Molecular Human Reproduction, Vol.17, No.5 pp.

275 285, 2011 Advanced Access publication on February 14, 2011 doi:10.1093/molehr/gar012

NEW RESEARCH HORIZON Review

Polar body-based preimplantation genetic diagnosis for Mendelian disorders

Anver Kuliev * and Svetlana Rechitsky
Reproductive Genetics Institute, 2825 North Halsted St., Chicago, IL 60657, USA *Correspondence address. Tel: + 1-773-472-4900; Fax: + 1-773-871-5221; E-mail: anverkuliev@hotmail.com

Submitted on December 16, 2010; resubmitted on January 21, 2011; accepted on February 7, 2011

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abstract: Introduced . 20 years ago, the use of polar bodies (PBs), involving sequential removal and genetic analysis of the rst (PB1)
and second (PB2) PB, provides the option for pre-embryonic diagnosis, when the objection to the embryo biopsy procedures makes preimplantation genetic diagnosis (PGD) non-applicable. PB-based approach has presently been utilized in PGD for genetic and chromosomal disorders, applied either separately, or together with embryo biopsy approaches, especially if there are two or more PGD indications. We present here the worlds largest experience of 938 PGD cycles for single-gene disorders performed by PB testing for 146 different monogenic conditions, which resulted in the birth of 345 healthy children (eight pregnancies are still ongoing), providing strong evidence that PBbased PGD is a reliable and safe procedure, with an extremely high accuracy rate of over 99%. With application of microarray technology, PB-based approach will be utilized for increasing number of indications, involving simultaneous testing for 24 chromosomes and single-gene disorders. Key words: rst and second polar body / single-gene disorders / indications for polar body analysis / pre-embryonic diagnosis / accuracy of PB analysis

Introduction
The use of polar bodies (PBs) as an earlier approach to prenatal diagnosis was proposed by the World Health Organization in the early 1980s (Kuliev et al., 1985), and applied clinically in 1990 (Verlinsky et al., 1990), representing now one of the established approaches to preimplantation genetic diagnosis (PGD) (PGDIS, 2008; ESHRE Consortium, 2011). The idea of performing PGD with the use of PB is based on the fact that the rst (PB1) and second (PB2) PB are the by-products of female meiosis, allowing the prediction of the resulting genotype of the maternal contribution to the embryos. Neither PB1, which is extruded as a result of the rst meiotic division, nor PB2, extruded following the second meiotic division, have any known biological value for pre- and post-implantation embryo development, and may be removed without any embryo damage and tested to evaluate the resulting embryo genotype. Initially, only PB1 was tested, based on the fact that in the absence of crossing-over, PB1 will be homozygous for the allele not contained in the oocyte and PB2 (Verlinsky and Kuliev, 1991). However, PB1 approach was not applicable for predicting the eventual genotype of the oocytes, if crossing-over occurred, because the primary oocyte in this case will be heterozygous for the abnormal gene. As the frequency of crossing-over varies with the distance between the locus and the centromere, the PB1 approach

appeared to be of a limited value, unless the oocytes can be further tested by PB2 analysis, to detect hemizygous normal oocytes resulting after meiosis II. Thus, PB-based PGD technique requires a sequential testing of PB1 and PB2 (Fig. 1) (Verlinsky and Kuliev, 2000). As will be demonstrated below, although PB approach may be sufcient to perform PGD with high accuracy, other approaches, such as single blastomere removal at the cleavage stage, or blastocyst biopsy, should be available to ensure PGD application in complex cases, and to avoid the transfer of affected embryos, determined by paternally derived mutations. In addition, these methods also provide a conrmatory diagnosis following the PB diagnosis. The choice of additional methods will differ depending on circumstances, and a reliable diagnosis may require using two or even three different methods, especially when there is more than one indication for PGD, such as PGD for single-gene disorders together with HLA typing, or preimplantation HLA typing together with aneuploidy testing. Combined testing is required with the expanding range of PGD indications when testing is performed for causative gene, linked markers, HLA typing and aneuploidy in the same case. In this paper, we will present the worlds largest experience of 938 PGD cycles for Mendelian disorders performed for 146 monogenic conditions with the use of PB approach, which resulted in preselection and transfer of embryos originating from mutation-free oocytes in 84%

& The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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of cases, demonstrating the safety, reliability and extremely high accuracy of the procedure.

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and Kuliev, 2000), the remaining 701 cases involved additional blastomere and/or blastocyst biopsy (De Boer et al., 2004).

Materials and Methods

As seen from Table I, 2158 PGD cycles were initiated from 1206 patients in the period of 1990 2010, of which 938 performed by the use of PB1 and PB2 sampling from 553 patients at risk for producing offspring with inherited disorders will be presented. While 237 of these cycles were done exclusively by the use of PB approach described elsewhere (Verlinsky

PB removal
PB1 and PB2 were removed following stimulation and oocyte retrieval using a standard IVF protocol. Following extrusion of PB1, the zona pellucida is opened mechanically using a microneedle, and PB1 aspirated into a blunt micropipette. Although this mechanical method is safe and highly efcient for PB removal, laser-assisted techniques were also applied in other centers (Montag et al., 2004). The oocytes were then inseminated with motile sperm, or using ICSI, and examined for the presence of pronuclei and extrusion of PB2, which were removed in the same manner as PB1. The biopsied oocytes were then returned to culture, checked for cleavage and transferred, depending on the genotype of the corresponding PB1 and PB2. Although PB1 and PB2 have no known biological signicance in pre- and post-implantation development, follow-up studies have been carried out to investigate possible detrimental effect of the procedure. These studies showed no differences in fertilization rate or blastocyst formation after PB1 removal in comparison with non-biopsied ooctyes: nor was any detrimental effect observed after PB2 removal or after PB1 and PB2 sampling followed by embryo biopsy, which was evident from cleavage rate, blastocyst formation and the number of cells in the respective blastocysts (Magli et al., 2004; Verlinsky and Kuliev, 2005; Cieslak et al., 2006).

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Pre-embryonic diagnosis
When required, PB testing was completed prior to embryo formation, for which purpose PB2 was removed and tested before pronuclei fusion, and the PB testing was completed at the same cycle. In such cycles, only zygotes originating from mutation-free oocytes were allowed to proceed to embryo development and transferred in the same cycle, avoiding the formation and possible discard of any affected embryo. This became realistic with the progress of completing the genetic analysis prior to pronuclei fusion. In such cycles, oocytes were obtained in a standard IVF protocol, from which PB1s were removed as usual 3.5 h after aspiration, and followed by PB2 sampling, 6.5 h after ICSI (Kuliev et al., 2006). The analysis for maternal mutation in the biopsied PB1 and PB2 were completed in , 9 h, while all the oocytes were still at the pronucleus stage; so, the affected oocytes were frozen at this particular stage, prior to the embryo formation, whereas the embryos originating from the mutationfree oocytes were allowed to develop and replaced after reaching the blastocyst stage.

Figure 1 Principle of PB-based PGD for single-gene disorders example of PGD for CFTR mutation. The large circle represents the genotype of oocyte obtained from heterozygous carrier of mutant gene prior to PB1 extrusion (metaphase I, MI). Other seven big circles show oocytes resulting from the rst (metaphase II, MII) and second meiotic divisions. Seven smaller circles show extruded PB1s, and ve smallest onesPB2s. N, represents normal; CF, affected (CFTR) allele; C1 and C2, possible outcomes from heterozygous MII oocyte after the second meiotic division. Shaded circles represent oocytes and PBs containing mutant gene. Upper portion shows extrusion of homozygous normal PB1, resulting in affected oocyte, conrmed by mutant PB2 (shaded small circle), while left-hand portion presents opposite sequence of events. Crossover situation evidenced by heterozygous PB1 is shown in middle, resulting in either affected (C1) (evidenced by normal PB2 extrusion) or normal (C2) oocyte (evidenced by abnormal PB2 extrusion).

DNA analysis
To perform genetic analysis, the biopsied PB1 and PB2 were placed directly into a lysis solution, consisting of 0.5 mcl 10 PCR buffer, 0.5 mcl 1% Tween 20, 0.5 mcl 1% Triton X-100, 3.5 mcl H2O and

Table I Clinical outcome of 2158 PGD cycles for Mendelian disorders.

Cells tested PB PB + BL Subtotal BL + BC Total Patient 131 422 553 653 1206 Cycle 237 701 938 1220 2158 Embryo transfers 188 602 790 988 1778 # of embryos transferred 379 1199 1578 1859 3437 Pregnancy 72 (38.3%) 257 (41.7%) 329 406 735 (41.3%) Birth 64 (34%) 281 (46%) (8) 345 (8)a 343 (46)a 688 (38.7%) (54)a

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PB, polar body; BL, blastomere; BC, blastocyst. a Ongoing clinical pregnancy.

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absence of ADO of either normal or mutant allele, and allows avoiding the potential misdiagnosis due to ADO. Although most of the transferred embryos were pre-selected using this particular strategy, the embryos originating from homozygous normal oocytes, inferred from homozygous mutant status of PB1 and hemizygous normal status of PB2, may also be transferred, provided that ADO could be excluded using sufcient number of linked polymorphic markers. Our preferred approach for avoiding misdiagnosis was a sequential genetic analysis of the PB1 and PB2 in PGD for maternally derived mutations. Detection of both mutant and normal alleles in the heterozygous PB1, together with the mutant allele in the corresponding PB2, leaves no doubt that the resulting maternal contribution to the embryo is normal, even without testing for the linked markers as a control. As mentioned, the mutation-free oocytes may be also predicted when corresponding PB1 is homozygous mutant, and corresponding PB2 is hemizygous normal (Fig. 1). However, if the corresponding PB1 is in fact heterozygous, but erroneously diagnosed as homozygous normal because of ADO of the normal allele, the extrusion of a normal allele with PB2 would result in misdiagnosis of the mutant oocyte as normal. Therefore, the embryos resulting from the oocytes with homozygous mutant PB1 cannot be acceptable for transfer, unless the heterozygous status of PB1 is excluded by the use of sufcient number of linked markers as described. The example of misdiagnosis, due to ADO of the normal allele in PB1, has been described earlier in a PGD for fragile site mental retardation 1 (FMR1) (Verlinsky and Kuliev, 2006). So, to completely avoid misdiagnosis, a sequential PB1 and PB2 may be required to combine with multiplex PCR to exclude the possibility of an undetected ADO in the actual heterozygous PB1. We previously demonstrated that more than half of ADOs can be detected by sequential PB1 and PB2 analysis; the remaining cases detected by multiplex PCR (Verlinsky and Kuliev, 2006). The other misdiagnosis was observed in PGD for myotonic dystrophy, which was also due to undetected ADO, as only one linked marker was available for testing, which is, of course, not sufcient for accurate diagnosis. So, overall, two misdiagnoses were observed in 790 PB-based PGD transfer cycles, suggesting an extremely high accuracy rate of 99.7%. This is in agreement with 99.4% accuracy rate per transfer cycles in our overall experience of PGD for Mendelian disorders. No misdiagnosis was observed also in a few previous case reports performed by PB biopsy approach (Tomi et al., 2006; Altarescu et al., 2008, 2009).

0.05 mcl Protenase K (20 mg/ml in a 0.5 ml PCR tube). After spinning down at a low speed in a microfuge for a few seconds, the samples were covered with one drop of mineral oil and incubated at 458C for 15 min in a thermal cycler. Proteinase K was then inactivated at 968C for 20 min, which was also the beginning of the hot start of the rst round PCR. Lower stringency and longer annealing time were used in the rst round PCR, with the introduction of the mixture of all outside primers for both mutant genes and polymorphic markers. Following the rst round PCR, separate aliquots were amplied in the second round PCR with specic inside primers for each site, using a higher stringency (Rechitsky et al., 2000). To avoid misdiagnosis due to preferential amplication, multiplex nested PCR was performed, involving a simultaneous detection of the mutant gene together with up to three or more highly polymorphic markers, closely linked to the gene tested. Only when the polymorphic sites and the mutation agreed, were embryos transferred. So, multiplex amplication allowed detecting allele-drop out (ADO) and prevented the transfer of misdiagnosed affected embryos. In addition to short tandem repeats (STRs) linked to the genes studied, STRs located on other chromosomes were also studied for testing of a possible contamination by extraneous DNA, and identication of the origin of individual embryos in the established pregnancies. A list of STRs, their sequences and PCR conditions for their analysis were described elsewhere (Verlinsky and Kuliev, 2006).

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Results and discussion

PGD outcome of PB testing
Table I presents our overall data of 2158 PGD cycles for single-gene disorders, which is the worlds largest experience in one centre. A total of 938 of these cycles were performed by the PB approach, involving the retrieval and testing of 9036 oocytes, of which 7841 (86.8%) were with both PB1 and PB2, with the results of sequential PB1 and PB2 testing obtained in 7650 (97.6%) of these oocytes. This made it possible to preselect for transfer as many as 1578 embryos originating from these oocytes (1.99 on the average), in 790 (84.2%) cycles, resulting in 329 pregnancies (41.6% per transfer cycle) and birth of 345 healthy children (eight clinical pregnancies are ongoing). Additional embryo biopsy was applied in 701 of these cases, to conrm the diagnosis when necessary, or identify unaffected embryos for transfer in the absence of embryos originating from mutation-free oocytes, such as heterozygous carrier embryos, originating from mutant oocytes. As mentioned, the type of additional tests differed depending on the type and number of indications for PGD and an example for PGD for congenital disorder of glycosylation [phosphomannomutase-2 (PMM2) gene] is shown in Fig. 2.

Autosomal recessive disorders

The list of Mendelian disorders for which PGD was performed by PB analysis is presented in Table II, showing that autosomal recessive disorders were the most frequent indication for PGD by PB analysis, performed for 81 recessive conditions in 504 (53.7%) of an overall 938 PB cycles. PB analysis is the method of choice for autosomal recessive disorders, because for avoiding the transfer of embryos with autosomal recessive conditions, it is sufcient to preselect the embryos originating from the mutation-free oocytes. The largest group of autosomal recessive disorders performed by PB analysis were hemoglobinopathies, applied extensively in Cyprus (Kuliev et al., 2005). As a result of PB testing performed in 504 PGD cycles for recessive conditions, a total of 882 (2.1 embryos on the average) unaffected

PB testing accuracy
Of a total of 9036 oocytes tested, amplication of both PB1 and PB2 was successful in 7650 PB1/PB2 sets, suggesting an extremely high (97.6%) amplication efciency. This allowed transferring embryos in 84.2% of initiated cycles, with the priority in preselection of mutationfree oocytes given to the oocytes with heterozygous PB1, i.e. with both normal and mutant genes amplied, ideal for further testing, despite the fact that their potential transfer depended entirely on the identication of mutant gene in the sequential analysis of PB2 (Fig. 1). In the absence of DNA contamination, this indicates the

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Figure 2 PGD by sequential PB1, PB2 and blastomeres analysis for congenital disorder of glycosylation (PMM2 gene). (A) Pedigree and table with PGD results of testing for PMM2 gene and six linked markers. As can be seen from pedigree, the mother and father are carriers of different mutations (P113L and R141H, respectively), their affected child being double-heterozygous for PMM2 mutation. (B) Sequential PB1 and PB2 analysis of 12 oocytes available for testing resulted in the prediction of oocyte status in 11 of them (PB2 of oocye # 3 failed to amplify). Of 12 PB1s tested, # 11 appeared to be homozygous normal, and # 13 homozygous mutant, conrmed by PB2 testing, and further testing by blastomere analysis in embryo # 11. The remaining oocytes were heterozygous; so based on the sequential testing of PB2 mutant, four of them (oocytes # 2, 5, 9 and 15) were predicted free of PMM2 mutation, requiring no further blastomere testing. (C) A follow-up blastomere testing of embryos originating from mutant oocytes or the oocyte with failed amplication of PB2, showing the preselection of additional six unaffected embryos, of which carrier embryo # 8 and embryo originating from oocyte 5, with heterozygous PB1 and mutant PB2, were transferred, resulting in unaffected ongoing pregnancy.

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Table II List of Mendelian disorders for which PGD performed by PB-based approach.
Genetic disorder ACYL-CoA DEHYDROGENASE, MEDIUM-CHAIN, DEFICIENCY ACYL-CoA DEHYDROGENASE, LONG-CHAIN, DEFICIENCY ACYL-CoA DEHYDROGENASE, VERY LONG-CHAIN; ACADVL ARGININOSUCCINIC ACIDURIA CEROID LIPOFUSCINOSIS, NEURONAL 2, LATE INFANTILE; CLN2 CITRULLINEMIA CONGENITAL ADRENAL HYPERPLASIA (CAH) DEAFNESS, NEUROSENSORY, AUTOSOMAL RECESSIVE 1; DFNB1 CYSTIC FIBROSIS; CF CYSTINOSIS, NEPHROPATHIC; CTNS ECTODERMAL DYSPLASIA, HYPOHIDROTIC EPIDERMOLYSIS BULLOSA DYSTROPHICA FANCONI ANEMIA, COMPLEMENTATION GROUP A FANCONI ANEMIA, COMPLEMENTATION GROUP F FANCONI ANEMIA, COMPLEMENTATION GROUP J GAUCHER DISEASE, TYPE I GLUTATHIONE SYNTHETASE DEFICIENCY GLYCOGEN STORAGE DISEASE II HEMOGLOBINALPHA LOCUS 1; HBA1 HEMOGLOBINBETA LOCUS; HBB HURLER SYNDROME HYPOPHOSPHATASIA, INFANTILE ISOVALERIC ACIDEMIA; IVA KRABBE DISEASE LEUKOENCEPHALOPATHY WITH VANISHING WHITE MATTER; VWM HOMOCYSTINURIA DUE TO DEFICIENCY OF N(5,10)-METHYLENETETRAHYDROFOLATE REDUCTASE ACTIVITY NEPHROSIS 1, CONGENITAL, FINNISH TYPE; NPHS1 NEUROPATHY, HEREDITARY SENSORY AND AUTONOMIC, TYPE III; HSAN3 OCULOCUTANEOUS ALBINISM, TYPE I; OCA1 OSTEOPETROSIS, AUTOSOMAL RECESSIVE POLYCYSTIC KIDNEY DISEASE, AUTOSOMAL RECESSIVE; ARPKD PROPIONIC ACIDEMIA SANDHOFF DISEASE SICKLE CELL ANEMIA SMITH-LEMLI-OPITZ SYNDROME; SLOS SPINAL MUSCULAR ATROPHY, TYPE I; SMA1 TAY-SACHS DISEASE; TSD THROMBOTIC THROMBOCYTOPENIC PURPURA, CONGENITAL; TTP TYROSINEMIA, TYPE I ZELLWEGER SYNDROME; ZS ATAXIA-TELANGIECTASIA; AT ADENOMATOUS POLYPOSIS OF THE COLON; APC ANGIOEDEMA, HEREDITARY; HAE BRAIN TUMOR, POSTERIOR FOSSA OF INFANCY, FAMILIAL BREAST CANCER, FAMILIAL BREAST-OVARIAN CANCER, FAMILIAL CARDIOMYOPATHY, DILATED, 1A; CMD1A Gene ACADM ACADL ACADVL ASL CLN2 ASS CYP21A2 GJB2 CFTR CTNS EDA COL7A1 FANCA FANCF FANCJ GBA GSS GAA HBA1 HBB IDUA ALPL IVD GALC EIF2B2 MTHFR NPHS1 IKBKAP TYR TCIRG1 PKHD1 PCCA HEXB HBB DHCR7 SMN1 HEXA ADAMTS13 FAH PEX1 ATM APC AD SMARCB1 BRCA1 BRCA2 LMNA Inheritance AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AR AD AD AD AD AD AD

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Table II Continued
Genetic disorder CHARCOT-MARIE-TOOTH DISEASE, AXONAL, TYPE 2E CHARCOT-MARIE-TOOTH DISEASE, DEMYELINATING, TYPE 1A CHARCOT-MARIE-TOOTH DISEASE, DEMYELINATING, TYPE 1B CRANIOFACIAL DYSOSTOSIS, TYPE I; (CFD1) DARIER-WHITE DISEASE; DAR DIAMOND-BLACKFAN ANEMIA; DBA DYSTROPHIA MYOTONICA 1 EPIPHYSEAL DYSPLASIA, MULTIPLE, 1; EDM1 HUNTINGTON DISEASE; HD LI-FRAUMENI SYNDROME 1; LFS1 LOEYS-DIETZ SYNDROME; LDS MACHADO-JOSEPH DISEASE; MJD MARFAN SYNDROME; MFS MIGRAINE, FAMILIAL HEMIPLEGIC, 1; FHM1 MULTIPLE ENDOCRINE NEOPLASIA, TYPE I; MEN1 NEUROFIBROMATOSIS, TYPE I; NF1 NEUROFIBROMATOSIS, TYPE II; NF2 OCULOCUTANEOUS ALBINISM, TYPE II; OCA2 OMENN SYNDROME OPTIC ATROPHY 1; OPA1 OSTEOGENESIS IMPERFECTA CONGENITA; OIC POLYCYSTIC KIDNEY DISEASE 1; PKD1 POPLITEAL PTERYGIUM SYNDROME; PPS RETINOBLASTOMA; RB1 SPINOCEREBELLAR ATAXIA 1; SCA1 SPINOCEREBELLAR ATAXIA 2; SCA2 SPINOCEREBELLAR ATAXIA 6; SCA6 SPINOCEREBELLAR ATAXIA 7; SCA7 STICKLER SYNDROME, TYPE I; STL1 SYMPHALANGISM, PROXIMAL; SYM1 TORSION DYSTONIA 1, AUTOSOMAL DOMINANT; DYT1 TREACHER COLLINS-FRANCESCHETTI SYNDROME; TCOF TUBEROUS SCLEROSIS TYPE 1 TUBEROUS SCLEROSIS TYPE 2 VON HIPPEL-LINDAU SYNDROME; VHL ADRENOLEUKODYSTROPHY; ALD AGAMMAGLOBULINEMIA, X-LINKED; XLA ALBINISM, OCULAR, TYPE I; OA1 ALPORT SYNDROME, X-LINKED; ATS CHARCOT-MARIE-TOOTH DISEASE, X-LINKED, 1; CMTX1 CHOROIDEREMIA; CHM EMERY-DREIFUSS MUSCULAR DYSTROPHY, X-LINKED; EDMD FABRY DISEASE FRAGILE SITE MENTAL RETARDATION 1 GRANULOMATOUS DISEASE, CHRONIC, X-LINKED; CGD HEMOPHILIA A HEMOPHILIA B Gene NEFL PMP22 MPZ FGFR2 ATP2A2 RPS19 DMPK COMP HTT TP53 TGFBR2 ATX3 FBN1 CACNA1A MEN1 NF1 NF2 OCA2 RAG1 OPA1 COL1A1 PKD1 IRF6 RB1 ATXN1 ATX2 CACNA1A SCA7 COL2A1 NOG DYT1 TCOF TSC1 TSC2 VHL ABCD1 BTK OA1 AMMECR1 GJB1 CHM EMD GLA FMR1 CYBB F8 F9 Inheritance AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD XL XL XL XL XL XL XL XL XL XL XL XL

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Table II Continued
Genetic disorder HYDROCEPHALUS, X-LINKED; L1CAM IMMUNODEFICIENCY WITH HYPER-IgM, TYPE 1; HIGM1 IMMUNODYSREGULATION, POLYENDOCRINOPATHY, AND ENTEROPATHY, X-LINKED; IPEX INCONTINENTIA PIGMENTI; IP MUCOPOLYSACCHARIDOSIS TYPE II (HUNTER) MUSCULAR DYSTROPHY, DUCHENNE TYPE; DMD MYOTUBULAR MYOPATHY 1; MTM1 NORRIE DISEASE; NDP ORNITHINE TRANSCARBAMYLASE DEFICIENCY PELIZAEUS-MERZBACHER-LIKE DISEASE; PMLD RETT SYNDROME; RTT WISKOTT-ALDRICH SYNDROME; WAS Gene L1CAM CD40LG FOXP3 IKBKG IDS DMD MTM1 NDP OTC PLP1 MECP2 WAS Inheritance XL XL XL XL XL XL XL XL XL XL XL XL

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Table III Results of 938 PGD cycles for Mendelian disorders performed by PB analysis.
Cells tested Autosomal recessive Polar bodies Polar bodies + blastomere/blastocyst Subtotal Autosomal dominant Polar bodies Polar bodies + blastomere/blastocyst Subtotal X-Linked Polar bodies Polar bodies + blastomere/blastocyst Subtotal Total
a b

Cycles

ETa

# Embryos

Pregnancy

Delivery

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115 389 504 46 118 164 76 194 270 938 94 334 428 37 105 142 57 163 220 790 199 683 882 78 207 285 102 309 411 1578 (1.99) 33 137 170 20 45 65 19 75 94 329 (41.6%) 32 158 190 17 47 64 15 76 91 345 (8)b (43.6%)

Embryo transfer. Ongoing clinical pregnancy.

embryos were preselected for transfer in 428 (84.9%) of these cycles, yielding 170 (39.7%) clinical pregnancies and 190 healthy children born, with no misdiagnosis (Table III). As can be seen from Fig. 2, presenting PB-based PGD for congenital disorder of glycosylation (mutation in PMM2 gene), the sequential PB1 and PB2 analysis provides a robust procedure for selection of embryos originating from mutation-free oocytes (oocytes # 2, 5, 9, 13 and 15). The case also shows the utility of additional blastomere testing to identify the unaffected embryos originating from the mutant oocytes, such as embryos # 4, 7, 8, 11 and 14. One of these embryos (carrier embryo # 8) was transferred, together with embryo # 5, originating from oocytes with heterozygous PB1 and mutant PB2, resulting in an unaffected ongoing singleton pregnancy.

X-linked disorders
X-linked conditions were the second largest group of indications for performing PB analysis, involving PGD for 24 different conditions (Table II). A total of 270 cycles were performed, which resulted in the transfer of 411 unaffected embryos (1.9 embryos on the average) in 220 (81.5%) cycles, yielding 94 clinical pregnancies and birth of 91 children (Table III), with one misdiagnosis for FMR1, mentioned. This involved indirect testing using linkage analysis, as no test is available for direct analysis of the expanded alleles. So, the mutation-free oocytes in this case were inferred from the presence of the linked markers for the normal allele, and misdiagnosis was due to the fact that the number of markers amplied was not sufcient for higher accuracy of diagnosis. However, despite predicted 10% error rate in one of the embryos,

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Figure 3 PB-based PGD for X-linked adrenoleukodystrophy (ABCD1 Gene). (A) Pedigree and table with PGD results of testing for ABCD1 gene and seven linked markers. Haplotypes of the mother and grandmother carrying a mutation are shown in red in the left columns and the normal haplotypes shown in black on the right columns. The only maternal brother is affected with mutant allele inherited from their mother (shown in red). On the left of the pedigree, an unaffected child, born as a result of PGD, is shown carrying only normal maternal allele (linked to seven markers). (B) As can be seen from the sequential analysis of PB1 and PB2, oocytes # 7 and # 8 were diagnosed as affected based on homozygous normal PB1 and mutant PB2, while oocyte # 3 was predicted normal, as evidenced by mutant PB1, conrmed by all seven linked markers and normal PB2, in agreement with all the linked markers. This embryo was transferred, resulting in normal singleton pregnancy and birth of the unaffected child, mentioned.

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Figure 4 PGD for myotonic dystrophy by sequential PB1 and PB2 analysis. (A) Pedigree showing haplotypes of the mother, who inherited an expanded allele of dystrophia myotonica-protein kinase (DMPK) gene (shown in red) from her father (normal haplotype is shown in black). Her two brothers are also affected, while the only sister is normal. (B) Sixteen oocytes were tested by PB1 (top) and PB2 (middle) analysis for the presence of the expanded allele in DMPK gene by six polymorphic markers. Embryos, originating from oocytes # 7, 9, 12, 14, 16, 22 and 23 were predicted normal based on the presence of the normal maternal chromosome (haplotype) (black bar). Embryos # 3, 5, 6, 10, 11, 13, 15 and 17 were affected, evidenced by the presence of the maternal affected haplotype (red bar). PB1 in the oocyte # 19 was heterozygous, but PB2 did not amplify; so genotype of these oocytes could not be predicted. One embryo originating from oocyte # 23 predicted free of expanded allele, based on the presence of heterozygous PB1 and hemizygous affected PB2, was transferred, resulting in singleton pregnancy and birth of a normal child (see pedigree). *Follow-up embryo biopsy required to check homozygous results for PB1.

the couple selected transferring this particular embryo, in addition to two embryos diagnosed with higher accuracy, which in fact turned out to be heterozygous because of an undetected ADO of both alleles linked to the normal gene (Verlinsky and Kuliev, 2006).

PB analysis is obviously the method of choice in PGD for X-linked disorders, because preselection of oocytes free of X-linked disorders allows avoiding any micromanipulation of the embryo. As seen from Fig. 3, demonstrating PB-based PGD for X-linked

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adrenoleukodystrophy, the embryos # 3, originating from the oocyte with mutant PB1 and normal PB2, conrmed with simultaneous testing of six linked markers, required no further testing and was transferred irrespective of gender or the paternal genetic contribution, resulting in birth of an unaffected child.

Kuliev and Rechitsky

Dominant disorders
In contrast to autosomal recessive and X-linked disorders, PB-based approach is applicable to only maternally derived dominant disorders. As listed in Table II, we performed PB-based PGD for 41 different dominant conditions of maternal origin; for such cases PB analysis was concentrated in detection and transfer of embryos deriving from oocytes with heterozygous PB1 and hemizygous mutant PB2. The example is demonstrated in Fig. 4, presenting PB-based PGD for myotonic dystrophy, in which the embryo originating from oocyte # 23 was predicted free from expanded allele, based on the presence of both normal and expanded allele in PB1 and an expanded allele in PB2. ADO of normal allele could not be totally excluded in cases of oocytes with homozygous mutant PB1 in oocytes # 3, 5 and 6; so, to transfer the embryos originating from these oocytes, the follow-up embryo biopsy was required, involving tracing the maternal and paternal normal haplotypes. PB approach was applied in a total of 164 PGD cycles for dominant disorders, which resulted in the transfer of 285 unaffected embryos (2.0 embryos on the average) in 142 (86.6%) cases, yielding 65 (45.8%) clinical pregnancies and birth of 64 children (Table III), with one misdiagnosis. The latter represented PGD for myotonic dystrophy, which is always a challenge, because the diagnosis is based on indirect testing, requiring sufcient number of linked markers, while only one such marker was available for testing.

may allow performing pre-embryonic diagnosis for any disease. On the other hand, PB approach is a component of PGD for couples with two or more PGD indications and should be available in testing of complex conditions, including Mendelian disorders and chromosomal aneuploidy, originating predominantly from female meiosis. A wider application of the PB approach to PGD of single-gene disorders may be expected with the present tendency of using PB biopsy for testing of chromosomal aneuploidy by FISH (Gianaroli et al., 2010), recently also with an application of microarray technology (Geraedts et al., 2010), which makes it possible to perform PGD simultaneously for multiple conditions, including chromosomal aneuploidy and singlegene disorders. So, the presented results of PB-based PGD demonstrates that this technique is highly efcient and reliable, providing extremely high accuracy of PGD for Mendelian disorders.

References
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De novo mutations
Because PB testing allows identifying the normal and mutant maternal haplotypes in the process of performing PGD procedure, it has a special utility in cases of maternally derived de novo mutations (DNM) of dominant origin, when neither origin, nor relevant haplotypes are available for tracing the inheritance of DNM in single cells biopsied from oocytes and embryos. We performed 151 PGD cycles for DNM, with the overall 84% success rate of identifying unaffected embryos for transfer, with the average 1.72 embryos per transfer (219 embryos transferred in 127 cycles), that resulted in 49.6% pregnancy rate and birth of 64 unaffected children, with no misdiagnosis observed in the follow-up analysis. In 54 (35.8%) of these cases, the testing was completed exclusively by sequential PB1 and PB2 testing. This was described in more detail elsewhere (Rechitsky et al., 2011).

Conclusion
Presented data show that PB-based approach is an integral part of PGD, which makes it possible to perform preselection of mutationfree oocytes and complete PGD prior to fertilization. This provides the possibility for pre-embryonic diagnosis for couples objecting to embryo biopsy because of their social or religious attitudes. Although the approach is currently limited to PGD for autosomal recessive conditions, X-linked disorders and dominant mutations of maternal origin, the future progress in testing of paternal mutations prior to fertilization

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