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RECENT ADVANCES IN REPRODUCTIVE GENETIC TECHNOLOGIES
Gene Levinson, Carolyn B. Coulam, W. Christine Spence, Richard J. Sherins and Joseph D. Schulman
Genetics & IVF Institute, 3015 Williams Drive, Fairfax, VA 22031, and Medical College of Virginia, Richmond, VA
From Biotechnology, September, 1995; with minor editorial modifications. Copyright © 1995, Gene Levinson and Nature Publishing. All rights reserved. |
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Abstract
Several technical advances offer new possibilities for the diagnosis and treatment of reproductive and genetic disorders. Intracytoplasmic sperm injection (ICSI) allows treatment of the numerous members of the infertile male population whose sperm cannot penetrate into the egg to initiate fertilization. Molecular genetic testing provides clients of reproductive age with additional information that permits prevention of genetic diseases such as fragile X syndrome, the leading cause of inherited mental retardation. Preimplantation genetic testing (PGT) offers prospective parents who carry genetic disorders the opportunity to have children of their own with greatly decreased risk of initiating a pregnancy involving an affected individual. Flow-cytometric sperm separation offers a new, effective approach for prevention of X-linked genetic disorders. Two major causes of recurrent pregnancy loss (RPL) involve recurrent trisomies and immunological disorders. Of the latter, 70% of studied populations of patients can attain live births with simple treatment protocols. Maternal serum assays involving multiple markers reduce both false positives and false negatives in detection of trisomies. Despite these advances in research, many safe and effective methods of diagnosis and treatment remain under-utilized in the clinical arena.
Keywords: intracytoplasmic sperm injection, molecular genetic testing, preimplantation genetic testing, clinical sperm separation, maternal alpha-fetoprotein assay, recurrent pregnancy loss, fetal cells in maternal blood.
Prospectus
A chasm separates biomedical discoveries from their implementation in clinical practice. In the fields of human reproduction and genetics, reasons include the pace of discovery in these rapidly moving fields, and the caution exercised by medical professionals and regulatory bodies. The purpose of this brief review is to evaluate several advances in reproductive technology that are known to the research community but that are not widely available as clinical services at the present time. Most represent active areas of service and research at the Genetics & IVF Institute (GIVF), and most are offered by GIVF to patients and clients from around the world. Although we have consulted with a number of scientific and medical colleagues in preparing this review, the views expressed herein are our own. Due to the unusually broad scope of this review, the bibliography is not intended to be exhaustive, and only a limited sampling of key citations is provided.
Male Infertility & Intracytoplasmic Sperm Injection (ICSI)
The classic or textbook approach to assessment of male fertility includes determination of the number and motility of the spermatozoa present in semen, and assessment of morphology. Evidence suggests, however, that these parameters do not provide the most sensitive criteria for clinical assessment of fertility (1,2). A series of functional capabilities is required for a sperm cell to reach, and ultimately penetrate and activate, the egg. Recent estimates suggest that only about 10% of male infertility is attributed to underproduction of sperm due to maturation arrest or germinal aplasia, and that only 10% more can be attributed to pure motility disorders. This means that approximately 80% of infertile men have disorders ranging from profound oligospermia (less than 50 million sperm per ejaculate) to failure of the sperm to acrosome react. The acrosome reaction allows the sperm to penetrate through the zona pellucida, to enter into the perivitelline space, and ultimately bind to the egg membrane or oolemma and penetrate into the egg.
In vitro acrosome reaction (IVAR) provides a better means of clinical assessment of male infertility, because it has an 80% negative predictive value. The IVAR test is easily learned by competent lab technicians, is amenable to quality control, and requires only standard lab equipment such as a fluorescence microscope and laboratory centrifuge. The method (3-5) was developed about 10 years ago by Overstreet and colleagues and has been clinically applied for most of that period at cutting-edge infertility centers.
Another approach to fertility assessment is the hamster oocyte penetration assay (HOP), in which (commercially available) frozen zona-free hamster oocytes are mixed with the human sperm sample to be tested; fertilization potential is then assessed by observing pronucleus formation in the hamster cell (6-10). Fact and opinion concerning the predictive value of both IVAR and HOP assays has been a subject of controversy in the literature (11-14).
One might argue that IVAR or HOP assays would have limited usefulness if most cases of severe male infertility were untreatable. This was true until 1992. In that year, a "seminal" paper in a July issue of Lancet (15) described a powerful new method that has revolutionized the treatment of male infertility. That method is intracytoplasmic sperm injection (ICSI) (16-18). ICSI allows fertility experts and embryologists to effectively treat the huge number of couples where the infertile male sperm cannot penetrate into the egg to initiate fertilization.
ICSI involves in vitro fertilization technology modified to include microinjection of a single sperm cell into each egg. This means that if as few as one viable sperm per available egg can be obtained from the semen, epididymis, or testes, then otherwise infertile men can now father children. ICSI allows IVF teams to treat couples with a male component of infertility who were previously either extremely difficult to treat, with poor outcomes, or frankly untreatable. This includes couples with unexplained failure-to-fertilize in IVF, including factors on both sides of the sexual aisle.
Although it is difficult to estimate the precise number of infertile males of reproductive age wishing to have children who could benefit from this technique, they number in the millions in the US alone, based on the estimated 5% of reproductive aged men who have involuntary infertility. ICSI can also benefit the additional group of post-vasectomy males who after vasectomy reversal often have diminished sperm quality, or who can avoid vasectomy reversal entirely through NSA (non-surgical sperm aspiration) and ICSI.
Molecular Testing for Genetic Disorders
From an ethical point of view, molecular testing for genetic disorders that may cause serious or fatal disease is widely accepted by the majority of citizens of the US and Canada (19), yet remains under-utilized in clinical practice. A summary of the most prevalent disorders and diagnostic capabilities for these disorders is shown in Table 1. The following paragraphs focus on reliable tests for serious disorders that, despite their high incidence, may not be well known to many readers.
Fragile X syndrome (fraX) is the most common cause of inherited mental retardation (20). FraX is caused by expansion of a triplet repeat in the FMR-1 gene (21-23) which reduces expression of the FMR-1 gene product in the brain of affected individuals. As is true of other triplet expansion disorders, fraX is found at high frequencies among a broad range of ethnic groups, and the mutations often grow worse (genetic "anticipation") over small numbers of generations. It is now thought that as many as 1/259 (F. Rousseau, pers. comm.) of the general population carries alleles --called fragile X premutations--that, when maternally transmitted, may expand to full mutations which can lead to profound mental retardation in the next generation. An earlier published estimate of this carrier frequency was 1/354 (24).The disease is more likely to cause profound mental retardation in boys than in girls: it is X-linked, although not fully recessive. The risk for expansion depends on the size of the premutation carried by the prospective mother (25), and also on the precise sequence of the expanded region (26).
Fortunately, there is a definitive test for fragile X premutations and full mutations (key technical papers are referenced in (27)). Full mutations are reliably detected by standard Southern blots. The precise size of premutations can be determined by PCR, although this is not the preferred method for reliable detection of full mutations. The test can be performed on either whole blood or cultured prenatal samples. Genetic counseling issues are complex when a full mutation is detected during pregnancy, and especially difficult in cases involving a female fetus, because the degree of mental retardation, if any, can be highly variable. As mentioned above, however, males are usually profoundly affected by full mutations.
Although it is well known among the Jewish community that Tay Sachs mutations (28) are frequent and preventable among people of Ashkenazi Jewish heritage, Gaucher disease (29- 33), which has a higher incidence in the same group (1/450; (34,35) ), has not received the same amount of attention, despite the fact that carriers can be identified by a standard blood test. A current method involves PCR amplification of DNA target sequences where the most frequent mutations are found (36). Also less well known is the fact that Ashkenazi Jews are at high risk for other recessive disorders, including a particular cystic fibrosis mutation known as W1282X (37,38), and Canavan disease (39-41), presumably because historically, some Jewish communities were small and genetically isolated. Similar trends are seen in other historically isolated groups-- for example, the thalassemias that are of disproportionately high frequency in persons of Greek origin (42,43). DNA-based tests are now available for these and many other disorders that afflict particular ethnic groups.
Questions of when, or even whether, to offer molecular genetic testing for certain diseases or carrier states have been foremost in the news of late, and generate considerable controversy (44). A key topic of debate is whether physicians, and especially their patients, are (a) able to properly evaluate specialized technical data, and (b) whether patients are able to properly assess the often complex ramifications of disease or carrier diagnosis--either their own, or that of family members. A related question is whether parents have the right to know about issues that could profoundly affect their unborn--or born-- offspring's health and well being. The view held by the authors is that prospective parents are usually capable of informed consent, and are entitled to obtain medical information related to their own health, or the health of their children.
The most extreme example of a decision by some professional personnel to WITHHOLD availability of a genetic test of known accuracy (45) concerns Huntington's disease, a dominant, usually fatal neurodegenerative disease with late adult onset. (Note that this example is NOT meant to be representative of typical issues confronting genetics professionals). Huntington's disease causes progressive mental deterioration that slowly increases, with age of onset usually in the 40s or 50s. Knowledge that a person does in fact have the Huntington mutation could represent a profound source of possible psychological stress for some individuals. Because of these concerns, a consortium of molecular geneticists has made a group decision NOT to offer this test to children under 18 years of age, even if it is requested by the parents, and even if the parents have themselves already undergone presymptomatic testing. Less well-known is the position of this group mandating that prenatal testing for Huntington's disease will only be given if the prospective parents promise--on a signed consent form-- that they will terminate the pregnancy if the disorder is found in the fetus. It is hard to imagine how such a position can long be sustained.
Preimplantation Genetic Testing
In 1989, a research team at Hammersmith Hospital in London demonstrated that genetic disorders can be detected in human in vitro fertilized embryos prior to transfer to the womb, thus providing a means for prevention of a limited number of genetic diseases -(46,47). In the years that have followed, several groups (48-51) have begun to offer preimplantation genetic testing (PGT) for an increasing number of serious genetic diseases, as well as gender-testing for prevention of X-linked recessive diseases (Table 1).
PGT was made possible by the advent of the polymerase chain reaction (PCR) (52-54), which revolutionized human clinical genetics (55). The use of nested primers, (or preamplification with random primers (56)) , has made possible reliable single-cell genetic analysis. Independent analysis of two blastomeres has improved the reliability of diagnosis for PGT (57,58). In principle, most disorders that can be detected by PCR can be detected in single embryonic cells. We have developed a fragile X test suitable for PGT that has now been clinically applied (27,59-61). We have recently performed the first successful PGT diagnosis for Marfan's syndrome, a dominant disease (in preparation), resulting in an ongoing pregnancy. Spinal muscular atrophy (SMA) is the second most frequent fatal genetic disorder in the US population (62-64)(cystic fibrosis has been number 1), and we are now poised to offer a PGT test for this disorder. The other disorders that have been commonly prevented by PGT include recessive X-linked disorders-- by testing the gender and selecting the unaffected female embryos for transfer-- and cystic fibrosis. In addition, the test has also been applied by a number of groups throughout the world to less common but serious genetic diseases.
More recently, fluorescence in situ hybridization (FISH) has also been used for analysis of both gender and aneuploidy (65-67). Observed preimplantation embryos often exhibit mosaicism, and these observations cannot be fully explained as FISH artifacts (68-72). It is currently unclear, however, whether mosaicism indicates abnormalities that will remain after development, since an alternative possibility is that such defective cells are eliminated during the process of embryogenesis. To make matters worse, the probes commonly used for aneuploidy detection in preimplantation embryos X, Y, 21, 13 and 18-- do not pick up the most common chromosomal defects in early embryos, such as trisomies 16 and 22 (73). The possibility for enhancement of success rates for IVF, or reduction of the rate of clinically important trisomies, is currently the subject of investigation by several teams.
Flow cytometric sperm separation for gender pre-selection, based on differential DNA content of the X and Y chromosomes (74), was first developed for animal husbandry by a group headed by Dr. Lawrence Johnson (75). The technique was adapted to human spermatozoa in a collaborative effort involving Dr. Johnson and the Genetics & IVF Institute (76). The Institute has licensed the patented technology for human applications. The human sperm separation technique was developed specifically for the prevention of X-linked recessive disorders carried by the mother: selection of X-bearing sperm would result in females who would not express these genetic conditions, and so would be free of disease. Common examples of serious X-linked recessives include hemophilia, Duchenne's muscular dystrophy and X-linked hydrocephalus; over 350 other X-linked diseases have been reported. The technique can also be applied to other conditions where gender is a medical issue. Human X and Y-bearing sperm cells are more difficult to separate than those of many other mammals because they differ by less than 3 % in their DNA content, and human sperm tend to be variable in shape and size.
Improvements in the flow-cytometric method have generated recent average purities exceeding 85% for X-bearing sperm. Both speed and purity are increasing rapidly through efforts currently underway at the Genetics & IVF Institute. The purity in each sort is rigorously tested daily by a rapid FISH procedure, using colored X and Y probes. The method is non-mutagenic by the Ames mutagenicity assay (77) and dozens of normal births have been demonstrated in animals, including cattle, swine, and rabbits (75,78-82). Preliminary data indicates that fertilization rates in vitro are about 65%, the same as those observed for normal IVF procedures. The method can also be applied to simpler, less costly, direct intra-uterine insemination (IUI).
The concept of separating X and Y-bearing spermatozoa is not new (83). In fact, human sperm separation, for purposes of family balancing, has been offered clinically for several years at dozens of clinics. Sperm separation by flow cytometry is, however, distinguished from other methods in two ways: first, the separation is based on sensitive analysis of DNA content. Second, enrichment of the sperm has been proven quantitatively by FISH analysis. When one considers the slight difference in the DNA content of human X- and Y-bearing sperm, it is easy to imagine that a method based on sensitive measurement of DNA differences in single cells would be more effective than those based on density centrifugation or swimming speed, and this is consistent with data from FISH analysis.
The flow cytometric separation method should be suitable for both intra-uterine insemination and for IVF. Recently, an historic birth of a karyotypically normal baby girl has resulted from the combined clinical deployment of flow cytometric sperm separation with PGT for the prevention of X-linked hydrocephalus, a recessive mutation carried by the mother (84). This represents the first human birth involving flow cytometric sperm separation, as well as the first example of the combination of PGT with a proven method of sperm enrichment, for the prevention of a genetic disorder. [More recent information on MicroSort sperm separation is available.]
It is estimated that 2-5% of reproducing women worldwide suffer from recurrent pregnancy loss (RPL). A classical diagnosis assumed RPL after a minimum of three consecutive losses, although a minority of doctors suggest that two or more consecutive losses may be a more appropriate cut-off for this diagnosis (85).
The first diagnostic question that arises in cases of RPL concerns the mechanism responsible, which can be divided into problems with the pregnancy involving chromosomes, and problems with the uterine environment, including anatomic, hormonal, or immunological problems. A classical view is that approximately six percent of RPL could be attributed to fetal karyotypic abnormalities arising from parental translocations. But recent data (86) suggest that additional factors represent far greater sources of fetal karyotypic abnormalities. One study of 90 spontaneous losses showed that 60% of the fetuses carried karyotypic abnormalities. These data suggest that a substantial proportion of RPL may be due to recurrent trisomies. The relation to maternal age in these cases remains unclear. In such cases, early diagnosis of fetal trisomy would provide the couples with information that is critical to evaluating their reproductive options, but this service is currently offered to a small fraction of those who could benefit from this knowledge.
For the immunologic disorders, treatment options for a physician include low-dose aspirin, heparin, prednisone, or intravenous immunoglobulin, depending on the particulars of the case. Such treatments have been shown to reduce pregnancy losses in certain situations (87). For both autoimmune and alloimmune disorders, approximately 70% of treated women go on to have a live birth.
Other recent studies have shown that the presence of anti-paternal antibodies is a function of the total number of weeks of previous pregnancy, whether successful or not, and that 77% of women who have had 160 total weeks of pregnancy (whether live born or lost) will have these antibodies. This suggests that the common use of alloimmunity as a marker for RPL may be incorrect. One recent study (88) suggests that elevated numbers of circulating natural-killer cells may provide a better diagnostic marker for alloimmunity leading to RPL; further research on this and other alternative markers is needed.
Meanwhile, at a time when these newer approaches are not widely available, other recent studies suggest that commonly applied treatments are of limited utility. In this category is the common practice of immunization of the mother with paternal immunoglobulins; numerous international studies have suggested that the therapeutic value of this treatment may be limited (89-93). There is also evidence that alloimmunity may increase with maternal exposure to paternal antigens, and that these treatment preparations may contain potent cytokines.
New Approaches to Maternal Serum Assays
Maternal serum levels of alpha-fetoprotein (MS-AFP) have traditionally provided a second-trimester screening test that can identify neural tube defects or chromosomal abnormalities such as Down syndrome (94-97) This test has become a standard of care.
Although MS-AFP is relatively well known to health care professionals, recent advances in this method are not universally applied. The MS-AFP test, when combined with the maternal serum markers unconjugated estriol and human chorionic gonadotropin--called AFP Plus or Triple Screen-- improves the sensitivity of the test significantly: from 25% in an earlier study, to 58% with AFP Plus. This improved method reduces the rate of false positives for the detection of Down Syndrome and other trisomies in the second trimester of pregnancy. Methods and strategies for first trimester screening are already under investigation, and include new markers such as pregnancy associated plasma protein A (PAPP-A (98)).
Fetal Cells in Maternal Blood?
Over the past 20 years, evidence for the presence of circulating fetal nucleated cells in maternal blood has been steadily accumulating (99-110). If a sufficient number of bona fide cells of fetal origin were present, it would in principle be possible to supplement existing methods of prenatal diagnosis with a method that holds no risk to the fetus. Existing methods of amniocentesis and chorionic villus sampling currently provide cells that can be cultured, and routinely allow complete karyotypic and genetic analysis. Such techniques are only used when medically indicated, however, because of the small but significant risk to the fetus associated with any invasive procedure. The hope has been that fetal cells would provide information, using the rapid technique of fluorescence in situ hybridization (FISH), related to the most common chromosomal aberrations, such as trisomy 21, which causes Down syndrome, which is more prevalent with advanced maternal age.
Most research efforts have focused on nucleated erythrocytes, which bear surface and internal antigens that facilitate isolation and proof of fetal origin. Research on other cells types has not been widely reproduced, although small numbers of such types as trophoblast, granulocytes and lymphocytes of fetal origin have been found. In recent months, new techniques have made it possible to obtain rigorous estimates of the number of bona fide fetal nucleated erythrocytes in maternal blood. These techniques rely on detection of highly specific fetal antigens, such as hemoglobin F1, and simultaneous typing with fluorescent Y-specific DNA probes. We have recently shown (109,110) with the combined use of erythroid colony assays and PCR that the vast majority, but not all, nucleated erythrocytes that circulate in maternal blood are in fact of maternal rather than fetal lineage. Combined use of hemoglobin F and X and Y-specific probes by our group has shown that samples of 15 cc. of maternal blood contain an average of only about 3 (three) nucleated erythrocytes of fetal origin when enriched by both negative and positive depletion. These and earlier studies by others (104,107) indicate that although there are cells of fetal origin in maternal blood, their usefulness for FISH assays for aneuploidy may be limited, barring significant breakthroughs. Improved methods for separation of whole blood may increase this number, although probably not by orders of magnitude. Improved methods for single-cell genetic analysis will also likely be of great importance. Recently, we have developed flow cytometric methods that are effective in sorting HbF-bearing cells (Keyvanfar et al., in preparation).
Summary & Prospectus
As news of rigorous, safe and effective new medical technologies is disseminated, both among healthcare professionals and their prospective patients, methods of diagnosis and treatment that are not universally applied should become more widely utilized. In accord with legal precedent in the United States, we believe that patients have the right to obtain complete and state-of-the-art information concerning the health of themselves, their families, and their children. Obviously, this right is compromised by real-world, serious issues concerning the cost and accessibility of high quality services. Problems exist in every sphere-- in the private sector, managed care, socialized medicine, and underdeveloped countries. At GIVF we have observed, anecdotally, an interesting development-- that insurance companies and employers are beginning to realize the substantial short-and long-term savings that accrue when they accede to patient requests for reimbursement for early diagnosis, treatment, and prevention of serious genetic disorders. Perhaps at last, economic values will become a driving force towards preventive medicine, a direction that has long been mandated by the ethics of compassionate medical care.
Acknowledgements
We are grateful to the following individuals for helpful, stimulating, and critical discussions related to the content of this manuscript: David Bick, Susan Black, Andy Dorfmann, Lee Fallon, Lawrence Johnson, Shirley Jones, Dixie King, Anne Maddalena, Michael Opsahl, and Michael Sisson.
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TABLE 1. (ASCII text, not formatted): Common Genetic Disorders.
Disease Incidence Ethnic predilection Diagnostic capabilities Ref
alpha-1 Antitrypsin 1/2500-5000 Caucasians 100% (for deficiency alleles Z & S) (111), Rare null allele cannot be detected (112) Canavan disease 1/5000 Ashkenazi Jewish 95% (2 common mutations) (40), (41) Cystic Fibrosis 1/2500 Caucasians 85% (~10 common mutations) (121), 95% (Ashkenazi Jewish population) (114) Duchenne 1/3500 males none 65% (deletions), 5% (duplications) (115) Muscular Dystrophy remaining 30%, linkage analysis Fragile X 1/1250 males none >99% (expansions) (116) 1/2500 females Gaucher 1/450 Ashkenazi Jewish 95% (4 common mutations) (34) Hemophilia A 1/5-10,000 none 50% of severe (inversion) (117) males remaining, linkage analysis Huntington 1/10,000 none >99% (expansions) (118) Myotonic 1/8000 none >99% (expansions) (119) Dystrophy Neuro- 1/3-5000 none linkage analysis (120) fibromatosis I Sickle Cell 1/600 African 100% (121) disease American Spinal Muscular 1/6000 none 98% (deletion) (64) Atrophy Tay-Sachs 1/3600 Ashkenazi Jewish 98% (3 common mutations) (122)
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