First Unaffected Pregnancy Using Preimplantation Genetic Diagnosis for Sickle Cell Anemia

ORIGINAL CONTRIBUTION

Kangpu Xu, PhD Context Sickle cell anemia is a common autosomal recessive disorder. However, pre- Zhong Ming Shi, MD implantation genetic diagnosis (PGD) for this severe genetic disorder previously has not been successful. Lucinda L. Veeck, MLT, DSc Objective To achieve pregnancy with an unaffected embryo using in vitro fertiliza- Mark R. Hughes, MD, PhD tion (IVF) and PGD. Zev Rosenwaks, MD Design Laboratory analysis of DNA from single cells obtained by biopsy from em- ICKLE CELL ANEMIA IS ONE OF THE bryos in 2 IVF attempts, 1 in 1996 and 1 in 1997, to determine the genetic status of each embryo before intrauterine transfer. most common human autosomal recessive disorders. It is Setting University hospital in a large metropolitan area. caused by a mutation substitut- Patients A couple, both carriers of the recessive mutation for sickle cell disease. Sing thymine for adenine in the sixth Interventions Standard IVF treatment, intracytoplasmic sperm injection, embryo bi- codon (GAG to GTG) of the gene for the opsy, single-cell polymerase chain reaction and DNA analyses, embryo transfer to uterus, ␤-globin chain on chromosome 11p, pregnancy confirmation, and prenatal diagnosis by amniocentesis at 16.5 weeks’ ges- thereby encoding valine instead of glu- tation. tamic acid in the sixth position of the Main Outcome Measure DNA analysis of single blastomeres indicating whether globin chain. The frequency of sickle cell embryos carried the sickle cell mutation, allowing only unaffected or carrier embryos trait (carrier status) among the African to be transferred. American population at birth is about Results The first IVF attempt failed to produce a pregnancy. Of the 7 embryos ana- 8%, and the incidence of sickle cell ane- lyzed in the second attempt, PGD indicated that 4 were normal and 2 were carriers; mia at birth is 0.16%, or 1 per 625 diagnosis was not possible in 1. Three embryos were transferred to the uterus on the births.1 Furthermore, the widespread fourth day after oocyte retrieval. A twin pregnancy was confirmed by ultrasonogra- presence of the sickle gene in other eth- phy, and subsequent amniocentesis revealed that both fetuses were unaffected and nic groups has also been confirmed.2 For were not carriers of the sickle cell mutation. The patient delivered healthy twins at 39 example, in urban centers in the United weeks’ gestation. States, nearly 10% of patients with vari- Conclusion This first unaffected pregnancy resulting from PGD for sickle cell ane- ous sickling disorders identify them- mia demonstrates that the technique can be a powerful diagnostic tool for carrier couples selves as non–African American.3 who desire a healthy child but wish to avoid the difficult decision of whether to abort Children affected with sickle cell ane- an affected fetus. mia experience recurrent episodes of pain JAMA. 1999;281:1701-1706 www.jama.com (during sickle cell crises) and increased rican American children aged 1 to 4 years Author Affiliations: The Center for Reproductive susceptibility to potentially life- due to sickle cell disease.4 Life expec- Medicine and Infertility and the Department of Ob- threatening conditions, including bac- stetrics and Gynecology, Weill Medical College of Cor- tancy for persons with sickle cell dis- terial infections, cerebrovascular acci- nell University, New York, NY (Drs Xu, Shi, Veeck, and ease varies and there is an age-related Rosenwaks); and the Department of Reproductive Ge- dents, and organ failure. According to US pattern in mortality rates: a peak in pa- netics, Wayne State University, Detroit Medical Cen- statistics collected between 1981 and ter, Detroit, Mich (Dr Hughes). tients younger than 5 years, with a 1992, there were 6.8 deaths per 1000 Af- Corresponding Author and Reprints: Zev Rosen- gradual increase starting in late adoles- waks, MD, The Center for Reproductive Medicine and 5 Infertility, Weill Medical College of Cornell Univer- cence. Progress in the treatment of sickle sity, 505 E 70th St, Room HT326a, New York, NY See also Patient Page. 6 cell disease has been slow. At present, 10021 (e-mail: [email protected]).

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there is no satisfactory treatment for the because she was carrying fetuses af- digested with the restriction enzyme sickling condition, although blood trans- fected with sickle cell anemia. Genetic DdeI (GIBCO/BRL, Rockville, Md) for fusion may reduce the risk of a first stroke diagnosis indicated that both female and 3 hours. Subsequently, 10 µL of di- in children7 and gene therapy holds male partners were carriers of the sickle gested product was run on a 10% acryl- promise for a curative approach.8 cell mutation. After extensive counsel- amide gel. On completion of electro- Early prenatal diagnosis of the dis- ing, the couple gave informed consent phoresis, the gel was stained with ease is critical because it allows a couple and elected to undergo preimplanta- ethidium bromide and photographed by to consider pregnancy termination as tion genetic diagnosis. The study was UV transillumination. As predicted, un- an option. The first DNA diagnostic approved by the Weill Medical Col- affected DNA showed 3 bands (201, 90, procedure for prenatal purposes was re- lege of Cornell University (New York, and 74 base pairs [bp]), carrier DNA ported 20 years ago.9 Subsequently, it NY) Institutional Review Board. showed 4 bands (291, 201, 90, and 74 was recognized that the mutation it- To confirm the genetic status of the bp), and an affected homozygous self affected the cleavage site of a re- couple and establish a protocol for sample showed only 2 bands (291 and striction enzyme, DdeI, that could rec- single-copy gene amplification from 74 bp). Testing of single lymphocytes ognize the DNA sequence of CTNAG single cells, blood was collected from from the male and female subjects (N = A, T, C, or G). While DNA from a both partners. Lymphocytes were iso- showed the same predicted patterns (4 normal allele (CTGAG) would be di- lated using Ficoll-Paque density gradi- bands of predicted sizes), together with gested by the enzyme, DNA from an af- ent separation (Pharmacia Biotech Inc, an unaffected DNA control (3 bands) fected allele in which A is substituted Piscataway, NJ) with the protocol pro- (FIGURE 1). by T (CTGTG) would not.10,11 The re- vided by the manufacturer. Single lym- The IVF procedure has been de- sulting differences between DNA frag- phocytes were loaded into 0.5-mL tubes scribed previously.20 Briefly, to ensure ment sizes can then be recognized by containing 5 µL of lysis buffer19 and that several embryos would become electrophoresis, thus forming the ba- stored at −20°C before trial testing. available for DNA analysis, multiple sis for diagnosis. With the advent of On the day of preliminary trial test- ovarian follicular development was ini- polymerase chain reaction (PCR), rapid ing, sample tubes were removed from the tiated with gonadotropin therapy. Af- DNA analysis methods have become freezer and heated to 65°C for 10 min- ter pituitary desensitization with go- available, and these techniques are now utes before they were placed back on ice. nadotropin hormone–releasing widely used for prenatal diagnosis.12-16 Five microliters of neutralization buffer hormone agonist (leuprolide acetate, An alternative and powerful diagnos- was added to each tube. A nested PCR TAP Pharmaceutical, Chicago, Ill), tic tool for identifying sickle cell status approach was used for the amplifica- ovarian stimulation was begun on day in embryos is preimplantation genetic di- tion of the region surrounding the sickle 3 of the ensuing menstrual cycle using agnosis (PGD), which became possible cell mutation. The primers used have intramuscular administration of a com- nearly a decade ago.17 Preimplantation been described previously.17,19 Polymer- bination of urofollitropin (75 U of genetic diagnosis takes advantage of as- ase chain reaction was performed after pure follicle-stimulating hormone) sisted reproductive techniques in con- adding a standard mixture of all com- and menotropins (150 U of follicle- junction with modern molecular meth- ponents, including 2.5 mmol of Mg2+, 0.2 stimulating hormone and luteinizing ods. With PGD, the genetic status of an mmol of dNTPs (containing dATP, hormone) (Serono Laboratories, Nor- embryo can be determined before trans- dCTP, dGTP, and dTTP, Perkin Elmer, well, Mass). Follicular growth was fer into the uterus after in vitro fertiliza- Foster City, Calif), 100 ng of primers, monitored by daily serum estradiol lev- tion (IVF), thus eliminating the risks of and2UofTaqpolymerase (AmpliTaq, els and pelvic ultrasonograms. To in- bearing a child with the disease. Perkin-Elmer). A hot start at 95°C was duce final oocyte maturation, 3300 IU While PGD for sickle cell anemia has applied for 3 minutes to ensure com- of human chorionic gonadotropin was been performed in the mouse model,18 plete denaturation of the template. For administered when 2 follicles of 18 mm routine clinical application in the hu- the PCR profile, the following param- in average diameter were observed on man previously has not been success- eters were used: 93°C denaturation for ultrasonogram. Transvaginal oocyte re- ful. In this article, we describe our ex- 30 seconds; 50°C for 40 seconds for an- trieval was performed 35 hours later. perience using PGD to determine the nealing; and 72°C for 45 seconds for ex- To avoid sperm contamination and pos- precise genetic status of embryos gen- tension. A total of 20 amplification cycles sible amplification of sperm DNA, in- erated by assisted reproduction for a were applied for outer primers. For the tracytoplasmic sperm injection was couple who are heterozygous carriers inner primer set, identical parameters used. After 16 hours of incubation, fer- of the sickle cell mutation. were used, except that the annealing tilization was confirmed by the identi- temperature was raised to 55°C and a to- fication of 2 pronuclei. Normally fer- METHODS tal of 40 cycles were used. tilized concepti were then transferred A 34-year-old female patient had un- Following the nested PCR amplifi- to droplets of human tubal fluid (made dergone 2 previous induced abortions cation, 18 µL of amplified product was on site) supplemented with 15% ma-

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ternal serum under mineral oil (ER female and 24 from the male, were suc- ase chain reaction amplification was Squibb & Sons Inc, Princeton, NJ). Bi- cessfully amplified. As predicted, 4 successful in 7 of 9 blastomeres (5 of 6 opsy was performed on the morning of bands of correct size were obtained from embryos). Amplification failed in 1 cell the third day after harvest. All em- both partners. An example of the gel is from embryo 5 and in 1 cell from embryos were maintained at 37°C in an at- shown in Figure 1. bryo 6. Restriction enzyme digestion mosphere of 5% carbon dioxide. Cleav- During the first IVF attempt in No- demonstrated that 4 were homozy- age rate and morphologic appearance vember 1996, 18 oocytes were re- gous unaffected, 2 were carriers were assessed daily. trieved. Four of 6 mature oocytes were (FIGURE 2), and 1 was of unknown sta- Blastomere biopsy was carried out in normally fertilized after single-sperm in- tus due to PCR amplification failure. On the early morning, approximately 65 jection by intracytoplasmic sperm in- the afternoon of day 4, all concepti that hours after oocyte collection. Briefly, a jection, yielding 4 embryos. underwent biopsy demonstrated fur- holding pipette was used to stabilize the Embryo biopsy was performed on all ther cleavage. Selection of embryos for embryo (at the 9 o’clock position). A 4 embryos on day 3 by removing a single transfer was based on PGD diagnosis, hole was made at the 3 o’clock posi- blastomere from each conceptus. Poly- growth rate, and morphology. Two un- tion by expelling a small amount of merase chain reaction and restriction en- affected embryos were of poor quality, acidified Tyrode solution (pH, 2.35) zyme analysis revealed that 1 was ho- displaying slow cleavage and multi- onto the zona pellucida through a small- mozygous unaffected, 2 were carriers, nucleation (embryo 2) or high frag- bore pipette. Reverse suction was ap- and 1 was homozygous affected. Trans- mentation (embryo 4) and therefore plied as soon as a hole of appropriate fer of 1 unaffected embryo on day 4 failed were not suitable for transfer (TABLE). size was created to reduce possible dam- to result in a pregnancy. Because there were only 3 high- age caused by exposing the blasto- The second IVF attempt was initi- quality transferable embryos—2 unaf- meres to the acidic solution. A large in- ated in August 1997, during which 16 fected (embryo 1 with 15 cells; em- ner-diameter biopsy pipette replaced the oocytes were retrieved. Of those, 8 were bryo 6 with 8 cells) and 1 carrier pipette containing the acidified Ty- mature and underwent intracytoplas- (embryo 8 with 10 cells) (FIGURE 3 and rode solution. Subsequently, 1 or 2 cells mic sperm injection. Seven concepti the Table)—and because the patient were aspirated, depending on the total cleaved at least once by the following was willing to accept a fetus of carrier cell number of the embryo. Blasto- day. On the morning of the third day, status, all 3 embryos were transferred. meres were rinsed in biopsy medium 2 cells were removed from 2 embryos A twin pregnancy was confirmed by 3 times before loading into a PCR tube and 1 cell from 5 embryos. Polymer- ultrasonography at 7 weeks. Amnio- containing 5 µL of lysis buffer. Tubes were processed and PCR amplifica- Figure 1. Establishment of Single-Cell PCR and Confirmation of the Carrier Status tion and restriction enzyme analysis of Female and Male by Restriction Enzyme Analysis were performed as described herein. Micromanipulated embryos were fur- M12345678910 ther cultured in medium droplets over- night. Embryo transfer was performed 364 bp in the afternoon of day 4. 291 bp Pregnancy was determined by serum ␤–human chorionic gonadotropin measurement on cycle days 28 and 201 bp 35, followed by ultrasonographic as- sessment at 7 weeks’ gestation. The genetic status of the fetuses was con- 90 bp firmed after amniocentesis by an 74 bp independent laboratory. Cultured amniocytes were also sent to our laboratory for follow-up PCR analysis. RESULTS

Polymerase chain reaction and restric- βA/βS βA/βA βS/βS Female Male tion enzyme analysis of single lympho- PCR indicates polymerase chain reaction; bp, base pair; and M, DNA size marker (100-bp ladder). Lanes 1, 3, cytes from both the female and male 5, 7, and 9 are PCR-amplified but undigested DNA (364 bp), and lanes 2, 4, 6, 8, and 10 are DdeI-digested partners clearly indicated that each car- DNA. Lanes 1 and 2 are from a known carrier DNA (␤A/␤s, 4 bands); lanes 3 and 4 are from a known unaf- ried the sickle cell mutation. Forty-six fected DNA (␤A/␤A, 3 bands); lanes 5 and 6 are from known affected DNA (␤s/␤s, 2 bands); lanes 7 and 8 are from the female partner; and lanes 9 and 10 are from the male partner. of 48 single lymphocytes, 24 from the

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centesis performed by an independent fected child, and half of the offspring rily applied for severe genetic disor- laboratory at 16.5 weeks revealed that may carry the mutation. Although pre- ders for which detailed genetic infor- neither fetus harbored the sickle cell natal testing is currently available, some mation is available. Normal pregnancies mutation. DNA analysis of amnio- couples have strong personal objec- following a search for specific muta- cytes shipped from the prenatal diag- tions to aborting affected fetuses. For tions have been reported for only a few nostic laboratory to our own labora- these couples, PGD provides a realis- genetic diseases, including cystic fibro- tory also showed that both fetuses were tic alternative to prenatal testing. sis22-24 and Tay-Sachs disease.25 unaffected (FIGURE 4). The patient de- Although the first pregnancy Despite the fact that sickle cell ane- livered healthy, unaffected fraternal achieved by PGD for sex determina- mia is one of the most common ge- twin girls at 39 weeks’ gestation. tion to avoid the transmission of a sex- netic disorders and detailed genetic in- linked disorder occurred nearly a de- formation is available,1,26 unaffected COMMENT cade ago,17 PGD for single-gene defects pregnancies following PGD for sickle After natural conception, couples who is still in the experimental stage be- cell anemia previously have not been carry autosomal recessive mutations cause of its complexity and technical reported. Lack of previous success in risk a 25% chance of delivering an af- difficulties. At present, PGD is prima- this area presumably is due to the length of time and effort required to over- come technical difficulties inherent in Figure 2. Restriction Analysis (DdeI) of PCR-Amplified DNA From Each Blastomere these procedures, as well as lack of From 6 Embryos available research funding. Our re-

M1 2 3 45678 M12345678 sults demonstrate that sickle cell anemia can be detected in single cells by PCR and restriction enzyme analysis and that unaffected pregnancies can be established by the transfer of embryos 364 bp of known genetic makeup that have un- 291 bp dergone biopsy. 201 bp The protocol used in this study was initially developed in the mouse model by Sheardown et al.18 In their investiga- 90 bp tion, 4 tandem copies of the human 74 bp ␤-globin gene were detected in trans-

βA/βA βA/βA βA/βS βA/βA βA/βA βA/βA βA/βS βA/βS genic mouse embryos. In humans, ␤-glo- 1 2 3 Control 1 2 3 Sperm bin is a single-copy gene. Under clinical PGD circumstances, single-cell PCR PCR indicates polymerase chain reaction; bp, base pair; and M, DNA size marker (100-bp ladder). For both panels, lanes 1, 3, 5, and 7 are PCR amplified but undigested DNA (364 bp) and lanes 2, 4, 6, and 8 are DdeI- requires an extremely sensitive proto- digested DNA. Left, Lanes 1 and 2 (products from the blastomere of embryo 1) = ␤A/␤A; lanes 3 and 4 (em- col. In this study, we initially tested the bryo 2) = ␤A/␤A; lanes 5 and 6 (embryo 3) = ␤A/␤s; and lanes 7 and 8 (unaffected DNA control) = ␤A/␤A. Right, Lanes 1 and 2 (products from the blastomere of embryo 4) = ␤A/␤A; lanes 3 and 4 (embryo 6) = ␤A/␤A; lanes 5 protocol on single lymphocytes iso- and 6 (embryo 8) = ␤A/␤s; and lanes 7 and 8 (products from spermatozoa of the male partner) = ␤A/␤s. Note lated from each member of the couple that in lane 8 on the left and lanes 2 and 4 on the right, a faint band (Ϸ125 bp) above 90 bp is believed to be the digested products from amplified DNA of the first PCR. It is seen only when too much DNA is used for at risk. Lysis buffer, reported to be bet- 19 enzyme digestion and does not interfere with diagnosis because of its intensity and size. ter for single-cell PCR, was used in-

Table. Detailed Information on Fertilization, Embryo Morphology, Biopsy, PGD Results, Intrauterine Transfer, and Implantation* Embryo No. of Blastomeres No. of Blastomeres PGD No. of Blastomeres Embryo No. (% Fragmentation Before Biopsy)† Removed Results (% Fragmentation on Day 4) Transfer Implantation 1 8 (5) 1 ␤A/␤A 15 (5) Yes Yes 2 4 (10) 2 ␤A/␤A 4 (10) No . . . 3 7 (20) 1 ␤A/␤S 10 (15) No . . . 4 5 (30) 1 ␤A/␤A 3 (20) No . . . 5 8 (10) 1 Unknown 6 (15) No . . . 6 7 (10) 2 ␤A/␤A 8 (10) Yes Yes 7‡ ...... 8 7 (10) 1 ␤A/␤S 10 (10) Yes No *PGD indicates preimplantation genetic diagnosis; ellipses, data not applicable. †Cytoplasmic fragmentation occurs as cells make and break contact during cleavage. The percentage of total fragmentation is defined as the percentage of total surface area covered with cytoplasmic fragments.21 ‡Oocyte not fertilized.

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stead of the freeze-thaw method.18 Us- ture32,33 may even further enhance em- ful diagnostic tool for carrier couples ing the modified protocol, successful bryo viability, thus increasing the preg- who desire a healthy child but wish to amplification was achieved in 46 (96%) nancy rate after PGD. avoid the difficult decision of whether of 48 single lymphocytes tested. This In summary, this is the first unaf- to abort an affected fetus. The proce- provided invaluable preliminary tech- fected pregnancy and delivery after suc- dure, successfully used in this case, may nical experience prior to executing the cessful PGD for sickle cell anemia. Our also be applied to other monogenic dis- PCR-PGD technique in the clinical set- results demonstrate that PGD for the de- orders and further supports the notion ting. However, PCR failure occurred in tection of sickle cell anemia is a power- that PGD is destined to be an integral 1 of the 9 cells undergoing biopsy. Di- agnostic failure in PCR-PGD could be Figure 3. Embryo Morphology Prior to Transfer on Day 4 due to a number of factors, including the absence of a nucleus, loss of the blastomere during handling, and blastomere mosaicism.27 While the current technique is apparently adequate, further modification and enhancement, such as the use of fluorescent PCR, may further improve the accuracy and effi- ciency of this method. 8 We performed biopsies on day 3 and 1 intrauterine transfer on day 4 because the biopsy, PCR, enzyme digestion, and electrophoresis could not be com- pleted within 10 hours. This change pro- 6 vided additional valuable hours for ac- curate diagnosis. Apparently, the extended in vitro culture did not compromise embryo viability. Because both

the morphology and genetic status of Numbers indicate embyos 1, 6, and 8. each of the transferred embryos were known, it is worth examining the char- acteristics of each embryo. On the morn- Figure 4. Polymerase Chain Reaction and Restriction Analysis From Amniocytes From the 2 Fetuses, Showing 2 Unaffected DNA Patterns ing of day 3, embryo 6 (Table) was com- posed of 7 blastomeres, 2 of which were M1234567 removed for testing. This accounts for more than one fourth of the embryo vol- ume. At the time of intrauterine trans- 364 bp fer on day 4, the conceptus had reached the 8-cell stage. This embryo im- planted, as indicated by its genetic sta- ␤A ␤A tus ( / ). It is known from animal 201 bp models that biopsy does not decrease implantation and subsequent live birth rates28 and that embryo biopsy does not necessarily impair subsequent in vitro 29 development in humans. This particu- 90 bp lar case unequivocally shows that the re- moval of 2 blastomeres from a 7-cell con- 74 bp ceptus did not compromise its developmental potential. Further- more, our results confirm that day-3 bi- DNA βA βA βA βA βA βA opsy and day-4 transfer is indeed a fea- DNA Fetus A Fetus B sible approach for PGD. Recent progress Bp indicates base pair; M, DNA size marker (100-bp ladder). Lane 1 shows unaffected DNA prior to DdeI di- in developing culture media30,31 and/or gestion; lanes 2 and 3, unaffected DNA control (␤A/␤A, 3 bands) after DdeI digestion; lanes 4 and 5, fetus A ␤A ␤A ␤A ␤A human autologous endometrial cocul- (unaffected, / , 3 bands); and lanes 6 and 7, fetus B (unaffected, / , 3 bands).

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aspect of assisted reproductive technol- able that with further refinements, PGD reen Moomjy, MD; Glenn L. Schattman, MD; and Steven D. Spandorfer, MD for support of this work. ogy. Given the current methods and rela- will certainly become an invaluable and We also acknowledge the embryological skills of Gian- tively high cost of the procedure, it is un- powerful diagnostic modality. piero D. Palermo, MD; Lisa Bovis, BS; Jianming Li, PhD; Deborah Liotta, BS; Julio Rimarachin, MD, PhD; Zhen likely that PGD will totally replace Acknowledgment: We thank Owen K. Davis, MD; Pak Ye, BS; and Nikica Zaninovic, MS. The editorial assis- prenatal testing. However, it is conceiv- Chung, MD; Ina Cholst, MD; Isaac Kligman, MD; Mau- tance of Donna Espenberg is gratefully appreciated.

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