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Picturing the Fertile Genome
New imaging technologies are predicting and improving the success of in vitro fertilization
By Roberta Friedman

Featured Article.

As researchers seek new ways to boost the success rates of in vitro fertilization (IVF), they are finding that most fertilized human eggs appear destined for the evolutionary trash heap. A surprising number of botched chromosomes in eight-day-old embryos may explain why IVF clinics often have to try and try again to start a viable pregnancy.

Embryo at three days (6-10 cell stage).

British scientists recently reported a new chromosome-imaging technique that may allow clinics to improve the odds for IVF success. And their findings highlight the possible reason for all-too-common failures: Only three of 12 embryos sampled from couples undergoing IVF had the proper complement of chromosomes, according to the study by Dagan Wells and colleagues at University College Medical School, in London.

IVF workers have long suspected that some human conceptions carry a number of genetic mistakes. The new approach, which involves a genome 'check-up' prior to the implantation of the embryo, reveals just how many mistakes can occur. "I don't think people suspected the error rate would be as high as 75 percent," says Harvey Stern, director of the preimplantation genetic diagnosis program at the Genetics and IVF Institute, in Virginia.

Embryos whose chromosomes all appear healthy have an increased potential of making a baby, says Stern, who contrasts this with the "horrendously chaotic chromosome patterns" seen in some embryos. The abundant errors probably cannot be attributed to the practice of fertilizing in a dish. Although the embryos were donated by couples undergoing IVF, who may have included older women with inferior eggs, or men with problems producing sperm, the fact that three of the embryos were normal suggests to Stern that biology rather than technology is to blame.

The blastomere biopsy showing an isolated blastomere to be analyzed. Each blastomere has a full complement of maternal and paternal chromosomes.

With techniques developed for IVF, scientists are casting a spotlight on our reproductive inefficiencies as a species. Wells points out there does seem to be a 'weeding out' of defects quite early in the stages of making a human baby. Barnyard animals are more successfully bred in a laboratory dish than are humans. Even our genetically close relatives—other primates—have a better track record in the dish. The IVF failure rate for humans is an order of magnitude higher than for other species. "I haven't heard a good explanation of why this is so," says Wells.

Laboratories are starting to routinely check chromosomes at a few days after an egg and sperm join, before the embryo is placed in the womb. The pre-implantation chromosome diagnosis provides more reassurance that the pregnancy might make it to term.

Prior approaches to pre-implantation genetic diagnosis have relied on fluorescent colored probes designed to seek out and stick to each normal chromosome. Wells' method amplifies all the genes in the sampled cell by copying its individual DNA molecules over and over, and analyzing the expanded sample. The technique is based on PCR, which is commonly used to identify mutations in cancer cells and was a cornerstone of the recent sequencing of the human genome.

Fluorescence in situ hybridization (FISH): Normal female carrying 2 copies of chromosomes 13, 18, 21 and X.

For pre-implantation diagnosis, it takes three days after an egg and sperm join in a dish before enough cells are available for scientists to sample without harm. Assured that the chromosomes are present in normal numbers, doctors can then implant an embryo that continues to grow in the womb. Compared to simply placing a fertilized egg in the woman without any clue to its viability, the checkup approach should lead to better success rates.

The human cell contains 23 pairs of chromosomes, with each parent contributing one chromosome to each pair. During cell division, there is a stage in which the chromosomes are easily seen, and most of the diagnostics to gauge the fertilized egg depend on that stage. But Wells' method is able to use cells at any point in the cell-division cycle.

The PCR-based, comparative genomic hybridization also allows every chromosome to be assessed, whereas other methods can assess only a few. "The nice part is, you can query all the chromosomes," says Stern. But queries can take several days—time that a developing embryo cannot afford to waste.

Researchers in Italy are using a wider palette of colored tags to label chromosomes.

A decision to implant must be made by day five, and an embryo cannot be put on hold while awaiting a healthy bill of chromosomes. Freezing the embryo would definitely reduce the implant rates further, says Stern. IVF can achieve a verifiable pregnancy with 25 to 30 percent of all implant attempts. (There is a 20 to 25 percent rate of loss after the pregnancy is confirmed.) Biopsies for pre-implantation diagnosis of chromosomes do not detract from this rate and even appear to boost IVF success.

Both Stern and Wells say that the new approach as it stands has important drawbacks. Aside from the time needed, it requires expertise in several highly specialized techniques. "I'm very keen not to sell this as a (means) to increase our pregnancy rates" just yet, Wells cautions. "We need to simplify and run it a little quicker." But he believes that can be done, estimating that by another 12 months a more streamlined protocol will be ready for testing.

While Wells' group tinkers with their technique, the ability to see chromosomes with colored probes has also made some advances. Researchers in Italy have announced they can use a wider palette of colored tags to label chromosomes. Their approach also has highlighted the high error rate in human conception. Nearly two-thirds of the 717 embryos they examined in vitro had chromosomal abnormalities, and, therefore, were not implanted.

Fluorescence in situ hybridization (FISH): Male monosomic 18: This affected male is missing a copy of chromosome 18. (Y chromosome is yellow).

So far, the Italian team, of the Societa Italiana Studi Medicina della Riproduzione, in Bologna, can label nine chromosomes, and they report a 22 percent success rate in getting embryos to implant in a woman's womb. Previously, their reported success rate was only 10 percent. American IVF centers use far fewer color probes in pre-implantation diagnosis, says Kenneth Burry, of Oregon Health Sciences University, in Portland.

The Oregon chromosome screen uses just five colored probes. Burry, who directs the Division of Reproductive Endocrinology and Infertility at OHSU, has been using the test for three years in research and expects that it would cost a minimum of $1,500 compared to the price of another IVF cycle.

Scott Sills, who heads a private IVF practice in Atlanta, says that his laboratory also looks at only four or five chromosomes at once, because of the difficulty in finding enough distinct colors. IVF workers would want to pick a color scheme that distinguishes each probe, and telling pink from magenta—or yellow from orange, for example—is difficult. "We have to hedge our bets," Sills says, "and put our money on" finding the most important chromosomal changes to IVF success.

The finding of so many chromosomal errors in human embryos has implications for cancer, and even for our survival as a species. "If we reduce our fecundity rate much more," Stern says, "if we pollute the air or (increase) cosmic rays, we are going to be as extinct as the dinosaurs, because we are kind of on the edge right now in terms of our reproduction."

"What we are learning about human embryos is pretty scary but also pretty fascinating," says Stern. He wonders if some embryos with abnormal cells survive. And he points out that the chaotic division of the genetically abnormal embryos is a lot like cancer. "If you can figure out the genes involved in eventually reining in these embryos," Stern says, this research might benefit cancer patients as well.

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Wells, D. and Delhanty, J.D. Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Mol Hum Reprod 6, 1055-1062 (November 2000).

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