|Courtesy of David Hill, ART Reproductive Center Inc.|
Two separated blastomeres subjected to FISH analysis to check the chromosomes.
Unlike past cases when parents selected a sibling who was an HLA match and could provide a bone marrow transplant, Adam was both a match and free of Fanconi anemia. As the family faced media scrutiny, concerns of selective breeding mingled with awe at this little-recognized ability to circumvent a lethal genetic legacy.
But PGD's implications transcend helping individuals. Within a family, the ability to choose the genes of the next generation can stamp out a particular disease. On a population level, this power can, over time, alter the gene pool. "This is an emergent technology with astonishing ramifications," says David Hill, scientific director of the ART Reproductive Center in Beverly Hills, Calif.
A PGD Progress Report
The technology is possible because of a basic fact of animal development called indeterminate cleavage. Like other vertebrates, an eight-celled human embryo, often called a pre-embryo, can continue developing even after a cell or two is removed. "In PGD, embryos obtained for in vitro fertilization [IVF] are biopsied, and their genetic composition determined. Only those free of genetic disease are transferred back to the woman," explained Lewis Krey, a professor of obstetrics and gynecology and cell biology and director of the IVF clinic at the New York University School of Medicine, at the annual meeting of the American Society of Human Genetics held in Philadelphia Oct. 3-8.
Allen Handyside and colleagues at Hammersmith Hospital in London in pioneered PGD in the early 1990s.2 It built on IVF, yet it also embraced the fledgling fluorescence in situ hybridization (FISH), a fast way to highlight specific chromosomes with tagged DNA probes, or "chromosome paints." "The first reports on PGD, about 10 years ago, were almost coincident with the broadening of FISH. It wasn't long until prenatal diagnosis centers were applying FISH to human pre embryos," recalls Hill. Embryology met genetics, and so was born a powerful new adjunct to IVF. "Today, about 50 centers worldwide offer it. More than 500 babies in total have been born without genetic disease," said Krey.
An International Working Group tracks clinical progress of PGD.3 Meanwhile, a literature trail monitors insights into early development gleaned from observations on spare embryos. And the professional composition of the field reflects the merger of medicine and basic science--reproductive endocrinologists and obstetrician/gynecologists work with geneticists and developmental biologists.
A Slow Start
Despite its technical success, PGD hasn't quite achieved the familiarity of amniocentesis. The reason is economic reality, according to Sandra Carson, a reproductive endocrinologist and director of ART at Baylor College of Medicine in Houston. "The baseline technology is here, but it is very expensive, and no insurance company covers it. In comparison, amnio is quick, you get an answer in a few weeks, and it takes 15 minutes to do," she says.
The patient pools for PGD and amnio differ in size and expectations. Whereas many women of "advanced maternal age" (over 35) seek amnio, those considering PGD either have a genetic disease in the family or have had recurrent spontaneous abortions, which is often due to aneuploidy (missing or extra chromosomes). This is a much smaller group. And the patients have different perceptions. "Infertile women have been through so much therapy already, they have a different attitude. When told they have a 35 percent chance of having a baby, they find that incredible. But to a fertile woman, that figure is quite low, especially considering that [patients] have to pay $8,000 to $10,000 out of pocket," Carson says.
PGD adds "quality control" to IVF while reducing the likelihood of having to remove some later embryos. Most patients at Reproductive Biology Associates in Atlanta, for example, are older women who have had several spontaneous abortions. "They want to screen embryos and put back the normal ones. Chromosome abnormalities are increased in this group, and to compensate, in the past we'd just transfer a lot more embryos and hope some would be normal. Now, with better ovulation drugs, we are getting more eggs and embryos, but we don't want to transfer 10 to 15 of them," says laboratory director J. David Wininger. PGD previews chromosomally doomed embryos when they are just balls of cells, not fetuses crowding out healthy siblings. (Another ART, polar body biopsy, circumvents the problem entirely by identifying eggs with abnormal chromosome numbers--another story.)
|Courtesy of J. David Wininger|
One cell is removed from an eight-cell embryo in preparation for preimplantation genetic diagnosis.
Probing Early Embryos
In PGD, timing is everything, and researchers are still working out the specifics. The earliest a cell can be sampled is three days post-IVF. Explains Hill, "It has to be at the six- to eight-cell stage, when cells start to form tight junctions between each other and communicate. At the eight-cell stage, the embryo's genome begins transcription. You can still pluck off one or two cells and the embryo continues to develop as if you didn't do a thing."
Hill begins the manipulation phase of the procedure by melting a hole in the surrounding zona pellucida, and gently aspirating a blastomere with a hollow glass tool. Next, he deposits the cell onto a microscope slide, where it dries. But unlike the cells sampled in amnio, a blastomere nucleus is not dividing, the chromosomes not condensed enough to be stained and visible. This is where FISH comes in, highlighting specific chromosomal sites in interphase (nondividing) nuclei. Alternatively, PCR is used to amplify specific disease- causing gene variants.
Next comes the choosing stage in this molecular version of artificial selection. Hill relates that on average, a couple produces 12 fertilized eggs from IVF. "Of these 12, say 10 are fertilized. We probably biopsy eight, and we can successfully interpret about six. For the other two, either nothing lights up, or there are too many lights and we can't differentiate chromosomes due to an unfortunate landing of DNA."
A new twist to PGD is to test and transfer a day 5 embryo--the 80- to120-celled blastocyst. By this time, survival is more likely. "Embryos stop developing at various times, for biological reasons. A lot stop at the morula stage," said Krey, referring to the solid ball of cells that precedes the hollowed-out blastocyst. Using a blastocyst avoids what Hill calls the "big black eye for ART"-- transferring too many early embryos, then having to remove some. But a blastocyst is harder to biopsy than a ball of eight cells, and it is more likely to be mosaic--that is, mutations can arise in either the cells destined to develop into the embryo (the inner cell mass) or in the surrounding trophectoderm cells that become the extraembryonic membranes. Mosaics can lead to false positives or negatives.
Success rates vary, but about two-thirds of couples eventually have a child, said Krey. A variation on the theme is offered for late-onset disorders such as Huntington disease, in which a couple wishes to avoid the condition in a child, but the affected parent, still presymptomatic, does not want to know his or her fate. The couple is only told that the embryo is free of the gene--not whether the parent will be affected.
|Courtesy of Vysis Inc.|
Genetic abnormalities detected with Vysis Inc.'s MultiVysion PGT technology.
Learning from Spare Embryos
While government bioethics committees debate manipulating human embryos, privately funded researchers can glean information from "spares," the fertilized ova that couples choose to discard, donate, or freeze. These glimpses into early human prenatal development sometimes correct long-held ideas. Consider a study from Magdelena Bielanska and co-workers in the departments of Obstetrics and Gynecology and Human Genetics at Royal Victoria Hospital at McGill University in Montreal.
The researchers examined the chromosomes of sperm from a man with the extra X chromosome of Klinefelter syndrome.4 Dogma held that many such sperm would have an extra X chromosome, which could lead to a preponderance of XXX and XXY offspring. Only 3.9 percent of the man's sperm had extra chromosomes, but examination of 10 of the man's spare embryos showed that half of them had "chaotic chromosome X, Y, and 18 patterns." So, even though most of his sperm were normal, his embryos weren't. The source of reproductive problems in Klinefelter disease, therefore, might not be in sperm, but in early embryos.
In another telling study, researchers at the Monash Institute of Reproduction and Development in Clayton, Victoria, Australia, used FISH to follow the fates of single blastomeres with abnormal chromosome numbers. Specifically, they wanted to see whether the abnormal cells preferentially wound up in the inner cell mass or trophectoderm. They not only found that development does not shunt the chromosomally correct cells into the embryo, but further learned that cells with extra chromosomes partake of the inner cell mass much more often than would result from chance.5
Experiments on freezing--cryopreservation--follow the fates of normal "spares." "I successfully thawed an embryo frozen for nine years, implanted it, and it made it to term. We don't know how long an embryo can stay in suspended animation like this. But I'll bet embryos can be frozen for much longer, maybe 1,000 years," says Hill. About a quarter of the couples coming to his center for IVF freeze spare embryos, he adds, often for sex selection--which opens a can of worms.
A Slippery Slope?
PGD for sex selection helps families where the woman carries an X-linked disease, and thus each son faces a one-in-two chance of inheriting it. An alternative is to use prenatal diagnosis to identify an affected male and end the pregnancy--perhaps using sperm selection to up the odds of conceiving a girl. But PGD is also used--some say, abused--to control the gender composition of a family. "From the dawn of time, people have tried to control the sex of offspring, whether that means making love with one partner wearing army boots, or using a fluorescence-activated cell sorter to separate X and Y bearing sperm. PGD represents a quantum leap in that ability--all you have to do is read the X and Y chromosome paints," says Hill.
While PGD used solely for family planning is certainly more civilized than placing girl babies outside the gates of ancient cities to perish, it disturbs many people. In a 1999 statement, the Washington-based American Society for Reproductive Medicine endorsed the use of PGD for sex selection to avoid passing on an X-linked disease,6 but the society discouraged use for family planning, citing "gender bias, harm to individuals, and ... inappropriate use and allocation of medical resources" as reasons. Still, Hill reports that many couples who freeze embryos "have no qualms about discarding the sex that they do not want," asking the ART center to do it. But keeping people from using PGD for this purpose will be difficult. "I think it is hard to put the genie back in the bottle," he adds. Ironically, Hill reports that about equal numbers of couples select boys or girls.
Some people fear that sex selection may be just the beginning. The influx of human genome data presages a potential proverbial slippery slope. Sums up Wininger: "DNA chips are the future. We will be able to screen if not the entire genome, certainly a lot more than the single gene defects and aneuploidy that we look at now. And it is not far off. It will be the next big thing."
Ricki Lewis (firstname.lastname@example.org) is a contributing editor for The Scientist.
1. D. Josefson, "Couple select healthy embryo to provide stem cells for sister," The British Medical Journal, 321:917, Oct. 14, 2000.
2. A. H. Handyside et al., "Pregnancy from biopsied human preimplantation embryos sexed by Y-specific DNA amplification," Nature, 244:768-70, 1990.
3. ESHRE PGD Consortium Steering Committee, "Preliminary assessment of data from January 1997 to September 1998," Human Reproduction, 14:3138-48, December 1999.
4. M. Bielanska et al., "Fluorescence in situ hybridization of sex chromosomes in spermatozoa and spare implantation embryos of a Klinefelter 46,XY/47,XXY male," Human Reproduction, 15:440-4, February 2000.
5. M.C. Magli et al., "Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro," Human Reproduction, 15:1781-86, August 2000.
6. ASRM Ethics Committee Supports Sex Selection to Prevent Genetic Diseases, www.asrm.org/Media/Press/ethics1099.html
PGD -- From Single Genes to Extra Chromosomes
Because many of the applications of PGD are for exceedingly rare conditions, centers often specialize in a few disorders, while also screening the five chromosomes most likely to be present in an extra copy (X, Y, 13, 18 and 21). Often, biopsy material is sampled at the center nearest the couple's home, and sent to the appropriate facility for the particular disease.
PCR is used to detect single-gene disorders, and FISH probes to check chromosomes. The gene/chromosome techniques overlap for identifying translocations, in which different chromosomes exchange parts. PCR amplifies the regions where the two different chromosomes meet. For triplet repeat disorders (such as fragile X syndrome, Huntington disease, and myotonic dystrophy) PCR coupled to fragment size analysis detects genes sufficiently expanded to cause disease.
So far, PGD has been used to detect: