The increased use of preimplantation genetic screening (PGS) (link!!!) in recent years has resulted in improved outcomes for couples with infertility who are going through IVF. It is now routine to transfer a single embryo and maintain excellent pregnancy rates and minimize the risk of miscarriage across age groups. But just when we thought the controversies surrounding PGS had been mostly resolved, over the last several months a new controversy has emerged. This latest one revolves around the issue of mosaicism or embryos that predicted to be a mix of normal and abnormal. High-profile articles have in the New York Times and the New England Journal of Medicine, focusing on the challenge of what to do with mosaic embryos created during IVF.
It’s important to realize that nothing has changed intrinsically about the embryos transferred in IVF; for decades we have been transferring unscreened embryos that included a mix of chromosomally normal (euploid), chromosomally abnormal (aneuploid) and mixed (mosaic) embryos. However, as genetic technologies with higher resolution have been applied to PGS, the ability to reliably detect more types of genetic errors, such as mosaicism, has improved. This has resulted in a shift in how we classify PGS-tested embryos: from the previous “normal or abnormal” dichotomy, to now a spectrum that includes a more ambiguous, “mosaic” designation. But before considering the challenges of mosaicism and what to do with predicted mosaic embryos, it is important to take a step back and carefully consider what we know, and what we don’t know, about mosaicism.
What Is Mosaicism?
Simply stated mosaicism is when an embryo, or person, is composed of two or more genetically different cell types. Depending on the chromosome involved, and the proportion of abnormal cells, mosaicism could lead to embryos that cannot implant, that miscarry or that result in pregnancies with abnormalities (mosaicism confined to the placenta has been associated with abnormal growth of the fetus).
The PGS Diagnostic Process
As currently performed, PGS involves using a laser to biopsy several cells (typically 5-10) from the outer layer of a blastocyst. This outer layer, known as the trophectoderm (TE), eventually becomes the placenta and membranes. A study performed at Reproductive Medicine Associates of New Jersey demonstrated that TE biopsy is safer than biopsying a single cell on day 3, and it’s also more accurate. A separate collection of cells called the inner cell mass (ICM) becomes the actual embryo, and ultimately the baby, and is not touched by the embryologist when TE biopsy is performed. Diagnosing mosaicism in a TE biopsy is very different from diagnosing it in an amniocentesis or blood test. Those latter tests involve actually culturing cells and visually inspecting the structure of the chromosomes in dozens of cells. Some cells may have the normal complement of 46 chromosomes (22 pairs of autosomes numbered 1-22 and the sex chromosomes X and/or Y), and some cells may have 45 (due to monosomy or 1 copy of a given chromosome) or 47 chromosomes (due to trisomy or 3 copies of a given chromosome). But the amount of DNA obtained in a TE biopsy for PGS is miniscule, just several picograms worth, and that creates a problem because the cells cannot be cultured or visually inspected. The DNA also cannot be directly analyzed from individual cells. Rather, the aggregate of the DNA from the biopsy is extracted from the cells and amplified (copied) millions of times over to be able to obtain a quantity sufficient for analysis. This amplification step and secondary analysis has the potential to introduce errors.
How We Detect Mosaicism
Several studies have shown that PGS can be accurately performed with microarrays, real-time polymerase chain reaction and next generation sequencing to select euploid embryos with a higher chance of implanting. A much greater challenge is to determine if there is a mixture of normal and abnormal cells within a single biopsy, indicating mosaicism. First of all, some mosaicism may not be captured by a single biopsy. What if there are nests of abnormal cells that fall outside the tiny TE biopsy? Even if the biopsy captures a mix of two different cell types, will our technologies accurately be able to detect that fact?
Several studies on the detection of mosaicism have focused on first conducting experiments with different cell lines in the laboratory. Mixtures of normal and abnormal cells are created and then the DNA is amplified and analyzed in a similar fashion to PGS. The relative change in the copy number calls can be plotted out for different levels of mosaicism. This can be translated to the results from biopsies on real embryos. If the result from amplified DNA from the embryo biopsy falls in the mosaic range then the embryo is now classified as mosaic and potentially abnormal. But these same embryos used to be called euploid (normal) or aneuploid (abnormal) in earlier versions of the PGS technology. Now they are being reclassified into this new category of mosaicism. Some embryos falling in this new category undoubtedly are truly mosaic and at risk for poorer outcomes. However, some may be truly uniformly euploid or aneuploid, but due to the limitations of amplification and genetic analysis now fall in the mosaic range. The prevalence of mosaic embryos is also controversial with some labs finding ~5% of embryos are mosaic and others up to 30%.
Reliance On A Single Biopsy
There have now been multiple publications showing that the transfer of “mosaic” embryos can result in the births of healthy euploid babies with no apparent evidence of mosaicism. These mosaic range embryos appear to have roughly half the chance of implanting as the normal-range embryos. But how do we know these embryos were truly mosaic, rather than euploid embryos that happened to fall in the mosaic range? Performing another biopsy and obtaining the same result might strengthen the diagnosis, but this introduces an extra intervention for the embryos and due to the nature of mosaicism, a normal result on a second biopsy cannot exclude mosaicism throughout the embryo. For this reason, some experts have expressed caution about making a diagnosis of mosaicism on a single trophectoderm biopsy. Rather, they argue we should call these type of embryos “mosaic range” or “at risk for mosaicism.”
So has this new categorization created more problems for PGS? There is concern that if it limits the pool of embryos available for transfer it could actually result in lowering the chance for a live birth from a given IVF cycle. Others argue that the normal embryos transferred after screening out abnormal embryos and mosaic embryos will be even more likely normal and more likely to result in the desired healthy baby that patients going through IVF desperately seek.
A Matter of Prioritization
Over the last few years several prospective studies have now shown that PGS can improve the chance of delivery, but 30-40% of apparently euploid embryos still fail to implant. While there are a multitude of factors that must align for a successful pregnancy to result after an IVF-embryo transfer cycle, could it be that some of these purportedly “normal” embryos that failed to implant really harbored chromosomally-abnormal cells that limited their potential? Even more controversial, could some of the normal embryos that implant really be harboring undetected mosaicism and could that have an impact on the obstetrical and long-term outcomes to the babies born through IVF? It is unlikely that the process of IVF and embryo culture actually increases mosaicism. So, since mosaicism is a very rare finding in ongoing pregnancies and newborns, perhaps these embryos rarely succeed in implanting and delivering any way. With this type of thinking, maybe these embryos should be given a chance if a couple has exhausted their ability to produce truly normal, euploid embryos. The introduction of this classification of embryos has the potential to further enhance selection for elective single embryo transfer, which is the ideal and safest way to perform IVF. If patients with multiple embryos available prioritize the transfer of the normal ones, outcomes may improve further. The next several years will be an exciting time as laboratory and clinical studies help to further elucidate this conundrum of how to diagnose mosaicism and what to do with the embryos deemed to be mosaic or at risk of mosaicism.