Authors: Javier Del Río and Sara Sanz
Genetic problems in the embryo are one of the most important causes of pregnancy loss and miscarriage. However, identifying embryo mosaicism as the cause of genetic problems during development is not an easy task.
WHAT IS A MOSAIC EMBRYO?
The term mosaicism refers to the presence of more than just one cell line, which present different chromosome count (1). Additionally, the most common situation in these cases is the presence of a mixture of distinct aneuploid cells, rather than of a variety between euploid and aneuploid cells. These are the embryos that may be at risk of misdiagnosis (2).
There are four possible types of mosaic embryos (3,4):
1. Embryos with a mix of euploid and aneuploid cells in the trophectoderm (TE), and with aneuploid cells in the inner cell mass (ICM).
2. Embryos with a mix of euploid and aneuploid TE cells and euploid ICM.
3. Embryos with euploid TE cells and aneuploid ICM.
4. Embryos with aneuploid TE cells and euploid ICM.
Even though there exists no specific cut-off to determine mosaicism, the Preimplantational Genetic Diagnosis International Society (PGDIS) suggests an embryo with more than 20% of aneuploid cells to be considered as mosaic. This means lower levels of mosaicism should be treated as normal (euploid) (5).
It has been traditionally thought that only genetic problems in the oocyte or the sperm could be responsible for embryo mosaicism. Nevertheless, it is currently postulated that this also occurs during the first mitotic divisions, when maternal transcripts control the cell cycle of the early embryo (6).
NORMAL INDICATIONS FOR CHROMOSOMAL TESTING TO DETECT EMBRYO MOSAICISM
1. Advanced maternal age. This is the most common cause for aneuploid problems (7). A recent study has shown that women between the ages of 35 and 43 years have more probabilities (an increase ranging from 28 to 78%) of presenting mis-segregation for the most clinically relevant aneuploidies, namely chromosomes 13,16,18,21 and 22 (8).
2. Severe male factor infertility. Even though levels of sperm aneuploidy are associated with increased levels of chromosomal abnormalities in embryos (9), such abnormalities could also arise from certain males who do not present any chromosomal abnormality a priori. Such could be specific cases of oligoastenozoospermic patients (10).
3. Recurrent implantation failure (RIF). In spite of the lack of specifications for such diagnosis, it is usually defined as the occurrence of three or more failed IVF attempts due to an unidentified cause. RIF is the usual diagnosis in those cases in which after a cumulative transfer of more than 10 good-quality embryos, the eventual result is IVF failure (7,11,12,13).
4. Recurrent miscarriage. The definition of this concept may vary for every country. However, generally speaking it can be defined as the occurrence of 3 or more consecutive miscarriages once pregnancy has reached at least 14 weeks (14). The main cause for this problem seems to be aneuploidy, which has been identified as the leading cause in a high percentage of miscarriages (15,16).
5. Previous trisomic pregnancy. Cases in which there has been a previous trisomic pregnancy entail higher probability of suffering from another aneuploid conception. Therefore, it is in this group of patients in which it would be beneficial to conduct a study to find out possible related causes (17).
BIOPSY TECHNIQUES TO STUDY A CASE OF MOSAICISM
Although there are different biopsy strategies, depending on the embryo stage, blastocyst biopsy is recommended over both polar body and blastomere biopsies in those cases in which mosaicism is suspected. Blastocyst biopsy is less invasive, it is possible to extract a higher number of cells, which increases the probability to confirm mosaicism, and it is cheaper than the other techniques due to the lower number of embryos required for biopsy (7). Furthermore, if cells with different chromosome complements are widely distributed throughout the trophectoderm, there might be a good chance of capturing a representative sample. If cells are clustered, mosaicism could not be easily detected, thus providing false normal results (18).
TECHNIQUES EMPLOYED TO DIAGNOSE MOSAICISM
The earliest trials of PGD involved the use of karyotyping and PCR. By the mid-90s, the use of cytogenetic techniques such as FISH allowed for the progress of preimplantation diagnosis. This very own approach was later shown to impose important technical limitations to the analysis, and so it was encouraged the development of new technologies that could minimize the errors in diagnosis (19).
Fluorescence in situ hybridization (FISH)
It allows for the analysis and identification of chromosomes or chromosome fragments with 5-10 fluorescently labelled molecular probes from one cell (blastomere) (Fig. 2). This cell can be biopsied from day-3 embryos, it could belong to the trophectoderm from blastocysts or it could also be a polar body biopsied from an oocyte or a zygote. Therefore, PGS–FISH diagnosis is limited to the most common abnormalities involving chromosomes 13, 15–18, 21, 22, X and Y. Some studies of preimplantation embryos diagnosed by using this technique estimate a 5-7% error caused by mosaicism when embryos are reanalyzed. Additionally, FISH is being rapidly replaced by other DNA analysis methods with higher efficiency (20,21).
Array Comparative Genomic Hybridization (aCGH)
This technique relies on whole genome amplification from one or more blastomeres (Fig. 3). It provides a quantitative analysis based on the comparison between the relative amount of tested DNA and the control DNA. Thus, chromosome imbalances such as aneuploidies, unbalanced translocations, deletions and duplications are easily detected. However, since balanced chromosome rearrangements such as reciprocal translocations or inversions do not affect copy number, such alterations cannot be identified (19,20). When blastomeres are analyzed by aCGH, the error rate measured is merely 2% (21,23). Notably, some studies have found that PGS-aCGH after blastocyst biopsy provides higher implantation and pregnancy rates than PGS-FISH (24).
Next Generation Sequencing (NGS)
Next Generation Sequencing belongs to the group of Massively Parallel Sequencing (MPS) methods that allow for parallel processing of an extremely large number of nucleic acid molecules (Fig. 4). As a result of sequencing on a microspace scale, it has been possible to drastically increase the amount of information collected during one test up to an entire human genome. Also, it is the only method that allows for analysis of all chromosomes (aneuploidies or translocations) and mutations responsible for any single-gene disease, just using one biopsy and in a single step (20). Although clinical results have documented high pregnancy rates following transfer of screened embryos, further data along with an extended use in clinical application are required to better define the role of NGS in PGS. Nevertheless, it seems that this method may actually lead to reduced costs per patient, thus allowing IVF couples a wider use of PGS for choosing the most competent embryo for transfer (26).
DIFFICULTIES WHEN MANAGING RESULTS
The detection of mosaicism at an early stage does not mean that it will spread along embryo development (27). However, the utilization of chromosome identification techniques as part of the IVF process makes it possible to identify embryos “at risk of mosaicism’’ in order to select those that are suitable for transfer (18).
Mosaic embryos are supposedly less competent than others due to a reduced implantation potential. Therefore, by discarding mosaic embryos implantation rates should be improved and, simultaneously, embryo loss rates reduced. Nevertheless, mosaic embryos may still have reproductive potential, and consequently they could still be viable. Furthermore, discarding embryos capable of producing healthy children will decrease pregnancy rates in those patients who get a low number of blastocysts in the pool of transferable embryos (18).
It is important to take into account the reaction of the patients when they are informed about their embryos being at risk for mosaicism, what may entail genetic abnormalities, reduce implantation rates, increase loss risk and even diminish obstetrical and neonatal outcomes. However, there is not a simple answer when patients decide to transfer a mosaic embryo; either way, the obstetrical team should be informed for future screens (18).
SUGGESTED GUIDELINES BY THE PREIMPLANTATINO GENETIC DIAGNOSIS INTERNATIONAL SOCIETY (PGDIS)
This article has been selected for publication in the Scientists in Reproductive Technologies (SIRT) Newsletter of The Fertility Society of Australia: DEL RÍO, J. and SANZ, S. (2017) Mosaic embryos are capable of producing healthy children. How to handle it? Fertility Society of Australia - SIRT Newsletter 4(20): 12-15.
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