This year marks the 40th anniversary of the first IVF birth. In the past four decades, assisted reproduction has made tremendous strides toward improving pregnancy outcomes for millions of patients facing infertility, recurrent miscarriage, and genetic abnormalities. While genetic testing of embryos has been around for most of the time IVF (in vitro fertilization) has been available, it has only been in the past few years that, due to improving techniques, preimplantation genetic testing (PGT) has become mainstream.
Traditional preimplantation genetic testing
Genetic testing falls under two broad categories. Preimplantation genetic diagnosis (PGD) is used for couples with a known or suspected genetic disorder such as cystic fibrosis, Tay Sachs disease, sickle cell anemia, or dozens of other specific mutations. Preimplantation genetic screening (PGS) tests for structural chromosomal abnormalities such as Down syndrome. In PGS, the goal is to assess whether entire chromosomes out of the 23 pairs that are normally found are either duplicated or deleted. This results in either one too many or one too few chromosomes in a pair, which is called aneuploidy.
While the reasons for conducting PGD are very specific – the known presence of a genetic defect – PGS has more varied indications, and can be used to reduce miscarriage rates and to select known normal embryos in women at risk for chromosomal abnormalities such as Down syndrome. The genetic abnormalities that PGD and PGS identify are major causes of embryo implantation failure and miscarriage, as well as causing possible birth defects in children successfully delivered.
Next generation sequencing can greatly improve embryo implantation
In order to biopsy the embryos, embryos must be given time to develop for five or six days after the eggs are harvested as part of IVF. By this time, some of the embryos will have reached the blastocyst stage and have started dividing into cell lines destined to become the baby and another group of cells destined to become the placenta.
About five cells from the group that will become the placenta, called the trophectoderm, are sampled. The biopsied cells are then sent to a reference laboratory where they will be broken down and analyzed using next generation sequencing (NGS), a sophisticated method that uses molecular techniques and advanced computers to determine the likelihood of chromosomal errors.
Until recently, NGS could only provide two results – normal or abnormal. For instance, if there was an extra chromosome in pair 21, the diagnosis would be Down syndrome. On the other hand, if the result were 46XX (normal female) or 46XY (normal male), the patient would be reassured that there was a very low likelihood of chromosomal error. Transfer of a single, normal embryo to a woman’s uterine cavity would increase successful implantation, perhaps as high as 70 percent, and reduce miscarriage risk.
The in-between state – and promise – of mosaicism
In the past year, NGS technology has advanced sufficiently such that there are now shades of gray in the formerly clear-cut division between normal and abnormal. This in-between state is called mosaicism. Embryos that are mosaic have different proportions of normal and abnormal cells. If a developing embryo (blastocyst) has about 120 cells, a low-level mosaic embryo would have mostly normal cells and a lower percentage of abnormal cells. A high-level mosaic blastocyst would have mostly abnormal cells and a lower percentage of normal cells.
Geneticists who work in this field have established the following criteria.
- If fewer than 20 percent of the cells in the blastocyst are abnormal, the embryo is labeled as normal.
- If 20 to 40 percent of the cells are abnormal, it is considered a low-level mosaic.
- When 40 to 80 percent of the cells are abnormal, it is labeled a high-level mosaic.
- If greater than 80 percent abnormal cells are present, it will be called abnormal.
Importantly, mosaicism happens after fertilization, rather than whole chromosome abnormalities such as Down’s syndrome which are due to abnormal egg development and therefore are present prior to fertilization. For this reason, mosaicism does not increase with the parents’ ages and occurs equally in all age groups.
When to transfer mosaic embryos, when not to
Some centers have already transferred such mosaic embryos when no other normal embryos were available for transfer. The concern, of course, is whether transferring these embryos could lead to living children with significant congenital health issues. These centers have found that while transferring most types of mosaic embryos is associated with lower pregnancy rates and higher rates of miscarriage, it is an all or none phenomenon and there is no increase in congenital abnormalities.
There are some caveats
Some types of mosaic embryos should never be transferred since they may lead to live births with potentially severely compromised outcomes. For instance, if the abnormal cells have an extra chromosome in pair 21, this can lead to a full-term Down syndrome child who could have potentially severe abnormalities. Similar full-term deliveries can also be found with extra chromosomes in pairs 13 and 18, and these should also never be transferred, even if determined to be low-level mosaics.
Extra chromosomes 2, 7, and 16 can lead to healthy normal children but are associated with intrauterine growth retardation that can severely shorten the pregnancy and lead to preterm delivery. Extra chromosomes 14 and 15 may be associated with a unique condition called uniparental disomy, where both of the chromosomes come from the same parent and therefore may be associated with specific neonatal anomalies.
Mosaic embryos with superfluous numbers of the remaining chromosomes may be considered for transfer if no normal embryos are available, since they either will lead to a successful outcome or failed implantation or miscarriage.
More mosaicism research is needed!
More research needs to be conducted to refine the state of knowledge in this area. For instance, there is an embryologic phenomenon called confined placental mosaicism. This is where abnormal cells are sequestered in the placenta and the fetus itself is normal. This likely occurs later in the pregnancy and not in the early blastocyst.
In summary, mosaicism is an old biologic process that can now be identified using PGS due to improved technology. While knowledge in this field is increasing rapidly, current recommendations include:
- Transfer of certain types of mosaic embryos should only be considered when no normal embryos are available.
- Transfer of mosaic embryos leads to lower pregnancy rates and higher miscarriage rates, but normal live births can occur.
- Thorough counseling with a genetic counselor and consent should be given.
- Amniocentesis should be performed later in the pregnancy to assess genetic risk.
- More research is needed!