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Chapter 23 Preimplantation genetic diagnosis
Chapter 23 Preimplantation genetic diagnosis BACKGROUND That preimplantation genetic diagnosis (PGD) would emerge first as a series of clinical experiments had been anticipated for more than a decade, but especially since the efficacy of in vitro fertilization (IVF) improved dramatically during the mid1980s. This mode of intervention is particularly attractive to couples who do not accept therapeutic abortion as a useful option after intrauterine diagnosis of genetic mutations. By the 1990s, the confluence of assisted reproductive technologies (ARTs) with molecular biology spawned the new era of human PGD. An increasing number of research laboratories have contributed to this nascent technology, which has already been employed to prevent several inherited diseases, including hemophilia, cystic fibrosis, and Tay-Sachs disease (Jones, 1987; Saiki, 1988; Summers, 1988; Ethics Committee, 1990; Gordon, 1990; Grifo, 1990; Handyside, 1990; Hardy, 1990; Monk, 1990; Simpson, 1990; Verlinsky, 1990; Morsy, 1992; Takeuchi, 1992; Gibbons, 1994). These ongoing clinical experiments using human preembryos were preceded by several decades of preclinical investigation, both in embryological animal models and evolving DNA amplification techniques coupled with methodologies for mutation detection. Chief among these preclinical databases were observations demonstrating the utility and safety of microbiopsy at various preimplantation stages. That is, a DNA sample could be acquired reliably via preembryo biopsy with minimal trauma to the preembryo during removal of one or two blastomeres or their nuclei, whether at the two-cell stage or at progressive stages of development through the formation of the blastocyst. Both the potential for ultimate viability and the normalcy of children derived from microbiopsied preembryos were preserved. It is imperative to recognize that the current status of human PGD remains in the realm of research. Ultimately, a much broader experience is required in order to offer a medical service for 648
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which an adequate database indicates reliable, unambiguous preembryo diagnosis to prevent inherited disease. Indeed, the consequences of an erroneous diagnosis can be tragic and far more devastating than failure to achieve pregnancy. Accordingly, an appropriate, cautious pace of progress has characterized the evolution of PGD thus far. With the efficacy of IVF, oocyte diagnosis has now become feasible in humans. Gamete aneuploidy is certainly a possible explanation for low fecundity rates of approximately 20% observed in normal cycles or in IVF. There appear to be differences between spermatozoa and oocytes in the frequency of chromosomal abnormalities. Appro ximately 5% to 13% of sperm possess chromosomal abnormalities, with structural abnormalities more common than numerical abnormalities (aneuploidies) in most series (Pellestor, 1991). Reports of oocyte abnormalities are considerably more variable, but in general, aneuploidy is more frequently observed than structural abnormalities (Martin, 1986; Wramsby, 1987; Van Blerkom, 1988; Tarin, 1991). There are several potential means to achieve PGD. One method involves polar body (PB) biopsy, the basic premise of which relies upon the events taking place in normal meiosis. In meiosis I, 46 chromosomes are present. These then replicate to produce a four-nuclei (4n) complement of genetic material. For an oocyte containing a normal (wild type) allele and a mutant allele, the first meiotic division results in one allele going to the oocyte and the other allele going to the PB. Each of these gametes contains a 2n complement of chromosomes. In the second meiotic division, the resulting cells possess a haploid number of chromosomes. If the oocyte is normal, the PB analysis by polymerase chain reaction (PCR) should be abnormal. Likewise, if the oocyte possesses the mutation, the PB will be normal. Performance of PCR on the first PB provides genetic diagnosis and spares the oocyte, which may be used for insemination (Coutelle, 1989). It must be stressed, however, that PB diagnosis can correctly identify mutant versus wild Fertility and Sterility
alleles only if no crossing-over occurs. Should crossing-over occur, both the oocyte and the PB will be heterozygous; thus the genotype of the embryo cannot be predicted. Crossing-over is much more common at the telomeric regions of chromosomes than at the centromeres; thus gene location on the chromosome becomes important. Polar body diagnosis offers the only practical method for gamete PGD because in sperm analysis such study destroys the gamete. Unlike meiosis in the male, which results in four haploid spermatozoa, meiosis in the female yields one mature oocyte and three PBs. Through the study of PBs, the genotype can be predicted for the PB, thereby sparing the secondary oocyte for fertilization. Only oocytes containing normal alleles would be used for fertilization, whereas those harboring mutant alleles would not be inseminated. INDICATIONS
There are more than 4,000 known genetic mutations associated with specific inherited human diseases, wherein aberrant nucleotide insertions or deletions are predicted to be causal. A dozen or so of these single gene defects are now amenable to preventive intervention by PGD, but many more could theoretically be detected by this method. An issue of complexity is that for many Mendelian mutations, such as cystic fibrosis, there are multiple molecular mutations. Specific nucleotide primer systems are often required to amplify each mutant region via PCR. Perhaps the technology will move toward simultaneous co-amplification of a number of potential genetic disease states. Determination of gender (XY or XX) can be accomplished also by molecular methods. However, some favor the use of fluorescent in situ hybridization without PCR for DNA amplification. Fluorescent in situ hybridization is also likely to be the preferred methodology for detecting an aneuploidy. With regard to conditions involving more than one gene (multifactorial, polygenic), such as spina bifida, diabetes, or cardiovascular disease, the diagnostic methodology for PGD is not available at present. For now, we must rely on a rudimentary understanding of Mendelian inheritance patterns to prevent single gene defects, for example, homozygous recessive diseases (25% risk of passage) like Tay-Sachs or cystic fibrosis, X-linked diseases that are passed from mothers to 50% of their sons, or dominant diseases such as Huntington's (50% risk of passage). Vol. 62, No.5, November 1994
RESERVATIONS
There is general agreement that prevention of inherited diseases by not transferring to the uterus those preembryos with specific genetic mutations is justifiable. Public support weighs heavily in favor of PGD when the birth of children who would experience severe health problems that would diminish their quality of life and longevity and the consequences of these factors on parents, siblings, and others close to them is examined alongside the opportunity to assure avoidance of this fate. Although some members of the Committee believe that nondisease uses of PG D are ethically unacceptable, other members would not exclude some uses of PGD in special circumstances for gender selection. All members of the Committee share the view that such uses should not occur without outside ethical review-national or local-of the need and circumstances. Some persons, however, may object to PGD even when its applications are limited to prevention of severely debilitating, even terminal, inherited diseases. Generally, public acceptance of these technologies for disease prevention rises with earlier intervention, parallel with opinions that the value of human life rises as development progresses from fertilization, implantation and viability to birth (see chapter 26). Reservations about PGD stem primarily from potential applications that depart from medical indications expressly for disease prevention. For example, gender, skin color, and "straight" teeth are characteristics of persons; these features are not diseases or significant risk factors that severely limit quality of life or longevity. Parental preferences based on such arbitrary or trivial factors that stem from certain "desirable" physical characteristics or perceived cognitive advantages are not yet acceptable rationales for intervention via PGD or prenatal diagnosis. Three primary factors may be useful in determining the appropriateness of PG D. Examples may clarify the principal factors involved. First, prevention of devastating inherited diseases that markedly curtail quality of life and longevity is justifiably the priority toward which society's stewardship of PGD should be directed. Tay-Sachs disease is a vivid example in that the risk element to carrier parents is 1 in 4, with affected children manifesting developmental problems from as early as 6 months of age and dying usually by 3 or 4 years of age. This scenario is destructive to the family and often fin ancially devastating as well. Prevention of Tay-Sachs Supplement 1
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and similar diseases by PGD provides a method of disease avoidance that is ethically acceptable and more desirable than prevention after pregnancy (later prenatal diagnosis and elective abortion). Second is the issue of gender selection. When this technology is used to prevent an X -linked genetic disease such as hemophilia or Duchenne's muscular dystrophy, public support is generally strong, especially when the diagnostic methodology includes identification of the "well" male preembryo via mutation detection. If unaffected male preembryos are discarded along with ones containing X-linked mutations, then the objection rate rises somewhat. However, the use oftechnology for gender selection technology solely to fulfill parental preferences raises many questions. Although so-called "family balancing" services are ardently sought by a growing number of prospective patients, the use ofPGD solely for gender selection is not acceptable. Some persons would argue that parental liberty should extend to the choice of gender, especially if the preconception or preimplantation method does not interrupt pregnancy. Many others would strongly oppose any use of PGD or other reproductive technology that would enable parents to control the gender of their offspring, except in cases of Xlinked inherited disease. The fear is that availability of preembryo gender selection, other than to prevent an X-linked inherited disease, would foster gender discrimination and anti-social trends that American law and governmental policies have sought to remedy throughout this century. The third factor is deference to be shown to the preembryo because of the sanctity of human life. As stated in chapter 1, human life in the form of a preembryo deserves respect and value above that accorded to other cells and tissues of human derivation which do not themselves possess the inherent potential of a new human being. Because of the preembryo's rudimentary status and the fact that preembryo discard in other circumstances is accepted (such as discard of unwanted frozen preembryos), a decision to discard preembryos because they have a serious genetic defect seems ethically
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acceptable. However, many persons would argue that respect for the sanctity of human life prohibits the discard of human preembryos simply because they do not contain some preferred trait or characteristic desired by parents. COMMITTEE RECOMMENDATIONS
The Committee finds strong ethical justification for PGD in the prevention of inherited diseases that severely deplete the quality of human life or longevity; individual couples or families should make this judgment. This position derives principally from recognition of the suffering of affected individuals and their families, as well as the impact on society. The Committee recognizes however that PGD is experimental technology and therefore should be carried out as a clinical experiment. The Committee finds highly problematic the use of gender selection to achieve "family balancing" or other preferential goals based on nondisease traits. However, it may be premature to declare that there are absolutely no circumstances under which gender selection should be used, regardless of the technology involved in achieving it. A special advantage of PGD is that it will preclude the need for a couple to undergo later prenatal diagnosis and terminate a pregnancy in case of a positive result. The appeal ofPGD is that its goal is the same as prenatal screening and abortion for genetic indications, but it achieves this end by screening preembryos rather than fetuses.*
* The attitude of this chapter toward terminating life is its weakest analytic component. For instance, it argues from "the fact that preembryo discard in other circumstances is accepted (such as discard of unwanted frozen preembryos)" to the ethical acceptability of discarding preembryos with genetic defects. This analysis does not take any account of the fact that many people would not accept the discard of unwanted or frozen preembryos, nor does it display any arguments to bolster its position. The AFS Ethics Committee is on record as saying that the preembryo deserves "special respect." How casual discard of unwanted preembryos is compatible with such respect is unclear. If preimplantation genetic diagnosis is embedded in and reinforces this loose evaluation of the preembryo, it too is vulnerable to powerful ethical objections. (RAM)
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which an adequate database indicates reliable, unambiguous preembryo diagnosis to prevent inherited disease. Indeed, the consequences of an erroneous diagnosis can be tragic and far more devastating than failure to achieve pregnancy. Accordingly, an appropriate, cautious pace of progress has characterized the evolution of PGD thus far. With the efficacy of IVF, oocyte diagnosis has now become feasible in humans. Gamete aneuploidy is certainly a possible explanation for low fecundity rates of approximately 20% observed in normal cycles or in IVF. There appear to be differences between spermatozoa and oocytes in the frequency of chromosomal abnormalities. Appro ximately 5% to 13% of sperm possess chromosomal abnormalities, with structural abnormalities more common than numerical abnormalities (aneuploidies) in most series (Pellestor, 1991). Reports of oocyte abnormalities are considerably more variable, but in general, aneuploidy is more frequently observed than structural abnormalities (Martin, 1986; Wramsby, 1987; Van Blerkom, 1988; Tarin, 1991). There are several potential means to achieve PGD. One method involves polar body (PB) biopsy, the basic premise of which relies upon the events taking place in normal meiosis. In meiosis I, 46 chromosomes are present. These then replicate to produce a four-nuclei (4n) complement of genetic material. For an oocyte containing a normal (wild type) allele and a mutant allele, the first meiotic division results in one allele going to the oocyte and the other allele going to the PB. Each of these gametes contains a 2n complement of chromosomes. In the second meiotic division, the resulting cells possess a haploid number of chromosomes. If the oocyte is normal, the PB analysis by polymerase chain reaction (PCR) should be abnormal. Likewise, if the oocyte possesses the mutation, the PB will be normal. Performance of PCR on the first PB provides genetic diagnosis and spares the oocyte, which may be used for insemination (Coutelle, 1989). It must be stressed, however, that PB diagnosis can correctly identify mutant versus wild Fertility and Sterility
alleles only if no crossing-over occurs. Should crossing-over occur, both the oocyte and the PB will be heterozygous; thus the genotype of the embryo cannot be predicted. Crossing-over is much more common at the telomeric regions of chromosomes than at the centromeres; thus gene location on the chromosome becomes important. Polar body diagnosis offers the only practical method for gamete PGD because in sperm analysis such study destroys the gamete. Unlike meiosis in the male, which results in four haploid spermatozoa, meiosis in the female yields one mature oocyte and three PBs. Through the study of PBs, the genotype can be predicted for the PB, thereby sparing the secondary oocyte for fertilization. Only oocytes containing normal alleles would be used for fertilization, whereas those harboring mutant alleles would not be inseminated. INDICATIONS
There are more than 4,000 known genetic mutations associated with specific inherited human diseases, wherein aberrant nucleotide insertions or deletions are predicted to be causal. A dozen or so of these single gene defects are now amenable to preventive intervention by PGD, but many more could theoretically be detected by this method. An issue of complexity is that for many Mendelian mutations, such as cystic fibrosis, there are multiple molecular mutations. Specific nucleotide primer systems are often required to amplify each mutant region via PCR. Perhaps the technology will move toward simultaneous co-amplification of a number of potential genetic disease states. Determination of gender (XY or XX) can be accomplished also by molecular methods. However, some favor the use of fluorescent in situ hybridization without PCR for DNA amplification. Fluorescent in situ hybridization is also likely to be the preferred methodology for detecting an aneuploidy. With regard to conditions involving more than one gene (multifactorial, polygenic), such as spina bifida, diabetes, or cardiovascular disease, the diagnostic methodology for PGD is not available at present. For now, we must rely on a rudimentary understanding of Mendelian inheritance patterns to prevent single gene defects, for example, homozygous recessive diseases (25% risk of passage) like Tay-Sachs or cystic fibrosis, X-linked diseases that are passed from mothers to 50% of their sons, or dominant diseases such as Huntington's (50% risk of passage). Vol. 62, No.5, November 1994
RESERVATIONS
There is general agreement that prevention of inherited diseases by not transferring to the uterus those preembryos with specific genetic mutations is justifiable. Public support weighs heavily in favor of PGD when the birth of children who would experience severe health problems that would diminish their quality of life and longevity and the consequences of these factors on parents, siblings, and others close to them is examined alongside the opportunity to assure avoidance of this fate. Although some members of the Committee believe that nondisease uses of PG D are ethically unacceptable, other members would not exclude some uses of PGD in special circumstances for gender selection. All members of the Committee share the view that such uses should not occur without outside ethical review-national or local-of the need and circumstances. Some persons, however, may object to PGD even when its applications are limited to prevention of severely debilitating, even terminal, inherited diseases. Generally, public acceptance of these technologies for disease prevention rises with earlier intervention, parallel with opinions that the value of human life rises as development progresses from fertilization, implantation and viability to birth (see chapter 26). Reservations about PGD stem primarily from potential applications that depart from medical indications expressly for disease prevention. For example, gender, skin color, and "straight" teeth are characteristics of persons; these features are not diseases or significant risk factors that severely limit quality of life or longevity. Parental preferences based on such arbitrary or trivial factors that stem from certain "desirable" physical characteristics or perceived cognitive advantages are not yet acceptable rationales for intervention via PGD or prenatal diagnosis. Three primary factors may be useful in determining the appropriateness of PG D. Examples may clarify the principal factors involved. First, prevention of devastating inherited diseases that markedly curtail quality of life and longevity is justifiably the priority toward which society's stewardship of PGD should be directed. Tay-Sachs disease is a vivid example in that the risk element to carrier parents is 1 in 4, with affected children manifesting developmental problems from as early as 6 months of age and dying usually by 3 or 4 years of age. This scenario is destructive to the family and often fin ancially devastating as well. Prevention of Tay-Sachs Supplement 1
658
and similar diseases by PGD provides a method of disease avoidance that is ethically acceptable and more desirable than prevention after pregnancy (later prenatal diagnosis and elective abortion). Second is the issue of gender selection. When this technology is used to prevent an X -linked genetic disease such as hemophilia or Duchenne's muscular dystrophy, public support is generally strong, especially when the diagnostic methodology includes identification of the "well" male preembryo via mutation detection. If unaffected male preembryos are discarded along with ones containing X-linked mutations, then the objection rate rises somewhat. However, the use oftechnology for gender selection technology solely to fulfill parental preferences raises many questions. Although so-called "family balancing" services are ardently sought by a growing number of prospective patients, the use ofPGD solely for gender selection is not acceptable. Some persons would argue that parental liberty should extend to the choice of gender, especially if the preconception or preimplantation method does not interrupt pregnancy. Many others would strongly oppose any use of PGD or other reproductive technology that would enable parents to control the gender of their offspring, except in cases of Xlinked inherited disease. The fear is that availability of preembryo gender selection, other than to prevent an X-linked inherited disease, would foster gender discrimination and anti-social trends that American law and governmental policies have sought to remedy throughout this century. The third factor is deference to be shown to the preembryo because of the sanctity of human life. As stated in chapter 1, human life in the form of a preembryo deserves respect and value above that accorded to other cells and tissues of human derivation which do not themselves possess the inherent potential of a new human being. Because of the preembryo's rudimentary status and the fact that preembryo discard in other circumstances is accepted (such as discard of unwanted frozen preembryos), a decision to discard preembryos because they have a serious genetic defect seems ethically
668
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acceptable. However, many persons would argue that respect for the sanctity of human life prohibits the discard of human preembryos simply because they do not contain some preferred trait or characteristic desired by parents. COMMITTEE RECOMMENDATIONS
The Committee finds strong ethical justification for PGD in the prevention of inherited diseases that severely deplete the quality of human life or longevity; individual couples or families should make this judgment. This position derives principally from recognition of the suffering of affected individuals and their families, as well as the impact on society. The Committee recognizes however that PGD is experimental technology and therefore should be carried out as a clinical experiment. The Committee finds highly problematic the use of gender selection to achieve "family balancing" or other preferential goals based on nondisease traits. However, it may be premature to declare that there are absolutely no circumstances under which gender selection should be used, regardless of the technology involved in achieving it. A special advantage of PGD is that it will preclude the need for a couple to undergo later prenatal diagnosis and terminate a pregnancy in case of a positive result. The appeal ofPGD is that its goal is the same as prenatal screening and abortion for genetic indications, but it achieves this end by screening preembryos rather than fetuses.*
* The attitude of this chapter toward terminating life is its weakest analytic component. For instance, it argues from "the fact that preembryo discard in other circumstances is accepted (such as discard of unwanted frozen preembryos)" to the ethical acceptability of discarding preembryos with genetic defects. This analysis does not take any account of the fact that many people would not accept the discard of unwanted or frozen preembryos, nor does it display any arguments to bolster its position. The AFS Ethics Committee is on record as saying that the preembryo deserves "special respect." How casual discard of unwanted preembryos is compatible with such respect is unclear. If preimplantation genetic diagnosis is embedded in and reinforces this loose evaluation of the preembryo, it too is vulnerable to powerful ethical objections. (RAM)
Fertility and Sterility