR Cycles to OR 1040 41 129 473 904 164 66 261 3078 ART method IVF 197 9 33 52 143 2 16 59 511 ICSI 835 32 94 417 725 161 50 187 2501 IVF + ICSI 2 0 1 2 7 0 0 1 13 Frozen 0 0 0 0 4 0 0 0 4 Frozen and ICSI 1 0 1 1 1 1 0 5 Unknown 5 0 0 1 24 0 0 14 444 Cancelled after OR 46 3 2 9 50 9 1 8 128 Cycles to PGS 994 38 127 464 854 155 65 253 2950 Zona breaching AT drilling 565 18 84 333 539 59 42 168 1808 Laser drilling 386 20 42 122 266 89 18 57 1000 Mechanical 30 0 1 8 12 7 5 14 77 Unknown 13 0 0 1 37 0 0 14 654 Biopsy method Polar body biopsy 693 20 46 12 24 0 3 4 1783 Cleavage aspiration 8143 18 81 423 724 118 60 243 24813 Cleavage extrusion 92 0 0 27 62 36 2 5 224 Cleavage flow 70 0 1 6 1 0 1 16 displacement Unknown 13 0 0 1 38 0 0 0 524 Embryology COC 10907 391 1311 6663 12600 2476 836 3377 38561 Inseminated 9466 324 1116 5586 10801 1976 745 2825 32839 Fertilized 6742 192 795 3979 7738 1315 547 2015 23323 Biopsied 5406 209 739 3017 5227 971 435 1545 17549 Successfully biopsied 5325 208 733 2921 5155 958 413 1519 17232 Diagnosed 45095 199 710 24555 47825 804 2815 12965 150365 Transferable 11365 64 229 8795 18915 357 2255 5725 53535 Transferred 13585 49 163 7205 13145 267 1335 4565 44605 Frozen 126 9 36 77 295 42 8 94 687 Clinical outcome Cycles to ET 677 28 96 358 643 133 60 193 2188 hCG positive 201 6 22 123 192 59 20 79 702 Positive heart beat 151 5 18 98 136 52 19 55 534 Clinical pregnancy 15/22 12/18 14/19 21/27 15/21 32/39 29/32 21/28 17/24 rate % per OR/% per ET

ART, assisted reproduction technique; AT, acid Tyrode’s; COC, cumulus oocyte complexes; ET, embryo transfer; OR, oocyte retrieval. Other—includes data with two indications. 1These data were not extracted from I–IV. 2These data were not extracted from I–III. 3One cycle had cleavage stage biopsy and polar body biopsy. 4Several cycles had incomplete results. 5Several cycles from one centre had no information on the number of embryos diagnosed, number embryos diagnosed as transferable, but patients did have embryos transferred. In these cases, undiagnosed or abnormal embryos were transferred. attribute it to a human error without discarding a FISH error or Seventeen misdiagnoses have been reported in six reports, embryonic mosaicism, because the diagnosis was established ten for PCR and seven for FISH. Except for one misdiagnosis on one cell. The trisomy 16 found in miscarriage material after for a reciprocal translocation where an inappropriate probe PGS for AMA and RIF occurred after first polar body biopsy scheme was used (Sermon et al., 2005; Mackie Ogilvie and only, in which clearly two signals for chromosome 16 had been Scriven, 2004; Kyu Lim et al., 2004), the reason for the misdi- found. The second trisomy 16 found in miscarriage material agnoses is difficult to establish. occurred after PGS at the cleavage stage. Re-probing of the fixed blastomere clearly showed two signals for chromosome 16. Although a FISH error seems unlikely, the cause of this Discussion error (mosaicism of the embryo, human error) cannot be traced This sixth report of the ESHRE PGD Consortium reports on an anymore. ever-increasing number of cycles, pregnancies and babies, 331 K.D.Sermon et al.

Table Vb. Cycles performed for PGS, data collection VI

Indication AMA AMA + AMA + RIF Recurrent RIF SMF No Other Total miscarriage miscarriage indication

Total number of cycles 478 66 197 251 462 188 43 37 1722 Number infertile 381 57 182 193 450 144 41 25 1473 Female age 414140363433333737 Cycles cancelled before OR 0 2 0 1 0 0 5 1 9 Cycles to OR 478 64 197 250 462 188 38 36 1713 ART method IVF 27 11 8 27 22 0 24 5 124 ICSI 443 51 183 213 435 188 14 31 1558 IVF + ICSI 5 0 1 8 1 0 0 0 15 Unknown 1 0 5 0 0 0 0 0 6 ICSI + frozen 2 2 0 2 4 0 0 0 10 Cancelled post OR 18 9 0 6 7 2 3 0 45 Cycles to PGD 460 55 197 244 455 186 35 36 1668 Zona breaching AT drilling 186 30 105 147 149 75 4 20 716 Laser drilling 257 23 88 90 256 111 31 14 870 Mechanical 17 2 4 7 50 0 0 2 82 Biopsy method PB biopsy 47 13 62 5 54 0 0 4 185 Cleavage aspiration 372 41 135 232 384 179 35 31 1409 Cleavage extrusion 41 1 0 7 17 7 0 1 74 Cleavage flow displacement 0 0 0 0 0 0 0 0 0 Embryology COC 4707 539 1980 3211 6540 2752 437 500 20666 Inseminated 3837 446 1623 2607 5344 2202 403 405 16867 Fertilized 2638 302 1122 1890 3775 1507 285 296 11815 Biopsied 2128 280 1063 1436 3065 1080 196 248 9496 Successfully biopsied 2109 280 1045 1431 3042 1073 194 239 9413 Diagnosed 1994 265 988 1321 2842 990 171 224 8795 Transferable 614 65 314 534 1053 431 91 91 3193 Transferred 480 54 254 357 709 311 54 58 2277 Frozen 80 7 35 95 156 43 24 14 454 Clinical outcome Cycles to ET 297 35 155 191 380 164 31 30 1283 hCG positive 74 7 36 75 120 70 13 14 409 Positive heart beat 56 6 27 56 92 52 11 11 311 Clinical pregnancy rate 12/19 9/17 14/17 22/29 20/24 28/32 29/35 31/37 18/24 (% per OR/% per ET) Number of fetal heartbeats 65 6 29 72 118 69 11 14 384 Implantation rate 14 11 11 20 17 22 20 24 17 (fetal heartbeats/100 embryos transferred)

AMA, advanced maternal age; ART, assisted reproduction technique; AT, acid Tyrode’s; COC, cumulus oocyte complexes; OR, oocyte retrieval; RIF, repeated implantation failure; SMF, severe male factor. from a still increasing number of participating centres. For this for X-linked diseases and for social sexing) remains stable. For report, the referrals are not included, because it was felt from the sexing for X-linked diseases, this reflects the fact that more previous reports that the data were biased and incomplete. and more specific PGD at the DNA level is offered. For social Indeed, only few centres reported all referrals, i.e. referrals for sexing, only two centres offer it, and this has remained the patients who did not come through for PGD because either it same for the last three reports. To be able to offer PGD for was technically impossible to help them, or for ethical reasons, monogenic diseases or chromosomal abnormalities, a close or reasons related to IVF e.g. AMA. collaboration between an IVF centre and a genetic laboratory is An interesting feature is the relative increase in the number mandatory. The higher technical expertise needed for this type of PGS cycles. This reflects the fact that many IVF laboratories of diagnosis explains why less centres are able to offer it. offer PGS, and PGS only, as an additional method to select the It becomes obvious that at birth PGD babies have character- best embryo for transfer. The ESHRE PGD Consortium Steer- istics that are comparable to those of ICSI babies. No preg- ing Committee would like to emphasize that they feel that there nancy complication or malformation at birth is particularly is a need for large randomized controlled trials to investigate occurring in the PGD population. The characteristics of the the (cost-)effectiveness of PGS for well-defined indications. babies at birth are also comparable, and the main complication Currently, there are insufficient data that demonstrate that PGS remains multiple pregnancies leading to morbidity and mortal- is indeed a cost-effective alternative for standard IVF (Staessen ity in PGD offspring. et al., 2004; Twisk et al., 2006). The ESHRE PGD Consortium has been active during the PGD for monogenic diseases and chromosomal abnormali- past year: the PGD Consortium guidelines for PGD and PGS ties have also been steadily increasing, whereas sexing (both were published in 2005 (Thornhill et al., 2005), and recently, a 332 ESHRE PGD Consortium-VI

Table VIa. PGD for social sexing, data collection I–V

Method for sexing FISH SS only FISH SS + PGS PCR Unknown Total

Total cycles 124 22 103 5 254 Number infertile 18 6 16 1 41 Female age 35 38 35 35 35 Cycles cancelled before OR 0 0 0 0 0 Cycles to OR 124 22 103 51 2541 ART method IVF 104 1 5 3 113 ICSI 17 21 88 2 128 Frozen 3 0 2 0 5 Frozen and IVF 0 0 1 0 1 Frozen and ICSI 0 0 5 0 5 Unknown and frozen 0 0 2 0 2 Cancelled after OR 3 6 5 14 Cycles to PGD 121 22 97 0 240 Zona breaching AT drilling 9 0 10 0 19 Laser drilling 112 0 0 0 112 Mechanical 0 22 87 0 109 Biopsy method Cleavage aspiration 120 0 10 0 130 Cleavage extrusion 1 22 87 0 110 Embryology COC 1463 301 1504 23 3291 Inseminated 1421 210 1122 19 2772 Fertilized 1022 128 730 11 1891 Biopsied 876 100 613 0 1589 Successfully biopsied 802 100 603 0 1505 Diagnosed 732 83 552 0 1367 Transferable 303 24 325 0 652 Transferred 178 23 248 0 449 Frozen2 99 1 49 03 149 Clinical outcome Cycles to ET 96 14 82 0 192 hCG positive 40 4 34 0 78 Positive heart beat 38 0 21 0 59 Clinical pregnancy rate % per OR/% per ET 31/40 20/26 23/31

ART, assisted reproduction technique; AT, acid Tyrode’s; COC, cumulus oocyte complexes; FISH, fluorescent in-situ hybridization; OR, oocyte retrieval. 1One natural cycle included. 2Eleven cycles with embryos frozen without biopsy or failed diagnosis included. 3Three embryos frozen without biopsy were not included.

Table VIb. Continued

Table VIb. PGD for social sexing, data collection VI FISH PCR Total

FISH PCR Total Embryology COC 1066 154 1220 Total cycles 73 7 80 Inseminated 832 117 949 Number infertile 1 0 1 Fertilized 566 78 644 Female age 38 43 38 Biopsied 426 52 478 Cycles cancelled before OR 1 0 1 Successfully biopsied 409 52 461 Cycles to OR 72 7 79 Diagnosed 393 52 445 Cycles cancelled before ART 0 0 0 Transferable 78 35 113 ART method Transferred 71 21 92 IVF 5 0 5 Frozen 8 12 20 ICSI 65 6 71 Clinical outcome ICSI + frozen 0 1 1 Cycles to ET 40 7 47 Unknown + frozen 2 0 2 hCG positive 16 4 20 Cancelled post OR 1 0 1 Positive heartbeat 12 3 15 Cycles to PGD 71 7 78 Clinical pregnancy rate (% per OR/% per ET) 17/30 43/43 19/32 Zona breaching Number of fetal heartbeats 14 7 21 AT drilling 0 0 0 Implantation rate (fetal hearts/100 20 33 23 Laser drilling 1 0 1 embryos transferred) Mechanical 70 7 77 Biopsy method ART, assisted reproduction technique; AT, acid Tyrode’s; COC, cumulus Cleavage aspiration 1 0 1 oocyte complexes; ET, embryo transfer; FISH, fluorescent in-situ hybridiza- Cleavage extrusion 70 7 77 tion; OR, oocyte retrieval.

333 K.D.Sermon et al.

Table VIIa. Evolution of pregnancy, data I–V Table VIIb. Evolution of pregnancy, data VI

Number of Number of Number of Number of pregnancies fetal sacs pregnancies fetal sacs Pregnancies 501 564 Pregnancies 1133 1279 6 FISH cycles 399 454 FISH cycles 883/1133 PCR cycles 102 110 6 PCR cycles 251/1133 Subclinical pregnancies1 46 Subclinical pregnancies1 166/1133 Extrauterine gestation 7 Clinical pregnancies 967 1279 Clinical pregnancies 448 564 Singletons 694/967 694/1279 Singletons 345 345 Twins 234/967 468/1279 Twins 90 180 Triplets 36/967 108/1279 Triplets 13 39 Quadruplet 0 0 Quadruplet 2/967 8/1279 7 Unknown 0 0 Unknown 1/967 1/1279 First trimester loss 45 67 First trimester loss 98/967 157/1279 Miscarriage 44 45 Miscarriage 88/9678 100/1279 Vanishing twins 21 Extrauterine pregnancy 10/9679 11/1279 TOP 12 12 Vanishing twins/triplets 46/1279 Ongoing pregnancies (>12 weeks) 403 497 Ongoing pregnancies >12 weeks 869 1122 Second trimester loss 4 5 Second trimester loss 26/869 36/1122 Miscarriage 3 4 TOP 13 13 Miscarriage 18/8692 27/1122 2 3 Reduction of multiple pregnancies 13 TOP after misdiagnosis 4/869 8/1122 Triplet to twin 4 4 4 TOP after amniocentesis 4/869 Triplet to singleton 4 8 Reduction5 1/1122 Twin to singleton 1 1 Reduction of multiple pregnancies 18/1122 Remaining data 399 479 Quadruplet to twin 4/1122 Singletons 321 321 Triplet to twin 7/1122 Twins 76 152 Triplet to singleton 6/1122 Triplet 2 6 Lost to follow-up 26 26 Twin to singleton 1/1122 Singletons 26 26 Normal evolution 843 1068 Twins 0 0 Lost to follow-up 39 51 Triplets 0 0 Deliveries 804 1017 Deliveries 373 453 Singletons 602/804 602/1017 Singletons 295 295 Twins 191/804 382/1017 Twins 76 152 Triplets 11/804 33/1017 Triplets 2 6 FISH, fluorescent in-situ hybridization; TOP, termination of pregnancy. FISH, fluorescent in-situ hybridization; TOP, termination of pregnancy. 1 1Subclinical pregnancy defined as pregnancy without any other clinical signs but positive Subclinical pregnancy defined as pregnancy without any other clinical signs, but positive serum hCG. serum hCG. 2 2 For encephalocoele. One triplet: fetal reduction, followed by amniocentesis and loss of remaining twin at 16 3 weeks (one fetal sac counted in reduction, two in miscarriage, one second trimester preg- For spina bifida. nancy loss after miscarriage counted). 3One misdiagnosis for sexing, FISH, female fetus, indication social sexing; one misdiag- nosis for beta thalassaemia, PCR; one misdiagnosis for myotonic dystrophy, PCR, one misdiagnosis after PGS, karyotype 45,X. Table IXa. Method of delivery and gestational age, data collection I–V 4TOP for abnormalities found after amniocentesis, not related to the PGD: trisomy 18, indica- tion for PGD parent carrier of reciprocal translocation not involving chromosome 18; one pol- Total Singletons Twins Triplets ymalformation, one cystic hygroma, failed karyotype, one Turner mosaic. 5 One misdiagnosis for sexing, PCR, indication Duchenne, twin pregnancy, selective termina- Number of deliveries 804 602 191 11 tion of male fetus. Cycle done in 1996, Y-specific amplification only. Method of delivery 6One fetal sac was tested by FISH and PCR. 7Number of fetal heart beats not known. Counted further as 1 fetal heart beat. Vaginal 333 294 38 1 8One miscarriage after amniocentesisis. Caesarian 385 255 121 9 9One heterotrophic gestation continued as singleton after reduction of extrauterine gesta- Vaginal and Caesarian 1 0 1 0 tion at 6 weeks. Unknown 85 53 31 1 Term at delivery Preterm 187 77 102 8 Term 474 420 52 2 Unknown 143 105 37 1 joint report of the European Society for Human Genetics (ESHG) and ESHRE was published (Soini et al., 2006). This report discusses the interface between genetics and assisted reproductive technology regarding technical, sociological, eth- Table IXb. Method of delivery and gestational age, data VI ical and legal issues and deals not only with PGD but also with Total Singleton Twin Triplet other topics such as screening of gamete donors and reproduc- tive tourism. Number of deliveries 373 295 76 2 Method of delivery The ESHRE PGD Consortium also aims at constantly Vaginal 174 156 18 0 improving the data collection and its processing. A major reor- Caesarean 177 119 56 2 ganization of the database is planned, and better follow-up of Vaginal and Caesarean 2 2 0 0 Unknown 20 18 2 0 data submission is now reassured by a permanent staff member Term at delivery at ESHRE. Quality assessment of captured FISH images is Preterm 105 48 55 2 planned, as well as retrospective analysis of the children born Term 233 217 16 0 Unknown 35 30 5 0 after PGD, as a preliminary study for the long-term child 334 ESHRE PGD Consortium-VI

Table Xa. Confirmation of diagnosis per fetal sac, data collection I–V Table Xb. Confirmation of diagnosis per fetal sac, data collection VI

Method Result Method Result

N Normal Abnormal N Normal Abnormal Unknown

Prenatal diagnosis1 Prenatal diagnosis1 FISH FISH CVS 49 49 0 CVS 7 7 0 Amnio 258 253 52 Amnio 64 61 12 23 Ultrasound 98 96 23 Ultrasound 146 144 24 0 Unknown 3 3 0 Total 217 212 3 2 Total 408 401 7 PCR PCR CVS 12 11 15 0 CVS 66 66 0 Amnio 17 17 0 0 Amnio 47 40 74 Ultrasound 11 11 0 0 Ultrasound 4 3 15 Total 40 39 1 0 Unknown 2 2 0 Post-natal diagnosis6 Total 119 111 8 FISH Post-natal diagnosis6 Karyo miscarriage 17 9 87 FISH Karyo postnatal 26 25 18 Karyo miscarriage 25 117 148 Physical examination 181 181 0 Karyo postnatal 47 459 210 Total 224 215 9 Physical examination 265 263 211 PCR Total 337 319 18 DNA test miscarriage 0 0 0 PCR Karyo miscarriage 3 3 0 Karyotype miscarriage 1 114 0 Physical examination 7 7 0 DNA test miscarriage 2 2 0 DNA test postnatal 6 6 0 DNA test postnatal 24 23 112 Karyotype 4 4 0 Sweat test 4 4 0 Sweat test 2 2 0 Physical examination 715 6113 Algo test 2 2 0 Karyotype 2 2 0 Unknown 5 5 0 Karyo + DNA 1 1 0 Total 29 29 0 Unknown 1 1 0 Total 42 40 2 CVS, chorionic villus sampling; FISH, fluorescent in-situ hybridization. 1FISH fetal sacs tested: 217/454; PCR fetal sacs tested: 40/110; Total prenatal CVS, chorionic villus sampling; FISH, fluorescent in-situ hybridization. testing: 257/564. 1FISH Fetal sacs tested: 408/991; PCR Fetal sacs tested: 119/289; Total prena- 2After PGD for inversion: 46,XX/47,XXX ongoing and live birth. tal testing: 527/1279 (one fetal sac had PCR and FISH at PGD). 3One amnio after omphalocoele found on US. Mors in utero. 2Two misdiagnoses: sexing, female fetus, social sexing (terminated): PGS: 4One encephalocoele, one spina bifida. Both TOP. 45,X (terminated). Trisomy 18 after PGD for reciprocal translocation. 5De novo translocation 46,XX,(13;20)(q14;p11). 46,XX,t(1;5)(q43;q13)de novo after PGD for 46,XY,t(1;11)(p12;q12) (born), 6FISH fetal sacs/babies tested: 183/454; PCR fetal sacs/babies tested: 20/110; 45,X[10]/46,XX[55] after PGS for repeated IVF failures (TOP). Total post-natal testing: 203/564. 3Polymalformation on US, normal karyotype, terminated. Cystic hygroma, 745,X (PGD for 45,XX,der(13;14)(q10;q10)), 47,XX,+16 (PGS for RIF), karyotype failed, PGD for 45,XX,der(13;21)(q10;q10). 45,XY (no more details given, PGD for 45,XY,der(13;14)(q10;q10)), 4Six misdiagnoses: XL Duchenne (selective reduction of one affected embryo 47,XY,+9 (PGS for RM), trisomy 4 (PGS for AMA), trisomy 16 (PGS for of twin pregnancy), beta-thalassaemia (terminated), myotonic dystrophy (ter- AMA and RIF), trisomy 16 (PGS for RM). One misdiagnosis: T13 after PGD minated), CF (born), XL (born), amyloid polyneuropathy for 45,XY,der(13;14)(q10;q10). (born), 46,XX,t(8;9)(p?23;p12) de novo after PGD for Duchenne muscular 8De novo translocation involving chromosome 20. dystrophy. 5Echogenic bowel at US, misdiagnosis for CF, born. 6Total FISH sacs/babies tested: 337/991; Total PCR sacs/babies tested: 42/ 289; Total postnatal testing: 379/1280. Table XIa. Data on live-born children, data collection I–V 7Normal karyotype in a fetus with multiple malformations, After amniocente- sis with normal karyotype. Total children born 990 8One trisomy 3, one trisomy 15, two trisomy 16, one trisomy 22, one mosaic Sex trisomy 22, one monosomy X, 47,XY,+D(3), one misdiagnosis, Male 410 47,XX,+der(22)t(11;22)(q23.3;q11.2)mat; parent carrier balanced translocation, Female 535 46,X,inv(Y)(p11q12); trisomy 16 after PGS with PB1 analysis; Unknown 45 45,XX,der(7)t(7;15)(p14;q11.2),-15de novo; 47,XX,+9, trisomy 22. Mean birth weight (g) 9One baby born with Fryns syndrome had a karyotype (normal result). Three Singletons 3255 544 twins with polymalformations had a normal karyotype. Twins 2431 304 10One misdiagnosis, trisomy 21 after aneuploidy screening, Triplets 1756 21 46,XX,t(1;5)(q43;q13)de novo already seen in prenatal. Mean birth length (cm) 11One baby with Fryns syndrome, one baby with Prune Belly syndrome (both Singletons 50 352 normal karyotype). Twins 47 153 12One CF carrier twin pregnancy—on PGD both diagnosed as homozygote normal. Triplets 44 6 13One twin after PGD for CF: one misdiagnosis, one healthy. 14After amniocentesis. 15One baby (PGD for DMD) had FISH + PCR, diagnosis confirmed by physical examination and amnio. Supplementary materials follow-up. With these measures, ESHRE hopes to help IVF Supplementary data are available at http:// humrep.oxfordjournals. and genetic centres to bring PGD up to today’s standards of org/. good laboratory and clinical practice. Table IIc. Chromosomal abnormalities analysed, data VI. 335 K.D.Sermon et al.

born after ICSI (1991–1999) and of 1995 infants born after IVF (1983– Table XIb. Data on live-born children, data collection VI 1999). Hum Reprod 17,671–694. ESHRE PGD Consortium Steering Committee (1999) ESHRE preimplantation Total children born 441 genetic diagnosis (PGD) consortium: preliminary assessment of data from Sex January 1997 to September 1998. Hum Reprod 14,3138–3148. Male 200 ESHRE PGD Consortium Steering Committee (2000) ESHRE preimplantation Female 238 genetic diagnosis (PGD) consortium: data collection II (May 2000). Hum Unknown 3 Reprod 15,2673–2683. Mean birth weight (g) ESHRE PGD Consortium Steering Committee (2002) ESHRE preimplantation Singletons 3214 (n = 260/295)1 genetic diagnosis consortium: data collection III (May 2001). Hum Reprod Twins 2404 (n = 112/152)1 17,233–246. Triplets 1372 (n = 6/6)1 Mean birth length (cm) Harper JC, Boelaert K, Geraedts J, Harton G, Kearns WG, Moutou C, Singletons 50 (n = 179/295)1 Muntjewerff N, Repping S, SenGupta S, Scriven PN et al. (2006) ESHRE Twins 45 (n = 88/152)1 PGD Consortium data collection V: cycles from January to December 2002 Triplets 40 (n = 3/6)1 with pregnancy follow-up to October 2003. Hum Reprod 21,3–21. Mean head circumference (cm) Kyu Lim C, Hyun Jun J, Mi Min D, Lee HS, Young Kom J, Koong MK and Singletons 34 (n = 132/285)1 Kang IS (2004) Efficacy and clinical outcome of preimplantation genetic Twins 32 (n=74/150)1 diagnosis using FISH for couples of reciprocal and Robertsonian transloca- Triplets 28 (n = 3/6)1 tions; the Korean experience. Prenat Diagn 24,556–561. Apgar scores Mackie Ogilvie C and Scriven PN (2004) Preimplantation genetic diagnosis Good 242/255 (PGD) for reciprocal translocations. Prenat Diagn 24,553–555. Poor 13/255 Sermon K, Moutou C, Harper J, Geraedts J, Scriven P, Wilton L, Magli MC, Michiels A, Viville S and De Die C (2005) ESHRE PGD consortium data 1Numbers between brackets indicate the number of newborns for whom infor- collection IV: May-December 2001. Hum Reprod 20,19–34. mation is available out of the total number of newborns. Soini S, Ibarreta D, Anastasiadou V, Ayme S, Braga S, Cornel M, Coviello DA, Evers-Kiebooms G, Geraedts J, Gianaroli L et al. (2006) The interface between assisted reproductive technologies and genetics: technical, social, Table IIIc. List of indications for which sexing has been ethical and legal issues. Eur J Hum Genet 14,588–645. Staessen C, Platteau P, Van Assche E, Michiels A, Tournaye H, Camus M, performed, data collection VI. Devroey P, Liebaers I and Van Steirteghem A (2004) Comparison of blasto- Table IVc. List of indications under ‘others’ for monogenic transfer with or without preimplantation genetic diagnosis for aneu- diseases with PCR, data VI. ploidy screening in couples with advanced maternal age: a prospective Table VIIIa. Complications in clinical pregnancies, data I–V. randomized controlled trial. Hum Reprod 19,2849–2858. Thornhill AR, deDie-Smulders CE, Geraedts JP, Harper JC, Harton GL, Table VIIIb. Complications in clinical pregnancies, data VI. Lavery SA, Moutou C, Robinson MD, Schmutzler AG, Scriven PN et al. Table XIIa. Congenital malformations and neonatal compli- (2005) ESHRE PGD consortium ‘Best practice guidelines for clinical pre- cations at birth, data collection I–V. implantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS)’. Hum Reprod 20,35–48. TableXIIb. Congenital malformations and neonatal compli- Twisk M, Mastenbroek S, van Wely M, Heineman MJ, Van der Veen F, cations at birth, data collection VI. Repping S (2006) Preimplantation genetic screening for abnormal number of chromosomes (aneuploidies) in in vitro fertilisation or intracytoplasmic sperm injection. Cochrane Database Syst Rev 25,CD005291. References Bonduelle M, Liebaers I, Deketelaere V, Derde M-P, Camus M, Devroey P and Van Steirteghem A (2002) Neonatal data on a cohort of 2889 infants Submitted on August 21, 2006; accepted on August 31, 2006

336 Human Reproduction Vol.21, No.1 pp. 3–21, 2006 doi:10.1093/humrep/dei292 Advance Access publication September 19, 2005.

ESHRE PGD Consortium data collection V: Cycles from January to December 2002 with pregnancy follow-up to October 2003

J.C.Harper1,12, K.Boelaert2, J.Geraedts3, G.Harton4, W.G.Kearns5, C.Moutou6, N.Muntjewerff3, S.Repping7, S.SenGupta1, P.N.Scriven8, J.Traeger-Synodinos9, K.Vesela10, L.Wilton11 and K.D.Sermon2

1UCL Centre for PGD, Department of Obstetrics and Gynecology, University College London, 86–96 Chenies Mews, London WC1E 6HX, UK, 2Centre for Medical Genetics, University Hospital and Medical School of the Dutch-speaking Brussels Free University (Vrije Universiteit Brussel, VUB), Laarbeeklaan 101, 1090 Brussels, Belgium, 3PGD working group Maastricht, Department of Clinical Genetics, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands, 4Genetics and IVF Institute, 3020 Javier Road, Fairfax Virginia, 22031, USA, 5Shady Grove Centre for Preimplantation Genetics, 15001 Shady Grove Road, Suite 400, Rockville, Maryland, 20850, USA, 6Service de la Biologie de la Reproduction, SIHCUS-CMCO, 19, Rue Louis Pasteur, BP120, 67303 Schiltigheim, France, 7Center for Reproductive Medicine, Academic Medical Center, Fertility Laboratory (A1-229), Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands 8Department of Cytogenetics, and Center for Preimplantation Genetic Diagnosis, Guy’s and St Thomas’ NHS Foundation Trust, Guy’s Hospital, St Thomas Street, London SE1 9RT, UK, 9Laboratory of Medical Genetics, University of Athens, St Sophia’s Children’s Hospital, 11527 Athens, Greece, 10Sanatorium Repromeda, Vinicni 235, 615 00 Brno, Czech Republic and 11Melbourne IVF, 320 Victoria Parade, 3002 East Melbourne VIC, Australia 12To whom correspondence should be addressed: [email protected]

The fifth report of the ESHRE PGD Consortium is presented (data collection V). For the first time, the cycle data were collected for one calendar year (2002) in the following October, so that data collection was complete for preg- nancies and babies. The data were collected using a Filemaker Pro database and divided into referrals, cycles, preg- nancies and babies. There are currently 66 active centres registered with the consortium; however, the data presented here were obtained from 43 centres and included 1603 referrals, 2219 cycles, 485 pregnancies and 382 babies born. The cycle data were divided into preimplantation genetic diagnosis (PGD) for inherited disorders (including chromosome abnormalities, sexing for X-linked disease and monogenic disorders), aneuploidy screening (PGS) and the use of PGD for social sexing. Data collection V is compared with the previous cumulative data collec- tion (I–IV), which comprised 4058 PGD/PGS cycles that reached oocyte retrieval.

Key words: aneuploidy screening/preimplantation genetic diagnosis/embryo biopsy/PGS/PGD

Introduction 2002) and is referred to as data V. Data VI (January to December The ESHRE PGD Consortium was established in 1997 and one 2003) has been collected since December 2004 and is currently of the aims was to collect detailed data on referrals, PGD/PGS being processed. Cycle data are now collected in the October for the cycles, pregnancies and babies born. There are currently 66 previous calendar year so that data collection is complete for preg- active centres registered with the consortium. This includes nancies and babies. In this report, two sets of data are presented. almost all active European Centres and additionally centres The first set, referred to as data collection I–IV, comprise the cumu- from Australia, Argentina, Israel, Korea, Taiwan and USA. lative data collections from the previous reports (ESHRE PGD Four sets of data have previously been published. The initial data Consortium Steering Committee, 1999, 2000, 2002; Sermon et al., collection included PGD cycles performed up to September 1998 2005). The second set is data collection V. The FileMaker Pro 6™ (ESHRE PGD Consortium Steering Committee, 1999), the second (FP6) database was used for the last two data-set collections. included cycles from September 1998 to May 2000 (ESHRE PGD Consortium Steering Committee, 2000), the third was from May Materials and methods 2000 to May 2001 (ESHRE PGD Consortium Steering Committee, 2002) and the fourth included data from May 2001 to December Data collection 2001 (Sermon et al., 2005). This is the first set of data that relates to Data I–III were collected using paper copy or Excel spreadsheets. cycles performed in a complete calendar year (January to December Data IV and V were collected using a FileMaker Pro 6 database

Published by Oxford University Press 2005 on behalf of the European Society of Human Reproduction and Embryology. 3 J.C.Harper et al. designed by C.Moutou, which contained the following tables: refer- delian disorders remained the same. Duchenne muscular dystro- rals, cycles, pregnancies and babies born. Details of the forms used phy, and fragile X syndrome were the most and how the database works were described in Sermon et al. (2005). frequent referrals for X-linked disorders, CF/CBAVD, β-thalas- saemia and spinal muscular atrophy (SMA) were the most fre- Results and discussion quent referrals for autosomal recessive disorders, and Huntington’s disease and myotonic dystrophy were the most fre- The results are shown in the tables and only highlights and quent autosomal dominant referrals. There were three referrals important trends are discussed in the text. Sixty-six active cen- for HLA testing, two of these because of Fanconi anaemia and tres are currently registered with the consortium. For 2002, 43 one in combination with sickle cell disease. It is interesting to centres contributed data (see Appendix), whereas 11 failed to note that there were only four referrals for social sexing. This is a do so. Seven centres are new members of the consortium and discrepancy with the number of cycles reaching OR for social five had not started their PGD activity in 2002. sexing, which was 72. However, in 120 cases the reason for refer- Clinical pregnancy rates (presence of a fetal heart) are ral was unknown (i.e. the data were not provide by the centre). expressed as a percentage of the cycles that reached oocyte Table III gives the reasons for PGD. Since a couple might retrieval (% per OR) and as a percentage of cycles that had an have indicated more than one reason, the total is >100%. In embryo transfer procedure (% per embryo transfer). Addition- comparison to previous collections, genetic risk and previous ally the % implantation rate (percentage of fetal heart beats out termination of pregnancy (TOP) have remained at the same of the total number of embryos transferred) for cycles of data level, while genetic risk and objection to TOP have decreased. collection V are presented. Genetic risk and sub- or infertility has increased together with age-related aneuploidy screening, i.e. PGS. Referral data V In line with past developments, referrals for chromosomal disor- Data on cycles ders increased and were the most frequent reason for referral Table IVa shows the cumulative data for collections I–IV. A (Table I). The vast majority of referrals were for aneuploidy total of 2000 cycles reached the stage of oocyte retrieval (OR) screening (Table II). Compared to the previous data collections for PGD (chromosome abnormalities, sexing for X-linked dis- there were fewer referrals for reciprocal and Robertsonian trans- ease and monogenic disorders), 1570 had an embryo transfer locations. The number of referrals for monogenic disorders procedure (77% per OR) and clinical pregnancy rates of 18% per (‘Autosomal recessive’ and ‘Autosomal dominant’ in Table I) did OR and 23% per embryo transfer procedure were obtained. not change and the most frequent referrals in each group of Men- These data are broken down in Tables Va, VIa and VIIa. A total of 1876 reached the stage of OR for PGS in data I–IV, 1342 hav- Table I. Referrals according to indication ing an embryo transfer procedure (71%) and clinical pregnancy rates of 18% per OR and 25% per embryo transfer procedure Data collection were obtained. These data are broken down in Table VIIIa. A III–IV V total of 182 cycles reached OR for social sexing, 131 cycles pro- duced embryos of the desired sex and clinical pregnancy rates of Chromosomal (numerical/structural) 1703 1150 X-linked 397 109 24% per oocyte retrieval and 34% per embryo transfer procedure Autosomal recessive 374 102 were obtained. These data are broken down in Table IXa. Autosomal dominant 316 111 Looking overall at these 4058 cycles that reached oocyte Mitochondrial 6 1 Two indications 11 2 retrieval, ICSI was used in the majority of cases. Acid Tyrode’s Y-chromosome deletion 3 4 drilling was performed in more cycles than drilling using the Social sexing 68 4 laser, and cleavage stage aspiration was still the most commonly Unknown 74 120 used biopsy method. Data has been obtained for 54 060 oocytes. Table IVb shows data collection V. A total of 2219 cycles was started. For PGD, 936 cycles were started, 868 reached Table II. Referrals for chromosomal disorders

Data collection Table III. Reasons for preimplantation genetic diagnosis (PGD) III–IV V Data collection Structural chromosomal aberrations Reciprocal translocation 372 126 III–IV V Robertsonian translocation 141 70 Inversion 20 8 Genetic risk and previous TOP 448 (15) 134 (8.4) Deletion 6 4 Genetic risk and objection to TOP 856 (29) 309 (19.3) Numerical chromosome aberrations Genetic risk and sub- or infertility 655 (22) 482 (30.1) Aneuploidy risk 1060 882 Genetic risk and sterilization 19 (0.6) 10 (0.6) 47,XXY; 47,XYY 28 13 Age-related aneuploidy 982 (33) 987 (61.6) Sex chromosomal mosaicism 33 11 Other 379 (13) 384 (24.0) Male meiotic abnormalities 20 11 Other 6 13 Values in parentheses are percentages. Unknown 17 12 Some couples had more than one reason for PGD, hence the total is >100%. TOP = termination of pregnancy. 4 ESHRE PGD Consortium data collection V

Table IVa. Overall cycle data collection I–IV Table IVb. Overall cycle data collection V

Indication PGD PGS PGD-SS Total Indication PGD PGS PGD-SS Total

Number infertile 577 1774 19 2370 Total cycles 936 1211 72 2219 Female age 33 35 35 35 Number infertile 417 1120 22 1559 Cycles to OR 2000 1876 182 4058 Female age 32 38 36 35 Cancelled after OR before IVF/ICSI 4 0 0 4 Cycles cancelled before OR 68 9 0 77 Assisted reproduction treatment Cycles to OR 868 1202 72 2142 IVF 278 355 109 742 Cancelled after OR before IVF/ICSI 2 0 0 2 ICSI 1684 1463 67 3214 Assisted reproduction treatment IVF + ICSI 1 10 0 11 IVF 91 156 4 251 Frozen 9 4 5 18 ICSI 760 1038 61 1859 Frozen and ICSI 3 1 1 5 IVF + ICSI 2 3 5 Cancelled ICSI 3 3 Unknown 1 1 Unknown 18 43 0 61 IVF + frozen 0 1 1 Cancelled after OR 88 77 14 179 ICSI + frozen 11 4 4 19 Cycles to PGS/PGD 1908 1799 168 3875 Unknown and frozen 2 2 4 FISH 1070 1799 115 2984 Cancelled after OR 73 51 0 124 PCR 838 0 53 891 Cycles to PGS/PGD 793 1151 72 2016 Zona breaching FISH 507 1151 28 1686 AT drilling 1291 1140 19 2450 PCR 286 0 44 330 Laser drilling 507 533 106 1146 Zona breaching Mechanical 96 61 43 200 AT drilling 412 668 0 1080 Unknown 14 65 0 79 Laser drilling 349 467 6 822 Biopsy method Mechanical 32 16 66 114 Polar body biopsy 11a 70a 081a Biopsy method Cleavage aspiration 1831a 1519a 125 3475a Polar body biopsy 14 108 122 Cleavage extrusion 51 143 43 237 Cleavage aspiration 745 962 5 1712 Cleavage flow displacement 2 16 0 18 Cleavage extrusion 34 81 67 182 Unknown 16 52 0 68 Embryology Embryology COC 11 558 14 187 1002 26 747 COC 27 397 24 374 2289 54 060 Inseminated 9732 11 909 742 22 383 Inseminated 23 621 20 930 2030 46 581 Fertilized 7151 8321 480 15 952 Fertilized 17 088 15002 1411 33 501 Biopsied 5363 6541 396 12 300 Biopsied 13 005 11 008 1193 25 206 Successfully biopsied 5290 6472 396 12 158 Successfully biopsied 12 708 10 760 1109 24 577 Diagnosed 4727 5991 351 11 069 Diagnosed 11 106 9045 1016 21 167 Transferable 1711 2211 183 4105 Transferable 4374 3625 469 8468 Transferred 1169 1528 145 2842 Transferred 3211 2932 304 6447 Frozen 160 251 21 432 Frozen 633 436 128 1197 Clinical outcome Clinical outcome Cycles to embryo transfer 603 846 61 1510 Cycles to embryo transfer 1570 1342 131 3043 HCG positive 198 271 22 491 HCG positive 458 431 56 945 Positive heart beat 152 198 15 365 Positive heart beat 362 336 44 742 [clinical pregnancy rate (%) per (18/25) (16/23) (21/25) (17/24) [clinical pregnancy rate (%) per (18/23) (18/25) (24/34) (18/24) OR/% per embryo transfer] OR/% per embryo transfer] Implantation rate (fetal hearts/ 15 17 14 16 embryos transferred) (%) PGD (preimplantation genetic diagnosis) column includes PGD for chromosome abnormalities, sexing for X-linked disease and PGD for PGD (preimplantation genetic diagnosis) column includes PGD for monogenic disorders; PGS = aneuploidy screening; PGD-SS = PGD chromosome abnormalities, sexing for X-linked disease and PGD for for sex selection. monogenic disorders. aFour cycles had polar body biopsy and cleavage stage biopsy. OR = oocyte retrieval; AT = acid Tyrode’s; COC = cumulus–oocyte OR = oocyte retrieval; AT = acid Tyrode’s. COC = cumulus-oocyte complexes. complexes; PGS = aneuploidy screening; PGD-SS = PGD for sex selection.

OR, 603 had an embryo transfer procedure (69% per OR) and Chromosomal abnormalities clinical pregnancy rates of 18% per OR and 25% per embryo Tables Va–c summarizes the 733 and 474 cycles collected for transfer procedure were obtained. These data are broken down data collections I–IV and V respectively. Table Vc lists the in Tables Vb, VIc and VIIc. A total of 1211 cycles was per- karyotypes for which PGD was offered in data V and can be formed for PGS, of which 1202 reached OR and 846 had an found in the electronic version of the paper available at embryo transfer procedure (70% per OR) and clinical preg- Human Reproduction online (http://humrep.oxfordjournals.org/). nancy rates of 16% per OR and 23% per embryo transfer pro- Overall there was very little difference between the two data cedure were obtained. These data are broken down in Table collections. Reciprocal translocation was the most frequent VIIIb. A total of 72 cycles reached OR for social sexing. class of chromosome aberration; ICSI was the predominant Embryos were transferred in 61 cycles and a clinical pregnancy mode of fertilization; acid Tyrode’s with cleavage aspiration rate of 21% per OR was obtained. These data are broken down was the predominant sampling method. A global average of in Table IXb. 14.7 and 13.7 COC per OR cycle was collected for the I–IV As above for data I–IV, in data V ICSI, acid Tyrode’s drilling and V data collections respectively; the fertilization rate (73 and cleavage stage aspiration were the most common methods and 74%), the proportion of successfully biopsied embryos used. Data are presented on 26 747 oocytes. which gave a diagnosis (90 and 91%), the proportion of 5 J.C.Harper et al.

Table Va. Preimplantation genetic diagnosis (PGD) for chromosomal abnormalities, data collection I–IV

Indication Robertsonian Robertsonian Reciprocal, Reciprocal, Sex chromosome Other Total translocations, translocations, male carrier female carrier abnormalities male carrier female carrier

Total cycles 120 110 182 162 103 56 733 Number infertile 96 38 66 35 90 20 345 Female age 33 32 33 32 34 33 32 Cycles cancelled before OR 4 2 5 3 4 2 20 Cycles to OR 116 108 177 159 99 54 713 Cancelled after OR before IVF/ICSI 0 0 1 0 3 0 4 Assisted reproduction treatment IVF 7 14 28 50 9 19 127 ICSI 107 92 147 108 87 35 576 IVF + ICSI 0 1 0 0 0 0 1 Frozen 0 0 1 1 0 0 2 ICSI and frozen 2 1 0 0 0 0 3 Cancelled after IVF/ICSI 3 2 6 1 3 3 18 Cycles to PGD 113 106 170 158 93 51 691 Zona breaching AT drilling 85 74 137 134 49 36 515 Laser drilling 28 29 26 16 20 11 130 Mechanical 0 3 7 8 24 4 46 Biopsy method Polar body biopsy 1 4 0 3 0 0 8 Cleavage aspiration 111 102 166 147 93 50 669 Cleavage extrusion 1 0 3 7 0 1 12 Cleavage flow displacement 0 0 1 1 0 0 2 Embryology COC 1714 1652 2806 2525 1074 715 10486 Inseminated 1452 1368 2404 2189 876 635 8924 Fertilized 999 1005 1805 1666 645 426 6546 Biopsied 647 800 1449 1420 479 343 5138 Successfully biopsied 630 780 1417 1391 475 330 5023 Diagnosed 522 708 1304 1292 420 299 4545 Transferable 196 199 289 287 192 123 1286 Transferred 165 171 249 251 140 88 1064 Frozen 4 13 7 0 13 2 39 Clinical outcome Cycles to embryo transfer 93 88 120 120 74 41 536 HCG positive 26 27 26 27 21 15 142 Positive heart beat 22 21 20 21 12 13 109 Clinical pregnancy rate (%) per OR/% per embryo transfer 19/24 19/24 11/17 13/18 12/16 24/32 15/20

OR = oocyte retrieval; AT = acid Tyrode’s; COC = cumulus–oocyte complexes.

successfully biopsied embryos with a transferable result (26 and laser. The biopsy was successful in 96% of embryos and in 25%), and the clinical pregnancy rate per OR (15 and 15%) 89% of embryos successfully biopsied a diagnosis was were similar. obtained. Of these embryos, 36% of embryos were diagnosed The relatively low pregnancy rates are likely to reflect as transferable and 77% cycles which reached OR resulted in that the low proportion of embryos with a transferable an embryo transfer procedure. Overall clinical pregnancy rates result limits the choice of embryo for transfer (∼1 in 4 of 19% per OR and 24% per embryo transfer procedure were embryos biopsied for all classes of chromosome aberration, obtained. Table VIb shows the indications for which sexing and only 1 in 5 embryos for reciprocal translocation cycles only was performed. This was mainly for haemophilia A (98 in particular). cycles), Duchenne muscular dystrophy (87 cycles), followed by X-linked mental retardation and retinitis pigmentosa (24 Sexing for X-linked disease cycles each). Table VIa shows the cumulative data I–IV for sexing only for Table VIc shows the data from collection V for sexing only X-linked disease using FISH and PCR. On average, 13.5 cases for X-linked disease using FISH and PCR. The most oocytes were collected per OR. FISH was used in the majority common indication in this group was Duchenne muscular dys- of cycles (n = 421) compared to PCR (n = 65). The majority of trophy (36 cycles), followed by haemophilia (22 cycles) and patients was fertile (88%). IVF was used in 10 cycles where Becker muscular dystrophy (nine cycles) (Table VId). A total the diagnosis was done by PCR. The consortium recommends of 127 cycles were in this group, with 118 cycles going to that when PCR is used, ICSI should be performed to prevent oocyte retrieval with an average of 12.6 oocytes per retrieval. paternal contamination (Thornhill et al., 2005). In this data col- The embryos in this group were mainly diagnosed using FISH lection, acid Tyrode’s was used for zona drilling more than the (97%). The biopsy was successful in 99% of embryos biopsied 6 ESHRE PGD Consortium data collection V

Table Vb. PGD for chromosomal abnormalities, data collection V

Indication Robertsonian Robertsonian Reciprocal, Reciprocal, Sex chromosome Other Total translocation, translocations, male carrier female carrier aneuploidy male carrier female carrier

Total cycles 73 57 125 146 41 32 474 Number infertile 64 32 82 67 37 21 303 Female age 36 33 34 34 33 33 33 Cycles cancelled before OR 4 3 6 9 1 2 25 Cycles to OR 69 54 119 137 40 30 449 Cancelled after OR before IVF/ICSI 0 0 0 0 2 0 2 Assisted reproduction treatment IVF 2 11 11 24 5 10 63 ICSI 65 42 105 108 32 20 372 IVF + ICSI 1 0 0 0 0 0 1 Unknown + frozen 0 0 0 0 1 0 1 ICSI + frozen 1 1 3 5 0 0 10 Cancelled after IVF/ICSI 8 5 13 16 4 1 47 Cycles to PGD 61 49 106 121 34 29 400 Zona breaching AT drilling 41 35 66 82 19 26 269 Laser drilling 20 12 33 38 13 2 118 Mechanical 0 2 7 1 2 1 13 Biopsy method Cleavage aspiration 59 45 98 117 34 29 382 Cleavage extrusion 2 4 8 4 0 0 18 Embryology COC 954 620 1684 1920 583 391 6152 Inseminated 810 517 1428 1671 467 347 5240 Fertilized 574 399 1060 1279 320 253 3885 Biopsied 382 342 841 1005 217 190 2977 Successfully biopsied 373 340 831 996 208 187 2935 Diagnosed 328 305 746 920 194 173 2666 Transferable 132 101 161 183 97 48 722 Transferred 93 73 136 152 62 40 556 Frozen 10 9 4 17 6 12 58 Clinical outcome Cycles to embryo transfer 42 37 73 84 28 19 283 HCG positive 14 14 24 22 10 3 87 Positive heart beat 12 11 16 16 8 3 66 Clinical pregnancy rate (%) per OR/% embryo transfer 17/29 20/30 13/22 12/19 20/29 10/16 15/23 Number of fetal hearts 16 13 17 19 9 5 79 Implantation rate (fetal hearts/embryos transferred) (%) 17 18 13 13 15 13 14

OR = oocyte retrieval; AT = acid Tyrode’s; COC = cumulus–oocyte complexes. and 93% of the embryos biopsied were diagnosed. A transfer embryo transfer procedures (82%) and clinical pregnancy rates was achieved in 84% of the cycles which went through PGD, of 20% per oocyte retrieval and 25% per embryo transfer pro- with an average of 1.9 embryos transferred in each cycle. The cedure were obtained. overall clinical pregnancy rate was 20% per OR and 25% per Important points to note from data I–IV are that only 59% embryo transfer, with an implantation rate of 17%. of embryos were diagnosed as transferable for recessive disor- ders, instead of the expected 75%. IVF was used as the method of fertilization in 26 cycles, again in contradiction to Monogenic disease recent recommendations (Thornhill et al., 2005). Acid Tyrode’s Table VIIa summarizes the cumulative data I–IV for PGD of drilling was used in the majority of cases with cleavage stage specific monogenic diseases. A breakdown is only presented aspiration. for the most common disorders. The most common autosomal Table VIIc summarizes data collection V. The same most recessive diseases were cystic fibrosis (247 cycles), β-thalas- common autosomal recessive and dominant disorders and saemia (103 cycles), spinal muscular atrophy (66 cycles) and specific diagnosis of X-linked diseases are found, as shown in sickle cell anaemia (15 cycles). The most common autosomal Table VIIa. Overall there was an average of 13 oocytes col- dominant diseases were myotonic dystrophy (160 cycles), lected per OR, biopsy was successful in 99%, diagnosis was Huntington’s disease (87 cycles) and Charcot–Marie–Tooth possible in 85% of embryos successfully biopsied, and 21% of disease (18 cycles). The most common specific diagnosis of cycles to OR resulted in a clinical pregnancy. Table VIId lists X-linked diseases were for fragile X (38 cycles), haemophilia A the diseases for data V which are included under ‘other’. (16 cycles) and Duchenne muscular dystrophy (26 cycles). Table Important points to note from data V are that five cycles VIIb lists the diseases which are included under ‘other’. A total had IVF instead of ICSI and laser drilling was the most com- of 909 cycles was started: 829 resulted in OR, 681 resulted in mon method used. Nineteen per cent of embryos tested for 7 J.C.Harper et al.

Table Vc. Chromosomal abnormalities analysed, data V (for web-based version only)

Female indication Cycles Female indication Cycles

45,X/46,XX 5 46,XX,t(3;16)(p25;q22) 3 45,X/47,XXX 1 46,XX,t(3;17)(p23;q23) 1 45,X/47,XXX/46,XX 8 46,XX,t(4;5)(q33;p15.31) 2 45,X/47,XXX/48,XXXX 1 46,XX,t(4;8)(p16.1;p23.1) 1 45,XX,der(13;14)(q10;q10) 39 46,XX,t(4;8)(q21.3;p21.3) 2 45,XX,der(13;15)(q10;q10) 7 46,XX,t(4;15)(p16;p13) 1 45,XX,der(13;21)(q10;q10) 5 46,XX,t(4;15)(q31.3;q22.1) 1 45,XX,der(13;22)(q10;q10) 1 46,XX,t(4;17) 1 45,XX,der(14;21)(q10;q10) 3 46,XX,t(4;18)(q24;p11.3) 4 45,XX,der(15;21)(q10;q10) 2 46,XX,t(5;13)(q33;q22) 1 45,XX,der(21;22)(q10;q10) 1 46,XX,t(6;7)(q15;q15) 1 46,X,del(X)(p22.1) 1 46,XX,t(6;12)(p11;q11) 1 46,X,der(X)t(X;Y)(p22.3;q12) 1 46,XX,t(6;19)(p22.1;q13.1) 2 46,XX,del(22)(q11.2q11.2) 3 46,XX,t(7;9)(q21;q34) 1 46,XX,ins(1;13)(p13;q13q22),t(3;6)(p12;p23) 1 46,XX,t(7;10)(q11.2;q26) 1 46,XX,inv(2)(p25q34) 1 46,XX,t(7;12)(p10;p10) 3 46,XX,inv(3)(p26 q13.2),t(13;20)(q31;p12) 1 46,XX,t(7;13)(q31.3;q21.3) 3 46,XX,inv(8)(p21.3q11.2) 1 46,XX,t(7;14)(p11;q13) 2 46,XX,inv(8)(p23.1q11.2) 1 46,XX,t(7;14)(q11.23;q22) 2 46,XX,inv(9)(p11q13) 6 46,XX,t(7;15)(p10;q10) 3 46,XX,inv(10)(p15q11.2) 1 46,XX,t(7;15)(p14;q11.2) 1 46,XX,inv(11)(p15.3q24.2) 1 46,XX,t(7;16)(q21.2;q23) 1 46,XX,inv(18)(p11.23q11.2) 2 46,XX,t(7;21)(p10;q10) 1 46,XX,t(1;5)(q23;p13.1) 1 46,XX,t(7;22)(p13;q11.2 1 46,XX,t(1;9)(p36.3;q34) 2 46,XX,t(8;11)(p21;p15) 1 46,XX,t(1;10)(q12;q11.2) 1 46,XX,t(8;12)(q11.2;q12) 1 46,XX,t(1;10)(q25;p11.2) 1 46,XX,t(8;13)(q24.1;q22) 1 46,XX,t(1;12)(p31;p13) 3 46,XX,t(8;19)(q21.3;q12) 1 46,XX,t(1;13)(p36.3;q12.3) 3 46,XX,t(9;10)(p13;q22) 1 46,XX,t(1;14)(p22;q24) 1 46,XX,t(9;11)(p21.3;q14.1) 1 46,XX,t(1;15)(q42;q22.1) 1 46,XX,t(9;13)(q12;p13) 1 46,XX,t(1;19)(q32.1;q13.1) 1 46,XX,t(9;15)(q34;q22) 1 46,XX,t(1;21)(p22;q21) 3 46,XX,t(9;16)(q34;p12) 1 46,XX,t(1;22)(p36.3;q11.2) 1 46,XX,t(10;13)(q22;q14) 2 46,XX,t(2;3)(q12;q25) 2 46,XX,t(10;15)(q23;q15) 2 46,XX,t(2;4)(p22.2;q33) 1 46,XX,t(10;16)(q24.2;p13.3) 2 46,XX,t(2;6)(p21;q25.1) 2 46,XX,t(10;17)(p10;p10) 4 46,XX,t(2;10)(q21;q26) 1 46,XX,t(10;18)(q?21;q?12) 2 46,XX,t(2;11)(q37.2;p12) 1 46,XX,t(11;17)(p15.3;q11.2) 1 46,XX,t(2;12)(q35;q24.1) 1 46,XX,t(11;18)(p14;q23) 1 46,XX,t(2;13)(p16;q31) 1 46,XX,t(11;22)(q23.3q11.2) 11 46,XX,t(2;13)(q23;q33) 1 46,XX,t(11;22)(q25;q12) 1 46,XX,t(2;13)(q36;q22) 2 46,XX,t(12;15)(q24,1;q24) 1 46,XX,t(2;14)(q21.3;q24.3) 3 46,XX,t(12;20)(q15;p11.2) 1 46,XX t(2;15)(p13;q22) 1 46,XX,t(12;21)(q15;q22) 3 46,XX,t(2;15)(p15;q24) 1 46,XX,t(13;18)(p11;q11.2) 1 46,XX,t(2;15)(q33;q11.2) 1 46,XX,t(13;21)(q14;q21) 1 46,XX,t(2;16)(q37;p11.2) 2 46,XX,t(13;21)(q22;q11.2) 2 46,XX,t(2;18)(p11.2;p11.3) 1 46,XX,t(14;18)(q23;q21.1) 3 46,XX,t(2;18)(q31;q23) 1 46,XX,t(14;18)(q24.2;p11.21) 2 46,XX,t(2;19)(q37.3;q13.1) 1 46,XX,t(14;18)(q24.3;q21.3) 1 46,XX,t(2;22)(p10;p10) 1 46,XX,t(14;22)(q24;q13) 1 46,XX,t(3;7)(q23;q31) 1 46,XX,t(19;22)(q13.4;q11.2) 1 46,XX,t(3;10)(q26.2;q21.21) 3 Robertsonian female 1 46,XX,t(3;14)(q13.2;q11.2) 1 Reciprocal female 3 46,XX,t(3;15)(q22;q21) 3

Male indication Cycles Male indication Cycles

45,X,del(Y)(q11.2q11.2),der(13;14)(q10;q10) 1 46,XY,t(5;20)(q33;q13.3) 1 45,XY,der(13;14)(q10;q10) 45 46,XY,t(6;9)(q23;q23) 1 45,XY,der(13;15)(q10;q10) 2 46,XY,t(6;10)(p11;p15) 1 45,XY,der(13;21)(q10;q10) 2 46,XY,t(6;11)(p22.2;q13.1) 1 45,XY,der(13;22)(q10;q10) 1 46,XY,t(6;13)(p22;q14.2) 1 45,XY,der(14;15)(q10;q10) 1 46,XY,t(6;15)(q27;q12) 1 45,XY,der(14;21)(q10;q10) 14 46,XY,t(6;20)(p21.3;q13.3) 1 45,XY,der(15;21)(q10;q10) 2 46,XY,t(7;13)(p21;q21.1) 2 45,XY,der(21;22)(q10;q10) 2 46,XY,t(7;16)(q22;q22) 2 45,XY,inv(9)(p11q13),der(13;14)(q10;q10) 1 46,XY,t(8;9)(p21;q22) 1 46,X,del(Y)(q11.2) 2 46,XY,t(8;10)(q13;p13) 1 46,X,inv(Y)(p11q12) 1 46,XY,t(8;11)(p21.1;p14.2) 1

8 ESHRE PGD Consortium data collection V

Table Vc. Continued

Male indication Cycles Male indication Cycles

46,X,t(Y;18)(q12;p11.2) 1 46,XY,t(8;11)(q21;q21) 1 46,XY,-21+,der(21;21)(q10;q10) 1 46,XY,t(8;14)(q21.2;q11.2) 1 46,XY,del(9)(?) 1 46,XY,t(8;14)(q22.1;q31) 2 46,XY,ins(7;6)(q22;q25.1q27) 1 46,XY,t(8;20)(p23.1;q13.1) 1 46,XY,inv(1)(p31.2q23) 1 46,XY,t(8;21)(p21.1;q22.3) 2 46,XY,inv(9)(p11q13) 3 46,XY,t(9;11)(q21.1;p14.3) 1 46, XY.ish t(2q;17q)(210E14-,B37c1+;B37c1-,210E14+) 1 46,XY,t(9;12)(p21;q13.1) 1 46,XY,t(1;2)(q25;q23) 1 46,XY,t(9;12)(p24;p11.2) 2 46,XY,t(1;3)(q44;q21) 1 46,XY,t(9;13)(p22;q12.3) 1 46,XY,t(1;4)(p32;q25 1 46,XY,t(9;13)(p23;q21.1) 2 46,XY,t(1;4)(q31;q35) 1 46,XY,t(9;13)(q33;q12) 3 46,XY,t(1;5)(q41;q33) 6 46,XY,t(9;14)(q32;p11.2) 1 46,XY,t(1;6)(p13.3;p22.2) 1 46,XY,t(10;12)(p13;p13.3) 1 46,XY,t(1;7)(p22;q35) 1 46,XY,t(10;17)(q22.1;q21.3) 3 46,XY,t(1;9)(p13.3;p13) 1 46,XY,t(10;19)(p11;p11) 1 46,XY,t(1;11)(p12;q12) 1 46,XY,t(10;19)(q21.2;p13.3) 1 46,XY,t(1;13)(q21;q11) 1 46,XY,t(11;12)(q13;p13) 1 46,XY,t(1;14)(q21;q32) 1 46,XY,t(11;16)(p15;q12) 2 46,XY,t(1;15)(q21;p11.2) 1 46,XY,t(11;19)(p11.2;q13.1) 2 46,XY,t(1;16)(p31;q22) 1 46,XY,t(11;22)(?;?) 1 46,XY,t(1;17)(p34;q25) 1 46,XY,t(11;22)(q23;q34) 4 46,XY,t(1;18)(p32;q23) 1 46,XY,t(11;22)(q23.3;q11.2) 6 46,XY,t(1;19)(q33;p11) 1 46,XY,t(12;16)(p11.2;p13.1) 2 46,XY,t(2;8)(p22;p23.1) 2 46,XY,t(13;14)(q21;q21) 2 46,XY,t(2;8)(q35;q11.2) 1 46,XY,t(13;18)(?;?) 2 46,XY,t(2;11)(q11.2;q23.1) 1 46,XY,t(14;15)(q32.1;q13) 1 46,XY,t(2;13)(p16;q31) 2 46,XY,t(14;17)(p?;q?) 1 46,XY,t(2;15)(q32;q26) 2 46,XY,t(14;20)(p11.2;q11.2) 1 46,XY,t(2;17)(q33;p11.2) 1 46,XY,t(2;18)(p23;q23) 1 46,XY,t(15;22)(p11.2;q12) 1 46,XY,t(2;20)(p10;q10) 1 46,XY,t(18;21)(p10;p10) 1 46,XY,t(2;22)(q37;q13) 3 47,XXY 14 46,XY,t(3;5)(p13;p15.1) 2 47,XXY/46,XY 3 46,XY,t(3;6)(q25;q23) 1 47,XY,+der( 15) 1 46,XY,t(3;6)(q26;q23) 1 47,XYY 6 46,XY,t(3;6)(q27.1;q21.1) 1 Robertsonian male 3 46,XY,t(3;7)(p14;q34) 1 Reciprocal male 3 46,XY,t(3;7)(q26.2;p14) 1 46,XY,t(3;14)(q25;q32.3) 1 Miscellaneous Cycles 46,XY,t(3;19)(p21;p13) 1 46,XY,t(4;8)(p16;q22.2) 1 45,XY,der(14;21)(q10;q10) and 45,XX,der(13;14)(q10;q10) 2 46,XY,t(4;12)(p16;q22) 1 46,XY,t(4;22)(q21;q13.3) 3 46,XY,t(5;6)(q35;p21.3) 2 46,XY,t(5;7)(q33;q22) 2 46,XY,t(5;8)(q3.1;q1.3) 1 46,XY,t(5;10)(p13;q23) 1

β-thalassaemia and HLA typing were diagnosed as suitable for Technical outcomes were good for all cycles. For data col- transfer which is almost exactly the expected percentage. lection V, >6500 embryos were biopsied and in 99% of cases this was successful. FISH analysis resulted in a diagnosis on 93% of embryos and 37% were chromosomally normal and Preimplantation genetic screening (PGS) suitable for transfer. The cumulative data for PGS from collections I–IV are shown The overall pregnancy result was 16% per OR and this in Table VIIIa; the breakdown is for cycles with one indica- ranged from 12% for AMA to 33% for SMF. A closer look at tion, i.e. advanced maternal age (AMA), recurrent miscarriage the cycle parameters of these two groups offers some explana- (RM), recurrent IVF failure (RIF) and severe male factor tion for the difference in pregnancy results. It is not surprising (SMF). Cycles with two indications are included in the ‘other’ that the AMA group had the highest mean maternal age of 41 column. In data collection V (Table VIIIb), the data have been years. The SMF group had the lowest mean maternal age of 32 divided to reveal multiple indications, e.g. AMA and RIF. The years. The AMA group fared poorly compared to the SMF use of PGS continues to increase with a total of 1990 cycles in group with fewer oocytes collected per PGS OR (9.6 versus data collections I–IV and 1211 cycles in data collection V 15.2), fewer embryos biopsied per PGS OR (4.4 versus 6.4) alone. In data collections I–IV, there was about the same and fewer embryos that were genetically suitable for transfer number of cycles for RIF and AMA, but in the most recent data (31 versus 36%). Of those that had PGS, fewer AMA than there were 418 cycles for AMA and only 275 for RIF. SMF patients had a transfer (60 versus 86%) and those that did 9 J.C.Harper et al.

Table VIa. Sexing only for X-linked disease using PCR or FISH, data Table VIb. List of indications for which sexing was performed, data collection I–IV collection I–IV

FISH PCR Total Indication Number of cycles

Total cycles 421 65 486 Haemophilia A 98 Number infertile 51 0 51 Duchenne muscular dystrophy 87 Female age 33 31 33 X-linked mental retardation 24 Cycles cancelled before OR 22 6 28 Retinitis pigmentosa 24 Cycles to OR 399 59 458 23 Assisted reproduction treatment Becker muscular dystrophy 17 IVF 115 10 125 Wiskott–Aldrich syndrome 13 ICSI 284 49 333 13 Cancelled after OR 20a 1b 21 Y deletion 11 Cycles to PGD 379 58 437 Ornithine transcarbamylase deficiency 10 Zona breaching Myotubular myopathy 9 AT drilling 236 50 286 9 Laser drilling 119 0 119 Hydrocephalus 9 Mechanical 24 8 32 Agammaglobulinaemia 9 Biopsy method Fabry disease 8 Cleavage aspiration 374 54 428 Charcot–Marie–Tooth disease 7 Cleavage extrusion 5 4 9 Chronic granulomatous disease 6 Embryology Menkes’ disease 6 COC 5354 808 6162 FG syndrome 6 Inseminated 4819 627 5446 X-linked autism 6 Fertilized 3420 501 3921 Löwe syndrome 5 Biopsied 2579 412 2991 Ectodermal dysplasia 4 Successfully biopsied 2490 376 2866 Choroideraemia 4 Diagnosed 2251 286 2537 2 Transferable 768 152 920 Kallman syndrome 2 Transferred 588 119 707 Lesch–Nyhan syndrome 2 Frozen 157c 56d 213e Epilepsy 1 Clinical outcome Coffin–Lowy syndrome 1 Cycles to embryo transfer 305 48 353 Barth syndrome 1 HCG positive 92 22 114 Ataxia 1 Positive heart beat 70 16 86 BRCA 1 1 Clinical pregnancy rate (%) per 18/23 27/33 19/24 Hypospadias 1 OR/embryo transfer (%) Exep macrosom males 1 Golabi–Rosen syndrome 1 aTwenty-seven embryos from two cycles frozen before biopsy due to Renal agenesis 1 hyperstimulation. 1 bTwenty embryos frozen before biopsy. Inversion X 1 cEleven cycles with embryos frozen without biopsy or after failed diagnosis Norrie disease 1 included. Skewed X inactivation 1 dThirteen cycles with embryos frozen without biopsy or failed diagnosis Sulphatidosis 1 included. Othersa 58 eTwenty-four cycles with embryos frozen without biopsy or after failed Total 486 diagnosis included. OR = oocyte retrieval; PGD = preimplantation genetic diagnosis; AT = acid aOthers include cycles with unknown or unclear indications. Tyrode’s; COC = cumulus–oocyte complexes. had fewer embryos transferred (1.6/embryo transfer versus 2.0/ syndrome, males whose semen analysis did not fulfil the WHO embryo transfer). These differences would make a significant con- criteria, testicular sperm extraction patients, altered male meio- tribution to the lower pregnancy rate observed in the AMA group, sis, altered FISH results, non-obstructive azoospermia, Y chro- particularly when outcomes are expressed per oocyte retrieval. mosome microdeletion and immature spermatids. The This year centres were asked how they define AMA, RIF, Consortium recognizes that there needs to be consistency in RM and SMF. For AMA, the majority of clinics defined this as these definitions for the purposes of data analysis and this will age >37 or >38 years, but several defined this as >35 years. For be addressed in the future. RIF, the majority of clinics defined this as three or more failed embryo transfer procedures (sometimes the definition adds Social sexing ‘with good quality embryos’) or >10 embryos replaced. How- Table IXa summaries data I–IV for social sexing. A total of ever, some clinics defined this as just two failed embryo trans- 182 cycles was started, with only 19 patients being infertile. Of fer procedures. The majority of clinics defined RM as three 182 cycles reaching OR, 1109 embryos were successfully previous miscarriages (sometimes the definition adds ‘consec- biopsied and 304 diagnosed as transferable. A total of 131 utive’). However, one clinic defined this as four previous mis- cycles reached embryo transfer with a clinical pregnancy rate carriages and several just as two previous miscarriages. Severe of 24% per OR and 34% per embryo transfer. male factor included many definitions; azoospermia, severe Table IXb summarizes data V for social sexing. A total of 72 oligoasthenoteratozoospermia, macrocephalic sperm, Klinefelter cycles was started, of which 22 included PGS. Only 22 patients

10 ESHRE PGD Consortium data collection V

Table VIc. Sexing only for X-linked disease using PCR or FISH, data Table VId. List of indications for which sexing has been performed, data collection V collection V

FISH PCR Total Indication Number of cycles

Total cycles 124 3 127 Duchenne muscular dystrophy 36 Number infertile 28 0 28 Haemophilia A + B 22 Female age 33 28 Becker muscular dystrophy 9 Cycles cancelled before OR 9 0 9 Y deletion 7 Cycles to OR 115 3 118 Retinitis pigmentosa 6 Assisted reproduction treatment Adrenoleucodystrophy 6 IVF 23 0 23 Menkes’ disease 6 ICSI 90 3 93 Inversion X 4 ICSI + frozen 1 0 1 X-linked mental retardation 3 Both IVF and ICSI 1 0 1 Unknown 3 Cancelled after OR 8 0 8 Alport 3 Cycles to PGD 107 3 110 Hunter’s disease 2 Zona breaching Chronic granulomatous 2 AT drilling 63 0 63 Lowes 2 Laser drilling 44 1 45 OTC 2 Mechanical 0 2 2 Falizaeus–Merzbacher 1 Biopsy method X-linked myotublar 1 Cleavage aspiration 104 2 106 Hoyeraal–Hreidarrson 1 Cleavage extrusion 3 1 4 Austism 1 Embryology Fragile X 1 COC 1433 55 1488 Sex chromosome mosaicism 1 Inseminated 1234 35 1269 Opitz–Kaveggia 1 Fertilized 902 25 927 X-linked haemophagocytic 1 Biopsied 713 19 732 Wiscott–Aldrich 1 Successfully biopsied 705 19 724 Klienfelter syndrome 1 Diagnosed 654 19 673 CMT 1 Transferable 255 8 263 X-linked chondrodysplasia 1 Transferred 169 8 177 X-linked retinoschisis 1 Frozen 22 0 22 LHON mitochondrial 1 Clinical outcome Total 127 Cycles to embryo transfer 89 3 92 HCG positive 29 0 29 OTC = ornithine transcarbamylase; CMT = charcot marie tooth disease; Positive heart beat 23 0 23 LHON = leber hereditary optic neuropathy. Clinical pregnancy rate (%) per 20/26 0 20/25 OR/% per embryo transfer Implantation rate (fetal heart 29/158a (18) 0 29/166a (17) beats/embryos transferred) (%) these different reports that the pregnancies obtained after PGD are quite comparable to those obtained after ICSI aEleven embryos transferred removed from calculation due to lack of informa- (Bonduelle et al., 2005), giving a first indication that embryo tion regarding the number of FHB in pregnancies resulting from the transfer of those embryos. biopsy is not detrimental to the course and outcome of preg- OR = oocyte retrieval; PGD = preimplantation genetic diagnosis; AT = acid nancy (Table Xa). As in ICSI, no particular complication Tyrode’s; COC = cumulus oocyte complexes. stands out, and the most important problem remains the multiplicity, causing most of the morbidity and mortality (Table XIa). With the introduction of single embryo transfer policies in many countries, this problem will decrease in the were infertile. The mean maternal age was 36 years. No cycles future. The most frequent mode of delivery was Caesarean were cancelled prior to OR. A total of 1002 oocytes was col- section (Table XIIa), both for the singletons as for the twins lected, and 396 embryos were successfully biopsied. In 93% of and triplets, and baby characteristics (birth weight, length, cycles, the zona was breached by mechanical means; and the gestational age at delivery) and malformations at birth are most used biopsy method was cleavage extrusion. In 61% of again quite similar to the characteristics of ICSI babies cycles PCR was used and in 39% FISH was used. In 11 cycles, (Table XIIIa, XIVa). embryos of the required sex were not obtained and the patients The most important factor for PGD in data collection I–IV did not have an embryo transfer procedure. No clinical preg- was the risk of misdiagnosis (Table XVa). Due to the com- nancies were obtained in the combined social sexing and PGS plexity of the data, it is not possible to calculate the error rate group. For the cases where FISH was used, a clinical preg- per fetal sac: several babies have been tested more than once, nancy rate of 50% was obtained. For the cases where PCR was e.g. with a prenatal diagnosis, and with a physical exam- used, a clinical pregnancy rate of 27% per OR and 29% per ination at birth. It is important, however, to take note of the embryo transfer procedure was obtained. An overall implanta- misdiagnoses that occurred, and to try and find the cause of tion rate of 14% was obtained. the misdiagnosis. In this way, future misdiagnosis can be avoided. Pregnancies and babies For data V, whereas 494 cycles ended in a positive HCG, In the four former data collections (I–IV), detailed data on only 485 pregnancies were reported. Fifteen pregnancies were 648 pregnancies have been collected. It is now clear from lost for follow-up after the cycle, and for 17 positive HCG, no 11 J.C.Harper et al. d b b 3 /25 734 b b HD = Huntington’s disease; HD = Huntington’s syndrome; DMD = Duchenne muscular dystrophy (specific); (specific); dystrophy DMD = Duchenne muscular syndrome; molysis molysis bullosa; DM1 = myotonic dystrophy; 10 18 3 10 26 38 16 102 909 a ophy; SC = sickle-cell anaemia; EB = epider cyte complex. 00 000 01 0000000 00000 83 47 14 8 127 75 10 15 3 10 23 30 15 80 -Thal SMA SC EB DM1 HD AP CMT ACH MS DMD FRAXA Haem β arcot–Marie–Tooth disease; ACH = achondroplasia; FRAXA = ACH fragile-X = achondroplasia; disease; arcot–Marie–Tooth b b c COC = cumulus–oo CF -thalassaemia; SMA = spinal muscular atr -thalassaemia; SMA = β embryo transferembryo 21/25 26/31 23/26 7/7 13/13 15/20 15/18 10/11 24/44 0 20/20 26/29 17/26 19/27 28/33 20 -thal = = -thal β fibrosis and fragile X syndrome. retrieval; AT = acid Tyrode’s; Cycles performed for single gene disorders using PCR, data collection I–IV nnw 00 000 0316 3000002 Cleavage aspiration 6 00002 Cleavage extrusionUnknown COCInseminatedFertilizedBiopsied biopsiedSuccessfully DiagnosedTransferableTransferredFrozen transfer to embryo Cycles HCG positive 194 beat heart Postive 11Clinical pregnancy rate (%) per OR/% per 1 1395 2528 9 2901 463 1050 1762 1413 0 1219 625 363 660 187 1188 741 752 473 0 189 94 440 405 355 119 78 247 364 174 139 83 145 299 159 3 161 1521 53 185 106 95 46 58 757 121 1728 83 935 83 1116 54 20 14 0 24 1065 29 443 62 37 34 93 673 766 8 8 111 14 23 0 15 78 634 176 272 82 451 209 107 28 213 1 380 66 3 0 15 137 177 78 20 0 68 111 1 10 61 3 97 11 0 112 32 67 25 19 63 9 293 55 21 348 79 25 0 23 10 164 17 270 296 208 33 8 9 12 64 4 15 0 131 192 4 154 58 176 0 3 164 36 1 3 1186 0 1420 25 71 138 113 132 10 10749 3 93 9251 4 117 4 638 903 54 21 1 72 0 52 4819 6621 0 0 36 41 23 644 5 4 28 2 4876 545 2 25 11 30 31 263 4024 6 185 6 2168 80 6 1440 6 6 681 4 3 4 64 26 29 381 167 202 IVFICSI 0518 3000002 5 00003 FrozenUnknown AT drilling 6226 0314 0100022 3000002 Laser drilling 4 00002 MechanicalUnknown 1201000 biopsy body Polar 202 0 90 148 57 56 1 3 78 15 4 2 6 33 8 0 16 0 138 9 7 0 5 8 78 0 0 0 78 9 0 52 0 17 35 40 0 0 10 3 0 0 10 0 2 13 0 21 0 0 3 32 0 0 8 2 0 15 0 9 8 0 11 86 0 19 775 0 15 1 0 0 40 1 38 490 1 258 7 7 18 Three cycles had both polar body biopsy and cleavage stage biopsy. stage cleavage and biopsy body polar both cycles had Three Eight cycles data. with missing Includes three HD with exclusion. One cycle for two indications: cystic Total cycles 247 103 66 15 8 160 87 AP = amyloid polyneuropathy, MS = Marfan’s syndrome; CMT = CMT Ch syndrome; MS = Marfan’s AP = polyneuropathy, amyloid Embryology Clinical outcome Table VIIa. a b c d Indication Autosomal recessive Autosomal dominant Specific sex-linked Other Total Female age 33333436323332283333326636283333 Number infertileFemale ORbefore Cycles cancelled ORCycles to treatment reproduction Assisted Cancelled ICSICancelled afterOR PGDCycles to Zona breaching 28 method Biopsy 75 12 47 4 219 5 91 0 0 3 62 0 8 0 211 0 15 16 7 8 11 0 84 6 6 56 144 0 5 0 1 14 81 0 7 0 1 8 10 3 0 0 132 12 17 0 0 0 78 3 3 0 0 10 10 3 0 15 1 23 0 2 2 3 14 36 0 0 0 10 0 16 0 0 23 8 94 0 13 0 32 80 829 181 0 4 16 0 88 0 780 0 6 3 49 Haem = haemophilia; OR = oocyte = haemophilia; OR Haem CF = cystic fibrosis (various mutations); (various mutations); fibrosis cystic CF = 12 ESHRE PGD Consortium data collection V

Table VIIb. List of indications for monogenic diseases listed as other in Table VIIa, data collection I–IV

Indication No. of cycles

Cycles without indication 7 21-Hydroxylase deficiency 2 Adrenoleukodystrophy 5 Agammaglobulinaemia 1 Alport syndrome 4 Adult polycystic kidney disease 1 BRCA1 1 Carbohydrate-deficient glycoprotein syndrome type 1A 3 Central core disease 3 Crouzon syndrome 2 Familial adenomatous polyposis coli (Gardner syndrome) 4 Fanconi anaemia 1 Gaucher disease 5 Glucose-6-phosphatase deficiency 1 Gorlin syndrome 2 HLA typing 3 Hunter syndrome (MPS II) 1 Hyperinsulinaemic hypoglycaemia PHH1 1 Infantile neuronal ceroid lipofuscinosis 2 Junctional epidermolysis bullosa 1 Lesch–Nyhan disease 1 Medium chain acyl-CoA dehydrogenase deficiency 1 MELAS (mitochondrial myopathy, encephalopathy, lactacidosis, ) 2 Metachromatic leukodystrophy 1 Neurofibromatosis 3 Oral–facial–digital syndrome type 1 3 Ornithine transcarbamylase deficiency 2 Osteogenesis imperfecta type I 3 Osteogenesis imperfecta type IV 4 Pelizaeus–Merzbacher disease 1 Retinoblastoma 1 RhD sensitization 4 Rhizomelic chondrodysplasia punctata 2 Skewed X inactivation 1 2 Spinal and bulbar muscular atrophy 1 Spinocerebellar ataxia type 7 1 3 Tay–Sachs disease 4 Tuberous sclerose 3 Tyrosinaemia 1 Von Hippel–Lindau disease 3 Waardenburg syndrome 1 Y deletion 3 ZFX/ZFY for sexing only 2 Total 102

data were reported (five centres). Thirteen pregnancies were as weight at birth, malformations and neonatal complications reported without a cycle, and 10 pregnancies were a result are comparable to data submitted in earlier data collections from a previous frozen cycle and are thus not included in the (Tables XIIb–XIVb). cycle database. The overall pregnancy data V are similar to the No misdiagnosis was reported for data collection V. How- data from previous data collections: multiplicity is still a major ever, it is noted that three de novo reciprocal translocations cause of complications, morbidity and mortality and measures were reported out of a total of 476 fetal sacs (1/159). In com- taken to avoid multiplets are still not transpiring in the data parison, on a large series of prenatal diagnosis, a prevalence of (Tables Xb, XIb, XIIb). No particular complication or malfor- 1/2000 amniocenteses was found (Warburton, 1991). This is mation stands out (Table XIIIb). the first report of a cluster of de novo reciprocal translocations. Of the 325 pregnancies in data collection V that ended in the Whether this is coincidence, or due to better follow-up of preg- birth of at least one baby, four deliveries were lost to follow- nancies and babies and reporting, will have to be investigated up, and no data were submitted on seven deliveries. Data were over the longer term (Table XVb). submitted on 314 deliveries. Data collection V is not remark- An attempt was made to calculate the singleton live birth able concerning the babies born: all baby characteristics such rates per OR (ideally live birth rates per started cycle should be 13 J.C.Harper et al. T = Charcot–Marie–Tooth T = Haem FRAXA f = haemophilia; OR = oocyte retrieval; AT = acid ACH (BMD) DMD e dystrophy (specific); Haem onic dystrophy; HD = Huntington’s disease; CM HD = Huntington’s dystrophy; onic DM1 CMT d dominat Specific sex-linked Others Total HD (HDexcl) c ecific); BMD = Beckerecific);muscular BMD = SMA SC b ar atrophy; SC = sickle-cell anaemia; DM1 = myot -thal + HLA) -thal + β 34 (0) 20/21 30/38 14/21 (0) 21/26 25/50 0 27/36 (0) 100 17/33 27/38 21/28 chenne muscular dystrophy (sp -Thal ( -Thal β a CF -thalassaemia; SMA = spinal muscul β red) (%)red) 13 26 (0) 12 13 19 (0) 15 25 0 (0) 21 40 10 20 17 -thal = = -thal β -thalassaemia. β Cycles performed for single gene disorders using PCR, data collection V PCR, data collection using gene disorders for single performed Cycles -thalassaemia and HLA typing in parentheses. β Number heartsfetal Number Implantation rate (fetal hearts/embryos transfer 12 18 (0) 6 2 9 (0) 8 1 0 5 (0) 2 1 9 73 IVFICSIFrozenAT drillingLaser drillingMechanical biopsy body Polar aspirationCleavage Cleavage extrusionCOCInseminatedFertilizedBiopsied biopsiedSuccessfully DiagnosedTransferableTransferred 3 66Frozen 0 transfer to embryo Cycles 35 (3) 0 19 42HCG positive 0Positive heartbeat 1 59 2 17 rate per transfer)OR/% per (% embryo pregnancy 13 (3)Clinical 3 19/25 28 (3) 0 29/ 0 2 (0) 24 364 747 0 10 (29) 181 837 1 12 (53) 376 555 10 365 487 21 (62) 0 44 (5) (35) 256 0 4 52 304 161 (29) 182 4 0 3 4 8 0 163 (26) 95 (5) 114 28 (5) 29 (1) 38(0) 11 135 262 2 35 (5) 2 0 70 (1) 12 13 17 360 4 41 181 98 26 4 (0) 3 (0) 137 3 (0) 16 (4) 10 (0) 116 1 (39) 13 (0) 241 7 (61) 449 55 33 4 120 4 0 566 (74) 42 79 24 0 (47) 318 0 4 33 205 14(1) 3 (39) 248 440 0 3 0 3 51 18 8 492 190 (38) 0 33 0 68 325 3 0 0 0 15 0 74 (13) 88 208 29 (5) 8(1) 9 5 59 14 8 34 2 178 1 (0) (1) 15 33 7 0 47 (9) 0 0 40 21 5 3 28 83 157(4) 91(3) 12 3 14 31 219 (7) 2 0 12 121(4) 0 0 12 3 (1) 2 53 6 (0) 9 (0) 2 33 95(4) 28 9 9 6 4 11 81(2) 32 0 0 21 110 3 295 61 3 12 142 8 0 2 58(1) 0 26 0 5 9 29 0 84 336 11(1) 2 0 1 1 7 185 396 1 1 24(1) 0 61 186 3 257 257 3223 5 46 0 0 0 1631 3918 5 188 2 2339 3 3 15 80 160 5 9(0) 5(0) 4(0) 1654 6 1 14 10 1388 17 82 12 24 0 2 2 726 46 228 4 3 2 436 12 9 9 80 82 63 Cycles for HD by exclusion. Includes one cycle for two indications: CF and social sexing. Includes two cycles for sickle cell and Includes one cyclefor X-linked dominant inheritance. Cycles for BMD in parentheses. disease; ACH = achondroplasia;syndrome; DMD = Du FRAXA = fragile-Xdisease; ACH = Tyrode’s; COC = cumulus–oocyte complexes. CF = cystic fibrosis (various mutations); (various mutations); fibrosis cystic CF = Total cyclesNumber infertile ageFemale Cycles cancelled before ORCycles to ORtreatment reproduction Assisted Cancelled after ORCycles to PGDZona breaching method Biopsy 8 32Embryology 77 3 (0) 22 (0) 33 38 (3) 69 34 (36) 35 (3) 6 2 63 4 27 5 (0)Clinical outcome 2 30 (3) 33 12 3 25 32 9 (1) 53 (6) 8 (1) 10 34 (32) 0 44 (5) 43 4 25 9 34 0 4 0 10 31 0 39 2 (0) 42 (5) 4 0 34 4 0 (1) 16 31(28) 1 (0) 4 36 3 4 4 0 (1) 15 34 2 0 3 1 35 13 0 1 2 (1) 15 0 (0) 34 36 0 12 3 335 33 2 33 3 0 34 12 301 0 86 32 1 283 18 Indication recessive Autosomal Autosomal Table VIIc. a b c d e f 14 ESHRE PGD Consortium data collection V

Table VIId. List of indications for monogenic diseases listed as other in Table VIIc

Indications No. of cycles

Adrenoleukodystrophy 1 Alport syndrome 2 BRCA1 5 Canavan disease 2 Central core disease 3 Epidermolysis bullosa simplex 1 Familial ademonatous polyposis (Gardner syndrome) 3 LCHAD 1 Lesch–Nyhan disease 1 Multiple exostoses 1 Norrie’s disease 1 Ornithine transcarbamylase deficiency 1 Osteogenesis imperfecta type I 1 PDH deficiency 1 Polycystic kidney disease 1 Retinoblastoma 4 Rhizomelic chondro dysplasia punctata 1 Spinocerebellar ataxia 3 (SCA3) 1 Spinocerebellar ataxia 7 (SCA7) 2 Severe combined immunodeficiency 1 Stickler syndrome 1 Von Hippel–Lindau 1 Total 36

LCHAD = long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency.

Table VIIIa. Cycles performed for preimplantation genetic screening, data collection I–IV

Indication AMA Recurrent miscarriage Recurrent IVF failure Severe male factora No indication Other Total

Total cycles 625 285 642 65 54 229 1900 Number infertile 596 229 637 65 53 194 1774 Female age 40 36 36 32 33 35 35 Cycles cancelled before OR 1 8 12 1 1 1 24 Cycles to OR 624 277 630 64 53 228 1876 Assisted reproduction treatment IVF 128 39 120 1 11 56 355 ICSI 490 235 476 63 42 157 1463 IVF + ICSI 1 2 6 0 0 1 10 Frozen 0 0 4 0 0 0 4 Frozen and ICSI 0 0 1 0 0 0 1 Unknown 5 1 23 0 0 14 43b Cancelled after OR 25 3 38 4 0 7 77 Cycles to PGS 599 274 592 60 53 221 1799 Zona breaching AT drilling 354 198 394 7 38 149 1140 Laser drilling 209 68 151 46 10 49 533 Mechanical 23 7 10 7 5 9 61 Unknown 13 1 37 0 0 14 65b Biopsy method Polar body biopsy 51c 7703270c Cleavage aspiration 472c 251 499 33 49 215 1519c Cleavage extrusion 57 14 42 26 1 3 143 Cleavage flow displacement 7 1 6 1 0 1 16 Unknown 13 1 38 0 0 0 52 Embryology COC 6933 4134 8708 952 699 2948 24 374b Inseminated 6143 3467 7479 735 628 2478 20 930 Fertilized 4427 2495 5395 464 467 1754 15 002 Biopsied 3580 1916 3495 326 365 1326 11 008 Successfully biopsied 3523 1827 3440 320 346 1304 10 760 Diagnosed 2815d 1461d 3186d 263 215d 1105d 9045d Transferable 582d 527d 1219d 129 195d 490d 3142d Transferred 971d 465d 881d 104 113d 398d 2932d Frozen 92 44 196 10 7 87 436

15 J.C.Harper et al.

Table VIIIa. Continued

Indication AMA Recurrent miscarriage Recurrent IVF failure Severe male factora No indication Other Total

Clinical outcome Cycles to embryo transfer 440 223 416 51 48 164 1342 HCG positive 130 74 118 21 18 70 431 Positive heart beat 103 61 86 19 17 50 336 Clinical pregnancy rate (%) per OR/% 17/23 22/27 14/21 30/37 32/35 22/30 18/25 per embryo transfer aThese data were not extracted from data I–III. bSeveral cycles had incomplete results. cOne cycle had cleavage stage biopsy and polar body biopsy. dSeveral cycles from one centre had no information on the number of embryos diagnosed, number of embryos diagnosed as transferable, but patients did have embryos transferred. In these cases, undiagnosed or abnormal embryos were transferred. AMA = advanced maternal age; OR = oocyte retrieval; AT = acid Tyrode’s; COC = cumulus–oocyte complexes. Other: includes data with two indications.

Table VIIIb. Cycles performed for preimplantation genetic screening, data collection V

Indication AMA AMA + AMA + RIF Recurrent RIF SMF No Other Total miscarriage miscarriage indication

Total number of cycles 418 43 129 198 275 101 13 34 1211 Number infertile 394 36 114 190 256 101 10 19 1120 Female age 41 40 40 36 35 32 33 35 38 Cycles cancelled before OR 2 2 0 2 1 1 0 1 9 Cycles to OR 416 41 129 196 274 100 13 33 1202 Assisted reproduction treatment IVF 69 9 33 13 23 1 5 3 156 ICSI 345 32 94 182 249 98 8 30 1038 IVF + ICSI 1 0 1 0 1 0 0 0 3 Unknown 0 0 0 0 1 0 0 0 1 Frozen embryos IVF + frozen 0 0 0 0 0 0 0 0 0 ICSI + frozen 1 0 1 1 0 1 0 0 4 Cancelled after OR 21 3 2 6 12 5 1 1 51 Cycles to PGS 395 38 127 190 262 95 12 32 1151 Zona breaching AT drilling 211 18 84 135 145 52 4 19 668 Laser drilling 177 20 42 54 115 43 8 8 467 Mechanical 7 0 1 1 2 0 0 5 16 Biopsy method Polar body biopsy 18 20 46 5 17 0 0 2 108 Cleavage aspiration 342 18 81 172 225 85 11 28 962 Cleavage extrusion 35 0 0 13 20 10 1 2 81 Cleavage flow displacement 0 0 0 0 0 0 0 0 0 Embryology COC 3974 391 1311 2529 3892 1524 137 429 14187 Inseminated 3323 324 1116 2119 3322 1241 117 347 11909 Fertilized 2315 192 795 1484 2343 851 80 261 8321 Biopsied 1826 209 739 1101 1732 645 70 219 6541 Successfully biopsied 1802 208 733 1094 1715 638 67 215 6472 Diagnosed 1694 199 710 994 1596 541 66 191 5991 Transferable 554 64 229 352 672 228 30 82 2211 Transferred 387 49 163 255 433 163 20 58 1528 Frozen 34 9 36 33 99 32 1 7 251 Clinical outcome Cycles to embryo transfer 237 28 96 135 227 82 12 29 846 HCG positive 71 6 22 49 74 38 2 9 271 Positive heart beat 48 5 18 37 50 33 2 5 198 Clinical pregnancy rate (%) per OR/% per 12/20 12/18 14/19 19/27 18/22 33/40 15/17 15/17 16/23 embryo transfer Number of fetal hearts 57 8 21 51 66 44 2 8 257 Implantation rate (fetal hearts/embryos transferred) (%) 15 16 13 20 15 27 10 14 17

AMA = advanced maternal age; RIF = repeated implantation failure; SMF = severe male factor; OR = oocyte retrieval; AT = acid Tyrode’s; COC = cumulus–oocyte complexes.

16 ESHRE PGD Consortium data collection V

Table IXa. Preimplantation genetic diagnosis (PGD) for social sexing, data Table IXb. Continued collection I–IV FISH PCR Total Method for sexing FISH PCR Unknown Total SS only SS + PGS Total cycles 118 59 5 182 Number infertile 18 0 1 19 Embryology Female age 35 34 35 35 COC 84 301 617 1002 Cycles cancelled before OR 0 0 0 0 Inseminated 78 210 454 742 Cycles to OR 118 59 5a 182a Fertilized 50 128 302 480 Assisted reproduction treatment Biopsied 36 100 260 396 IVF 102 4 3 109 Successfully biopsied 36 100 260 396 ICSI 13 52 2 67 Diagnosed 32 83 236 351 Frozen 3 2 0 5 Transferable 16 24 143 183 Frozen and ICSI 0 1 0 1 Transferred 12 23 110 145 Cancelled after OR 3 6 5 14 Frozen 6 1 14 21 Cycles to PGD 115 53 0 168 Clinical outcome Zona breaching Cycles to embryo transfer 6 14 41 61 AT drilling 9 10 0 19 HCG positive 3 4 15 22 Laser drilling 106 0 0 106 Positive heart beat 3 0 12 15 Mechanical 0 43 0 43 Clinical pregnancy rate (%) 50/50 0/0 27/29 21/25 Biopsy method per OR/% per embryo transfer Cleavage aspiration 115 10 0 125 Implantation rate (fetal hearts/ 33 0 15 14 Cleavage extrusion 0 43 0 43 embryos transferred)(%) Embryology COC 1379 887 23 2289 PGS = aneuploidy screening; SS = social sexing; OR = oocyte retrieval; Inseminated 1343 668 19 2030 AT = acid Tyrode’s; COC = cumulus–oocyte complexes. Fertilized 972 428 11 1411 Biopsied 840 353 0 1193 Successfully biopsied 766 343 0 1109 Table Xa. Evolution of pregnancy, data collection I–IV Diagnosed 700 316 0 1016 Transferable 287 182 0 469 No. of pregnancies No. of fetal sacs Transferred 166 138 0 304 Frozenb 93 35 0c 128 Pregnancies 648 803 Clinical outcome FISH cycles 485/648 PCR cycles 163/648 Cycles to embryo transfer 90 41 0 131 a HCG positive 37 19 0 56 Subclinical pregnancies 53/648 Positive heart beat 35 9 0 44 Clinical pregnancies 595 803 Clinical pregnancy rate (%) 30/39 15/22 0 24/34 Singletons 417/595 417/803 per OR/% per embryo transfer Twins 149/595 298/803 Triplets 28/595 84/803 aOne natural cycle included. Quadruplet 1/595 4/803 bEleven cycles with embryos frozen without biopsy or failed diagnosis included. First trimester loss 67/595 111/803 cThree embryos frozen without biopsy were not included. Miscarriage 62/595 71/803 OR = oocyte retrieval; AT = acid Tyrode’s; COC = cumulus oocyte complexes. Extrauterine pregnancy 5/595 5/803 Vanishing twins/triplets 35/803 Ongoing pregnancies >12 weeks 528 692 Second trimester loss 16/528 23/692 Miscarriage 10/528b 16/692b Table IXb. Preimplantation genetic diagnosis (PGD) for social sexing, data c collection V TOP after misdiagnosis 4/528 4/692 TOP after amniocentesisd 2/528 2/692 e FISH PCR Total Reduction 1/692 Reduction of multiple pregnancies 10/692 SS only SS + PGS Quadruplet to twin 2/692 Triplet to twin 5/692 Total cycles 6 22 44 72 Triplet to singleton 2/692 Number infertile 0 6 16 22 Twin to singleton 1/692 Female age 31 38 36 36 Normal evolution 512 659 Cycles cancelled before OR 0 0 0 0 Lost to follow-up 33/512 42/659 Cycles to OR 6 22 44 72 Deliveries 479 617 Assisted reproduction treatment Singletons 350/479 350/617 IVF 2 1 1 4 Twins 120/479 240/617 ICSI 4 21 36 61 Triplets 9/479 27/617

Frozen 0 0 0 0 a IVF + frozen 0 0 1 1 Subclinical pregnancy defined as pregnancy without any other clinical signs, but positive serum HCG. ICSI + frozen 0 0 4 4 b Unknown + frozen 0 0 2 2 One triplet: fetal reduction, followed by amniocentesis and loss of remaining Cancelled after OR 0 0 0 0 twin at 16 weeks (one fetal sac counted in reduction, two in miscarriage, one second trimester pregnancy loss after miscarriage counted). Cycles to PGD 6 22 44 72 c Zona breaching TOP = termination of pregnancy. One misdiagnosis for sexing, FISH, female β AT drilling 0 0 0 0 fetus, indication social sexing; one misdiagnosis for -thalassaemia, PCR; one Laser drilling 6 0 0 6 misdiagnosis for myotonic dystrophy, PCR, one misdiagnosis after PGS, karyotype 45,X. Mechanical 0 22 44 66 d Biopsy method Trisomy 18 after amniocentesis, indication for PGD parent carrier of recipro- cal translocation not involving chromosome 18; one polymalformation. Cleavage aspiration 5 0 0 5 e Cleavage extrusion 1 22 44 67 One misdiagnosis for sexing, PCR, indication Duchenne, twin pregnancy, selec- tive termination of male fetus. Cycle done in 1996, Y-specific amplification only. 17 J.C.Harper et al.

Table Xb. Evolution of pregnancy, data collection V Table XIa. Continued

No. of No. of Complication Incidence pregnancies fetal sacs Singletons Twins Triplets Pregnancies 485 476 (n = 65 (n = 24 (n = 3 FISH cycles 398/485a 389/476a patients) patients) patients) PCR cycles 88/485a 88/476a Subclinical pregnancies 113/485 Intrauterine death 0 2 0 Clinical pregnancies 372 476 Intrauterine growth retardation 6 1 0 Singletons 277/372 277/476 OHSS 1 0 0 Twins 85/372 170/476 Oligohydramnios 1 1 0 Triplets 8/372 24/476 Placenta accreta 3 0 0 Quadruplet 1/372 4/476 Placenta praevia 4 0 0 Unknown 1/372 (1)/476b Polyhydramnios 0 1 0 First trimester loss 31/372 46/476 Pre-eclampsia and hypertension 8 4 0 Miscarriage 26/372c 29/476 Premature rupture of the membranes 0 5 0 Vanishing twins 11/476 Preterm contractions 18 7 0 Extrauterine gestation 5/372d 6/476 Preterm dilatation 1 2 0 Ongoing pregnancies (>12 weeks) 341 430 Preterm labour 13 7 0 Second trimester loss 10/341 13/430 Psychological problems 1 0 0 Miscarriage 8/341 11/430 Pyelonephritis 1 0 0 TOPe 2/341 2/430 Toxoplasmosis maternal problem of 010 Reduction of multiple pregnancies 8/430 asphyxia and shock lung Triplet to twin 2/430 Twin to twin transfusion 0 1 0 Triplet to singleton 4/430 Total 92 36 3 Quadruplet to twin 2/430 Normal evolution 331 409 HELLP = haemolysis, elevated liver enzymes, low platelet count; OHSS = Singletons 258/331 258/409 ovarian hyperstimulation syndrome. Twins 71/331 142/409 Triplet 3/331 9/409 Lost to follow-up 7/331 9/409 Singletons 6/331 6/409 Twins 0/331 0/409 Table XIb. Complications in clinical pregnancies (total n = 61 patients), data Triplets 1/331 3/409 collection V Deliveries 325 400 Singletons 252/325 252/400 Complication Incidence Twins 71/325 142/400 Triplets 2/325 6/400 Singletons Twins Triplets (n = 45 (n = 13 (n = 3 aOne fetal sac was tested with FISH and PCR. patients) patients) patients) bNumber of fetal heartbeat (FHB) not known; counted further as one FHB. cOne miscarriage after amniocentesis. Abruptio placentae, retroplacental 200 dOne heterotopic gestation continued as singleton after reduction of extrauter- haematoma ine gestation at 6 weeks. Antepartum haemorrhage 1 0 0 eOne termination of pregnancy (TOP) for cystic hygroma, failed karyotype, Bleeding 13 1 0 one TOP for Turner mosaic at amniocentesis. Cholestase 0 0 1 Diabetes mellitus 4 1 0 Oedema 1 0 0 Emesis 12 4 0 Gastrointestinal problems 2 0 0 HELLP syndrome 3 0 0 Intrauterine growth retardation 0 1 0 Table XIa. Complications in clinical pregnancies (total n = 92 patients), data OHSS 1 1 0 collection I–IV Pregnancy-induced hypertension 1 0 0 Placenta praevia (+ bleeding) 3 (+3) 0 0 Complication Incidence Pre-eclampsia and hypertension 4 2 0 Premature rupture of the membranes 0 0 0 Singletons Twins Triplets Preterm contractions 11 6 0 (n = 65 (n = 24 (n = 3 Preterm dilatation 2 2 0 patients) patients) patients) Preterm labour 4 2 0 Twin to twin transfusion 0 0 2 Abortion risk 1 0 0 Uterine bleeding post-partum 1 0 0 Abruptio placentae, retroplacental 400Total 65 (+3) 20 3 haematoma Anaemia 1 0 0 HELLP = haemolysis, elevated liver enzymes, low platelet count; OHSS = Bleeding 13 2 0 ovarian hyperstimulation syndrome. Cerclage 3 0 2 Chorioamnionitis 2 0 0 Diabetes mellitus 4 2 0 Oedema 1 0 0 calculated but this is impossible because of the obvious under- Extrauterine pregnancy followed by 100 salpingectomy reporting of cancelled cycles). Only fresh cycles and pregnan- Gastrointestinal problems 1 0 1 cies with information up to and shortly after birth were taken HELLP syndrome 3 0 0 into account. Of the 1993 selected cycles, 455 resulted in a Idiopathic thrombocytopeny 1 0 0 positive HCG, 112 of which were biochemical pregnancies. Of 18 ESHRE PGD Consortium data collection V

Table XIIa. Method of delivery and gestational age, data collection I–IV Table XIVa. Congenital malformations and neonatal complications at birth, data collection I–IV Total Singletons Twins Triplets Malformations No. delivered 479 350 120 9 No data available 177/608 Method of delivery No malformations 403/431 Vaginal 204 177 27 0 Total malformations 28/431 Caesarean 227 145 74 8 Major malformations at birth Vaginal and Caesarean 1 0 1 0 Bilateral clubfoot 1 singleton Unknown 47 28 18 1 1 twin Term at delivery Cleft lip and palate 1 singleton Preterm 116 46 64 6 Congenital hip luxation 1 singleton Term 321 280 39 2 Cystic mass abdomen 1 twin Unknown 42 24 17 1 Exencephalia 1 twin Fryns syndrome, neonatal death 1 singleton Hydrocephaly 1 singleton Large cavernous haemangioma 1 singleton Table XIIb. Method of delivery and gestational age, data collection V Pes equinovarus 1singleton Phocomelia and pulmonary deficiency 1 singleton Total Singleton Twin Triplet Prune belly syndrome and stillbirth 1 twin Stillborn at 28 weeks (no details) 1 twin No. of deliveries 325 252 71 2 Unilateral intrauterine torsio testis 1 singleton Method of delivery Minor malformations Vaginal 129 117 11 1 ASD 1 twin Caesarean section 158 110 47 1 Bilateral hydrocoele 1 singleton Vaginal + Caesarean section 0 0 0 0 Capillary haemangioma 2 singletons, 2 twins Unknown 38 25 13 0 Cryptorchidy 1 singleton Gestational age at delivery Mongolian spot 1 singleton Preterm 71 31 38 2 Pyelourethral junctional stenosis 2 twins At term 153 140 13 0 Sacral dimple 1 singleton Unknown 101 81 20 0 Syndactyly digit iv–v 2 singletons Uniumbilical artery 1 singleton Neonatal complications No data available 169/608 Table XIIIa. Data on live-born children, data collection I–IV No neonatal complications reported 389/439 Neonatal complications reported 50/439 Total no. of children born 608 Stay at NCU (<1 week to 1 months) 3 singletons, 2 twins Sex Intrauterine death at 31 weeks, 3 triplets (1 pregnancy) Male 241 241/573 prematurity in two other children Female 332 332/573 Apnoea 1 twin Unknown 35 Dysmature 6 twins (3 pregancies) Mean birthweight (g) 2900 n = 534 Feeding problems 2 twins (2 pregnancies) Singletons 3266 n = 319 Fryns syndrome and neonatal death 1 singleton Twins 2477 n = 194 Gastro-oesophagal reflux 1 singleton Triplets 1756 n = 21 Neonatal deaths <7 days 3 Unknown n = 74 Pneumothorax 1 twin Mean birth length (cm) 48 n = 322 Prematurity and neonatal complication 5 singletons, 5 twins, 6 triplets Singletons 50 n = 219 (intubation, NCU) Twins 47 n = 97 Prune belly syndrome in one twin and 2 twins Triplets 44 n = 6 prematurity in the other twin (28 weeks) Unknown n = 286 Respiratory problems 1 singleton, 2 twins Stillborns 4 twins, 2 singletons

Table XIIIb. Data on live-born children, data collection V

Total children born 382 the remainder, four pregnancies were lost to follow-up, 32 Sex Male 169 were lost during pregnancy, and a total of 378 babies were Female 203 born from 307 deliveries. These were 238 singletons (all live Unknown 10 born), 134 twins (one stillborn) and six triplets (three stillborn). Mean birth weight (g) Singletons 3240 (n = 225/247) This results in a live birth rate of 15% per OR (307/1993) and a Twins 2350 (n = 110/132) singleton live birth rate per OR of 12% (238/1993). Mean birth length (cm) Singletons 49 (n = 133/247) Twins 47 (n = 56/132) Mean head circumference General remarks Singletons 34 (n = 106/247) Due to the large amount of work the data collection involves, Twins 32 (n = 45/132) Apgar scores this data collection is 1 year behind its expected publication Good 188/189 date. The Steering Committee of the Consortium are striving to Poor 1/189 ensure that data are published within a year of collection. It is Numbers in parentheses indicate the number of newborns for whom informa- planned that data VI (2003) will be published before the end of tion is available out of the total number of newborns. 2005 and data VII in 2006. 19 J.C.Harper et al.

Table XIVb. Congenital malformation and neonatal complications at birth, Table XVa. Confirmation of diagnosis per fetal sac, data collection I–IV data collection V Method Result No malformation data available 25/382 No malformation 338/382 n Normal Abnormal Singletons 226/247 Twins 109/132 Prenatal diagnosis Triplet 3/3 FISH Babies with malformation 19/382 CVS 40 40 0 Major Amniocentesis 179 176 3a Absence of corpus callosum, hemivertebra 1 singleton Ultrasound 8 7 1b Absence of corpus callosum, kidney dilatation, 1 singleton Unknown 3 3 0 growth retardation Total 230 226 4 Laryngomalacia, receding chin, strawberry naevus 1 singleton PCR Ventricular septum defect 1 singleton CVS 53 53 0 Ventricular septum defect, retrognatia 1 singleton Amniocentesis 32 26 6c Bilateral cryptorchidia 1 twin Ultrasound 4 3 1d Cleft lip and palate, pulmonary atresia 1 twin1 Unknown 2 2 0 Cleft lip and palate, tetralogy of Fallot 1 twina Total 91 84 7 Cleft lip, cardiopathy, short femur 1 twin Postnatal diagnosis Pulmonary hypoplasia 1 twin FISH Minor Karyotype miscarriage 16 7 9e Congenital hip luxation 1 singleton, 1 twin Karyotype postnatal 31 30f 1g Heart murmur 1 singleton Physical examination 97 95 2h Hydrops fetalis 1 twin Total 144 132 12 Pre-auricular tags 1 singleton PCR Pyelo-urethral junction stenosis 1 twin DNA test miscarriage 2 2 0 Sacral dimple 1 singleton DNA test postnatal 17 16 1i Single umbilical artery, disappearing hip luxation 1 singleton Sweat test 4 4 0 Unknown 1 singleton Physical examination 2 1 1j No neonatal data available 45/382 Karyotype 2 2 0 No neonatal complications 314/382 Karyotype + DNA 1 1 0 Singletons 215/247 Unknown 1 1 0 Twins 96/132 Total 29 27 2 Triplet 3/3 Neonatal complications 23/282 Prenatal diagnosis: FISH fetal sacs tested = 230/602; PCR fetal sacs tested = Bronchopulmonar dysplasia 1 singleton 91/201; total prenatal testing = 321/803. Growth retardation, mental retardation 1 singleton Postnatal diagnosis: total FISH sacs/babies tested = 143/602; total PCR Infection (1 meconium aspiration sepsis) 3 singletons sacs/babies tested = 29/201; total postnatal testing = 172/803. IUGR due to HELLP syndrome 1 singleton aTwo misdiagnoses: sexing, female fetus, social sexing (terminated): PGS: Neonatal care, tube feeding 1 week 1 singleton 45,X (terminated). Trisomy 18 after PGD for reciprocal translocation. Dysmaturity 2 twins bPolymalformation on ultrasound, normal karyotype, terminated. Hypoglycaemia 1 twin cSix misdiagnoses: XL Duchenne (selective reduction of one affected embryo Hypoglycaemia and feeding problems 1 twin of twin pregnancy), β-thalassaemia (terminated), myotonic dystrophy Neonatal care 1 singletonc, 3 twinsb (terminated), cystic fibrosis (born), XL retinitis pigmentosa (born), amyloid Prematurity 2 twins polyneuropathy (born). Neonatal death dEchogenic bowel at ultrasound, misdiagnosis for cystic fibrosis, born. Cleft lip, cardiopathy, short femur 1 twin eOne trisomy 3, one trisomy 15, two trisomy 16, one trisomy 22, one mosaic Pulmonar hypoplasia 1 twin trisomy 22, one monosomy X, 47,XY,+D(3), one misdiagnosis Stillborn at 22 weeks 2 twins 47,XX,+der(22)t(11;22)(q23.3;q11.2)mat; parent carrier balanced Stillborn at 25 weeks 2 twins translocation. fOne baby born with Fryns syndrome had a karyotype (normal result). aTwo babies from the same twin. gOne misdiagnosis, trisomy 21 after aneuploidy screening. bTwo babies from the same twin. hOne baby with Fryns syndrome, one baby with prune belly syndrome cSingleton with ventricular septum defect and retrognatia. (both normal karyotype). iOne CF carrier twin pregnancy: on PGD both diagnosed as homozygote normal. jOne twin after PGD for CF: one misdiagnosis, one healthy. For the first time we have expressed the clinical preg- CVS = chorionic villous sampling. nancy rates per embryo transfer procedure and also the implantation rates. Furthermore, we have presented prelim- inary results for live birth rates per OR. For data collection VI, we intend to publish live birth rates per oocyte retrieval remained low. The consortium will continue to collect data to and per embryo transfer procedure broken down for each support long-term evaluation of PGD/PGS and to promote indication. good practice. It appears that since 1997 there have been no major changes In 2005, the consortium published detailed guidelines on in the methodologies applied to most stages of the PGD/PGS PGD and PGS (Thornhill et al., 2005). Our aim is to update procedure. Technology has facilitated some minor modifi- these guidelines on a regular basis. cations, e.g. in mode of zona drilling, with laser drilling In 2005, a protocol for a long-term follow-up of PGD babies becoming increasingly frequent. It is encouraging that no born has been prepared and it is hoped that most of the centres misdiagnoses were reported between January and December registered with the consortium will be involved in this import- 2002, but disappointing that the overall pregnancy rates ant study. 20 ESHRE PGD Consortium data collection V

Sermon K, Moutou C, Harper J, Geraedts J, Scriven P, Wilton L, Magli M-C, Table XVb. Confirmation of diagnosis per fetal sac, data collection V Michiels A, Viville S and De Die C (2005) ESHRE PGD Consortium data collection IV: May–December 2001. Hum Reprod 20,19–34. Method Result Thornhill AR, deDie-Smulders CE, Geraedts JP, Harper JC, Harton GL, Lavery SA, Moutou C, Robinson MD, Schmutzler AG, Scriven PN, Sermon KD n Normal Abnormal and Wilton L (2005) ESHRE PGD Consortium Best Practice Guidelines for Clinical Preimplantation Genetic Diagnosis (PGD) and Preimplantation Prenatal diagnosis Genetic screening (PGS). Hum Reprod 20,35–48. FISH Warburton D (1991) De novo balanced chromosome rearrangements and extra CVS 9 9 0 a marker chromosomes identified at prenatal diagnosis: clinical significance Amnio 79 77 2 and distribution of breakpoints. Am J Hum Genet 49,995–1013. Ultrasound 90 89 1b Total 178 175 3 Submitted on August 1, 2005; accepted on August 8, 2005 PCR CVS 13 13 0 c Amnio 15 14 1 Appendix. Centres that contributed data to collection V Ultrasound 0 0 0 Total 28 27 1 Argentina: Fecunditas, Buenos Aires (Roberto Coco); Australia: Mel- Postnatal diagnosis bourne IVF, Melbourne (Leeanda Wilton), University of Adelaide, FISH Dept of Ob/Gyn, Adelaide (Nicole Hussey); Belgium: Centre for d,e f Karyotype miscarriage 9 4 5 Medical Genetics, Vrije Universiteit Brussel, Brussels (Karen Sermon), Karyotype postnatal 16 15g 1h Physical examination 168 168 0 Infertility Centre, Ghent University Hospital, Ghent (Josiane Van de Total 193 187 6 Elst), Leuven Insititute for Fertility and Embryology, Leuven, PCR (Gunther Van Kerkhoven), Hopital Erasme, Université Libre de Brux- Karyotype miscarriage 1 1i 0 j elles, Brussels (Serena Emiliani); Denmark: Aarhus University Hospi- Physical examination 5 50 tal, Aarhus (Johnny Hindkjaer); Finland: Helsinki University Central DNA test postnatal 7k 70 Total 13 13 0 Hospital, Helsinki (Christel Hyden-Granskog), AVA-Clinic, Tampere (Paivi Salin) ; France: Service de la Biologie de la Reproduction, Prenatal diagnosis: FISH fetal sacs tested: 178/389; PCR fetal sacs tested: 28/ SIHCUS-CMCO, Strasbourg (Stéphane Viville); Germany: Univer- 88; total prenatal testing: 206/476. sity of Bonn, Bonn (Marcus Montag), Centre for Gynecological Postnatal testing: FISH fetal sacs/babies tested: 193/389; PCR fetal sacs/ babies tested: 13/88; total post-natal testing: 205/426. , Reproductive Medicine and Human Genetics, a46,XX,t(1;5)(q43;q13) de novo after PGD for 46,XY,t(1;11)(p12;q12) (born), Regensburg (Andreas Hehr); Greece: IVF and Genetics, Athens 45,X[10]/46,XX[55] after PGS for repeated IVF failures (termination of (Elena Kontogianni); Laboratory of Medical Genetics, University of pregnancy). b Athens, St. Sophia’s Children’s Hospital, 11527 Athens, Greece Cystic hygroma, karyotype failed, PGD for 45,XX,der(13;21)(q10;q10). (Emmanuel Kanavakis and Joanne Traeger-Synodinos); Italy: SISMER, c46,XX,t(8;9)(p?23;p12) de novo after PGD for Duchenne muscular dystrophy. dNormal karyotype in a fetus with multiple malformations. Bologna (Luca Gianaroli), Reproductive Medicine, European Hospital, eAfter amniocentesis with normal karyotype. Rome (Marcello Iacobelli), HERA-UMR, Catania (Sandrine f46,X,inv(Y)(p11q12); trisomy 16 after PGS with PB1 analysis; Chamayou); Israel: The Danek Gertner Institute of Human Genet- 45,XX,der(7)t(7;15)(p14;q11.2),-15 de novo; 47,XX,+9, trisomy 22. ics, Sheba Medical Center (Ayala Aviram-Goldring), IVF Unit, gThree twins with polymalformations (see Table XIV) had a normal karyotype. h46,XX,t(1;5)(q43;q13) de novo already seen in prenatal. Hadassah Medical Organisation, Jeruzalem (Alex Simon), Tel-Aviv iAfter amniocentesis. Sourasky Medical Center, Tel-Aviv (Yuval Yaron); Korea: Cha jOne baby (PGD for DMD) had FISH + PCR, diagnosis confirmed by physical General Hospital, Seoul (Sook Hwan Lee and Mi-Kyung Chung); examination and amnio. Samsung Cheil Hospital, Seoul (Inn Soo Kang); The Netherlands: k β Three for CF, two for SMA, one for SC and one for -thal. Erasmus Medical Center, IVF lab, Rotterdam (Elena Martini), CVS = chorionic villous sampling. Center for Reproductive Medicine, Academic Medical Center, Amsterdam (Sjoerd Repping), PGD Working Group Maastricht, Acknowledgements Maastricht (Edith Coonen); Portugal: Faculty of Medicine of Porto- Hospital Sao Joao, Porto (Filipa Carvalho); Spain: Instituto Dexeus, We would like to thank ESHRE for its continuing support of this work Barcelona (Anna Veiga), Unitat de Biologia Cel.lular, Univ. Autonoma and to all participating centres. Barcelona, Barcelona (Josep Santalo), Instituto Valenciano de Infer- tilidad, Valencia (Carmen Rubio), Fundacion Jimenez Diaz, Madrid References (Esther Fernandez); Sweden: Karolinska Hospital, Stockholm (Elisabeth Bonduelle M, Wennerholm UB, Loft A, Tarlatzis BC, Peters C, Henriet S, Blennow); Sahlgrenska Hospital, Goteborg (Charles Hanson); Taiwan: Mau C, Victorin-Cederquist A, Van Steirteghem A, Balaska A, Emberson Chang Chung Memorial Hospital and Medical College, Tao-Yuan JR and Sutcliffe AG (2005) A multi-centre cohort study of the physical (Chun-Kai Chen); Turkey: American Hospital, Istanbul (Nesrin health of 5-year-old children conceived after intracytoplasmic sperm injec- tion, in vitro fertilization and natural conception. Hum Reprod 20,413–419. Ercelen); UK: University College London, London (Joyce Harper), ESHRE PGD Consortium Steering Committee (1999) ESHRE Preimplantation Center for Preimplantation Genetic Diagnosis, Guy’s and St Thomas’ Genetic Diagnosis (PGD) Consortium: preliminary assessment of data from NHS Foundation Trust, London (Peter Braude), Hammersmith January 1997 to September 1998. Hum Reprod 14,3138–3148. Hospital, London (Stuart Lavery), School of Biology, University of ESHRE PGD Consortium Steering Committee (2000) ESHRE Preimplantation Leeds (Marc Robinson); USA: Baylor College of Medicine, Houston, Genetic Diagnosis (PGD) Consortium: data collection II (May 2000). Hum Texas (Sallie McAdoo), Genetics and IVF Insitute, Fairfax, Reprod 15,2673–2683. ESHRE PGD Consortium Steering Committee (2002) ESHRE Preimplantation Virginia (Gary Harton), Shady Grove Centre for Preimplantation Genetic Diagnosis (PGD) Consortium: data collection III (May 2001). Hum Genetics, Rockville, Maryland (William Kearns); Jones Institute for Reprod 17,233–246. Reproductive Medicine, Norfolk, Virginia (Sue Gitlin).

21 RBMOnline - Vol 12. No 3. 2006 389 Reproductive BioMedicine Online; www.rbmonline.com/Article/2160 on web 30 January 2006

Letter Central data collection on PGD and screening

To the Editor

We have read with interest, the commentary in the December amount of data has already been used extensively not only as 2005 issue of Reproductive Biomedicine Online, by Baruch and standard reference in the literature, but also in policy-making of colleagues that reports on an ef fort to collect information about individual centres concerning PGD/PGS and in counselling of preimplantation genetic diagnosis (PGD) and preimplantation patients willing to embark in PGD/PGS. genetic screening (PGS) (Baruch et al. , 2005). The authors state that no comprehensive data are available about the use of Last but not least, in view of the importance for society of PGD/PGS, its accuracy, or the health outcomes of babies born collecting and analysing data on PGD/PGS, we welcome the following PGD/PGS. Although we applaud their ef fort, we American initiative and invite them to a fruitful collaboration. would like to draw to your and your readers’ attention the fact that there is already an extensive worldwide data collection on Sjoerd Repping 1, Joep Geraedts 2, Paul Scriven 3, Gary PGD/PGS coordinated by the ESHRE PGD Consortium. Harton4, Katerina Veselá 5, W illiam Kearns 6, Stéphane Viville7, Kar en Sermon8 The ESHRE PGD Consortium was established in 1997 with the aim to (i) survey the availability of PGD/PGS, (ii) collect 1Centre for Reproductive Medicine, Academic Medical prospectively and retrospectively data on accuracy, reliability Centre, Amsterdam, The Netherlands; 2PGD Working Group, and ef fectiveness of PGD/PGS, (iii) initiate follow-up studies, University Hospital, Maastricht, The Netherlands;3Cytogenetics (iv) produce guidelines and recommended PGD/PGS protocols Department and Centre for Preimplantation Genetic Diagnosis, and (v) formulate a consensus on the use of PGD/PGS. Guy’s and St Thomas’ NHS Foundation Trust, London, UK; Concurrent with these goals, the Consortium has, amongst 4Genetics and IVF Institute, Fairfax Vir ginia, USA;5Sanatorium others, published guidelines on how to practice PGD/PGS Repromeda, Brno, Czech Republic; 6Shady Grove Centre for (Thornhill et al. , 2005), is involved in international policy- Preimplantation Genetics, Rockville Maryland, USA; 7Service making and is currently initiating an extensive follow-up of de la Biologie de la Reproduction, Schiltigheim, France; children born from PGD/PGS cycles. 8Research Centre Reproduction and Genetics, Vrije Universiteit Brussel, Belgium The main ef fort of the Consortium, however, has been to collect data on PGD/PGS cycles. The aim for the data collection was Refer ences that it should be comprehensive, from the first contact with the patient to the birth of the baby. This approach entails a Baruch S, Adamson GD, Cohen J et al. 2005 Genetic testing of certain amount of commitment from the centres involved. embryos: a critical need for data. Reproductive BioMedicine Despite this unpaid workload, there are currently more than Online 11, 667–670. 50 centres participating in the Consortium, including centres ESHRE PGD Consortium Steering Committee 2002 ESHRE from Australia, South America, Europe and the USA, and any Preimplantation Genetic Diagnosis Consortium: data collection III centre in the world involved in PGD/PGS can easily become a (May 2001). Human Repr oduction 17, 233–246. Consortium member (http://www.eshre.com). Geraedts J, Handyside A, Harper J et al. 2000 European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis Consortium Steering Committee. ESHRE Since 1999, the ESHRE PGD Consortium has been collating preimplantation genetic diagnosis (PGD) consortium: data data on referrals, cycles, pregnancies and babies born after collection II (May 2000). Human Repr oduction 15, 2673–2683. PGD/PGS. In the beginning the data collection used hard copy Geraedts J, Handyside A, Harper J et al. 1999 ESHRE Preimplantation forms and Excel spreadsheets. Starting from data collection IV, Genetic Diagnosis (PGD) Consortium: preliminary assessment a FileMaker Pro database is in use that allows an automatic of data from January 1997 to September 1998. ESHRE PGD calculation of results and linking of the referral, cycle, pregnancy Consortium Steering Committee. Human Repr oduction 14, and baby data of one and the same patient. In total, five data 3138–3148. Harper JC, Boelaert K, Geraedts J et al. 2006 ESHRE PGD collections have been published thus far and the next will be Consortium data collection V: cycles from January to December published in 2006 (Geraedts et al. , 1999, 2000; ESHRE PGD 2002 with pregnancy follow-up to October 2003. Human Consortium, 2002; Harper et al. , 2006; Sermon et al. , 2005). Reproduction 21, 3–21. Cumulatively until January 2003, the ESHRE PGD database Sermon K, Moutou C, Harper J et al. 2005 ESHRE PGD Consortium holds data on 6200 PGD/PGS cycles with a total of over 37,000 data collection IV: May–December 2001. Human Repr oduction biopsied embryos (Harper et al. , 2006). Furthermore, data on 20, 19–34. the neonatal outcome of nearly 1000 children born from PGD/ Thornhill AR, deDie-Smulders CE, Geraedts JP et al. 2005 PGS have been collected and published. ESHRE PGD Consortium ‘Best Practice Guidelines for Clinical Preimplantation Genetic Diagnosis (PGD) and Preimplantation Genetic screening (PGS)’. Human Repr oduction 20, 35–48. The ESHRE PGD Consortium database easily allows for all calculations that were pointed out as important by Baruch and colleagues, including live birth rate per oocyte retrieval, misdiagnosis rate, and health of of fspring. In fact, this vast 389