PREIMPLANTATION GENETIC DIAGnosis

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1 ORIGINAL CONTRIBUTION Preimplantation HLA Testing Yury Verlinsky, PhD Svetlana Rechitsky, PhD Tatyana Sharapova, MS Randy Morris, MD Mohammed Taranissi, MD Anver Kuliev, MD, PhD For editorial comment see p Context Preimplantation genetic diagnosis (PGD) has become an option for couples for whom termination of an affected pregnancy identified by traditional prenatal diagnosis is unacceptable and is applicable to indications beyond those of prenatal diagnosis, such as HLA matching to affected siblings to provide stem cell transplantation. Objective To describe preimplantation HLA typing, not involving identification of a causative gene, for couples who had children with bone marrow disorders at need for HLA-matched stem cell transplantation. Design, Setting, and Participants HLA matching procedures conducted at a single site during in an in vitro fertilization program for 9 couples with children affected by acute lymphoid leukemia, acute myeloid leukemia, or Diamond-Blackfan anemia requiring HLA-matched stem cell transplantation. In 13 clinical cycles, DNA in single blastomeres removed from 8-cell embryos following in vitro fertilization was analyzed for HLA genes simultaneously with analysis for short tandem repeats in the HLA region to select and transfer only those embryos that were HLA matched to affected siblings. Main Outcome Measures Results of HLA matching and pregnancy outcome. Results As a result of testing a total of 199 embryos, 45 (23%) HLA-matched embryos were selected, of which 28 were transferred in 12 clinical cycles, resulting in 5 singleton pregnancies and birth of 5 HLA-matched healthy children. Conclusion This is the first known experience of preimplantation HLA typing performed without PGD for a causative gene, providing couples with a realistic option of having HLA-matched offspring to serve as potential donors of stem cells for their affected siblings. JAMA. 24;291: PREIMPLANTATION GENETIC DIAGnosis (PGD) has become available as an alternative to prenatal diagnosis in order to avoid the risk for pregnancy termination, because PGD allows selection of unaffected embryos before a pregnancy is established. Despite the need for ovarian stimulation and in vitro fertilization (IVF) to be part of the procedure, PGD has become an acceptable method for avoiding the birth of children with genetic disorders. 1-3 Introduced more than a decade ago, PGD has been applied in thousands of clinical cycles. 4 At present, more than different genetic conditions are indications for PGD, including a few novel indications for which traditional prenatal diagnosis has never been used. 5-9 This includes preimplantation HLA matching combined with PGD, not only to allow couples to have an unaffected child, but also to select a potential donor progeny for stem cell transplantation. 8 The approach has previously been applied to avoid the birth of a child with Fanconi anemia (FA), which is a severe autosomal recessive disorder characterized by inherited bone marrow failure requiring bone marrow or cord blood transplantation from an HLAmatched sibling. 8 This approach resulted in the birth of an HLA-matched child free of FA whose cord blood stem cells were transplanted to the affected sibling with FA, resulting in a successful hematopoietic reconstitution. HLA matching would not be considered appropriate for prenatal diagnosis because of the potential for termination of pregnancy, which could not be justified for the reason of HLA incompatibility. We describe the first clinical experience of preimplantation HLA matching without PGD of a causative gene, demonstrating the feasibility of this novel approach for stem cell transplantation in siblings with bone marrow failure. METHODS Setting The preimplantation testing procedures were conducted at the Reproductive Genetics Institute (RGI), Chicago, Ill. The RGI was established in 1989 and in 19 was designated as a Pan American Health Organization/ World Health Organization Collaborating Center for Prevention of Genetic Disorders. The study was ap- Author Affiliations: Reproductive Genetics Institute, Chicago, Ill (Drs Verlinsky, Rechitsky, Morris, and Kuliev and Ms Sharapova); and Assisted Reproduction and Gynaecology Center, London, England (Dr Taranissi). Corresponding Author: Anver Kuliev, MD, PhD, Reproductive Genetics Institute, 2825 N Halsted St, Chicago, IL 6657 (anverkuliev@hotmail.com). 24 American Medical Association. All rights reserved. (Reprinted) JAMA, May 5, 24 Vol 291, No

2 Figure 1. Set of Polymorphic STR Markers in HLA Region of Chromosome 6 Used for Preimplantation HLA Typing Chr 6 p q HLA Region 6p21.3 Genes HLA-F HLA-A HLA-E HLA-C HLA-B HLA-DR HLA-DQ Class II Class III Class I STRs D6S461 D6S276 D6S299 D6S464 D6S15 D6S36 D6S1624 D6S258 D6S248 MOG a,b,c,d RF D6S265 D6S51 MIB MICA,b,c,d N-2 D6S273 DN LH1 DQ-CAR II DQ-CAR G51152 D6S2447 TAP1 Ring 3CA D6S439 D6S291 D6S426 proved by the RGI institutional review board, which is composed of individuals who are independent of the RGI and which includes 2 obstetricians; 1 clinical geneticist physician; 1 internist; 1 anesthesiologist; 1 individual with a master s degree in genetic counseling; 1 medical laboratory technologist; 2 individuals with doctorate degrees in public health and psychology, respectively; 3 individuals with master s degrees in public relations, industrial relations, and nursing, respectively; and a church pastor. Of these, 6 are women. The institutional review board approval covers protocol and consent forms, which allows publication of the preimplantation genetic diagnosis data presented herein. All sets of parents provided written informed consent for preimplantation genetic diagnosis testing. IVF Protocol Thirteen IVF cycles (initial cycles did not have the desired outcome for 4 couples) were initiated in at the RGI for 9 couples from the United States and England who had a child affected with acute lymphoid leukemia or acute myeloid leukemia (11 cycles), or Diamond- Blackfan anemia (DBA) (2 cycles). These conditions require bone marrow transplantation and also have been successfully treated by cord blood transplantation. 1 Although mutation testing also may be required in combination with HLA typing in DBA, which can be caused by mutations in the gene encoding ribosomal protein S19 on chromosome 19 (19q13.2) or in another gene mapped to chromosome 8 (8p23.3-p22), the majority of DBA cases are sporadic with no mutation detected, 11 such as in both cases included in this study (testing for Graphic representation of short tandem repeats (STRs) in HLA classes I, II, and III on chromosome 6. All STR markers are dinucleotide repeats (AC)n, except for DQ- CARII, which is (CTG)n; TNF b,c,d (CT)n; 62 (TC- CA)n; MICA (GCT)n; D6S51 (CA-GA)n; MOG d (CTC)n. The STRs needed for identification of matching are shown in relation to genes in the HLA region, ordered from telomere (top) to centromere (bottom), allowing accurate HLA typing and identification of possible recombinations, which may lead to misdiagnosis. Markers with asterisks indicate possible ambiguity in localization. the mutation was performed elsewhere). Each of the couples desired to have another child a desire separate from the hope of having another child who could potentially serve as a donor of stem cells for the affected sibling, for whom an acceptable HLA match had not been found. As there is no institutional setting for identification of couples at need for preimplantation HLA testing, all couples requested the test on their own, having learned of the availability of the technique through their physicians or the Internet. A standard protocol for IVF, which includes a psychological evaluation performed by a psychologist at the RGI, was used in combination with a micromanipulation procedure to remove single blastomeres from the cleaving embryos at day 3, as described elsewhere. 12 HLA genes from the blastomeres were tested simultaneously with short tandem repeats in the HLA region 13 using a multiplex heminested polymerase chain reaction (PCR) system 12,14 involving only closely linked polymorphic short tandem repeat markers located throughout the HLA region, as shown in FIGURE 1 (HLA genes: HLA-A1, -A2, -A3, -A24; -A32; HLA- C4, -C6; HLA-B27, -B18, -B35, -B51, -B57; HLA-DRB1 * 1, -DRB1 * 7, -DRB1 * 1, and -DRB1 * 11. Short tandem repeats: D6S461; D6S276 * ; D6S299; D6S464 * ; D6S15; D6S36 * ; D6S1624 * ; D6S258; D6S248 * ; MOG a,b,c,d; RF; D6S265; D6S51; MIB; MICA;,b,c,d; 62; 82-1; 9N-2; D6S273 * ; DN; LH1; DQ- CAR II; DQ-CAR; G51152; D6S2447; TAP1; Ring 3CA; D6S439 * ; D6S291; and D6S426). Figure 1 presents positions of closely linked short tandem repeats throughout the HLA region ordered from telomere (top) to centromere (bottom), allowing accurate HLA typing and identification of possible recombinations, which may lead to misdiagnosis. An example of typing for a marker is given in FIGURE 2. For each family we selected heterozygous alleles and markers not shared by the parents. Such markers provide the information about the origin of chromosome 6. A haplotype analysis for father, 28 JAMA, May 5, 24 Vol 291, No. 17 (Reprinted) 24 American Medical Association. All rights reserved.

3 mother, and the affected child was performed for each family prior to preimplantation HLA typing. This allowed avoidance of misdiagnosis due to preferential amplification and allele drop out exceeding 1% in PCR analysis of single blastomeres, 12,14 potential recombination within the HLA region, and a possible aneuploidy or uniparental disomy of chromosome 6, which may also affect the diagnostic accuracy of HLA typing of the embryo. The multiplex nature of the first round of PCR analysis required a similar annealing temperature for the outside primers. Thirty cycles of PCR were performed with a denaturation step at 95 C for 2 seconds, annealing at 62 C to 5 C for a minute, and elongation at 72 C for 3 seconds. Twenty minutes of incubation at 96 C were performed before starting cycling. After cycling, 1 minutes of elongation were performed at 72 C. Annealing temperature for the second round was programmed at 55 C (details of the method have been published 8,12 ).The applied strategy provided a % HLA match, because the selected embryos had the same paternal and maternal chromosome 6 as the affected siblings. Two to 3 embryos determined to be HLA compatible with the affected sibling were transferred; the other HLA- Figure 2. Example of Typing for Short Tandem Repeat Markers Showing a Capillary Electrophoregram of Fluorescently Labeled Polymerase Chain Reaction Products of the MIB Marker Father Mother Affected Child HLA-Matched Child.78 bp bp bp bp bp bp bp bp Size, bp Table 1. Preimplantation HLA Typing Resulting in Birth of a Child HLA Matched to a Sibling With Acute Lymphoid Leukemia a Embryo No. HLA Genes and STRs Father Mother Affected Child D6S276* 13/ / 13/118 13/118 13/ 144/ 13/ / 13/ 13/ 144/13 118/ 144/ HLA-A 3/2 1/32 3/2 3/ADO 3/32 1/32 FA 1/32 3/32 3/32 1/3 2/32 1/32 D6S51 163/ / /167 ADO/ / / /ADO 148/ / / / / /163 HLA-B ADO/27 27/18 ADO/27 FA FA 27/18 ADO/27 27/18 ADO/18 ADO/18 27/57 27/18 27/18 MIB 118/ / /114 ADO/ / / /114 FA 118/ / / / /124 /12 /12 /12 /12 /12 /12 /12 /12 /12 /12 / 12/12 /12 D6S273* 275/ / / / / / / / / / / / /271 HLA-DRB1 ADO/1 11/1 ADO/1 ADO/1 ADO/1 11/1 ADO/1 11/1 ADO/1 FA 11/7 1/1 11/1 G /28 181/ /28 ADO/28 181/ / /28 ADO/ / / /181 28/ /191 Ring 3CA 162/162 16/ / / /155 16/ /162 FA 162/ /155 16/ /155 16/155 D6S /13 14/ /13 14/13 144/144 14/ /13 14/ /13 144/144 14/144 13/144 14/144 Predicted genotype Nonmatch Match Nonmatch Nonmatch Recombinant b Nonmatch Match Nonmatch Recombinant c Match Nonmatch Recombinant b Nonmatch NA NA NA Abbreviations: ADO, allele drop out; FA, failed amplification; NA, not applicable; STR, short tandem repeat. a Paternally derived markers in each cell are shown on the left and maternally derived markers on the right (not applicable to father and mother). Paternally and maternally derived matched markers are shown in boldface. The numbers represent the size of the polymerase chain reaction products in base pairs, corresponding to the number of STR repeats or an HLA allele name. Of note, for ADO/27 entries for HLA-B, and for the ADO/114 entry for MIB, for which both parents share the same repeat sequence, the other genetic information points to a genotype of maternal origin, with ADO involving the paternal marker. b Recombination detected between D6S426 and Ring 3CA. c Recombination detected between D6S276* and D6S American Medical Association. All rights reserved. (Reprinted) JAMA, May 5, 24 Vol 291, No

4 matched embryos were frozen for future availability. As per the parents written informed consent, some of the HLA-incompatible embryos not chosen for freezing, and those with inconclusive results, underwent PCR analysis of the whole embryo to corroborate the diagnosis based on blastomere analysis. A follow-up assessment of the HLA match was conducted in the established pregnancies using chorionic villus sampling or amniocentesis. Since the leukemias and the DBA in the affected siblings were sporadic and not associated with chromosome 6, there was no reason for concern that the HLAmatched children would be at risk for the same disease as the affected siblings. RESULTS A total of 199 embryos were tested for the 9 couples in 13 clinical cycles performed by the time the data were submitted for publication (15 embryos per cycle on the average), of which 45 (23%) were determined to be HLA matched to the affected siblings. The HLAmatched embryos were available for transfer in all but 1 cycle, for which there were no matched embryos. Overall, 28 HLA-matched embryos were transferred in 12 clinical cycles, resulting in 5 singleton pregnancies (42%) and births of 5 HLA-matched children. Of course, some couples require multiple cycles of IVF to achieve pregnancy. These results suggest that testing of approximately 1 embryos per cycle allows for selection of a sufficient number of the HLA-matched embryos for transfer to achieve a pregnancy and birth of an HLA-matched progeny. The usefulness of detection of recombination within the HLA region is demonstrated in TABLE 1, describing the results of HLA typing of 1 of the cycles resulting in the birth of a child HLA matched to a sibling with acute lymphoid leukemia. Of 1 embryos tested simultaneously for 11 alleles within the relevant HLA region in this family, recombination between the alleles for the D6S426 and Ring 3CA markers was observed in embryos 4 and 9, and between D6S276 * and D6S51 in embryo 7. Of the remaining 7 embryos, 3 were fully matched (embryos 2, 6, and 8), while the other 4 were HLA incompatible with the affected sibling, as seen from the haplotypes of the mother, father, and affected child. FIGURE 3 presents the results of preimplantation HLA matching for DBA. One embryo with maternal recombination (embryo 8) and another with both paternal and maternal recombination (embryo 16) (1 in the allele for the Ring 3CA marker and the other in the allele for D6S426) were detected in testing of 16 embryos (examples of different HLA typing results are shown). In addition, there was another em- Figure 3. Preimplantation HLA Typing for Diamond-Blackfan Anemia, Resulting in Birth of an HLA-Matched Child STR or Gene STR or Gene D6S276 D6S258 HLA-A D6S265 HLA-C HLA-B HLA-DRB1 Ring 3CA D6S A 2 A A A B D6S276 D6S258 HLA-A D6S265 HLA-C HLA-B HLA-DRB1 Ring 3CA D6S426 Affected Child Embryo 1 Embryo 5 Embryo 8 Embryo A 2 A 3 A 2 A 3 A 2 A 24 A 2 A 24 A 2 A B A B HLA Match Trisomy Recombinant Double Recombinant Short tandem repeat (STR) and gene order for haplotypes of mother, father, and affected child. Examples of different results of HLA typing of biopsied blastomeres are shown. Embryo 1 is HLA matched to the affected sibling. Embryo 8 shows maternal recombination and embryo 16 shows double recombination involving both the paternal and maternal alleles. Embryo 5 shows an extra maternal chromosome consistent with trisomy 6. Markers with asterisks indicate possible ambiguity in localization. Genes with asterisks indicate allelic variants. 282 JAMA, May 5, 24 Vol 291, No. 17 (Reprinted) 24 American Medical Association. All rights reserved.

5 bryo with trisomy 6 (embryo 5) with an extra maternal chromosome 6, making this and the other 2 above also unacceptable for transfer. However, 5 embryos appeared to be HLA matched, of which 2 were transferred, resulting in the birth of an HLA-matched child. The relevance of aneuploidy testing for chromosome 6 is seen from the results of HLA typing in another cycle, resulting in birth of an infant who was HLA matched to the sibling with acute lymphoid leukemia (FIGURE 4). Two of 1 embryos tested in this case (examples of different HLA typing results are shown) appeared to have only maternally derived chromosomes 6; 1 had only 1 maternal chromosome (embryo 1), and the other had 2 maternal chromosomes, representing uniparental maternal disomy of chromosome 6 (embryo 2). In addition, recombination between D6S291 and class II HLA alleles was evident in embryo 7, which was also unacceptable for transfer. Of the remaining 7 embryos, only 2 (only embryo 4 is shown) were HLA matched to the affected sibling and transferred, resulting in the birth of an HLA-matched child. Of only 7 embryos available for testing in another case of DBA, 4 were HLA nonmatched, including the one with recombination between the alleles for the TAP1 and Ring 3CA markers (embryo 8). The remaining 3 HLA-matched embryos (embryos 2, 3, and 9) were transferred, resulting in a singleton pregnancy and birth of an HLA-matched child (TABLE 2). In the remaining cycle performed for acute lymphoid leukemia and resulting in the birth of an HLAmatched child, 6 HLA-matched embryos were selected from 11 tested, with no evidence of recombination or aneuploidy. The 5 children born as a result of preimplantation HLA matching (mean age, approximately 1 year) are healthy and are comparable to more than children born after preimplantation genetic diagnosis procedures, including more than 4 children born after preimplantation genetic diagnosis procedures at our center. The mean birth weight percentile was 47% and the mean birth length percentile was 57%. The 5 children born after preimplantation genetic diagnosis procedures in the current study were confirmed to be HLA matched (per HLA typing of blood) to their affected siblings. One sibling with DBA received transplantation and is no longer red blood cell transfusion dependent, whereas the others are in preparation for transplantation or are in remission. COMMENT The data herein show the potential feasibility of preimplantation HLA match- Figure 4. Preimplantation HLA Typing for Acute Lymphoid Leukemia, Resulting in Birth of an HLA-Matched Child STR D6S36 D6S1624 MOG a D6S51 62 D6S273 LH1 G51152 TAP1 D6S STR D6S36 D6S1624 MOG a D6S51 62 D6S273 LH1 G51152 TAP1 D6S291 Affected Child Embryo 1 Embryo 2 Embryo 4 Embryo 5 Embryo 6 Embryo Monosomy 6 Uniparental Disomy HLA Match Maternal Match Paternal Match Recombinant Short tandem repeat (STR) order for haplotypes of mother, father, and affected child. Examples of different results of HLA typing of biopsied blastomeres are shown. Embryo 4 is HLA matched to the affected sibling. Embryo 1 has no paternal chromosome present (monosomy 6). Embryo 2 shows only maternal chromosomes being present (uniparental disomy). Embryo 7 is both a paternal and maternal nonmatch, the latter being due to maternal recombination. The other 2 embryos are nonmatches, embryo 5 being a maternal match only and embryo 6 being a paternal match only. Markers with asterisks indicate possible ambiguity in localization. 24 American Medical Association. All rights reserved. (Reprinted) JAMA, May 5, 24 Vol 291, No

6 ing for couples having a child affected with a bone marrow disorder, who may wish to have another child as a potential HLA-matched donor of stem cells for transplantation to the affected sibling. As our data show, HLA-matched embryos were selected and transferred in all but 1 cycle, resulting in pregnancies and birth of HLAmatched children in 42% of the transferred cycles, much higher than the pregnancy rate observed in women undergoing IVF. 15 Despite the relatively high rate of preferential amplification and allele drop out observed in PCR analysis of single blastomeres and potential for recombination within the HLA region described in our material, and a high rate of mosaicism for aneuploidies at the cleavage stage, 16 the introduced approach appeared to be highly accurate in the selection of HLAmatched embryos for transfer. This approach involves a multiplex PCR analysis involving testing for HLA alleles together with short tandem repeat markers within HLA and flanking regions, allowing the avoidance of misdiagnosis due to allele drop out, aneuploidy, or recombination of HLA alleles. Recombination in the HLA region was observed in 4.3% of blastomeres tested, suggesting the existence of a few areas in the HLA region that are prone to a higher recombination rate, which should be detected in order to avoid the risk for misdiagnosis (Verlinsky et al, unpublished data, September 23). On the other hand, aneuploidy of chromosome 6 was detected in 6.4% of blastomeres, including trisomy (2.2%) and monosomy 6 (4.2%) (Verlinsky et al, unpublished data, September 23). As mentioned, preimplantation HLA matching has previously been used together with PGD for FA to select and transfer unaffected embryos, avoiding affected pregnancy and also producing a potential donor progeny for stem cell transplantation for the affected sibling with FA. 8 In contrast, no testing for a causative gene was performed in the present series of couples, the sole objective being to identify the HLA-matched embryos. Although still highly controversial and even not allowed in some countries, this appeared to be a reasonable option for couples, because only a limited number of the embryos resulting from a hormonal hyperstimulation in IVF are actually selected for transfer anyway. Therefore, instead of selecting embryos for transfer based on morphologic criteria, those unaffected embryos representing an HLA match are selected. There are important ethical issues involving the selection of embryos with different normal parameters such as HLA type; however, preimplantation HLA typing allows for the avoidance of the ethical dilemma of therapeutic cloning. 17,18 The decision about preimplantation HLA typing is presently left with individuals and clinicians, as it is still expensive (approximately $3 on average for preimplantation HLA typing alone [the cost of IVF can exceed $ in the United States and is covered by insurance in only some states; HLA testing is not covered]) and not obligatorily covered by health insurers. Our results also demonstrate the prospect for the application of this approach to other conditions requiring an Table 2. Preimplantation HLA Typing for Diamond-Blackfan Anemia a Short Embryo No. Tandem Repeats Father Mother Affected Child D6S / / / / b / / / / / /229 b D6S276* 143/ / / /ADO 143/ / / / / /143 D6S / / / / / / / / / /132 D6S248* 253/ / / / / / / / / /278 MOG a /169 / / /169 / / /169 / /169 /169 RF 275/ /ADO 257/ / / / / / / /269 9N-2 129/ / / /127/ / / / / / /133 D6S273* 276/27 276/27 274/27 274/27 276/27 274/27 274/27 276/274 27/27 276/27 LH1 163/ / / / / / / / / /168 D6S2447 / / 151/ 151/ / 151/ /151 / / / TAP1 25/22 25/22 25/22 25/22 25/22 25/27 25/27 25/25 27/22 25/22 Ring 3CA /155 / / / / / 155/ /155 /155 /155 D6S439* 125/ / / / / / / / / /125 D6S / / / / / / / / / /123 D6S / / /129 FA 129/142 b 144/ / / / /129 Predicted genotype Match Match Nonmatch Nonmatch Recombinant c Match Nonmatch Nonmatch NA NA NA Abbreviations: ADO, allele drop out; FA, failed amplification; NA, not applicable. a Paternally derived markers in each cell are shown on the left and maternally derived markers on the right (not applicable to father and mother). Paternally and maternally derived matched markers are shown in boldface. The numbers represent the size of the polymerase chain reaction products in base pairs, corresponding to the number of STR repeats. Of note, for marker 9N-2 the 3 numbers suggest the possibility of genetic material from 2 maternal chromosomes, but this cannot be so because other markers indicate the presence of only 1 maternal chromosome 6. Thus, the genotype is considered to be of an unknown nature. b Of note, there is recombination between alleles for the D6S461 and D6S276* markers in the affected child, as well as in embryo 9 (which has paternal recombination between these markers vs the child, which has maternal recombination); embryo 9 also has recombination between alleles for D6S291 and D6S426 markers. However, because these markers are outside the HLA region, and other markers are in agreement with the match, the embryo was considered to be HLA matched to the affected child. c Recombination detected between TAP1 and Ring 3CA. 284 JAMA, May 5, 24 Vol 291, No. 17 (Reprinted) 24 American Medical Association. All rights reserved.

7 HLA-compatible donor for stem cell transplantation. This provides a realistic option for those couples who are parents of an affected child, who desire to have another child, and who as a wholly separate issue hope that the subsequent child might serve as a donor of stem cells for the affected sibling. In addition to sporadic forms of DBA, as well as leukemia, the method may potentially be applied to other conditions; for example, the method might be used by parents who have unsuccessfully sought an HLA-compatible donor for a child with other types of cancer. These expanding indications make preimplantation testing a complement to traditional prenatal diagnosis, allowing parents to avoid inherited conditions and pregnancy termination. At the same time, the evidence suggests that it may now be possible for a pregnancy to have genetic characteristics that may be beneficial for affected individuals in the family. Author Contributions: Dr Kuliev, as principal investigator of this study, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analyses. Study concept and design: Verlinsky, Kuliev. Acquisition of data: Rechitsky, Sharapova, Morris, Taranissi. Analysis and interpretation of data: Rechitsky, Kuliev. Drafting of the manuscript: Kuliev. Critical revision of the manuscript for important intellectual content: Verlinsky, Rechitsky, Sharapova, Morris, Taranissi. Obtained funding: Verlinsky. Administrative, technical, or material support: Verlinsky, Sharapova, Morris, Taranissi. Study Supervision: Verlinsky, Rechitsky, Kuliev. Funding/Support: This study was funded by the Reproductive Genetics Institute. Role of the Sponsor: The Reproductive Genetics Institute sponsored the design and conduct of the study; the collection, management, analysis, and interpretation of the data; and the preparation, review, and approval of the manuscript. REFERENCES 1. International Working Group on Preimplantation Genetics. 21 Preimplantation Genetic Diagnosis Experience of Three Thousand Clinical Cycles. Report of the 11th Annual Meeting International Working Group on Preimplantation Genetics, in conjunction with the 1th International Congress of Human Genetics; May 15-19, 21; Vienna, Austria. Reprod BioMed Online. 21;3: Geraedts J, Handyside A, Harper J, et al, European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis Consortium Steering Committee. ESHRE preimplantation genetic diagnosis (PGD) consortium: data collection II (May 2). Hum Reprod. 2;15: Kuliev A, Verlinsky Y. Current features of preimplantation genetic diagnosis. Reprod BioMed Online. 22;5: Verlinsky Y, Cohen J, Munne S, et al. Over a decade of preimplantation genetic diagnosis experience a multicenter report. Fertil Steril. In press. 5. Rechitsky S, Verlinsky O, Chistokhina A, et al. Preimplantation genetic diagnosis for cancer predisposition. Reprod BioMed Online. 22;4: Verlinsky Y, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis for early onset Alzheimer disease caused by V717L mutation. JAMA. 22;287: Abou-Sleiman PM, Apessos A, Harper JC, Serhal P, Delhanty JD. Pregnancy following preimplantation genetic diagnosis for Crouson syndrome. Mol Hum Reprod. 22;8: Verlinsky Y, Rechitsky S, Schoolcraft W, Strom C, Kuliev A. Preimplantation diagnosis for Fanconi anemia combined with HLA matching. JAMA. 21;285: Verlinsky Y, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis for sonic hedgehog mutation causing familial holoprosencephaly. N Engl J Med. 23;348: Cord blood banking. Bull World Health Organ. 1998;76: Online Mendelian Inheritance in Man (OMIM). Diamond-Blackfan anemia, autosomal dominant. Online Mendelian Inheritance in Man (OMIM 1565, OMIM 259). Available at: Accessed April 8, Verlinsky Y, Kuliev A. Atlas of Preimplantation Genetic Diagnosis. London, England: Parthenon Publishing Group; Foissac A, Salhi M, Cambon-Thomsen A. Microsatellites in the HLA region: 1999 update. Tissue Antigens. 2;55: Rechitsky S, Strom C, Verlinsky O, et al. Accuracy of preimplantation diagnosis of single-gene disorders by polar body analysis of oocytes. J Assist Reprod Genet. 1999;16: Assisted reproductive technology in the United States: 1997 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril. 2; 74: Munne S. Preimplantation genetic diagnosis of numerical and structural chromosome abnormalities. Reprod BioMed Online. 22;4: Damewood MD. Ethical implications of a new application of preimplantation diagnosis. JAMA. 21; 285: Edwards RG. Ethics of preimplantation diagnosis: recordings from the Fourth International Symposium on Preimplantation Genetics. Reprod BioMed Online. 23;6: American Medical Association. All rights reserved. (Reprinted) JAMA, May 5, 24 Vol 291, No