Article Preimplantation HLA typing with aneuploidy testing

Transcription

1 RBMOnline - Vol 12. No Reproductive BioMedicine Online; on web 10 November 2005 Article Preimplantation HLA typing with aneuploidy testing Dr Svetlana Rechitsky is a graduate of Kharkov University s Genetics Faculty, and received her PhD in Experimental Molecular Embryology from the Second Moscow Medical Institute in She moved to the Reproductive Genetics Institute in 1989 to head the DNA laboratory, which has performed the largest preimplantation genetic diagnosis (PGD) series for single gene disorders, with PGD design for the majority of these disorders developed for the first time. She has published more than 30 papers in the field of PGD, including a key contribution to the recently published Atlas of Preimplantation Genetic Diagnosis. Dr Svetlana Rechitsky Svetlana Rechitsky, Anver Kuliev 1, Tatyana Sharapova, Katya Laziuk, Seckin Ozen, Irina Barsky, Oleg Verlinsky, Ilan Tur-Kaspa, Yury Verlinsky Reproductive Genetics Institute, Chicago, IL, USA 1 Correspondence: 2825 North Halsted Street, Chicago, IL 60657, USA. anverkuliev@hotmail.com Abstract Preimplantation HLA typing has been introduced for the treatment of affected siblings, requiring HLA-identical stem cell transplantation. This was applied either in combination with preimplantation genetic diagnosis (PGD) to ensure that the preselected HLA-matched embryos were also free of the genetic disorder, or without PGD, with the only purpose of selecting and transferring the HLA-matched embryos. Because patients requesting preimplantation HLA typing are usually of advanced reproductive age, aneuploidy testing allows not only the avoidance of the birth of children with chromosomal disorders, but also improvement of the reproductive outcome, which is still not sufficiently high in preimplantation HLA typing at the present time. This study presents the results of the first experience of preimplantation HLA typing combined with aneuploidy testing, demonstrating feasibility and impact of aneuploidy testing on the accuracy and outcome of preimplantation HLA typing. Of a total of 138 cycles performed, 87 were combined with PGD and 52 without testing for the causative gene, of which aneuploidy testing was performed in 27 cycles, allowing the preselection and transfer of only those HLA-matched embryos that were also euploid. Although the euploid HLA-identical embryos were available for transfer in only half of these cycles, pregnancy and birth of unaffected HLA-identical children were observed in approximately half of these cycles, suggesting the potential usefulness of incorporating aneuploidy testing into preimplantation HLA typing. Keywords: accuracy, PCR-based aneuploidy testing, PGD, preimplantation HLA typing, reproductive outcome Introduction The first case of preimplantation human leukocyte antigen (HLA) typing was performed in combination with preimplantation genetic diagnosis (PGD) for Fanconi anaemia complementation group C (FA-C), and resulted in successful haematopoietic reconstitution in the affected sibling by transplantation of stem cells obtained from the HLA-matched offspring resulting from PGD (Verlinsky et al., 2000, 2001). To improve access to HLA-identical bone marrow transplantation in sporadic bone marrow failures, this approach was then applied with the sole purpose of ensuring the birth of HLA-identical offspring, not involving PGD, which also resulted in radical treatment of a sibling with a sporadic Blackfan Diamond anaemia (BDA) by stem cell transplantation from an HLA-identical child born following preimplantation HLA typing (Velinsky et al., 2004). Eventually, preimplantation HLA typing became one of the most attractive indications for PGD, currently performed with or without testing for the causative gene (Kuliev and Verlinsky, 2004; Rechitsky et al., 2004; Van develde et al., 2004; Kahraman et al., 2005; Kuliev et al., 2005). However, because most patients requesting preimplantation HLA typing are of advanced reproductive age, the outcome of the procedure has not yet been sufficiently high, many patients still undergoing two or more attempts before they become pregnant and deliver an HLA-identical offspring. Therefore, testing for age-related aneuploidy may appear useful for improving the reproductive outcome of preimplantation HLA typing, which will also minimize the risk of delivering a child with chromosomal disorders, providing reassurance for patients who are usually concerned about their pregnancy outcomes. 89

2 90 This paper describes the first practical experience of preimplantation HLA typing, combined with aneuploidy testing, demonstrating its feasibility and impact on the accuracy and outcome of preimplantation HLA typing. Materials and methods A total of 138 preimplantation HLA typing cycles for 66 couples were performed, including 87 cycles from 41 couples combined with PGD and 52 from 25 couples without testing for causative gene (Table 1). Aneuploidy testing is currently offered as an integral component of preimplantation HLA typing to patients of advanced reproductive age, and has presently been applied in 27 cycles from 14 couples (Table 2). There were no cases of ovarian hyperstimulation syndrome. The indications for preimplantation HLA typing were distributed as follows: 55 cycles from 27 couples were performed for thalassaemia (see also Kuliev et al., 2005), 18 from seven couples for FA-C and Fanconi anaemia complementation group A (FA-A), one for Wiscott Aldrich syndrome (WAS), four from two couples for X- linked adrenoleukodystrophy (X-ALD), two from a single couple for X-linked hyperimmunoglobulin M syndrome (HYGM), and seven from three couples for X-linked hypohidrotic ectodermal dysplasia with immune deficiency (HED-ID), incontinentia pigmenti (IP) and inherited form of BDA (Table 3) (see also Rechitsky et al., 2004). Among the sporadic and acquired bone marrow failures, for which preimplantation HLA typing was performed in 52 cycles from 25 couples, were leukaemias and Blackfan Diamond anaemia, also requiring HLA-identical stem cell transplantation (see also Verlinsky et al., 2004). Preimplantation HLA typing was performed as described previously in detail (Rechitsky et al., 2004; Verlinsky and Kuliev, 2004). Primer sequences and polymerase chain reaction (PCR) conditions for PGD of the above conditions and HLA were also reported (Rechitsky et al., 2004), except for FA-A, which are presented in Table 4. The copy number of chromosomes, in which causative gene tested is located, was analysed using corresponding satellite markers, as well as the copy number of chromosome 6, in which the HLA genes are localized. A set of short tandem repeats (STR) throughout HLA region and chromosome specific satellite makers, allowed the identification of monosomy, trisomy or uniparental disomy of chromosome 6, as well as the possibility of recombination, which may strongly affect the accuracy of HLA matching (Rechitsky et al., 2004; Verlinsky et al., 2004). The information on the copy number of chromosome 6 and other chromosomes in which the causative genes tested are located was also useful for avoiding misdiagnosis due to mosaicism, which is known to be prevalent at the cleavage stage (Munné, 2002). In some cases, when the results of PCR based aneuploidy testing were not available, fluorescence insitu hybridization (FISH) analysis was performed as described elsewhere (Verlinsky and Kuliev, 2005). Although both the PCR-based aneuploidy testing and FISH are reliable, the PCR method allows the analysis of the copy number of chromosomes in the same multiplex PCR reaction for causative gene analysis, while an additional cell is required to perform FISH analysis. Aneuploidy testing was performed in patients aged 35 years and older by adding primers for chromosome specific microsatellite markers to the multiplex PCR protocols worked out for a specific genetic disorder or HLA typing. Although patients were informed about the poor outcomes of the procedure after 40 years of age, it was still performed on patient request. The primers for PCR based aneuploidy testing are shown in Table 5, listing polymorphic markers used for identification of the copy number of chromosomes 13, 16, 18, 21, 22 and X. The strategy for HLA typing depended on the mutation origin, number of the causative gene mutations tested, and the requirement for aneuploidy testing. For testing of maternal mutations and maternal HLA match, the first polar body (PB1) was removed following maturation (after PB1 extrusion), followed by the second polar body (PB2) removal next day after fertilization of oocytes and following PB2 extrusion. Single blastomeres were removed from the 8-cell embryos for testing of both paternal and maternal mutations and paternal and maternal HLA match. A haplotype analysis for father, mother and affected child was performed for each family prior to preimplantation HLA typing. This allowed the detection and avoidance of misdiagnosis due to preferential amplification and allele dropout (ADO), affecting the diagnostic accuracy of PGD and HLA typing. A multiplex hemi-nested PCR was used, with the first-round of PCR requiring similar annealing temperature of the outside primers. Informed consent was given by all patients involved in the study. This was based on the Institutional Review Board approval, that in cases of unaffected HLA embryos, they would be preselected for transfer back to the patients, in cases of unaffected but non-hla-matched embryos, they would be frozen for future possible use by the couple, and in cases of prediction of mutant embryos, they would be exposed to the confirmatory analysis using the genomic DNA from these embryos to evaluate the accuracy of the single cell based PGD. Results The results of the preimplantation HLA-typing experience, representing the world s largest series, are presented in Tables 1 3. Of 630 embryos tested in 87 PGD cycles performed for 41 couples carrying the above-mentioned mutations, 428 unaffected embryos were detected, of which 90 were also HLAmatched to the siblings (14.3%). Only 77 were acceptable for transfer in 53 of 87 PGD cycles (1.5 embryos per transfer on average), yielding 14 unaffected HLA-matched pregnancies (26.6% per transfer) and birth of 10 healthy children HLAmatched to affected siblings, with two pregnancies still ongoing following confirmation of PGD by prenatal diagnosis. Of 428 embryos tested for HLA type only, in 52 cycles from 25 couples, 413 were with conclusive results, of which 84 were predicted to be HLA matched to the affected siblings (19.6%) (Table 2). Of these embryos, 52 were transferred in 34 cycles, resulting in 13 clinical pregnancies and birth of 12 HLA-identical children. Of these embryos, 204 were also tested for aneuploidies, resulting in prediction of 36 HLA-matched embryos (17.6%), of which only 21 were also euploid (10.3%). Nineteen were acceptable for transfer in only 13 (48%) of 27 cycles (1.5 embryos per transfer on average), resulting in seven

3 (54%) clinical pregnancies, and birth of six HLA identical children. Aneuploidy testing is currently performed in increasing numbers of preimplantation HLA typing cycles combined with PGD, as previously described in PGD for thalassaemia (Kuliev et al., 2005). One of such cases combined with PGD for FA-A is presented in Figures 1 and 2. As parents were carriers of different mutations (R95I-W5 and R1080L), both PB and blastomere analysis were performed, so mutation free oocytes might be selected (oocytes nos. 4, 7, 9, 10 and 14) prior to blastomere analysis (Figure 1B), which was then used mainly for selection of paternally derived mutation-free embryos, originating from mutant oocytes (embryos 6 and 11) (Figure 2F) and HLA matching, simultaneously with linked marker analysis and aneuploidy testing (Figure 1). Aneuploidy testing identified three abnormal embryos (embryos 2, 14 and 16), including one with trisomy X, and two with complex monosomies. In addition, three embryos with monosomy 6 were detected in the process of HLA typing, including two embryos with other chromosomal abnormalities (embryos 2 and 16), and one missing only chromosome 6 (embryo 13). As can be seen from Figure 1, only one euploid embryo (embryo 11) appeared to be unaffected and HLA matched to the affected sibling, and was transferred back to the patient, resulting in the birth of a healthy child, following confirmation of FA-A-free status and HLA matching to the affected sibling with FA-A by CVS (Figure 2). The first case of preimplantation HLA typing for two affected siblings in the family at a time, performed simultaneously with aneuploidy testing, is presented in Figure 3. The couple had two thalassaemic children, each having different HLA types, and inherited two different mutations from parents, IVS2 1 from the father and IVS1 5 from the mother, so two different HLA-matched progeny were required for bone marrow transplantation for the siblings. Of seven embryos tested, six were with the complete results, of which one contained only one copy of chromosome 11 (embryo 2), the other was homozygous affected (embryo 6), and four unaffected, including one carrier of a paternally derived thalassaemia mutation (embryo 9); the others were free of either maternal or paternal mutation. HLA typing showed that one of the embryos was matched to one of the affected siblings, another to the other affected sibling (embryos 4 and 9), and three were non-matched (embryos 6, 7 and 8), including one with single chromosome 6 (embryo 6). A single aneuploid oocyte suggesting trisomy 22 in the resulting embryo was detected by FISH analysis of PB1 and PB2 (excluded from further analysis of the causative gene and HLA type), in addition to the embryos with monosomy 11 (embryo 2) and monosomy 6 (embryo 6) already mentioned. Two unaffected HLA-matched aneuploidy free embryos (embryos 4 and 9) were transferred, resulting in a singleton pregnancy and birth of a healthy baby, HLA matched to one of the affected siblings with thalassaemia. Table 1. Overall results of preimplantation human leukocyte antigen (HLA) typing with and without preimplantation genetic diagnosis. Testing Patient/ No. No. embryos Pregnancy/ cycle transfers transferred birth (%) a HLA plus PGD 41/ /10 (2) c (26.4) HLA b 25/ /12 (39) Total 66/ /22 (2) c (31) a Implantation rates were not available for all cycles as some were performed in other clinics. b Includes 27 cycles combined with aneuploidy testing shown in Table 2. c Ongoing pregnancies. Table 2. Outcome of preimplantation HLA typing with and without aneuploidy testing. HLA HLA plus Total aneuploidy testing Patient/cycle 11/25 14/27 25/52 Total embryos Matched embryos (36) a 69 (84) a Non-matched embryos Transfers (%) 21 (84) 13 (48) 34 No. embryos transferred 33 (1.6) b 19 (1.4) b 52 Pregnancy (%) 6 (28.5) 7 (53.8) 13 (38) Birth a Includes all HLA-matched embryos irrespective of whether they were euploid or not. b Values are numbers unless stated otherwise. 91

4 Table 3. Results and outcome of preimplantation HLA typing combined with PGD. Disease Patient/ No. Normal embryos Abnormal embryos Embryo Pregnancy/ cycle embryos Match Non-match Match Non-match transfers/ birth studied no. embryos Thalassaemia 27/ /57 8/6 (1) a FANCA, 7/ /12 4/2 (1) a FANCC WAS 1/ /1 0 ALD 2/ Hyper-IgM 1/ /2 1/1 HED+ID; IP 2/ /3 1/1 BDA 1/ /2 0 Total (%) 41/ (14) /77 (1.4) 14/10 (2) a (26.4) b a Ongoing pregnancies. b Pregnancy rate Table 4. Primers and polymerase chain reaction (PCR) conditions used for preimplantation genetic diagnosis of Fanconi anaemia complementation group A (FANCA). Gene/polymorphism Upper primer Lower primer Annealing temperature (ºC) FANCA; R 951 W; Outside: 5 GTGTGTTCTGTCCTCATT 5 GATTATAGGTGTGAGCCA HpyCH4 III cuts normal GTATT 3 TCATG 3 allele; heminested PCR Inside: 5 GTGTGTTCTGTCCTCATT 5 AGGGTAGCTCTTTTCAAC 50 GTATT 3 ACTTA 3 FANCA; R 1080 L; Bsr I Outside: 5 CCTTCCCAGGAGCACTT 5 CTCTAGGACCGTCATGAG cuts mutant allele; Hph I CC 3 ATGCT 3 cuts normal allele; Inside: 5 CCTTCCCAGGAGCACTT 5 TGCCCAGGTGGTAGTAG 55 heminested PCR CC 3 GTGTC 3 HphI mis Inside: 5 CCTTCCCAGGAGCACTT 5 TGCCCAGGTGGTAGTAGGT 55 CC 3 GTTC 3 BsrI mis FANCA; intron I (CA)n; Outside: 5 CTGCTAAAGATATAGTTAGT 5 GTGCTGCGATTATAGGCGT heminested FL-PCR ACATGC 3 Inside: 5 CTGCTAAAGATATAGTTAGT 5 FAM GCCAATTTCTAGTCAT 55 ACATGC 3 TTGTTTC 3 FANCA; intron 27 SNP Outside: 5 GTGTGGGCTGTTGATGGTC 5 AAAGGCTGGCTACGTCC (-36 G/T); HhaI cuts G ; TG 3 TCC 3 heminested PCR Inside: 5 GTGTGGGCTGTTGATGGT 5 CGGGCCTCTGAGAACAAT 53 CTG 3 CT 3 D16S520; heminested Outside: 5 CGGTATTAGCAATCAGGG 3 5 CTTACCAACCTCCACAGC FL-PCR Inside: 5 FAM TTTCCAGAGAAAAAG 5 CTTACCAACCTCCACAGC 3 55 AACAC 3 D16S3026; heminested Outside: 5 CTCCCTGAGCAACAAACA 3 5 CGCCTGATTTAGGCTTT FL-PCR Inside: 5 FAM CGTGGCTTCACTAAAA 5 CGCCTGATTTAGGCTTT 3 55 CA 3 D16S3407; heminested Outside: 5 TCAGCCACACAGATAAACC 3 5 TTGTTTCCAATGTGTCTCC FL-PCR Inside: 5 HEX TGAGTTTGTTTTTTTCT 5 TTGTTTCCAATGTGTCTCC 3 55 GCT 3 92

5 Table 5. Primers for polymerase chain reaction aneuploidy testing showing polymorphic markers used for identification of the copy number of chromosomes tested. Chrom- Polymorphic Cytogenetic Hetero- Size Upper primer Lower primer Annealing osome repeat location zygosity temperature index (ºC) 13 D13S284; 13q Outside: 5 CCAAGAGTGT 5 GGCTAACATCGA (CA)n; 212 CCTCTGTTGCA 3 AGGGAGGT 3 heminested Inside: 5 Hex GGTGGAAACA 5 GGCTAACATCGAA 55 GAATTCATTCAAC 3 GGGAGGT 3 D13S631; 13q31 13q Outside: 5 ACCATTCCACTCC 5 GAAAAATAATTTC (GATA)n; 136 AGCCTG 3 TGGGGGTG 3 heminested Inside: 5 Hex GGCAACAAG 5 GAAAAATAATTTC 55 AGCAAAACTCTGT 3 TGGGGGTG 3 D13S742; 13q11-13q Outside: 5 TAAAAATCTTATTC 5 CACCTCCAGACTT (GAAA)n; 384 CTAACTGGG 3 CCCAATTC 3 heminested Inside: 5 Hex GGAAATAGGT 5 CACCTCCAGACTT 55 TGTACACCCATTG 3 CCCAATTC 3 D13S813; 13q32-13q Outside: 5 CCGCAGAGCTTTT 5 CGTTGTTGACTGA (ACT)n; 199 CCTAGTTAC 3 CTGAATACTG 3 heminested Inside: 5 Fam GGACATCAGG 5 CGTTGTTGACTGA 55 GAAGATTTAGTAA 3 CTGAATACTG 3 D13S1282; 13pter-13qter Outside: 5 GTAAAACCTGCC 5 GATATTATTGATAG (CA)n; 145 TAGTAGCATACAC 3 ATTCTTGAAATGA 3 heminested Inside: 5 GTAAAACCTGCCT 5 Fam CATTACTACCA 55 AGTAGCATACAC 3 TGTGGCTAGTTAAG 3 D13S1267; 13pter-13qter Outside: 5 CGAGTTGGCAG 5 TGTATGACAGAC (CA)n; 88 ACCTCAGAC 3 TGAGGAAAGGC 3 heminested Inside: 5 Hex ATTTATCCACA 5 TGTATGACAGACT 55 TCTCTACTACTGCG 3 GAGGAAAGGC 3 16 D16S520; 16q qter Outside: 5 CGGTATTAGCAAT 5 CTTACCAACCTCC (CA)n ; 170 CAGGG 3 ACAGC 3 heminested Inside: 5 Fam TTTCCAGAGA 5 CTTACCAACCTCC 55 AAAAGAACAC ACAGC 3 D16S p Outside: 5 CTTCATACAAAAC 5 TTCTTTTTGTAGC (CA)n ; 162 AGGCTTGAAAG 3 ATGTATGTGAAA 3 heminested Inside: 5 CTTCATACAAAAC 5 Fam CTGTTTGCCT 55 AGGCTTGAAAG 3 GCCTATTTGATA 3 D16S pter-16qter Outside: 5 GTAACCCAGTCT 5 GGGTGGCCAAG (CA)n; 155 TGACCAATGAG 3 GTGTTTG 3 heminested Inside: 5 Fam GGAAAGATGT 5 GGGTGGCCAAG 55 CTCCTTCCTCATAG 3 GTGTTTG 3 D16S 291; 16p Outside: 5 TGCAGCCTCAGT 5 TGCTGGGATTAC (CA)n; 138 TGTGTTTC 3 AGGCATG 3 heminested Inside: 5 TGCAGCCTCAGTT 5 Fam AAGGCTGGC 55 GTGTTTC 3 AGAGGAGGTGA 3 D16S 521; 16p Outside: 5 GGGCGACAGAG 5 GCCTTACAAATGT (CA)n; 161 CGAGACTC 3 CGTATTCACCAT 3 heminested Inside: 5 GGGCGACAGAGC 5 Hex AACGGAGTTT GAGACTC 3 ACAACCAAAATGC 3 55 D16S 3082; 16pter-16qter Outside: 5 GCGGAAATAACG 5 TCCAGGCTGTG (CA)n; 164 GTGACACT 3 AGGGGCT 3 heminested Inside: 5 GCGGAAATAACG 5 Fam CTGGGACTG 55 GTGACACT 3 CTGTCTCCTCTTAT 3 D16S 3395; 16pter-16qter Outside: 5 GCCAGAAGCCAT 5 CCTGGCAGTAA (ATT)n; 123 AGTTTCTAACC 3 GTCCTGAAAGAA 3 heminested Inside: 5 Hex GCAGAGTTCT 5 CCTGGCAGTAAGT 55 GGGAGGTGGAC 3 CCTGAAAGAA 3 18 D18S61 18q Outside: 5 AGGACTCCCAAA 5 ACTAACTGGAA (CA)n; 162 CTACATTCTTC 3 ATTTGTTGCTTC 3 heminested Inside: 5 AGGACTCCCAAA 5 Hex ACTCAGGAGC 55 CTACATTCTTC 3 ATGGTTATGTTTA 3 Continued on page 94 93

6 Continued from page D18S66 18q Outside: 5 AAGTTAGAGCAAG 5 GCAGCCTCGG (CA)n; 166 TCCCTGCC 3 AGAAACG 3 heminested Inside: 5 AAGTTAGAGCAA 5 Hex CGATTCCCGG 55 GTCCCTGCC 3 ACGGTTCT 3 D18S pter 18qter Outside: 5 TGTGAACATTTTA 5 ATTCTTCAATATA (CA)n; 154 GAGTTTGTAGTAC 3 CCCAGTTAATTG 3 heminested Inside: 5 TGTGAACATTTTA 5 Hex CCCGCCTGTAC 55 GAGTTTGTAGTAC 3 TGCCTGA 3 D18S pter 18qter Outside: 5 CACTCAGTTTGTG 5 GCAAATGGAGA (CA)n; 138 GTAATTTGTTAT 3 GACTTGGTCT 3 heminested Inside: 5 CACTCAGTTTGTG 5 Hex CCTGCCTTTC 55 GTAATTTGTTAT 3 CTAGTGGGT 3 D18s386; 18q q Outside: 5 CACTTGGAACC 5 TAGAAGTCATACA (GAAA)n; 401 CCCAGC 3 TTAAAAAGTGATAA 3 heminested Inside: 5 Hex CTGGGCAACA 5 TAGAAGTCATACA 55 GAGTGAGATTT 3 TTAAAAAGTGATAA 3 21 D21S268 21q qter Outside: 5 GCAACAGAGTG 5 TCCCCGCTG (CA)n 151 AGACAGGCTC 3 GCAGTGTAT 3 heminested Inside: 5 GCAACAGAGTGA 5 Hex AGTTTGTTCA 55 GACAGGCTC 3 CATCCTTGCC 3 D21S pter 21qter Outside: 5 ATACCTTCTCTAGA 5 CCCTTATCAACC (GATA)n 237 AATGATTCTGT 3 TGCCTGG 3 heminested Inside: 5 ATACCTTCTCTAG 5 Hex ATGGCTGCCA 55 AAATGATTCTGT 3 CATGAAAAC 3 D21S pter 21qter Outside: 5 TGTTCCCGAAA 5 ATATTCCTACAATG (GATA)n 286 TGATCTTGT 3 TATAAGTATTCTAAT 3 heminested Inside: 5 TGTTCCCGAAAT 5 Hex TCTCTACATAT 55 GATCTTGT 3 TTACTGCCAACAC 3 D21S pter 21qter Outside: 5 CCCGAGGGTCT 5 AGGAAAACTTT (CA)n 118 GACCACA 3 GGGACTCTTCAT 3 heminested Inside: 5 CCCGAGGGTCT 5 Hex CCAACTGACT 55 GACCACA 3 CCCAAACAACC 3 D21S pter 21qter Outside: 5 TGGATAGATAGTA 5 CCAGGCTTTC (GATA)n 198 GATAAATGGATGG 3 TGCCCACT 3 heminested Inside: 5 TGGATAGATAGTA 5 Hex CCCACTCCCA 55 GATAAATGGATGG 3 GCCTTCTAA 3 22 D22S277 22q Outside: 5 GACTTGTCAGATT 5 ATTCTGTACTTACA (CA)n 146 CTTGTGTGGTAG 3 AAAGTAACATATCC 3 heminested Inside: 5 GACTTGTCAGATT 5 Hex TAATAAATGCT 55 CTTGTGTGGTAG 3 CACAAGGGTGT 3 D22S282 22q Outside: 5 CATCCCCCTAT 5 TGCTGGGATT (CA)n 158 GCCCACC 3 ACAGGTGTGAG 3 heminested Inside: 5 CATCCCCCTATG 5 Hex CTGGCCTTCA 55 CCCACC 3 CTGTGGGGT 3 D22S283 22pter 22qter Outside: 5 ACTGCCATTTG 5 GACTTTCTGAG (CA)n 140 AGCAGGATG 3 CCACGGAGAT 3 heminested Inside: 5 Hex CCAACCAGC 5 GACTTTCTGAGC 55 ATCATCATCTAC 3 CACGGAGAT 3 D22S423 22q13.122q Outside: 5 TTGTCATTTT 5 GAGTGACTGAGT (CA)n CCCATCCCTG 3 AAATGTAGTGCTT 3 heminested Inside: 5 TTGTCATTTTCCC 5 Hex CTGACTCGTTT 55 ATCCCTG 3 AGGTCATGGT 3 D22S pter 22qter Outside: 5 AGATGAATGAATA 5 GTTTTACTAGGC (CA)n 141 AAGAAAATGTGG 3 ATAATCGTTTCA 3 heminested Inside: 5 AGATGAATGAATA 5 Hex TCATGTTGTT 55 AAGAAAATGTGG 3 TAAAGTGGCAGG 3 D22S535 22pter 22qter Outside: 5 AGTGGTTTCCAAG 5 CAGCTGCCAG (ATC)n 114 CCGCC 3 CCACACTG 3 heminested Inside: 5 Fam CATCAGGGTT 5 CAGCTGCCAGCC 55 TGGTACTTAGCA 3 ACACTG 3 Continued on page 95

7 Continued from page 94 D22S689 22pter 22qter Outside: 5 CCAACTCACATGA 5 AGTGAGACCC (GATA)n 216 GATAATACTTTATG 3 CATCTCAATCTG 3 heminested Inside: 5 Fam CTGCCAAAGA 5 AGTGAGACCCC 55 ATATCTAGGACATC 3 ATCTCAATCTG 3 X DXS8061 Xpter Xqter Outside: 5 GTCCATGAGGTA 5 GTCACAGTGA heminested 128 TCCAAACAGG 3 CTCCATCCCAT 3 Inside: 5 GTCCATGAGGTAT 5 Hex GCTTGTAAT 55 CCAAACAGG 3 CTCAACCCCTA DXS1055 Xp11.4 Xp Outside: 5 CTCTATGGGATAC 5 CCCATCTAGGTT (CA)n 110 ACTGTTCTGGG 3 TATGTAAGTACACT 3 heminested Inside: 5 CTCTATGGGATAC 5 Fam GGAATGCAT 55 ACTGTTCTGGG 3 CCCCATCATTAA 3 DXS8083 Xpter Xqter Outside: 5 CAGAATGAAGAG 5 TTATGTTCTTTG (CA)n 133 ACAACCTGTTAAT 3 CCCACTTTTT 3 heminested Inside: 5 Hex ACTATTCATCT 5 TTATGTTCTTT 55 GACAAGGGACCA 3 GCCCACTTTTT 3 DXS1068 Xp11.4 Xp Outside: 5 GGTCACGCCT 5 CTGAGAACACG (CA)n 140 AAGATGGAGT 3 CTGTTTTTTAC 3 heminested Inside: 5 Fam ATTATGACTA 5 CTGAGAACACG 55 AGGTTCTAGGGAC CTGTTTTTTAC 3 AC 3 DXS8025 Xpter Xqter Outside: 5 CAGCAGAGCAA 5 ATATCCTTTCA (CA)n 200 GACTCTGTCTC 3 CCTCCCTTTTC 3 heminested Inside: 5 CAGCAGAGCAAG 5 Hex GTGCTGCATT 55 ACTCTGTCTC 3 CTGGGTAATT 3 XY DXYS154 Xqter Xqter; Outside: 5 ACTCTCACCTAT 5 AAGTGATCC (CA)n Yqter Yqter 178 CCTATTCAACTTA 3 TCCCGCTTC 3 heminested Inside: 5 Hex ACATGATATTA 5 AAGTGATCCTC 50 TATGTAGAAAATCC 3 CCGCTTC 3 Amelogenin Xp22.2 Yp11.2 NA 100 Outside: 5 ACCTCATCCTG 5 AGGCTTGAGGC nested 104 GGCACCCTGG 3 CAACCATCAG 3 Inside: 5 CCCTGGGCTCTG 5 Fam ATCAGAGCTTA 50 TAAAGAATAGTG 3 AACTGGGAAGCTG 3 95

8 96 Figure 1. Preimplantation human leukocyte antigen (HLA) typing combined with aneuploidy testing and preimplantation genetic diagnosis (PGD) for Fanconi anaemia complementation group A (FANCA). (A) Family pedigree with HLA haplotype analysis based on parental (1.1 and 1.2) and affected child s (2.1) genomic DNA testing. HLA markers order is presented on the upper left. CVS = chorionic villus sampling. (B) Polar body analysis of 15 oocytes, 14 of which were with results, including four embryos (oocytes 4, 7, 9, 10) free of maternal mutation. (C) Results of blastomere mutation analysis (top), HLA typing (middle) and aneuploidy testing (bottom), showing that only embryo 11 is unaffected, euploid and HLA matched to the affected child (2.1), which was transferred resulting in birth of a healthy baby boy (2.2), representing a potential stem cell donor for the sibling. ET, embryo transfer.

9 Figure 2. Preimplantation genetic diagnosis (PGD) for Fanconi anaemia complementation group A (FANCA) by blastomere analysis of maternal and paternal mutations and confirmation of results by chorionic villus sampling (CVS). (A) Position of two different mutations and informative linked polymorphic markers used in PGD. (B) Artificially created restriction site primer (ACRS) was used in the second round of heminested polymerase chain reaction (PCR) to detect the maternal mutation. (C) Restriction maps for the maternal mutation, with ACRS introducing BsrI restriction site to the mutant maternal sequence, which creates two fragments of 119 and 21 bp in the mutant allele, leaving the normal allele uncut. (D, E) Restriction site and restriction map for HpyCH4III enzyme, creating two fragments of 120 and 24 bp in the normal paternal gene, leaving the paternal mutant allele uncut. (F) Left: blastomere analysis of the maternal mutation, using polyacrylamide gel electrophoresis of PCR product from blastomeres digested with BsrI restriction enzyme for the maternal (R 1080) mutation, showing four embryos containing maternal mutation (embryos 1, 2, 3, 6, and 11); the latter embryo appeared to be free of paternal mutation (see below), also HLA matched, euploid (see Figure 1), and was transferred resulting in birth of unaffected child. Right: blastomere analysis of paternal mutation, using polyacrylamide gel electrophoresis of PCR product from blastomeres digested with HpyCH4III restriction enzyme for the paternal (R 951 W) mutation, showing seven embryos containing the paternal mutation (embryos 1, 3, 4, 8, 10, 13 and 18), and the remaining eight being free of the paternal mutation, including embryo 11, mentioned above. (G) Chorionic villus sampling (CVS) confirmation of heterozygous unaffected status of the fetus, originating from the transfer of embryo 11. L, ladder (weight standard, 100 bp); F, father; M, mother; C, child; ET, embryo transfer. 97

10 98 Figure 3. Preimplantation genetic diagnosis (PGD) for beta-thalassaemia combined with human leukocyte antigen (HLA) and aneuploidy testing. Upper panel: family pedigree with HLA haplotype analysis based on parental (1.1 and 1.2) and two affected children s (2.1 and 2.2) genomic DNA testing. HLA markers order is presented on the upper left. HBB, haemoglobin-beta locus. Lower panel: results of HLA typing (top), mutation analysis (middle) and aneuploidy testing (bottom), showing that embryos 4 and 9 are unaffected, as well as euploid and HLA matched, embryo 4 being HLA identical to one of the affected children (2.1), and embryo 9 to the other (2.2). ET, embryo transfer.

11 Discussion The presented data suggest the potential utility of aneuploidy testing in preimplantation HLA typing, allowing the avoidance of transfer of those HLA identical embryos that are chromosomally abnormal, which are destined to be lost anyway either before or after implantation. It was previously demonstrated that more than half of embryos obtained from IVF patients of advanced reproductive age are chromosomally abnormal (Munné, 2002; Kuliev et al., 2003), making it obvious that aneuploidy testing should allow avoidance of transferring these embryos, which may be expected to contribute to the improvement of the pregnancy outcome of IVF patients of advanced reproductive age. Alternatively, incidental transfer of aneuploid embryos in the absence of chromosomal testing should lead to implantation and pregnancy failures in preimplantation HLA typing cycles, or may compromise the pregnancy outcome through spontaneous abortions. It was previously reported that aneuploidy testing improves pregnancy outcome through the improvement of implantation rate and reduction of spontaneous abortions, despite transferring fewer embryos (Gianaroli et al., 1999; Munné et al., 1999, 2003). Improvement in reproductive outcome was particularly obvious in comparison with the previous obstetric history of the same patients prior to undergoing PGD (Gianaroli et al., 2004; Verlinsky et al., 2005). Analysing the outcomes of hundreds of PGD cycles for chromosomal aneuploidies, strong differences were observed, including a 5-fold increase in implantation rate, more than 2-fold reduction of spontaneous abortion rate and more than 2-fold increase in take-home baby rate after PGD, compared with that prior to PGD. This is in accordance with data on the prevalence of chromosomal aneuploidies in pre- and post-implantation development, as at least every second oocyte and preimplantation embryo is aneuploid (Munné et al., 2002; Kuliev et al., 2003), compared with only 1 in 10 in recognized pregnancies, suggesting that the majority of the remaining embryos are eliminated before or during implantation. Although more data are still needed to further prove the impact of aneuploidy testing on the outcomes of preimplantation HLA typing, the presented data suggest that approximately half of aneuploidy free embryo transfers following preimplantation HLA matching resulted in pregnancy and birth of HLA-matched children, compared with 28.5% pregnancy rate following the transfer of HLA- matched embryos not tested for aneuploidy. In a comparable number of cycles performed with or without aneuploidy testing, despite unavailability of aneuploidy free embryos for transfer in over half of the cycles, compared with only 26% of cycles without aneuploidy testing, comparable numbers of pregnancies and births of HLA-matched children were observed, indicating a possible clinical relevance of avoiding chromosomally abnormal embryos from transfer in preimplantation HLA typing. The presented cases of aneuploidy testing applied in preimplantation HLA typing combined with PGD (Figures 1 3) also demonstrate feasibility of an additional analysis for a copy number of chromosomes in testing for causative gene, together with linked polymorphic markers, and HLAtyping, which allowed the preselection of those unaffected HLA matched embryos which were also free of aneuploidy. This might, however, require sequential PB1 and PB2 analysis, to identify the mutation-free oocytes, prior to blastomere analysis, as described in the case of preimplantation HLA typing combined with aneuploidy testing and PGD for FA-A. Of 15 oocytes and embryos tested in this case, four embryos were aneuploid, five were compound heterozygous (affected), four were homozygous normal, four were unaffected carriers of maternal or paternal mutations, and only one was unaffected HLA-identical embryo, which was also euploid. This single embryo was transferred, yielding an unaffected pregnancy and birth of a healthy HLA-identical child free of FA-A, providing a potential donor of HLA-compatible stem cell transplant for the affected brother with FA-A (Figure 1). Both the PCR-based aneuploidy testing and FISH are reliable, but the PCR method allows the analysis of the copy number of chromosomes in the same multiplex PCR reaction for causative gene analysis, while an additional cell is required to perform FISH analysis. Also of special interest is the other case of preimplantation HLA typing combined with aneuploidy testing, performed for a couple with two thalassaemic children, each with different HLA types, both requiring bone marrow transplantation. As seen from Figure 3, despite the availability of only seven embryos for testing, two unaffected HLA-identical embryos, which were also euploid, were selected, one for each of the affected siblings. This represents the first attempt at preimplantation HLA typing to obtain a potential donor progeny for both siblings with different HLA haplotypes, although unfortunately only one of the transferred embryos resulted in pregnancy and the birth of an unaffected child HLA matched to one of the siblings. The presented experience is the world s largest series, involving preimplantation HLA typing of 138 cycles, which has resulted in the transfer of HLA-identical embryos in approximately 80% of cycles, yielding 27 unaffected HLA- matched pregnancies and birth of 22 healthy children, with two pregnancies still ongoing following confirmation of PGD diagnosis. Of a total of 1058 embryos tested, 159 (15%) were identified for possible transfer, which is less than might be expected both in HLA typing (25%), and in combined PGD and HLA typing (18.7%). This may be due to the addition of aneuploidy testing, expected to identify at least 50% chromosomally abnormal embryos in patients of advanced reproductive age, therefore lowering each of the above probabilities by half. In fact, the mean number of embryos for transfer was only 1.4, which also reflects the lower probability of identification of HLA-matched unaffected embryos free of aneuploidy, also taking into consideration the average number of available embryos with results, usually much lower in women of advanced reproductive age (under 10 embryos on average). With one in two embryos expected to be aneuploid, one in four HLA-matched and three of four unaffected in autosomal recessive conditions, the overall probability of finding a suitable embryo for transfer could not be expected to be higher than 1 in 10 embryos. So with the availability of only 8.8 embryos on average with conclusive results in this material, only one HLA-matched unaffected euploid embryo would have been expected to be available for transfer. In fact, with the present tendency of limiting the transfer to only one blastocyst, to avoid multiple pregnancies, the availability of a single euploid embryo for transfer is quite sufficient to obtain a clinical pregnancy and birth of an HLA-identical progeny for stem cell transplantation for the affected siblings. 99

12 The usefulness of aneuploidy testing is also obvious for diagnostic accuracy improvement, as the error in the copy number of chromosomes may lead to misdiagnosis in testing for the causative gene and HLA typing. For example, the data further confirm an approximately 6% aneuploidy rate for chromosome 6 (data not shown), which could affect the HLA-typing results. Comparable aneuploidy rates for other chromosomes in which causative genes tested are located, such as beta-globin gene in chromosome 11, may also affect PGD results (Kuliev et al., 2005). Thus, in addition to avoidance of the transfer of chromosomally abnormal embryos, the testing for the copy number of chromosomes may become an important requirement for achieving the accuracy of PGD and preimplantation HLA typing. The follow-up studies of the mutant oocytes and embryos and the pregnancy outcomes did not find any misdiagnosis, suggesting an extremely high specificity and sensitivity of the presently used molecular genetic analysis. In conclusion, despite ethical issues involved in preimplantation HLA typing (Edwards, 2004; Forst, 2004; Robertson, 2005), the presented results show the increasing attractiveness of this option for couples with affected children requiring HLA compatible stem cell transplantation. With introduction of aneuploidy testing, this may also expand the practical application of preimplantation HLA typing to patients of advanced reproductive age, allowing improvement of their chances to become pregnant and deliver an HLA-matched progeny for stem cell transplantation in the affected siblings. Acknowledgements Munné S 2002 Preimplantation genetic diagnosis of numerical and structural chromosome abnormalities. Reproductive BioMedicine Online 4, Munné S, Sandalinas M, Escudero T et al Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reproductive BioMedicine Online 7, Munné S, Magli C, Cohen J et al Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Human Reproduction 14, Rechitsky S, Kuliev A, Tur-Kaspa I et al Preimplantation genetic diagnosis with HLA matching. Reproductive BioMedicine Online 9, Robertson JA 2003 Extending preimplantation genetic diagnosis: the ethical debate. Ethical issues in new uses of preimplantation genetic diagnosis. Human Reproduction 18, Van de Velde H, Georgiou I, De Rycke M et al Novel universal approach for preimplantation genetic diagnosis of β-thalassemia in combination with HLA matching of embryos. Human Reproduction 19, Verlinsky Y, Kuliev A 2005 Atlas of Preimplantation Genetic Diagnosis, 2nd edn. Taylor and Francis, London and New York, p Verlinsky Y, Rechitsky S, Sharapova T et al Preimplantation HLA typing. Journal of the American Medical Association 291, Verlinsky Y, Rechitsky S, Schoolcraft W et al Preimplantation diagnosis for Fanconi anemia combined with HLA matching. Journal of the American Medical Association 285, Verlinsky Y, Rechitsky S, Schoolcraft W et al Designer babiesare they reality yet? Case report: simultaneous preimplantation genetic diagnosis for Fanconi anemia and HLA typing for cord blood transplantation. Reproductive BioMedicine Online 1, 31. Received 3 August 2005; refereed 12 September 2005; accepted 30 September We are grateful to our colleagues in DNA and embryology laboratories, and also to our genetic counsellors, C. Lavin, R. Beck, R. Genoveze and D. Pauling for their participation in acquisition of the data and technical assistance. References 100 Edwards RG 2004 Ethics of PGD: thoughts on the consequences of typing HLA in embryos. Reproductive BioMedicine Online 9, Fost NC 2004 Conception for donation. Journal of the American Medical Association 291, Gianaroli L, Magli MC, Ferraretti A 2004 The beneficial effects of PGD for aneuploidy support extensive clinical application. Reproductive BioMedicine Online 10, Gianaroli L, Magli MC, Ferraretti AP, Munne S 1999 Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with poor prognosis: identification of the categories for which it should be proposed. Fertility and Sterility 72, Kahraman S, Karlilaya G, Sertyel S et al Clinical aspects of preimplantation genetic diagnosis of single gene disorders combined with HLA typing. Reproductive BioMedicine Online 9, Kuliev A, Verlinsky Y 2004 Preimplantation HLA typing and stem cell transplantation. Reproductive BioMedicine Online 9, Kuliev A, Rechitsky S, Verlinsky O et al Preimplantation diagnosis and HLA typing for haemoglobin disorders. Reproductive BioMedicine Online 11, Kuliev A, Cieslak J, Illkewitch Y, Verlinsky Y 2003 Chromosomal abnormalities in a series of 6733 human oocytes in preimplantation diagnosis of age-related aneuploidies. Reproductive BioMedicine Online 6,