Single Cell Sequencing Technique May Double IVF Success

Tue, 01/14/2014 - 9:32am
Cynthia Fox, Science Editor

Sperm injection into oocyte.A new gene sequencing approach may give in vitro fertilization (IVF) doctors a potentially less invasive, and more successful, IVF method, according to a new study in Cell.

Indeed, the team behind the study believes the new approach may double IVF success rates. The approach has generated much buzz, though some are skeptical of success rate claims.

“It is definitely scientifically interesting, but clinically it doesn’t offer much that is new, I think,” contends New York University IVF specialist Jamie Grifo.

The team from Harvard and Peking Universities was led by Peking University’s Jie Qiao. They found that, by using an improved genetic sequencing approach called MALBAC (multiple annealing and looping-based amplification cycles) on external polar bodies of embryos, they made accurate genetic diagnoses while avoiding the removal of key cells that become the embryo, or embryonic supporting structures (like placentas), for preimplantation genetic diagnosis (PGD).

PGD involves removing cells from embryos– destined to become either the body of the embryo, or its supporting structure, like the placenta– on day three or five. These cells are analyzed for chromosomal abnormalities and/or genetic mutations. Couples increasingly use this IVF approach when they want to avoid passing down devastating genetic diseases to their children.

When a given embryo’s analyzed cell(s) are found to be mutation-free, the embryo is implanted in the mother’s uterus, generally safely.

But it is still inefficient to fully sequence genomes in single cells this way. (Most PGD involves analysis of a few selected genes or chromosomes.) Whole genome sequencing can be preferable, as it clearly yields far more data in one fell swoop, showing both chromosomal abnormalities and DNA sequence variations.

The inefficiency of standard techniques for whole genome sequencing in single cells is due to a problem called “amplification bias.” Generally, only about 70 percent of the genome in single cells is sequenced evenly, at best, using standard methods in single cells (and generally, that figure can hover at 40 percent).

With MALBAC, that figure can rise to 93 percent, the Harvard/Peking team has reported in a past issue of Science. MALBAC can also be used to analyze how rapidly mutations accumulate.

For the current paper, the team created fertilized embryos from eggs (oocytes) donated by eight mothers. They then sequenced the female pro-nucleus, which is the mother’s genetic contribution, consisting of one copy of each gene. The team compared this sequence to that of polar bodies, two cells consisting of maternal DNA attached to the embryo that do not become part of the growing baby.

The polar bodies and pro-nucleus, all told, contain four copies of each of a woman’s genes— two each from her father and her mother. The team found that the genetic sequence of the polar bodies accurately predicted the genetic sequence of the pro-nucleus. They did this by counting which three versions of a gene were contained among the polar bodies, and thus figuring out which version of the gene must be represented in the pro-nucleus.

The team was able to check for aneuploidies, or large chromosomal abnormalities, that lead to miscarriages, in addition to disease-causing genes in the maternal DNA. The researchers also sequenced the pro-nuclei of the egg cells to demonstrate that the technique accurately reflects their genes.

“This is a very good scientifically sound paper,” Grifo says. “It should help us, among other things, understand meiosis better.”

Meiosis is the stage of oocyte development when chromosome number is halved, to prepare the oocyte for fertilization.

However, contends Grifo, “this is not a substitution for trophectoderm biopsy.” Trophectoderm biopsy involves removing cells from the five-day-old embryo, for genetic analysis, that form only extra-embryonic, supporting structures like the placenta.

With polar bodies, he notes, you are only analyzing the mother’s contribution. You miss the father’s genetic contribution, including his chromosomal abnormalities.

Furthermore, the study is too small to be conclusive.  “Many of the abnormalities you find in polar bodies later get corrected in the embryo,” Grifo says. “So you would always have to do the more standard biopsies, anyway. Polar bodies over-predict abnormalities.”

PGD is common. Many clinics in respected IVF circles offer it. Grifo’s group at NYU has done over 1,000 PGD cycles, for example. He has used the approach, not just to identify certain disease genes, but to winnow out embryos from older women containing too many chromosomal abnormalities. “You can almost wipe out the age effect by selecting normal embryos,” he says.

The bottom line, for Grifo: “The paper is interesting as no one has ever used polar bodies for sequencing before, just for technologies like FISH (fluorescent in situ hybridization) and SNPs (single nucleotide polymorphisms). Scientifically, it is an important paper because it shows some of the abnormalities in meiosis. But clinically it doesn’t solve the problem of finding the chromosomally normal embryo. It helps you get there, but not more than, say, trophectoderm biopsy on day five or six.”




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