What is Preimplantation Genetic Screening (PGS) for Chromosomal Abnormalities?

The interpretation of mosaicism among tiny preimplantation embryo–babies is complicated both for doctors and patients. The “Mosaic Embryo” is an embryo with both types of cells: normal and abnormal ones. Therefore, mosaic embryos represent a third category between normal (euploid) and abnormal (aneuploid) embryos. Some of the mosaic embryos have perfect chances to turn into the wonderful healthy fetuses. The others may cause the early or late pregnancy loss or even more complexities. This category of mosaic embryos may be characterized by decreased implantation and pregnancy potential with an increased risk of genetic abnormalities to the fetus and adverse pregnancy outcomes. But every case of embryonic mosaicism is unique and should be investigated prior to embryo implantation.

Many preimplantation embryos designed during IVF Treatment cycles contain two or more cytogenetically distinct cell lines. This phenomenon, known as chromosomal mosaicism, can involve the presence of cells with different types of abnormality [aneuploidy], the absence of any normal embryonic cells or a mixture of normal [euploid] and abnormal [aneuploid] embryonic cells. To Glance “Inside” the Tiny Sparkling Embryos and to find out are they normal, abnormal, or mosaic, – the Preimplantation Genetic Screening was designed.

Wondering how to interpret “Preimplantation Genetic Screening?” Preimplantation Genetic Screening (PGS) for aneuploidy is a test to be performed on embryos during IVF Treatment to screen for chromosomal abnormalities.

Recent studies have represented that among tiny mosaic embryo–dudes and embryo–ladies, or embryos with a mix of normal (euploid) and abnormal (aneuploid) cells, many still have the potential to implant in the womb and develop into chromosomally healthy fetuses, and, surely, turn in healthy newborn–bundles!

The interpretation of mosaicism among preimplantation embryos is complicated thing both for doctors and couples. Doctors should identify those mosaic embryos that won’t have the developmental abnormalities. Couples should make the serious decision concerning the Preimplantation Genetic Screening (PGS) testing that reveals the abnormalities in the genetic content. And this decision should be made only after the counselling.

Genetic Counselling is exclusively designed for giving the couples the most transparent and inclusive information about whether or not to pursue PGS. The pre–test (pre–PGS) counseling should include a discussion about the frequency of mosaic results, the challenges associated with the interpretation of these results, the possibility of a false positive diagnosis of embryonic mosaicism, and the limited predictive data available. The Genetic Counsellor should also say that the priority for Embryo Transfer should be given to mosaic embryos with low mosaicism levels.

Part 1: Mosaic Embryos: ‘Who’ or ‘What’ Are They? Is it Possible to Transfer Them?

1.1. What is an ‘EMBRYO’?

The assembly of a new life first depends on the union between a spermatozoon and an oocyte culminating in fertilization. If the fertilization occurs, the embryo appears. Fertilization is a mysterious phenomenon that turns two cells into a tiny embryo–dude or a tiny embryo–lady, or if two oocytes are fertilized than…two tiny embryo–dudes. Or two tiny embryo–ladies. Or if three oocytes are fertilized than three tiny embryo–babies: two tiny embryo–dudes and one tiny embryo–lady. Many versions can be inserted here.

1.2. How does the embryo appear in a natural way?

Wondering how does fertilization happen? Usually, a spermatozoon and an oocyte ‘meet’ in one of the two Fallopian tubes that connect the ovaries to the womb (uterus). Wondering how does the embryo–baby normally ‘appears’ and implants in the uterus? The tiny embryo–dude or the tiny embryo–lady (fertilized oocyte) then moves down the Fallopian tube by being wafted by fine hairs inside the tubes until it reaches the womb (uterus) two, three or four days later. Once there, this tiny embryo–bundle wonders where it should cuddle up itself in this new place. And it implants, attaching itself to the womb lining and that is where it usually continues to grow and develop.

The ability of the Fallopian tube to transfer the early embryo into the uterus is an excessive modality for a successful pregnancy. Apparently, structural abnormalities and functional abnormalities of the Fallopian tube will interfere with the Embryo Transfer process that can lead to tubal pregnancy.

1.3. How does the embryo appear in case if it is IVF Treatment cycle? 

In Vitro Fertilization Treatment cycle is designed to create the embryos in the laboratory and after that to transfer them to the mother’s uterus. In other words, fertilization [or EMBRYO ‘CREATION’] happens in the laboratory.

Embryologists will ‘design’ your tiny embryo–ladies and embryo–dudes in the laboratory. They will take the retrieved oocytes and toss them in a Petri dish with your husband’s sperm and let them do their thing. Another option is called Intracytoplasmic Sperm Injection (ICSI), where the Embryologists manually fertilize the oocytes with the sperm individually, but it is for extreme cases only.

The embryo will appear inside your uterus via the catheter. Your tiny scared embryo–lady or embryo–dude is taken out of the Petri–dish and waits for a ‘BIG TRANSFER’. If it is the time to replace this small embryo–bundle from the tube inside your uterus, it is placed inside a flexible catheter. The procedure of Embryo Transfer takes only several minutes. It takes all of three minutes to insert a weird kind of catheter, get it to where it needs to be, accurately place your little embryo inside your uterus, and that is all. YES, and it has to ‘LEARN’ so many things inside. It wonders: ‘Where am I?’ ‘What has happened?’ ‘Everything is pulsating around me…’ ‘Should I curl up here or there?’ ‘Oh, it is better on the left side?’ ‘I am scared. I will just cuddle up to that warm place and sleep there.’

1.4. Embryo classification [Classifying mosaicism in embryos]

Most Fertility Clinics and labs have the strong exclusion/inclusion criteria for the embryo classification. They classify embryos as “normal” (euploid) or “abnormal” (aneuploid). Normal embryos should implant without complications. They (euploid embryos) should be preferentially transferred over mosaic embryos. Abnormal embryos may implant, but even in this case, the miscarriage will occur. And mosaic embryos are the most mysterious ones. They not only have both normal and abnormal cells. That means that they may correct themselves and become the viable healthy fetuses or may cause the miscarriages and variable abnormalities.

Embryonic mosaicism occurs when the embryo has multiple genotype content. That means that two or more cell populations with different genotypes are present within the same embryo. 

New diagnostic techniques for Preimplantation Genetic Screening (PGS), such as Next–Generation Sequencing, have led to increased revealing and reporting of mosaicism. Most experts do not recommend transferring the mosaic embryos because of the lower implantation rates, increased risks of miscarriages, and increased risks of genetic abnormalities. But the interpretation of mosaicism is complicated because the transfer of some mosaic embryos has resulted in healthy fetuses and births of healthy babies. Do you remember that sometimes the mosaic embryos can correct themselves?

1.5. What are the Mosaic Embryos?

The ‘Mosaic Embryo’ is the term that is assigned to that embryo that has both normal and abnormal cells, that are revealed during Preimplantation Genetic Screening (PGS) Testing. In the case of embryonic mosaicism, the testing discloses the percentage of the cells with different genetic complements and shows the potential embryonic viability.

Not only IVF embryos’ may have mixed chromosomal contents, one embryo can be normal, another embryo can be abnormal, and the third embryo can be mosaic, but also those tiny embryo–dudes and embryo–ladies that appeared naturally. Wondering what does that mean ‘mosaic embryo has mixed chromosomal content’? The mosaic embryo contains some cells with a normal number of chromosomes and others with an abnormal number of chromosomes.

Mosaic embryos may represent a third category of embryos being between normal and abnormal ones. Wondering why do they represent the third category? They represent the third category because of the mysterious self–correction phenomenon. The experts say that mosaic cleavage stage embryos left in extended culture have been shown to self–correct to euploid blastocysts (normal embryos) in nearly 50% of cases. Therefore, this pattern of mysterious embryonic mosaicism, which is characterized by the presence of two or more genetically distinct cell lineages, typically one with a chromosome abnormality and the other with a normal chromosome composition, has become a controversial topic with the spirited debate over their potential viability.

1.6. What does the Mosaic Embryo look like inside?

Metaphorically saying, have you ever seen the pearl rondelle beads accurately covered ‘curled up’ in translucent package or envelope? If you have never even envisioned that, Google the rondelle pearl beads ‘curled up’ in translucent package or envelope. This is how your handsome embryo–dude or gorgeous embryo–lady looks like inside. If we are talking about the normal [healthy] embryo, this pearl content is accurately shaped, ivory–colored and has no ‘dark–blue’ [abnormal] pearls.

The mosaic embryo is a phenomenon. This tiny bundle can be envisioned as mixed pearls: ivory and dark–blue pears in the same line accurately covered ‘curled up’ in translucent package or envelope. It doesn’t have the pure chromosomal content. It has the affected [or even mixed] chromosomal content. But this affected content has its own categories as, in future, if the mosaic embryo corrects itself, the affected mosaic cells can be ‘TRANSFERRED’ to the placenta and won’t affect the tiny fetus, or, in the other case, they can turn the tiny embryo in ‘MOSAIC FETUS’ with many abnormalities. And the hardest question is should you give this little one the chance?

1.7. Does the mosaicism affect all embryonic cells?

Mosaic embryos can be categorized according to the percentage of abnormal cells in the biopsy, the chromosomes involved, and the types of abnormalities (full chromosome, the segment of a chromosome, single chromosome involved or multiple).

The mosaic embryo–dudes and embryo–ladies that have several chromosomes affected by mosaic abnormality [mosaic aneuploidy] have significantly lower implantation rates than any other category of mosaic embryos. In contrast, embryos with a mosaic segmental abnormality have a capacity to implant.

1.8. How do they ‘REVEAL’ the mosaic embryos? 

Mosaic preimplantation embryos contain two or more cell lines with different chromosome content, the consequence of errors in chromosome segregation occurring during mitotic divisions. In other words, their chromosome content is mixed. The scientists have designed advanced techniques to identify those tiny mosaic embryos. Wondering which ones?

In the past, to identify the embryonic mosaicism, the experts used Fluorescence In Situ Hybridization (FISH), a method favored because it represented the information on the chromosomal content of each cell. Using this method, the experts usually examined only about one–third to one–half of the chromosomes in each cell.

At present, the experts use the most advanced and accurate techniques for detecting the embryonic mosaicism.

Molecular cytogenetic techniques (for example, array Comparative Genome Hybridization [aCGH], Single–Nucleotide Polymorphism [SNP] array, quantitative Polymerase Chain Reaction [qPCR], Next–Generation Sequencing [NGS]) have the significant advantage over FISH technique. Wondering which one? Molecular cytogenetic techniques represent the information on the copy number of all 24 types of the chromosome.

Alternatively, the experts may use comprehensive chromosome screening technologies. These technologies involve the analysis of blastocyst biopsy samples, typically composed of 5 embryonic cells, which are not separated but instead are analyzed as a single entity. Although the presence of a mixture of normal and aneuploid cells in the testing sample can sometimes be detected with the use of methods such as aCGH, qPCR, and SNP array, they are relatively insensitive for this purpose. If the ideal results are obtained, mosaicism associated with proportions of aneuploid cells ranging from 40% to 60% can be detected.

The most accurate method to detect embryonic mosaicism is the new advanced technique of high–resolution Next–Generation Sequencing (hr–NGS). hr–NGS technique accurately detects embryonic mosaicism (aneuploid embryonic cells) from 20% to 80%. This method is utterly sensitive for detecting minor lines in mixed cell populations compared with aCGH (but several experts suggest aCGH as it has 100% specificity and sensitivity).

1.9. Why should they validate the ‘embryonic mosaicism’? 

Embryonic mosaicism is closely associated with the pregnancy loss. It is proven that the embryos with a mosaic biopsy miscarry more often and implant less frequently, although it is also clear that some of the mosaic embryos can correct themselves and turn into the viable fetuses.

Currently, there is no technique that detects the embryonic mosaicism with 100% specificity and sensitivity. To confirm that the embryo is ‘mosaic’ or ‘normal’, sometimes more than one technique should be used. The embryos categorized by hr–NGS as abnormal [aneuploid] or normal [euploid] are recommended to be confirmed by the aCGH technique. Mosaic embryos (20%–80% abnormal cells estimated by hr–NGS technique) may show the different results. On the other hand, hr–NGS technique detects many more mosaics than the aCGH technique.

Embryonic mosaicism can be revalidated with the use of the aCGH and the mosaicism may be not reliably detected. On the contrary, those embryos that are categorized as ‘NORMAL’ with the use of hr–NGS technique, after the revalidation with the use of the aCGH technique, may have the mosaic chromosomal content.

The fact that most of the mosaic embryos detected by hr–NGS are classified as ‘NORMAL’ when analyzed using the aCGH technique, indicates that validation of the embryonic mosaicism should be done.

Some of the experts may say that validation would result in fewer embryos receiving a “normal” diagnosis after PGS–A, but, this strong criterion may also result in transferring the most viable embryos with accurate chromosomal content and decreasing the miscarriage rates.

1.10. Embryo Selection: which mosaic embryos are better to transfer?

Chromosomal abnormalities that occur in mosaic embryos can be categorized as ‘monosomies’ and ‘trisomies’ Normally, there are 46 chromosomes arranged in 23 pairs. Twenty–two chromosomal pairs are called ‘autosomes’, and one pair, number 23, is the sex chromosomes. Any variation from this gold pattern causes chromosomal abnormalities.

A chromosome from any of the pairs may be duplicated (trisomy) or absent (monosomy); an entire set of 23 chromosome pairs can be duplicated three (triploidy) or more (polyploidy) times; or one arm or part of one arm of a single chromosome may be missing (deletion).

The experts recommend that the percentage of mosaicism be considered in Embryo Selection decisions. It must be also noted that the proportion of aneuploidy should be less than 20 percent because the higher percentage of aneuploidy is associated with the adverse outcomes. The higher percentage of aneuploidy not only disturbs but also may disrupt the delicate balance of genetic content.

They also recommend the preferential transfer of embryos showing mosaic monosomies over mosaic trisomies. The embryos showing mosaic euploid/monosomy are preferable to euploid/trisomy, given that monosomic embryos (excepting 45,X) are not viable. 

Another recommendation given by the experts is to transfer some mosaic embryos in preference to others, depending on the type of chromosome involved. The most problematic mosaic embryos that shouldn’t be transferred are those that have affected chromosomes 14 and 15; chromosomes 2, 7, and 16; and chromosomes 13, 18, and 21.

Mosaic embryos involving chromosomes 14 and 15 will cause the genetic disorder, called ‘disomy’. It is the condition of having a chromosome represented twice in a chromosomal complement. Mosaic embryos that have abnormalities in chromosomes 2, 7, and 16 are associated with intrauterine growth retardation. And mosaic embryos with affected chromosomes 13, 18, and 21 are considered to be problematic because in trisomic form these abnormalities can potentially reach term and the abnormalities they cause are beyond the imagination.

Part 2: Mosaic Embryo: Should You Give This Little One the Chance?

2.1. Why does the mosaicism happen? 

You won’t believe but the embryos are prone to mosaicism. Embryonic mosaicism was found to result from mitotic errors occurring after fertilization (after the embryo is created). As an early embryo (the fertilized oocyte) develops into a fetus, cells that initially acquired the extra chromosome give rise to new and larger populations of cells with the extra chromosome. All cells produced from the initial abnormal cell have the trisomy, but cells produced from the normal cells have the usual 46 chromosomes.

Or, there is another version of the mosaicism. The embryonic mosaicism happens because the sperm or oocyte itself had an extra copy of the chromosome. Normally, an early embryo (fertilized oocyte) contains 46 chromosomes. Mosaicism occurs in case if an early embryo (fertilized oocyte) contains 47 chromosomes. This occurs as a result of trisomy, in which there is an extra copy of a chromosome appears in an embryonic cell. In some cases, only some of the embryonic cells have the extra chromosome. Why does an extra copy of a chromosome appear in some embryonic cells? This happens in case if the sperm or oocyte itself had an extra copy of the chromosome, and the extra copy was not passed on to all cells after fertilization.

And the most wonderful thing is that the mosaic embryos may correct themselves. Isn’t it amusing?

2.2. Is it possible to reveal the mosaicism in an early embryo? 

Yes, it is possible to reveal the mosaicism in an embryo. Preimplantation Genetic Screening (PGS) was exclusively designed for the detection of the embryonic mosaicism in a single biopsy. The testing, usually performed on day 5 of embryo development, is completed by taking a tiny biopsy of the embryonic cells and examining the genetic makeup of the cell. During the examination, embryologists can detect, and often diagnose, potential chromosomal abnormalities.

2.3. Embryo selection: which mosaic embryos are better to transfer?

Chromosomal abnormalities that occur in mosaic embryos can be categorized as ‘monosomies’ and ‘trisomies’ Normally, there are 46 chromosomes arranged in 23 pairs. Twenty–two chromosomal pairs are called ‘autosomes’, and one pair, number 23, is the sex chromosomes. Any variation from this gold pattern causes chromosomal abnormalities.

A chromosome from any of the pairs may be duplicated (trisomy) or absent (monosomy); an entire set of 23 chromosome pairs can be duplicated three (triploidy) or more (polyploidy) times, or one arm or part of one arm of a single chromosome may be missing (deletion).

The experts recommend that the percentage of mosaicism be considered in embryo selection decisions. It must be also noted that the proportion of aneuploidy should be less than 20 percent because the higher percentage of aneuploidy is associated with the adverse outcomes. The higher percentage of aneuploidy not only disturbs but also may disrupt the delicate balance of genetic content.

They also recommend the preferential transfer of embryos showing mosaic monosomies over mosaic trisomies. It should be also noted that if the mosaic embryo doesn’t correct itself, the consequences can be fatal, including the early or late pregnancy loss, physical and cognitive defects, morbidity and mortality.

2.4. Genetic counseling is highly recommended

Because mosaic embryos are less likely to produce a viable pregnancy your doctor may recommend you not to transfer them. Normal embryos should be preferentially transferred over mosaic embryos. In case if you would decide to transfer the tiny mosaic embryo–dude or gorgeous mosaic embryo–lady, you should know that this small bundle of nerves has the chances as it may correct itself. If the transfer of the mosaic embryo is being considered, you should have also the genetic counseling about potential benefits and risks that are closely associated with this issue.

CONCLUSION: 

The mosaic embryo is a phenomenon as it contains both normal and abnormal embryonic cells. And it is difficult to say, which embryonic cells will prevail. This mystery hides either miracle or frustration. Therefore, prior to transfer the tiny mosaic embryo, it should be accurately validated.

The use of only one advanced technique for the detection of mosaicism and/or the application of diagnostic criteria that categorize most mosaic embryos as either normal or abnormal has negative clinical consequences. Mosaic embryos may be categorized as abnormal [aneuploidy], leading to potentially viable embryos being discarded, or as entirely normal, carrying an elevated risk of aneuploid pregnancy.

Every chromosome can be associated with an abnormal phenotype when in mosaic form, the spectrum extending from apparently normal to severely affected/lethal. However, as mentioned above, the types of mosaicism observed during preimplantation embryonic development, and those that affect the fetus or the newborn might represent different phenomena. This is probably one of the most important questions remaining to be answered by the experts.