SHOULD WE TEST THE FIRST POLAR BODY OR THE EMBRYO

SHOULD WE TEST THE FIRST POLAR BODY OR THE EMBRYO L. Gianaroli, C.M. Magli, A.P. Ferraretti Reproductive Medicine Unit - Via Mazzini, 12-40138 Bologna sismer@sismer.it

WOMEN S REPRODUCTIVE HEALTH IN THE 21 st CENTURY - Increased lifespan - Decreased fertility in European population Increased maternal age Increased number of divorces Increased male infertility - Increased demand of genetic health - Increased demand of prevention of disorders ONE BABY ONE HEALTHY BABY

INTERNATIONAL WORKING GROUP ON PGD > 7.000 cycles Aneuploidy HLA-typing Monogenic disorders Translocations Kuliev and Verlinsky: Thirteen years experience of preimplantation diagnosis: report of the Fifth International Symposium on Preimplantation Genetics. Reprod Biomed Online 8, 229-235, 2004. VISION 2000

Present Clinical Applications -aneuploidies -translocations -monogenic diseases -mitochondrial diseases -HLA -cancer risk -late onset diseases Risk for aneuploidy

INCIDENCE OF ANEUPLOIDY DURING DEVELOPMENT Most common aneuploidies % X Y 21 22 16 21 22 15 16 21 22 45,X +16 +21 +22 (10%) (50%) +13 +18 +21 +13 +18 +21 XXX XXY XYY Sperm Oocytes Embryos Spontaneous Stillbirths Livebirths abortions % Biochemical pregnancies a a P<0.05 Conventional cycles PGD cycles n=109 n=67 a VISION 2000

THE ORIGIN OF HUMAN TRISOMY Trisomy N. Cases Paternal (%) MI MII Maternal (%) MI MII Postzygotic (%) 2 18 28-54 13 6 7 14 - - 17 26 57 15 34-15 76 9-16 104 - - 100 - - 18 143 - - 33 56 11 21 642 3 5 65 23 3 22 38 3-94 3 - XXY 142 46-38 14 3 XXX 50-6 60 16 18 Hassold and Hunt (2001) Nature Reviews Genetics VISION 2000

BLASTOCYST DEVELOPMENT ACCORDING TO THE CHROMOSOMAL CONSTITUTION 1124 cultured embryos BLASTOCYST DEVELOPMENT n =427 NO BLASTOCYST DEVELOPMENT n =697 NORMAL 31 P <0.001 MONOSOMY TRISOMY HAPLOIDY POLYPLOIDY COMPLEX ABNORM 2 4 16 24 30 P <0.05 45 16 18 12 5 3 NORMAL MONOSOMY TRISOMY HAPLOIDY POLYPLOIDY COMPLEX ABNORM % % Vision 2000 0.96-12.03

BLASTOCYST DEVELOPMENT AFTER ONE CELL BIOPSY 112 hrs post insemination n=1434 80 e FISH normal % d FISH abnormal 60 e c d b 40 20 a b c a 0 4 5 6 7 8 9 10 11 12 Number of cells at 62 hrs post insemination abcde P<0.001 Vision 2000 0.96-12.03

EMBRYO CLEAVAGE AFTER ONE BLASTOMERE BIOPSY 88 hrs post insemination 60 % 50 40 30 20 10 No cleavage Cleavage of <50% cells Morulas 0 4 5 6 7 8 9 10 11 12 Number of cells at 62 hrs post insemination Vision 2000 0.96-12.03

Pregnancy Rate according to female age PR Age (yrs) 41 40 38 32 35 P<0.05 24 < - - - > 18 24 4 4 Pre Law Post Law VISION 2000 10/03/04-3/05

Abortion Rate according to female age AR Age (yrs) P<0.05 < - - - Pre Law Post Law VISION 2000 10/03/04-3/05

Age distribution in the treated patients ( 2004-2005 ) % of cycles Years < - - > VISION 2000 10/03/04-3/05

AFTER THE LAW Egg retrieval Cryopreservation of all oocytes in OHSS risk cycles Cumulus removal 1 Polar Body FISH analysis Insemination of 3 mature oocytes Results before insemination Embryo culture Transfer of all vital embryos Vision 2000

CHROMOSOMAL ANALYSIS ON OOCYTES INCIDENCE OF ANEUPLOIDY IN RELATION TO MATERNAL AGE n=1130 % a b ab 35 36-37 38-39 40-42 43 yrs a P<0.01 b P<0.05 Vision 2000 3.04-12.04

Years,, France : mean age at motherhood,, All births,,,, First births, Calendar year

%,,,,,,,,,, < - - - - - >= Years * * Età materna media al parto in Emilia Romagna anni 1981-2000

- - - Medie età materna popolazione generale Emilia R. Medie età materna S. Down Emilia R.

CHROMOSOMAL ANALYSIS ON OOCYTES FREQUENCY OF ANEUPLOIDY IN OOCYTES ACCORDING TO AGE 30 No. observations= 5.650 c % 25 ab 20 15 10 b c a 13 16 18 21 22 a P<0.005 b P<0.05 c P<0.025 5 0 35 36-37 38-39 40-42 43 yrs VISION 2000 03.04-12.04

CHROMOSOMAL ANALYSIS ON OOCYTES INCIDENCE OF ANEUPLOIDY IN RELATION TO PATIENTS CHARACTERISTICS n=1130 % ab b a a P<0.05 b P<0.25 Maternal age 38 yrs Recurrent abortions Repeated cycles Controls Vision 2000 3.04-12.04

CHROMOSOMAL ANALYSIS ON OOCYTES DISTRIBUTION OF ANEUPLOIDY PER ANALYZED CHROMOSOME IN RELATION TO PATIENTS CHARACTERISTICS n=1130 Recurrent abortions P<0.05 P<0.01 P<0.05 P<0.005 P<0.005 Maternal age Repeated cycles Controls 13 16 18 21 22 Chromosomes No. observations: 5.650 Vision 2000 3.04-12.04

CHROMOSOMAL ANALYSIS ON 1 st POLAR BODY Team - clinicians Team - biologists Oocyte Pick Up IVF laboratory Cytogenetic laboratory Oocyte screening 1 hour MI, GV In Vitro Maturation MII MII PB Biopsy Fixation Hybridization probes for chromosomes 13 16 18 21 22 Insemination of Chromosomally Normal Oocytes 6 hrs after oocyte pick up Results VISION 2000 10/03/04-3/05

CHROMOSOMAL ANALYSIS ON 1 st POLAR BODY MECHANICAL OPENING OF OOCYTE ZONA PELLUCIDA VISION 2000

CHROMOSOMAL ANALYSIS ON 1 st POLAR BODY 1 ST POLAR BODY BIOPSY VISION 2000

CHROMOSOMAL ANALYSIS ON 1 st POLAR BODY CHROMOSOMAL ANALYSIS OF 1 ST POLAR BODY 13 16 18 21 22 VISION 2000

CHROMOSOMAL ANALYSIS ON 1 st POLAR BODY ICSI AFTER 1 ST POLAR BODY BIOPSY VISION 2000

CHROMOSOMAL ANALYSIS ON 1 st POLAR BODY No. cycles 87 No. patients 75 Age (MSD) 37.84.3 No. previous cycles (MSD) 2.22.6 No. oocytes 698 No. biopsied oocytes (%) 564 (81) No. diagnosed oocytes (%) 527 (93) No. oocytes normal for the chromosomes 13, 16, 18, 21, 22 218 (41) No. inseminated oocytes 207 No. fertilized oocytes (%) 136 (66) No. generated embryos (%) 129 (95) No. transferred cycles (%) 66 (76) No. clinical pregnancies (%) 14 (21) No. spontaneous abortions 1 No. ectopic pregnancies 1 Implantation rate (%) 14.6 Magli MC, Ferraretti AP, Crippa A, Lappi M, Feliciani E, Gianaroli L (2005) Impact of chromosomal analysis of first polar body in immature oocytes generated by stimulated cycles. Fertil.Steril VISION 2000 10/03/04-3/05

% CHROMOSOMAL ANALYSIS ON 1 st POLAR BODY INCIDENCE OF ANEUPLOIDY IN RELATION TO THE MATURATION STAGE 87 cycles a a a P<0.005 384 MII 146 MI 34 GV Maturation stage 1 hr after oocyte pick up Magli MC, Ferraretti AP, Crippa A, Lappi M, Feliciani E, Gianaroli L (2005) Impact of chromosomal analysis of first polar body in immature oocytes generated by stimulated cycles. Fertil.Steril VISION 2000 10/03/04-3/05

CHROMOSOMAL ANALYSIS ON 1 st POLAR BODY ACCORDING TO THE MATURATION STAGE MII MI+GV In vitro matured P No. biopsied oocytes 384 180 No. diagnosed oocytes (%) 366 (95) 161 (89) 0.025 No. FISH normal (%) 169 (46) 49 (30) 0.005 No. FISH abnormal (%) 197 (54) 112 (70) 0.005 No. missing/extra chromatid (%) No. missing/extra chromosome (%) No. complex abnormalities (%) 108 (55) 38 (34) 0.001 9 (5) 5 (4) 80 (40) 69 (62) 0.001 Magli MC, Ferraretti AP, Crippa A, Lappi M, Feliciani E, Gianaroli L (2005) Impact of chromosomal analysis of first polar body in immature oocytes generated by stimulated cycles. Fertil.Steril VISION 2000 10/03/04-3/05

CHROMOSOMAL ANALYSIS ON 1 st POLAR BODY FREQUENCY OF ANEUPLOIDY FOR THE DIFFERENT CHROMOSOMES 13 16 18 21 22 P In vivo matured 2.5 a 3.3 b 2.5 4.3 ab 3.4 ab P<0.005 In vitro matured 6.7 cd 3.9 cf 3.6 de 6.3 5.7 ef ce P<0.025 d P<0.01 f P<0.05 P<0.001 P<0.05 P<0.01 Magli MC, Ferraretti AP, Crippa A, Lappi M, Feliciani E, Gianaroli L (2005) Impact of chromosomal analysis of first polar body in immature oocytes generated by stimulated cycles. Fertil.Steril VISION 2000 10/03/04-3/05

CHROMOSOMAL ANALYSIS ON 1 st POLAR BODY OOCYTE DEVELOPMENT IN RELATION TO THE MATURATION STAGE MII MI+GV In vitro matured No. inseminated oocytes 160 47 No. fertilized oocytes (%) 107 (67) 29 (62) No. generated embryos (%) 102 (95) 27 (93) No. grade I embryos (%) 60 (59) 10 (37) No. transferred embryos (%) 81 22 Magli MC, Ferraretti AP, Crippa A, Lappi M, Feliciani E, Gianaroli L (2005) Impact of chromosomal analysis of first polar body in immature oocytes generated by stimulated cycles. Fertil.Steril VISION 2000 10/03/04-3/05

OOCYTE CRYOPRESERVATION No. Thawed cycles 86 Age (MSD) 34.24.6 No. Thawed oocytes 289 No. Survived oocytes (%) 214 (74) No. Biopsied oocytes - No. FISH normal oocytes (%) - No. Inseminated oocytes 210 No. Fertilized oocytes 164 (78) No. Embryos 143 (87) No. Transferred cycles (%) 72 (84) No. Transferred embryos 128 No. Clinical pregnancies 8 (%) per transferred cycle (11.1) (%) per thawed cycle (9.3) Implantation rate (%) (4.9) No. Abortions 1 32 35.34.2 243 201 (83) 184 79 (43) 82 58 (71) 50 (86) 23 (72) 40 6 (26.1) (18.8) 12.8 1 Vision 2000 3/04-7/10/05

Aim of the study - To analyze the morphology of pronuclei in zygotes which were generated by oocytes diagnosed as normal by first polar body analysis. - To relate the obtained configurations to those which had been described as predictive of euploidy in a previous study, in which the chromosomal complement had been tested by embryo biopsy. VISION 20005

MATERIALS and METHODS Between March 2004 and November 2005: 272 patients (age 37.5 4.2 years) 308 assisted cycles in combination with the screening of aneuploidy on first PB Indication to aneuploidy screening were: Maternal age 36 years 3 3 previous IVF failures 3 spontaneous abortions Vision 2000

MATERIALS and METHODS One hour after collection,, oocytes were denuded by Hyase and PB biopsy on MII started immediately 21 15 13 18 16 22 Up to 3 chromosomally normal oocytes were selected for ICSI At 16 hours after insemination,, oocytes were checked for the presence and morphology of pronuclei, nucleoli and polar body Vision 2000

MATERIALS and METHODS Five patterns of Pronuclear Morphology were described according to the position and size of pronuclei A B C D E Juxtaposed centralized Juxtaposed peripheral Centralized and separated Unequal in size Fragmented Gianaroli et al. (2003) Pronuclear morphology and chromosomal condition as scoring criteria for embryo selection. Fertil Steril 80,341-349. Vision 2000

MATERIALS and METHODS 5 patterns of Nucleolar Morphology were described according to the position and size of nucleoli within pronuclei 1 2 3 4 5 Gianaroli et al. (2003) Pronuclear morphology and chromosomal condition as scoring criteria for embryo selection. Fertil Steril 80,341-349. Vision 2000

MATERIALS and METHODS 3 patterns for the position of the second Polar Body were described in relation to the longitudinal axis of pronuclei Gianaroli et al. (2003) Pronuclear morphology and chromosomal condition as scoring criteria for embryo selection. Fertil Steril 80,341-349. Vision 2000

RESULTS A total of 618 pronuclear zygotes were scored Pronuclear morphology: A B C D E In singamy n=516 (83%) n=33 (5%) n=16 (3%) n=20 (3.5%) n=13 (2%) n=20 (3.5%) 4c-1 F0 (day 2) n=170 n=154 (30%) n=7 (21%) n=1 (6%) n=4 (20%) n=2 (15%) n=2 (10%) P<0.001 Vision 2000 03/04-11/05

RESULTS A total of 618 pronuclear zygotes were scored Nucleolar morphology 1 2 3 4 5 In singamy n=177 (29%) n=243 (39%) n=126 (21%) n=26 (4%) n=26 (4%) n=20 (3.5%) n=48 (27%) n=79 (33%) 4c-1 F0 (day 2) n=170 n=34 (27%) n=4 (15%) n=3 (11%) n=2 (10%) P<0.001 Vision 2000 03/04-11/05

RESULTS A total of 618 pronuclear zygotes were scored Second Polar Body position: In singamy n=299 (48%) n=286 (46%) n=13 (2%) n=20 (3.5%) n=71 (24%) 4c-1 F0 (day 2) n=170 n=93 (32%) n=2 (15%) n=2 (10%) Vision 2000 03/04-11/05

PRONUCLEAR ZYGOTE CONFIGURATIONS OOCYTES DIAGNOSED AS CHROMOSOMALLY NORMAL AFTER 1 st PB BIOPSY n=517 a b c % A1 A2 A1 A2 A3 A3 A4 A4 A2 N zygotes= 117 109 78 87 62 44 10 9 1 a A1, A2 vs. A1, A2 P<0.001 b A1, A2 vs. A3, A3 P<0.001 c A3, A3 vs. A4, A4, A2 P<0.001 Vision 2000 03.04-12.05

PRONUCLEAR ZYGOTE CONFIGURATIONS CHROMOSOMAL STATUS IN PREIMPLANTATION EMBRYOS c d e n=1198 % a b a b A1 A2 A1 A2 A3 A3 A4 A4 A2 A5 A1 A3 A5 A4 A5 N embryos 164 116 128 121 129 156 139 109 19 27 21 17 11 35 6 a P<0.001 b P<0.005 c For chromosomally normal embryos A1, A2, A1, A2 vs. A3, A3 P<0.001; d For chromosomally normal embryos A3, A3 vs. A4, A4, A2, A5, A1, A3, A5, A4, A5 P<0.001 e For complex abnormalities A1, A2, A1, A2, A3, A3 vs. the others P<0.001 Vision 2000 03.04-12.05

59 GESTATIONAL SACS OF KNOWN PRONUCLEAR ZYGOTE CONFIGURATION A1 A2 A1 A2 A3 A3 B2 B2 C5 Total 43 gestational sacs (73%) 13 gestational sacs (22%) 3 gestational sacs (5%) 2 4 2 1 3 1 0 1 1 9 gestational sacs (60%) No. gestational sacs in PB patients (n=15) 4 gestational sacs (27%) 2 gestational sacs (13%) No. gestational sacs in PGD patients (n=44) 9 11 8 6 5 4 1 0 0 34 gestational sacs (77%) 9 gestational sacs (21%) 1 gestational sac (2%) Vision 2000 03.04-12.05

PGD - AS MATERNAL AGE 36 YEARS % b a c d a b Abnormal embryos Transferred cycles Pregnancy rate Implantation rate c d 36-37 38-39 40-42 43 Years n = 92 n = 100 n = 168 n = 75 N cycles ab P <0.001 cd P <0.05 Gianaroli et al. Clinical value of preimplantation genetic diagnosis. Placenta 2003 VISION 2000 0.96-12.02

FREQUENCY OF CHROMOSOMAL ANEUPLOIDIES ACCORDING TO MATERNAL AGE 50 45 40 No. observations: 10.392 35 30 25 20 15 10 5 0 36-37 38-39 40-42 43 years Gianaroli et al. Clinical value of preimplantation genetic diagnosis. Placenta 2003 13 16 15 22 21 P<0.01 n=2191 P<0.05 n=2155 P<0.001 n=1899 P<0.001 n=1988 P<0.001 n=2159 VISION 2000 0.96-12.02

31% Complex abnormalities 3% Polyploidy 3% Haploidy 186 CHROMOSOMAL ANALYSIS ON EMBRYOS n=5115 37% Euploidy 13% Monosomy 14% Trisomy 2% Monosomy+ trisomy 123 Tris 21 100 Tris 22 97 Tris 16 90 Tris 13 83 Tris 15 49 Tris 18 825 (16%) potential implantation 66 Mon 21 31 Mon X Vision 2000 9.96-12.03

23% Complex abnormalities 3% Polyploidy 2% Haploidy CHROMOSOMAL ANALYSIS ON EMBRYOS EMBRYOS GENERATED FROM 193 PREGNANT PATIENTS 36 208 pregnancies, 1237 embryos vs. non pregnant patients P<0.005 vs. non pregnant patients P<0.001 45% Euploidy 11% Monosomy 15% Trisomy 1% Monosomy+ trisomy 33 Tris 21 30 Tris 16 23 Tris 13 22 Tris 15 21 Tris 22 12 Tris 18 196 (16%) potential implantation 14 Mon 21 5 Mon X n=5115 Gianaroli L, Magli MC, Ferraretti AP, Tabanelli C, Trengia V, Farfalli V, Cavallini G(2005) The beneficial effects of PGD for aneuploidy supports extensive clinical application. Reprod. Biomed. Online. Vision 2000 9.96-12.043

INCIDENCE OF TRANSLOCATIONS NEONATAL 0.2% INFERTILE COUPLES 0.6% SEVERE MALE INFERTILITY 3.1% RECURRENT SPONTANEOUS ABORTIONS 9.2% VISION 2000

TRANSLOCATION CARRIERS INFERTILE COUPLES REPRODUCTIVE HISTORY Robertsonian Reciprocal No. of patients 34 37 No. of patients with previous abortions (%) 10 (29) 13 (35) No. of abortions (MSD) 1.81 2.31.4 No. of patients with severe male factor 18 11 VISION 2000 9/96-life

PGD FOR TRANSLOCATIONS Robertsonian Reciprocal No. cycles 50 52 Age 35.24.0 35.24.8 No. generated embryos 280 329 No. diagnosed embryos 234 262 FISH normal (%) 58 (25) a 38 (14) a FISH abnormal (%) 176 (75) b 224 (86) b No. transferred cycles (%) 34 (68) c 16 (31) c No. transferred embryos 1.5 0.7 1.4 0.5 No. clinical pregnancies (%) 15 (44) 6 (38) No. abortions 5* 2** Implantation rate (%) (36.0) (31.8) Take-home baby rate / patient (%) (29.4) (13.5) *1 after amniocentesis, normal karyotype; 1 t(13;14) was 47XY,21; 1 t(13;14) was 47XY,22 ** unbalanced ab <0.01 c <0.001 VISION 2000 9/96-life

PREGNANCY RATE IN RELATION TO THE PROPORTION OF NORMAL / BALANCED EMBRYOS Robertsonian Reciprocal % 20 >20 - <50 50 Proportion of normal / balanced embryos VISION 2000 9/96-life

PREGNANCY RATE IN RELATION TO THE PROPORTION OF NORMAL / BALANCED EMBRYOS The pregnancy after PGD in traslocation carriers is proportional to the % of normal / balanced embryos. The analysis of gametes could have a prognostic value on the chances of pregnancy after PGD Sperm analysis on male carriers Preliminary exam for PGD cycle VISION 2000 9/96-life

Società Italiana di Studi di Medicina della Riproduzione Analisi citogenetica per la traslocazione 46 XY t(14;20)(p11.2;q11.2) su cellule germinali maschili tramite F.I.S.H. (Fluorescence In Situ Hybridization) Sig. Data 04-10-2005 Data di nascita Campione di spermatozoi ottenuto ( ) per eiaculazione ( ) mediante recupero chirurgico dall apparato genitale in data 16-09-2005 e 22-09-2005 Accettazione in data 22-09-2005 N di cellule spermatiche analizzate: 107 N di cellule spermatiche diagnosticate: 107 (100 %) N di cellule spermatiche normali / bilanciate 81 (75,70 %) N di cellule spermatiche non bilanciate 26 (24,30 %) Utilizzate nel primo pannello le sonde specifiche per le regioni telomeriche 14q, 20p Utilizzate nel secondo pannello le sonde specifiche per le regioni centromeriche: 15 (controllo), 20 No. unbalanced sperm Diagnosi 14q 20p 15 20 N spermatozoi (%) Normali 1 1 1 1 81 (75,70) t(14;20) (p11.2;q11.2) Segregazione 2:2, adiacente I t(14;20) (p11.2;q11.2) Segregazione 2:2, adiacente II 2 0 1 1 19 (17,76) 1 1 1 0 7(6,54) Totale 107

PGD FOR TRANSLOCATIONS INTERCHROMOSOMAL EFFECT Rob. Rec. P No. cycles 50 30 Age 35.24.0 34.75.0 No. diagnosed embryos 234 166 FISH normal (%) 58 (24) 23 (14) 0.025 FISH abnormal (%) 176 (76) 143 (88) 0.025 for chromosomes transl. related (%) 43 (24) 67 (47) 0.001 mixed (%) 64 (37) 37 (26) N.S. for chromosomes transl. non related (%) 46 (26) 23 (16) 0.05 haploidy/polyploidy (%) 23 (13) 16 (11) N.S. Modified from: Gianaroli L, Magli MC, Ferraretti AP, Munnè S, Balicchia B, Escudero T, Crippa A. (2002) Possible interchromosomal effect in embryos generated by gametes from translocation carriers. Hum Rep 17, 3201-3207. VISION 2000 9/96-life

PGD FOR ANEUPLOIDY Indication No. cycles No. FISH diagnosed embryos % FISH normal % clinical pregnancies % implantation Maternal age36 years 534 2675 31 26 17.9 Repeated cycles3 121 633 41 38 26.3 Gonosomal mosaicism 65 350 36 51 36.1 Robertsonian translocations 43 214 24 48 38.6 Reciprocal translocations 41 201 13 33 25.0 Recurrent abortions 2 110 589 32 29 21.3 Azoospermia 87 381 33 25 18.5 Poor responders3 oocytes 80 141 25 18 14.3 VISION 2000 9.96-12.03

PGD FOR ANEUPLOIDY N patients 738 N cycles 1027 Age (M SD) 37.33.7 N diagnosed embryos 5102 FISH normal (%) 1676 (33) FISH abnormal (%) 3426 (67) Monosomy trisomy (%) 1432 (42) N transferred cycles (%) 697 (68) N transferred embryos (M SD) 1.7±0.7 N clinical pregnancies (%) 203 (29) N spontaneous abortions (%) 35 (16) Implantation rate (%) 20.7 Implantation rate / pregnant patient (%) 65.2 Vision 2000 0.96-12.03

% CHROMOSOMAL ABNORMALITIES AND CELLULAR STAGE a NORMAL KARYOTYPE n=4249 a b 4 5 6 7 8 9 10 11 12 Number of cells at 62 hrs post insemination 778 834 880 683 934 57 70 7 6 b c Number of embryos c a P<0.001 b P<0.001 c P<0.05 Vision 2000 0.96-12.03

13 16 18 21 22 X Y 15 17 VISION 2000

MALE CONTRIBUTION TO ANEUPLOIDY PGD for aneuploidy 201 patients 261 cycles 3 repeated IVF cycles (n=121) MESA-TESE > 1 IVF failure (n=52) No indications (n=88) X Y 13 15 16 18 21 22 Maternal age35 yrs Normal karyotype Vision 2000 0.96-12.03

% a P<0.025 bcd P<0.001 CHROMOSOMAL ANALYSIS ON SPERM CHROMOSOMALLY NORMAL SAMPLES a b c d a n=331 Normospermic OAT sev. OAT MESA TESE Modified from Gianaroli L, Magli MC, Ferraretti AP (2005) Sperm and blastomere aneuploidy detection in reproductive genetics and medicine. J. Histochem. Cytochem. b c d Vision 2000 4.03-12.04

CHROMOSOMAL ANALYSIS ON SPERM INCIDENCE OF ANEUPLOIDY PER SINGLE CHROMOSOME n=331 % XY Modified from Gianaroli L, Magli MC, Ferraretti AP (2005) Sperm and blastomere aneuploidy detection in reproductive genetics and medicine. J. Histochem. Cytochem. Vision 2000 4.03-12.04

% CHROMOSOMALLY ABNORMAL EMBRYOS CHAOTIC MOSAICS Normo Normo TMC TMC MESA TESE TESE IVF ICSI 0.5x10 6 <0.5x10 6 obstructive non-obstr. n=127 n=38 n=63 n=81 n=20 n=6 n=42 a P<0.001 a Vision 2000 0.96-12.03

% CHROMOSOMALLY ABNORMAL EMBRYOS GONOSOMAL ANEUPLOIDY ab a b Normo Normo TMC TMC MESA TESE TESE IVF ICSI 0.5x10 6 <0.5x10 6 obstructive non-obstr. a P<0.025 Vision 2000 0.96-12.03

PGD FOR ANEUPLOIDY Polar body Polar body and blastomere Blastomere No. cycles 19 32 62 Age (MSD) 38.44.0 38.43.6 38.13.4 Previous IVF cycles (MSD) 2.72.7 3.92.0 3.12.3 No. oocytes 199 327 No. 2PN biopsied 108 238 - FISH normal (%) 29 (27) 48 (20) - FISH abnormal (%) 69 (64) 114 (48) - No result (%) 10 (9) 76 (32) - No. biopsied embryos - 67 376 FISH normal (%) - 31 (46) 117 (31) FISH abnormal (%) - 36 (54) 258 (69) No result (%) - 0 1 No. embryos transferred 20 50 76 No. cycles transferred (%) 13 (68) 29 47 No. clinical pregnancies (%) 3 (23) 10 (35) 17 (36) Implantation rate (%) 15.0 26.0 25.0 No. Abortions 0 1* 1 *ectopic VISION 2000 9.96-07.03

ANEUPLOIDY PGD SINGLE GENE DISORDERS VISION 2000

I PB BIOPSY EMBRYO BIOPSY PROS -BY PRODUCT ANALYSIS -NO MOSAICISM RISK -NPP -FEMALE AND MALE GENETIC MATERIAL -REPETEABILITY OF THE TEST -IMPACT ON PREDICTION OF VIABILITY -SET IMPLEMENTED CONS -ONLY FEMALE GENETIC MATERIAL -SHORT AMOUNT OF TIME -SKILLED EMBRYOLOGISTS -PIF -RISK OF MISDIAGNOSIS -REDUCTION OF EMBRYONIC MASS

PATIENT SELECTION MATERNAL AGE 36 yrs 3 FAILED IVF CYCLES ALTERED KARYOTYPE TRANSLOCATION CARRIERS RECURRENT ABORTIONS MESA TESE POOR RESPONDERS TRANSFER IVF PGD + NO TRANSFER PGD FOR MONOGENIC DISEASES ADROLOGIST NO PREGNANCY PREGNANCY FISH ON SPERMATOZOA HIGH % FISH ABNORMAL TRANSFER NORMAL % FISH ABNORMALITIES DONOR SPERM FISH NORMAL PB BIOPSY OOCYTE DONATION 8 cells EMBRYO COLTURE FISH ABNORMAL NO TRANSFER

SHOULD WE TEST THE FIRST POLAR BODY OR THE EMBRYO L. Gianaroli, C.M. Magli, A.P. Ferraretti Reproductive Medicine Unit - Via Mazzini, 12-40138 Bologna sismer@sismer.it

-higher is the success rate of a routine program lower is the need for AS -higher is the incidence of difficult patients higher is the need for AS -higher is the interest to costumize the service higher is the need for AS