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Original Contribution |

First Unaffected Pregnancy Using Preimplantation Genetic Diagnosis for Sickle Cell Anemia FREE

Kangpu Xu, PhD; Zhong Ming Shi, MD; Lucinda L. Veeck, MLT, DSc; Mark R. Hughes, MD, PhD; Zev Rosenwaks, MD
[+] Author Affiliations

Author Affiliations: The Center for Reproductive Medicine and Infertility and the Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, New York, NY (Drs Xu, Shi, Veeck, and Rosenwaks); and the Department of Reproductive Genetics, Wayne State University, Detroit Medical Center, Detroit, Mich (Dr Hughes).


JAMA. 1999;281(18):1701-1706. doi:10.1001/jama.281.18.1701.
Text Size: A A A
Published online

Context Sickle cell anemia is a common autosomal recessive disorder. However, preimplantation genetic diagnosis (PGD) for this severe genetic disorder previously has not been successful.

Objective To achieve pregnancy with an unaffected embryo using in vitro fertilization (IVF) and PGD.

Design Laboratory analysis of DNA from single cells obtained by biopsy from embryos in 2 IVF attempts, 1 in 1996 and 1 in 1997, to determine the genetic status of each embryo before intrauterine transfer.

Setting University hospital in a large metropolitan area.

Patients A couple, both carriers of the recessive mutation for sickle cell disease.

Interventions Standard IVF treatment, intracytoplasmic sperm injection, embryo biopsy, single-cell polymerase chain reaction and DNA analyses, embryo transfer to uterus, pregnancy confirmation, and prenatal diagnosis by amniocentesis at 16.5 weeks' gestation.

Main Outcome Measure DNA analysis of single blastomeres indicating whether embryos carried the sickle cell mutation, allowing only unaffected or carrier embryos to be transferred.

Results The first IVF attempt failed to produce a pregnancy. Of the 7 embryos analyzed in the second attempt, PGD indicated that 4 were normal and 2 were carriers; diagnosis was not possible in 1. Three embryos were transferred to the uterus on the fourth day after oocyte retrieval. A twin pregnancy was confirmed by ultrasonography, and subsequent amniocentesis revealed that both fetuses were unaffected and were not carriers of the sickle cell mutation. The patient delivered healthy twins at 39 weeks' gestation.

Conclusion This first unaffected pregnancy resulting from PGD for sickle cell anemia demonstrates that the technique can be a powerful diagnostic tool for carrier couples who desire a healthy child but wish to avoid the difficult decision of whether to abort an affected fetus.

Figures in this Article

Sickle cell anemia is one of the most common human autosomal recessive disorders. It is caused by a mutation substituting thymine for adenine in the sixth codon (GAG to GTG) of the gene for the β-globin chain on chromosome 11p, thereby encoding valine instead of glutamic acid in the sixth position of the globin chain. The frequency of sickle cell trait (carrier status) among the African American population at birth is about 8%, and the incidence of sickle cell anemia at birth is 0.16%, or 1 per 625 births.1 Furthermore, the widespread presence of the sickle gene in other ethnic groups has also been confirmed.2 For example, in urban centers in the United States, nearly 10% of patients with various sickling disorders identify themselves as non–African American.3

Children affected with sickle cell anemia experience recurrent episodes of pain (during sickle cell crises) and increased susceptibility to potentially life-threatening conditions, including bacterial infections, cerebrovascular accidents, and organ failure. According to US statistics collected between 1981 and 1992, there were 6.8 deaths per 1000 African American children aged 1 to 4 years due to sickle cell disease.4 Life expectancy for persons with sickle cell disease varies and there is an age-related pattern in mortality rates: a peak in patients younger than 5 years, with a gradual increase starting in late adolescence.5 Progress in the treatment of sickle cell disease has been slow.6 At present, there is no satisfactory treatment for the sickling condition, although blood transfusion may reduce the risk of a first stroke in children7 and gene therapy holds promise for a curative approach.8

Early prenatal diagnosis of the disease is critical because it allows a couple to consider pregnancy termination as an option. The first DNA diagnostic procedure for prenatal purposes was reported 20 years ago.9 Subsequently, it was recognized that the mutation itself affected the cleavage site of a restriction enzyme, DdeI, that could recognize the DNA sequence of CTNAG (N=A, T, C, or G). While DNA from a normal allele (CTGAG) would be digested by the enzyme, DNA from an affected allele in which A is substituted by T (CTGTG) would not.10,11 The resulting differences between DNA fragment sizes can then be recognized by electrophoresis, thus forming the basis for diagnosis. With the advent of polymerase chain reaction (PCR), rapid DNA analysis methods have become available, and these techniques are now widely used for prenatal diagnosis.1216

An alternative and powerful diagnostic tool for identifying sickle cell status in embryos is preimplantation genetic diagnosis (PGD), which became possible nearly a decade ago.17 Preimplantation genetic diagnosis takes advantage of assisted reproductive techniques in conjunction with modern molecular methods. With PGD, the genetic status of an embryo can be determined before transfer into the uterus after in vitro fertilization (IVF), thus eliminating the risks of bearing a child with the disease.

While PGD for sickle cell anemia has been performed in the mouse model,18 routine clinical application in the human previously has not been successful. In this article, we describe our experience using PGD to determine the precise genetic status of embryos generated by assisted reproduction for a couple who are heterozygous carriers of the sickle cell mutation.

A 34-year-old female patient had undergone 2 previous induced abortions because she was carrying fetuses affected with sickle cell anemia. Genetic diagnosis indicated that both female and male partners were carriers of the sickle cell mutation. After extensive counseling, the couple gave informed consent and elected to undergo preimplantation genetic diagnosis. The study was approved by the Weill Medical College of Cornell University (New York, NY) Institutional Review Board.

To confirm the genetic status of the couple and establish a protocol for single-copy gene amplification from single cells, blood was collected from both partners. Lymphocytes were isolated using Ficoll-Paque density gradient separation (Pharmacia Biotech Inc, Piscataway, NJ) with the protocol provided by the manufacturer. Single lymphocytes were loaded into 0.5-mL tubes containing 5 µL of lysis buffer19 and stored at −20°C before trial testing.

On the day of preliminary trial testing, sample tubes were removed from the freezer and heated to 65°C for 10 minutes before they were placed back on ice. Five microliters of neutralization buffer was added to each tube. A nested PCR approach was used for the amplification of the region surrounding the sickle cell mutation. The primers used have been described previously.17,19 Polymerase chain reaction was performed after adding a standard mixture of all components, including 2.5 mmol of Mg2+, 0.2 mmol of dNTPs (containing dATP, dCTP, dGTP, and dTTP, Perkin Elmer, Foster City, Calif), 100 ng of primers, and 2 U of Taq polymerase (AmpliTaq, Perkin-Elmer). A hot start at 95°C was applied for 3 minutes to ensure complete denaturation of the template. For the PCR profile, the following parameters were used: 93°C denaturation for 30 seconds; 50°C for 40 seconds for annealing; and 72°C for 45 seconds for extension. A total of 20 amplification cycles were applied for outer primers. For the inner primer set, identical parameters were used, except that the annealing temperature was raised to 55°C and a total of 40 cycles were used.

Following the nested PCR amplification, 18 µL of amplified product was digested with the restriction enzyme DdeI (GIBCO/BRL, Rockville, Md) for 3 hours. Subsequently, 10 µL of digested product was run on a 10% acrylamide gel. On completion of electrophoresis, the gel was stained with ethidium bromide and photographed by UV transillumination. As predicted, unaffected DNA showed 3 bands (201, 90, and 74 base pairs [bp]), carrier DNA showed 4 bands (291, 201, 90, and 74 bp), and an affected homozygous sample showed only 2 bands (291 and 74 bp). Testing of single lymphocytes from the male and female subjects showed the same predicted patterns (4 bands of predicted sizes), together with an unaffected DNA control (3 bands) (Figure 1).

Figure 1. Establishment of Single-Cell PCR and Confirmation of the Carrier Status of Female and Male by Restriction Enzyme Analysis
Graphic Jump Location
PCR indicates polymerase chain reaction; bp, base pair; and M, DNA size marker (100-bp ladder). Lanes 1, 3, 5, 7, and 9 are PCR-amplified but undigested DNA (364 bp), and lanes 2, 4, 6, 8, and 10 are DdeI-digested DNA. Lanes 1 and 2 are from a known carrier DNA (βAs, 4 bands); lanes 3 and 4 are from a known unaffected DNA (βAA, 3 bands); lanes 5 and 6 are from known affected DNA (βss, 2 bands); lanes 7 and 8 are from the female partner; and lanes 9 and 10 are from the male partner.

The IVF procedure has been described previously.20 Briefly, to ensure that several embryos would become available for DNA analysis, multiple ovarian follicular development was initiated with gonadotropin therapy. After pituitary desensitization with gonadotropin hormone–releasing hormone agonist (leuprolide acetate, TAP Pharmaceutical, Chicago, Ill), ovarian stimulation was begun on day 3 of the ensuing menstrual cycle using intramuscular administration of a combination of urofollitropin (75 U of pure follicle-stimulating hormone) and menotropins (150 U of follicle-stimulating hormone and luteinizing hormone) (Serono Laboratories, Norwell, Mass). Follicular growth was monitored by daily serum estradiol levels and pelvic ultrasonograms. To induce final oocyte maturation, 3300 IU of human chorionic gonadotropin was administered when 2 follicles of 18 mm in average diameter were observed on ultrasonogram. Transvaginal oocyte retrieval was performed 35 hours later. To avoid sperm contamination and possible amplification of sperm DNA, intracytoplasmic sperm injection was used. After 16 hours of incubation, fertilization was confirmed by the identification of 2 pronuclei. Normally fertilized concepti were then transferred to droplets of human tubal fluid (made on site) supplemented with 15% maternal serum under mineral oil (ER Squibb & Sons Inc, Princeton, NJ). Biopsy was performed on the morning of the third day after harvest. All embryos were maintained at 37°C in an atmosphere of 5% carbon dioxide. Cleavage rate and morphologic appearance were assessed daily.

Blastomere biopsy was carried out in the early morning, approximately 65 hours after oocyte collection. Briefly, a holding pipette was used to stabilize the embryo (at the 9 o'clock position). A hole was made at the 3 o'clock position by expelling a small amount of acidified Tyrode solution (pH, 2.35) onto the zona pellucida through a small-bore pipette. Reverse suction was applied as soon as a hole of appropriate size was created to reduce possible damage caused by exposing the blastomeres to the acidic solution. A large inner-diameter biopsy pipette replaced the pipette containing the acidified Tyrode solution. Subsequently, 1 or 2 cells were aspirated, depending on the total cell number of the embryo. Blastomeres were rinsed in biopsy medium 3 times before loading into a PCR tube containing 5 µL of lysis buffer. Tubes were processed and PCR amplification and restriction enzyme analysis were performed as described herein.

Micromanipulated embryos were further cultured in medium droplets overnight. Embryo transfer was performed in the afternoon of day 4.

Pregnancy was determined by serum β–human chorionic gonadotropin measurement on cycle days 28 and 35, followed by ultrasonographic assessment at 7 weeks' gestation. The genetic status of the fetuses was confirmed after amniocentesis by an independent laboratory. Cultured amniocytes were also sent to our laboratory for follow-up PCR analysis.

Polymerase chain reaction and restriction enzyme analysis of single lymphocytes from both the female and male partners clearly indicated that each carried the sickle cell mutation. Forty-six of 48 single lymphocytes, 24 from the female and 24 from the male, were successfully amplified. As predicted, 4 bands of correct size were obtained from both partners. An example of the gel is shown in Figure 1.

During the first IVF attempt in November 1996, 18 oocytes were retrieved. Four of 6 mature oocytes were normally fertilized after single-sperm injection by intracytoplasmic sperm injection, yielding 4 embryos.

Embryo biopsy was performed on all 4 embryos on day 3 by removing a single blastomere from each conceptus. Polymerase chain reaction and restriction enzyme analysis revealed that 1 was homozygous unaffected, 2 were carriers, and 1 was homozygous affected. Transfer of 1 unaffected embryo on day 4 failed to result in a pregnancy.

The second IVF attempt was initiated in August 1997, during which 16 oocytes were retrieved. Of those, 8 were mature and underwent intracytoplasmic sperm injection. Seven concepti cleaved at least once by the following day. On the morning of the third day, 2 cells were removed from 2 embryos and 1 cell from 5 embryos. Polymerase chain reaction amplification was successful in 7 of 9 blastomeres (5 of 6 embryos). Amplification failed in 1 cell from embryo 5 and in 1 cell from embryo 6. Restriction enzyme digestion demonstrated that 4 were homozygous unaffected, 2 were carriers (Figure 2), and 1 was of unknown status due to PCR amplification failure. On the afternoon of day 4, all concepti that underwent biopsy demonstrated further cleavage. Selection of embryos for transfer was based on PGD diagnosis, growth rate, and morphology. Two unaffected embryos were of poor quality, displaying slow cleavage and multinucleation (embryo 2) or high fragmentation (embryo 4) and therefore were not suitable for transfer (Table 1). Because there were only 3 high-quality transferable embryos—2 unaffected (embryo 1 with 15 cells; embryo 6 with 8 cells) and 1 carrier (embryo 8 with 10 cells) (Figure 3 and Table 1)—and because the patient was willing to accept a fetus of carrier status, all 3 embryos were transferred.

Figure 2. Restriction Analysis (DdeI) of PCR-Amplified DNA From Each Blastomere From 6 Embryos
Graphic Jump Location
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-digested DNA. Left, Lanes 1 and 2 (products from the blastomere of embryo 1)=βAA; lanes 3 and 4 (embryo 2)=βAA; lanes 5 and 6 (embryo 3)=βAs; and lanes 7 and 8 (unaffected DNA control)=βAA. Right, Lanes 1 and 2 (products from the blastomere of embryo 4)=βAA; lanes 3 and 4 (embryo 6)=βAA; lanes 5 and 6 (embryo 8)=βAs; and lanes 7 and 8 (products from spermatozoa of the male partner)=βAs. Note that in lane 8 on the left and lanes 2 and 4 on the right, a faint band (≈125 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 enzyme digestion and does not interfere with diagnosis because of its intensity and size.
Table Graphic Jump LocationTable. Detailed Information on Fertilization, Embryo Morphology, Biopsy, PGD Results, Intrauterine Transfer, and Implantation*
Figure 3. Embryo Morphology Prior to Transfer on Day 4
Graphic Jump Location
Numbers indicate embyos 1, 6, and 8.

A twin pregnancy was confirmed by ultrasonography at 7 weeks. Amniocentesis performed by an independent laboratory at 16.5 weeks revealed that neither fetus harbored the sickle cell mutation. DNA analysis of amniocytes shipped from the prenatal diagnostic laboratory to our own laboratory also showed that both fetuses were unaffected (Figure 4). The patient delivered healthy, unaffected fraternal twin girls at 39 weeks' gestation.

Figure 4. Polymerase Chain Reaction and Restriction Analysis From Amniocytes From the 2 Fetuses, Showing 2 Unaffected DNA Patterns
Graphic Jump Location
Bp indicates base pair; M, DNA size marker (100-bp ladder). Lane 1 shows unaffected DNA prior to DdeI digestion; lanes 2 and 3, unaffected DNA control (βAA, 3 bands) after DdeI digestion; lanes 4 and 5, fetus A (unaffected, βAA, 3 bands); and lanes 6 and 7, fetus B (unaffected, βAA, 3 bands).

After natural conception, couples who carry autosomal recessive mutations risk a 25% chance of delivering an affected child, and half of the offspring may carry the mutation. Although prenatal testing is currently available, some couples have strong personal objections to aborting affected fetuses. For these couples, PGD provides a realistic alternative to prenatal testing.

Although the first pregnancy achieved by PGD for sex determination to avoid the transmission of a sex-linked disorder occurred nearly a decade ago,17 PGD for single-gene defects is still in the experimental stage because of its complexity and technical difficulties. At present, PGD is primarily applied for severe genetic disorders for which detailed genetic information is available. Normal pregnancies following a search for specific mutations have been reported for only a few genetic diseases, including cystic fibrosis2224 and Tay-Sachs disease.25

Despite the fact that sickle cell anemia is one of the most common genetic disorders and detailed genetic information is available,1,26 unaffected pregnancies following PGD for sickle cell anemia previously have not been reported. Lack of previous success in this area presumably is due to the length of time and effort required to overcome technical difficulties inherent in these procedures, as well as lack of available research funding. Our results 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 of known genetic makeup that have undergone biopsy.

The protocol used in this study was initially developed in the mouse model by Sheardown et al.18 In their investigation, 4 tandem copies of the human β-globin gene were detected in transgenic mouse embryos. In humans, β-globin is a single-copy gene. Under clinical PGD circumstances, single-cell PCR requires an extremely sensitive protocol. In this study, we initially tested the protocol on single lymphocytes isolated from each member of the couple at risk. Lysis buffer, reported to be better for single-cell PCR,19 was used instead of the freeze-thaw method.18 Using the modified protocol, successful amplification was achieved in 46 (96%) of 48 single lymphocytes tested. This provided invaluable preliminary technical experience prior to executing the PCR-PGD technique in the clinical setting. However, PCR failure occurred in 1 of the 9 cells undergoing biopsy. Diagnostic failure in PCR-PGD could be 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 efficiency of this method.

We performed biopsies on day 3 and intrauterine transfer on day 4 because the biopsy, PCR, enzyme digestion, and electrophoresis could not be completed within 10 hours. This change provided additional valuable hours for accurate diagnosis. Apparently, the extended in vitro culture did not compromise embryo viability. Because both the morphology and genetic status of each of the transferred embryos were known, it is worth examining the characteristics of each embryo. On the morning of day 3, embryo 6 (Table 1) was composed of 7 blastomeres, 2 of which were removed for testing. This accounts for more than one fourth of the embryo volume. At the time of intrauterine transfer on day 4, the conceptus had reached the 8-cell stage. This embryo implanted, as indicated by its genetic status (βAA). It is known from animal models that biopsy does not decrease implantation and subsequent live birth rates28 and that embryo biopsy does not necessarily impair subsequent in vitro development in humans.29 This particular case unequivocally shows that the removal of 2 blastomeres from a 7-cell conceptus did not compromise its developmental potential. Furthermore, our results confirm that day-3 biopsy and day-4 transfer is indeed a feasible approach for PGD. Recent progress in developing culture media30,31 and/or human autologous endometrial coculture32,33 may even further enhance embryo viability, thus increasing the pregnancy rate after PGD.

In summary, this is the first unaffected pregnancy and delivery after successful PGD for sickle cell anemia. Our results demonstrate that PGD for the detection of sickle cell anemia is a powerful diagnostic tool for carrier couples who desire a healthy child but wish to avoid the difficult decision of whether to abort an affected fetus. The procedure, successfully used in this case, may also be applied to other monogenic disorders and further supports the notion that PGD is destined to be an integral aspect of assisted reproductive technology. Given the current methods and relatively high cost of the procedure, it is unlikely that PGD will totally replace prenatal testing. However, it is conceivable that with further refinements, PGD will certainly become an invaluable and powerful diagnostic modality.

Motulsky AG. Frequency of sickling disorders in US blacks.  N Engl J Med.1973;288:31-33.
Rogers ZR, Powars DR, Kinney TR, Williams WD, Schroeder WA. Nonblack patients with sickle cell disease have African βs gene cluster haplotypes.  JAMA.1989;261:2991-2994.
Powars DR. Sickle cell disease in nonblack persons.  JAMA.1994;271:1885.
Davis H, Gergen PJ, Moore Jr RM. Geographic differences in mortality of young children with sickle cell disease in the United States.  Public Health Rep.1997;112:52-58.
Platt OS, Brambilla DJ, Rosse WF.  et al.  Mortality in sickle cell disease: life expectancy and risk factors for early death.  N Engl J Med.1994;330:1639-1644.
Cohen AR. Sickle cell disease: new treatments, new questions.  N Engl J Med.1998;339:42-44.
Adams RJ, McKie VC, Hsu L.  et al.  Prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial doppler ultrasonography.  N Engl J Med.1998;339:5-11.
Gay JC, Phillips III JA, Kazazian Jr HH. Hemoglobinopathies and thalassemia. In: Rimoin DL, Connor JM, Pyeritz RE, eds. Emery and Rimon's Principles and Practice of Medical Genetics3rd ed. New York, NY: Churchill Livingstone Inc; 1996:1599-1626.
Kan YW, Dozy AM. Antenatal diagnosis of sickle-cell anaemia by DNA analysis of amniotic-fluid cells.  Lancet.1978;2:910-912.
Chang JC, Kan YW. A sensitive new prenatal test for sickle-cell anemia.  N Engl J Med.1982;307:30-32.
Orkin SH, Little PFR, Kazazian Jr HH, Boehm CD. Improved detection of the sickle mutation by DNA analysis.  N Engl J Med.1982;307:32-36.
Saiki RK, Scharf S, Faloona F.  et al.  Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.  Science.1985;230:1350-1354.
Embury SH, Scharf SJ, Saiki RK.  et al.  Rapid prenatal diagnosis of sickle cell anemia by a new method of DNA analysis.  N Engl J Med.1987;316:656-661.
Chehab F, Doherty M, Cai S, Kan YW, Cooper S, Rubin EM. Detection of sickle cell anemia and thalassemias.  Nature.1987;329:293-294.
Kulozik AE, Lyons J, Kohne F, Bartram CR, Kleihauer E. Rapid and non-radioactive prenatal diagnosis of β-thalassemia and sickle cell disease: application of the polymerase chain reaction (PCR).  Br J Haematol.1988;70:455-458.
Wu DY, Ugozzoli L, Pal BK, Wallace RB. Allele-specific enzymatic amplification of β-globin genomic DNA for diagnosis of sickle cell anemia.  Proc Natl Acad Sci U S A.1989;86:2757-2760.
Handyside AH, Kontogianni EH, Hardy K, Winston RM. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification.  Nature.1990;344:768-770.
Sheardown SA, Findlay I, Turner A.  et al.  Preimplantation diagnosis of a human β-globin transgene in biopsied trophectoderm cells and blastomeres of the mouse embryo.  Hum Reprod.1992;7:1297-1303.
Li H, Cui X, Arnheim N. Analysis of DNA sequence variation in single cells.  Methods Enzymol.1991;2:49-59.
Davis O, Rosenwaks Z. In vitro fertilization. In: Adashi EY, Rock JA, Rosenwaks Z, eds. Reproductive Endocrinology, Surgery, and Technology.Philadelphia, Pa: Lippincott-Raven Publishers; 1996:2319-2334.
Veeck LL. An Atlas of Human Gametes and Conceptuses. London, England: Parthenon Publishing; 1999:48.
Handyside AH, Lesko JG, Tarin JJ, Winston RM, Hughes MR. Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis.  N Engl J Med.1992;327:905-909.
Liu J, Lissens W, Devroey P, Van Steirteghem A, Liebaers I. Polymerase chain reaction analysis of the cystic fibrosis ΔF508 mutation in human blastomeres following oocyte injection of a single sperm from a carrier.  Prenat Diagn.1993;13:873-880.
Ao A, Ray P, Harper J.  et al.  Clinical experience with preimplantation genetic diagnosis of cystic fibrosis (ΔF508).  Prenat Diagn.1996;16:137-142.
Gibbons WE, Gitlin SA, Lanzendorf SE, Kaufmann RA, Slotnick RN, Hodgen GD. Preimplantation genetic diagnosis for Tay-Sachs diseases: successful pregnancy after pre-embryo biopsy and gene amplification by polymerase chain reaction.  Fertil Steril.1995;63:723-728.
Platt OS, Dover GJ. Sickle cell disease. In: Nathan DG, Oski FA, eds. Hematology of Infancy and Childhood4th ed. Philadelphia, Pa: WB Saunders Co; 1995:732-782.
Harper JC, Coonen E, Handyside AH, Winston RML, Hopman AHN, Delhanty JDA. Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos.  Prenat Diagn.1995;15:41-49.
Takeuchi K, Sandow BC, Morsy M, Kaufmann RA, Beebe SJ, Hodgen GD. Preclinical models for human pre-embryo biopsy and genetic diagnosis, I: efficiency and normalcy of mouse pre-embryo development after different biopsy techniques.  Fertil Steril.1992;57:425-430.
Hardy K, Martin KL, Leese HJ, Winston RML, Handyside AH. Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage.  Hum Reprod.1990;5:708-714.
Barnes F, Crombie A, Gardner DK.  et al.  Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching.  Hum Reprod.1995;10:3243-3247.
Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfer.  Fertil Steril.1998;69:84-88.
Barmat LI, Liu H-C, Spandorfer SD.  et al.  Human preembryo development on autologous endometrial coculture versus conventional medium.  Fertil Steril.1998;70:1109-1113.
Menezo YJR, Bellec V, Zaroukian A, Benkhalifa M. Embryo selection by IVF, co-culture and transfer at the blastocyst stage in case of translocation.  Hum Reprod.1997;12:2802-2803.

Figures

Figure 1. Establishment of Single-Cell PCR and Confirmation of the Carrier Status of Female and Male by Restriction Enzyme Analysis
Graphic Jump Location
PCR indicates polymerase chain reaction; bp, base pair; and M, DNA size marker (100-bp ladder). Lanes 1, 3, 5, 7, and 9 are PCR-amplified but undigested DNA (364 bp), and lanes 2, 4, 6, 8, and 10 are DdeI-digested DNA. Lanes 1 and 2 are from a known carrier DNA (βAs, 4 bands); lanes 3 and 4 are from a known unaffected DNA (βAA, 3 bands); lanes 5 and 6 are from known affected DNA (βss, 2 bands); lanes 7 and 8 are from the female partner; and lanes 9 and 10 are from the male partner.
Figure 2. Restriction Analysis (DdeI) of PCR-Amplified DNA From Each Blastomere From 6 Embryos
Graphic Jump Location
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-digested DNA. Left, Lanes 1 and 2 (products from the blastomere of embryo 1)=βAA; lanes 3 and 4 (embryo 2)=βAA; lanes 5 and 6 (embryo 3)=βAs; and lanes 7 and 8 (unaffected DNA control)=βAA. Right, Lanes 1 and 2 (products from the blastomere of embryo 4)=βAA; lanes 3 and 4 (embryo 6)=βAA; lanes 5 and 6 (embryo 8)=βAs; and lanes 7 and 8 (products from spermatozoa of the male partner)=βAs. Note that in lane 8 on the left and lanes 2 and 4 on the right, a faint band (≈125 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 enzyme digestion and does not interfere with diagnosis because of its intensity and size.
Figure 3. Embryo Morphology Prior to Transfer on Day 4
Graphic Jump Location
Numbers indicate embyos 1, 6, and 8.
Figure 4. Polymerase Chain Reaction and Restriction Analysis From Amniocytes From the 2 Fetuses, Showing 2 Unaffected DNA Patterns
Graphic Jump Location
Bp indicates base pair; M, DNA size marker (100-bp ladder). Lane 1 shows unaffected DNA prior to DdeI digestion; lanes 2 and 3, unaffected DNA control (βAA, 3 bands) after DdeI digestion; lanes 4 and 5, fetus A (unaffected, βAA, 3 bands); and lanes 6 and 7, fetus B (unaffected, βAA, 3 bands).

Tables

Table Graphic Jump LocationTable. Detailed Information on Fertilization, Embryo Morphology, Biopsy, PGD Results, Intrauterine Transfer, and Implantation*

References

Motulsky AG. Frequency of sickling disorders in US blacks.  N Engl J Med.1973;288:31-33.
Rogers ZR, Powars DR, Kinney TR, Williams WD, Schroeder WA. Nonblack patients with sickle cell disease have African βs gene cluster haplotypes.  JAMA.1989;261:2991-2994.
Powars DR. Sickle cell disease in nonblack persons.  JAMA.1994;271:1885.
Davis H, Gergen PJ, Moore Jr RM. Geographic differences in mortality of young children with sickle cell disease in the United States.  Public Health Rep.1997;112:52-58.
Platt OS, Brambilla DJ, Rosse WF.  et al.  Mortality in sickle cell disease: life expectancy and risk factors for early death.  N Engl J Med.1994;330:1639-1644.
Cohen AR. Sickle cell disease: new treatments, new questions.  N Engl J Med.1998;339:42-44.
Adams RJ, McKie VC, Hsu L.  et al.  Prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial doppler ultrasonography.  N Engl J Med.1998;339:5-11.
Gay JC, Phillips III JA, Kazazian Jr HH. Hemoglobinopathies and thalassemia. In: Rimoin DL, Connor JM, Pyeritz RE, eds. Emery and Rimon's Principles and Practice of Medical Genetics3rd ed. New York, NY: Churchill Livingstone Inc; 1996:1599-1626.
Kan YW, Dozy AM. Antenatal diagnosis of sickle-cell anaemia by DNA analysis of amniotic-fluid cells.  Lancet.1978;2:910-912.
Chang JC, Kan YW. A sensitive new prenatal test for sickle-cell anemia.  N Engl J Med.1982;307:30-32.
Orkin SH, Little PFR, Kazazian Jr HH, Boehm CD. Improved detection of the sickle mutation by DNA analysis.  N Engl J Med.1982;307:32-36.
Saiki RK, Scharf S, Faloona F.  et al.  Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.  Science.1985;230:1350-1354.
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