Fetal and neonatal alloimmune thrombocytopenia is the most common cause of severe fetal and neonatal thrombocytopenia and occurs in 1 of 1,200 pregnancies. Fetal and neonatal alloimmune thrombocytopenia is caused by maternal sensitization to paternally derived antigens expressed on fetal platelets. These maternal immunoglobulin G antibodies can cross the placenta and bind fetal platelets, causing their destruction.1 Incompatibility of antihuman platelet antigens is the most frequent cause of fetal and neonatal alloimmune thrombocytopenia. Multiple different antigen incompatibilities have been described, with the majority being caused by antihuman platelet antigen-1a (77%).2 Antihuman platelet antigen-1 incompatibility is attributable to a single amino acid difference in the platelet membrane glycoprotein IIIa gene (INTEGRIN; BETA-3; ITGB3), leucine instead of Proline at codon 33, a difference caused by a single nucleotide variant (ITGB3 T196C, rs5918).3 Approximately 2% of the caucasian population is homozygous for the variant human platelet antigen-1b allele (C) and can produce antibodies against the more common human platelet antigen-1a allele (T). Intracranial hemorrhage has been reported in 7–26% of newborns with fetal and neonatal alloimmune thrombocytopenia,4 with a fatal outcome in approximately one third of these cases5 and long-lasting and severe sequelae for the survivors. If such a complication occurs, then the chance of recurrence in the next pregnancy is high in untreated antigen-positive siblings.6 Because there is no routine screening, human platelet antigen incompatibility is usually identified only after the first fetal and neonatal alloimmune thrombocytopenia case in a family. After identifying an affected neonate, antenatal management in subsequent pregnancies is considered to be the only therapeutic option. Administration of intravenous immunoglobulin (IVIG) to the mother (1–2 g/kg/wk), with or without dexamethasone or prednisone, was the first modality of antenatal therapy to prevent intracranial hemorrhage.7 However, using this therapy, numerous studies still report a constant rate of intracranial hemorrhage of 2.7–2.9%.6 Monitoring treatment using routine weekly intrauterine fetal blood sampling has been abandoned because of its many risks, eg, puncture-site hemorrhage, emergency cesarean deliveries, exsanguination and fetal demise, and further maternal sensitization. Its use now is reserved for sampling before platelet intrauterine transfusion or when contemplating vaginal delivery, and it is individualized based on experience and product availability for each institution.8,9 Because the invasive approach to the antenatal intrauterine therapy is associated with both fetal and maternal complications, investigators have sought various alternatives.
Some have suggested risk stratification of patients, limiting invasive fetal blood sampling to women with a history of severe allergic response or neonatal intracranial hemorrhage, and using empiric therapy in women with a history of successful response to IVIG.2,10,11 Moreover, the response to treatment may be affected by many additional factors, such as maternal human leukocyte antigen and possible ABO incompatibility.12 Fetal genotyping in couples with known human platelet antigen incompatibility identifies carriers of antiplatelet antibodies13 but does not eliminate the need for therapy. For patients who have previously experienced severe allergic responses, extensive IVIG treatment may not be an option. For this selected group of couples, preimplantation genetic diagnosis for the selection of fetuses that are not at risk would completely alleviate the need for potentially hazardous antenatal invasive procedures or treatment that may compromise both fetal and maternal health.
Preimplantation genetic diagnosis was developed two decades ago for couples at genetic risk for having pregnancies with potentially affected embryos. Preimplantation genetic diagnosis is performed by analyzing blastomeres (one cell of a six- to eight-cell embryo), polar bodies (extruded by the oocyte), or more recently by blastocyst biopsy14 for Mendelian and chromosomal disorders.15 Developed first for severe genetic disorders, preimplantation genetic diagnosis was subsequently applied for human leukocyte antigen matching, Rh incompatibility, and Kell incompatibility.16,17 Although, in theory, preimplantation genetic diagnosis could be accomplished using mutation analysis alone, because of allele drop-out this would be accompanied by a high error rate that could reach 20%.18 Allele drop-out is present in single-cell polymerase chain reaction and is attributable to amplification of only one of two alleles. Therefore, preimplantation genetic diagnosis protocols include several linked polymorphic microsatellite markers flanking the disease gene to minimize misdiagnosis attributable to allele drop-out.19,20
We were prompted to develop a reliable preimplantation genetic diagnosis protocol for human platelet antigen-1 incompatibility by a family in whom antenatal treatment was not possible because of the mother's hypersensitivity to IVIG. Preimplantation genetic diagnosis for human platelet antigen-1 incompatibility eliminated the need for antenatal therapy and resulted in the birth of a healthy newborn.
MATERIALS AND METHODS
A couple in which the woman (32 years old) was homozygous for the human platelet antigen-1b (C/C) allele and the man (34 years old) was heterozygous for the human platelet antigen-1a/1b (C/T) alleles presented to our preimplantation genetic diagnosis unit. After a pregnancy and vaginal delivery of a healthy boy, a second pregnancy ended in a spontaneous abortion at 10 weeks. The next child was born by vaginal delivery and intracranial hemorrhage was diagnosed with a platelet count of 60,000 platelets/microliter. This sequence of events raised the suspicion of fetal and neonatal alloimmune thrombocytopenia, which was subsequently confirmed by the identification of maternal antihuman platelet antigen-1a antibodies (by MAIPA assay). Genetic testing by single-strand conformation polymorphism analysis revealed incompatibility between the parents; the mother was homozygous for the human platelet antigen-1b/b allele and the father and neonate were heterozygous (human platelet antigen-1a/b). Prenatal genetic diagnosis during the fourth pregnancy showed incompatibility and the expectant mother received 10 treatments of IVIG, but a severe allergic reaction developed that required intensive care and respiratory support because of anaphylactic shock. The child was born prematurely at 30 weeks of gestation by cesarean delivery with a platelet count of 110,000 platelets/microliter. Because IVIG could not be administered for further pregnancies, the couple optioned for preimplantation genetic diagnosis for the next pregnancy.
In vitro fertilization (IVF) treatment including ovarian stimulation and oocyte retrieval was performed using the long downregulation protocol as previously described.21 Intracytoplasmic sperm injection blastomere biopsy and embryo culture were performed as previously described.22
Genetic testing for rs5918 (T196C) of the ITGB3 gene in conjunction with haplotype analysis using microsatellite markers was performed with DNA extracted from peripheral blood cells using high salt precipitation.23 Fourteen markers surrounding the ITGB3 gene were selected carefully to exclude duplicated regions that could interfere with accurate analysis (Fig. 1). Eight informative polymorphic microsatellite markers were identified flanking the ITGB3 gene (D17S920, D17S791, D17S1834, human platelet antigen-AC2 [chr 17:42683943], D17S1859, human platelet antigen-AC5 [chr 17:43462414], human platelet antigen-AC7 [chr 17:43696707], and human platelet antigen-TG1 [chr 17:43907747], UCSC genomic browser March 2006 [NCBI36/hg18])24 (Fig. 1) and were used for haplotype construction.
Individual blastomeres underwent biopsy and were transferred to tubes containing 5 microliters of proteinase K lysis buffer25 and incubated at 45°C for 15 minutes, followed by inactivation at 94°C for 15 minutes. A multiplex polymerase chain reaction for all eight informative markers was performed in single cells (Fig. 2); 1.5 microliters from each reaction were used as a template for hemi-nested polymerase chain reaction with one primer, 5′ fluorescently labeled with 6-FAM, HEX, or TAMRA, for an additional 35 cycles for each of the eight individual markers. Reaction products were diluted and run on an ABI Prism 3100xl Avant automated sequencer and analyzed using GeneMapper software ABI. Preimplantation genetic diagnosis is a routine clinical procedure in our institution; therefore, no Institutional Review Board approval was required.
Eight informative markers were identified using genomic DNA before the preimplantation genetic diagnosis cycle and were used in conjunction with the C196T variant for single-cell analysis. In one IVF–preimplantation genetic diagnosis cycle, 28 oocytes were retrieved; all were mature (MII oocytes) and underwent intracytoplasmic sperm injection. Twenty-four oocytes fertilized, and eight zygotes were cryopreserved before biopsy. Twelve embryos reached the six- to eight-cell stage on day 3 and underwent biopsy. Three embryos were found to be homozygous for human platelet antigen-1b (C/C) and suitable for transfer, seven were heterozygous for human platelet antigen-1b/1a (C/T), and therefore not suitable for transfer, and two showed total amplification failure. Two of the three homozygous human platelet antigen-1b/1b homozygous embryos were transferred, resulting in an uncomplicated vaginal birth of a healthy boy weighing 3,260 g with a platelet count of 280,000/microliters at birth. The genotype was confirmed postnatally. The overall combined allele drop-out rate for all reactions was 10%.
We present the development of a reliable preimplantation genetic diagnosis method for preventing fetal and neonatal alloimmune thrombocytopenia syndrome in cases in which the proband is homozygous for human platelet antigen-1b/1b and the spouse is heterozygous for human platelet antigen-1a/1b. Approximately 10% of human platelet antigen-1b homozygous women who had been pregnant with a human platelet antigen-1a fetus develop antibodies to human platelet antigen-1a.26 Although fetal and neonatal alloimmune thrombocytopenia occurs in only 1 out of 1,200 pregnancies,27 it can have severe fetal implications. Without treatment, approximately 10–20% of affected fetuses will have development of intracranial hemorrhage, with up to 50% of these occurring in utero.28 Usually, after birth of a child with petechiae or other signs of bleeding, the diagnosis of fetal and neonatal alloimmune thrombocytopenia is confirmed by antihuman platelet antigen identification and demonstration of genetic incompatibility between parents. The main reason that fetal and neonatal alloimmune thrombocytopenia is not considered for prophylactic treatment, similar to hemolytic disease of the newborn, is because of the fact that self-immunization against human platelet antigen-1a only occurs during the first incompatible pregnancy.29 Intravenous immunoglobulin therapy is currently the standard care for pregnancies at risk,27 sometimes in conjunction with fetal blood sampling for fetal platelet count. Whereas fetal blood sampling is an invasive procedure, it can reduce the number of IVIG infusions because only when the fetal platelet count is less than 100,000/microliter is the IVIG treatment administered. Undesirable effects from IVIG treatment occur in less than 5% of patients. Common adverse effects occur soon after infusions and include headache, flushing, chills, myalgia, wheezing, tachycardia, lower back pain, nausea, and hypotension.30 Anaphylactic reactions have rarely occurred in patients receiving IVIG therapy.31 In couples in which a human platelet antigen-1b/b homozygous woman has development of alloimmune response against the human platelet antigen-1a antigen, and when the available treatment is not recommended because of allergic reactions, preimplantation genetic diagnosis is the most attractive option to avoid a fetus or neonate with thrombocytopenia. Although fertile couples undergoing preimplantation genetic diagnosis may experience physical and psychological stress associated with IVF, the knowledge that only human platelet antigen-compatible embryos will be transferred should alleviate emotional stress caused by possible autoimmune pregnancy. In this case, the first born son's human platelet antigen genotype was human platelet antigen-1a/1b (C/T), and the subsequent pregnancy resulted in a fetus that had development of intracranial hemorrhage in utero. In the next pregnancy, which was found to be incompatible as well, the proband received IVIG from week 24 of gestation, but a severe allergic reaction developed and the treatment had to be stopped at 30 weeks of gestation. The child was born asymptomatic with mild thrombocytopenia (100,000 platelets/microliter). For the next pregnancy, the couple opted to perform preimplantation genetic diagnosis.
Although costs of preimplantation genetic diagnosis and IVIG treatment for fetal and neonatal alloimmune thrombocytopenia vary in different countries, Thung et al27 reported the cost-effectiveness of empiric IVIG treatment for human platelet antigen with and without fetal blood sampling in the United States and found an average cost of $88,850 per woman receiving empiric therapy compared with $76,341 when fetal blood sampling is measured. The cost of one preimplantation genetic diagnosis cycle in the United States was reported by Turkaspa et al31 as between $15,000 and $20,000 and includes pre-IVF screening ($1,000), medications ($1,500–$5,000), cost of the IVF cycle ($12,000), genetic system set-up ($1,000–$2,000), biopsy of embryos ($1,500), and genetic analysis of embryos ($3,000). Even if three cycles are required to achieve a pregnancy, this is less costly than the IVIG treatment suggested by Thung et al.27 In Israel, the estimated costs of IVIG are $70,000–$90,000 per human platelet antigen patient for 10- to 13-week therapy,33 whereas the costs of one IVF–preimplantation genetic diagnosis cycle (at our center, including set-up) are only $7,000. The economics of the cost-to-benefit ratio in Israel makes preimplantation genetic diagnosis an even more attractive option.
Molecular analysis for human platelet antigen-1 in single cells was already reported in 19943 as a proof of principle that human platelet antigen-1 status determination from single cell analysis is reliable. The protocol used only the discrimination between the T and C alleles of the rs5918 variant in the ITGB3 gene. Preimplantation genetic diagnosis analysis using only the mutation or allele variant is the main cause of misdiagnosis because of allele drop-out in single cells. The addition of at least three polymorphic markers in conjunction with the mutation has been demonstrated to reduce the risk of misdiagnosis attributable to allele drop-out to almost 0%.34 We elected to include eight polymorphic markers in our analysis. Our overall allele drop-out rates (10%) were lower than those described in the literature for blastomere analysis.34 Although, statistically, 50% of the embryos would be predicted to be human platelet antigen-1b, on preimplantation genetic diagnosis analysis only three out of the 12 embryos were found to be human platelet antigen-1b (C/C) and suitable for transfer. Two were transferred, resulting in birth of a healthy boy (human platelet antigen-1b homozygote [C/C]).
This is a report of preimplantation genetic diagnosis for fetal and neonatal alloimmune thrombocytopenia resulting in the birth of a healthy newborn using a strategy that includes multiple markers in conjunction with the rs5918 variant. This method is an alternative for women who cannot receive IVIG treatments during pregnancy and may be considered in cases involving a history of severe fetal and neonatal alloimmune thrombocytopenia.
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