**5. Mosaicism**

Mosaicism is defined as the presence of different cell lines in the same embryo. Two different kinds of mosaicism can occur: diploid/aneuploid mosaic with a mix of aneuploid and euploid cell lines and aneuploid mosaic with a mix of cell lines with different chromosomal abnormalities. There can be various types of aneuploidies in mosaic embryos: single chromosome loss or gain, complex or structural aneuploidies [155]. The origin of mosaicism is related to mitotic errors happening after fertilization at the third division stage. These mitotic errors, taking place before DNA duplication, are basically: anaphase delay, mitotic non-disjunction, accidental chromosome demolition, or premature cell division. The aneuploid cells rate depends on the time at which mitotic error happens; in embryos in which errors take place at the second cleavage stage, there will be a higher percentage of aneuploid cells [156,157]. Occasionally, mosaicism may derive from a meiotic non-disjunction event, causing a trisomic conceptus, followed by a post-zygotic event (trisomy rescue) [158,159].

FISH was first technique used to investigate the frequency of embryonic mosaicism: studies involving this method reported mosaicism rates that varied from 30% to 90% at cleavage stage, and from 18% to 46% at blastocyst stage [160]. More clear information regarding the mosaicism rate was obtained with the introduction of recent molecular techniques for genetic testing, such as SNP array, aCGH, and NGS. The mosaicism rates detected using these analyses are lower than those reported using FISH. At cleavage stage, the incidence of mosaic diploid-aneuploid varies from 15% to 71%; at blastocyst stage, the rates are lower, with an incidence ranging from 3.9% to about 33% [65,161]. Moreover, mosaic diploid/aneuploid embryos rate, decreases with maternal age: in women, less than 35 years old, the rate is 26.6%, and in >42 years old women it is 10.5% [162].

Studies regarding the distribution of mosaic cells in the blastocyst have been conducted to establish whether there is a preferential distribution between TE and inner cell mass and a recent one demonstrated that, in low-level mosaicism, there is a preferential allocation in TE, but, in high-level mosaicism, the aneuploidies seems to be uniformly distributed in the whole embryo [163]. An important question, still open, regarding mosaic embryos, is whether data obtained from a small portion of TE cells may predict the whole embryo asset. It has been hypothesized that the mosaicism rate deduced from a single TE biopsy might not be representative of the remaining TE cells or of the inner cell mass [164].

Mosaic embryos have not been considered to be suitable for transfer and they were discarded, while considering them as aneuploid embryos. Mosaicism was supposed to be responsible for altered embryo development, thus leading to implantation failure, or resulting in congenital malformation, mental retardation, and uniparental disomy [164].

Greco et al. [165] were the first, in 2015, to demonstrate that mosaic diploid/aneuploid embryos were suitable for implantation, giving birth to healthy babies. In his study, 18 diploid/aneuploid mosaic embryos, diagnosticated by aCGH, were transferred in women who did not have any euploid blastocyst for frozen embryo transfer. The implantation rate of these embryos was 44%, with a live birth rate of 33%. Every fetus was confirmed through chorionic villi to have a normal karyotype. Since then, a great number of studies involving the transfer of mosaic embryos were developed and they all confirmed the capability of these embryos to lead to healthy live births [166,167]. Table 1 reassume the clinical outcomes after mosaic blastocysts transfer presented in the most relevant papers [155,157,165–167].


**Table 1.** Clinical outcomes after transfer of mosaic embryos.

Several studies have demonstrated that chromosomal mosaicism extent significantly affect clinical results. Spinella et al. [155] proved that blastocysts with a mosaicism rate lower than 50% had significantly higher ongoing pregnancies (40.9% vs. 15.2%) and live births (38% vs. 19%) when compared to those with a mosaicism rate over 50%. These findings were confirmed by the paper of Viotti et al. from 2019 [168]. This study assessed that complex and single segmental mosaics had better outcomes than those with multiple gain or losses of whole chromosomes [168]. Victor et al. [167] assessed that the mosaicism rate is not correlated with clinical outcomes, reporting any significant difference in embryos with mosaicism rate lower than 40% or included between 40% and 80%. Clinical outcomes were only affected by maternal age and the type of mosaicism involved, with better results in young women and segmental aneuploidies.

A still unsolved question is whether the transfer of mosaic embryos might result in congenital abnormalities or in the birth of affected babies. Over 200 pregnancies have been reported, obtained after the transfer of mosaic embryos; 42 gave birth to healthy children [155,165,167] and the other were, at the moment of the studies publication, still ongoing and apparently normal [166–168]. However, this number is still not high enough to draw any conclusion and future studies are needed. Due to the lack of these data, a transfer involving mosaic embryos should only be performed when unavoidable and after appropriate counseling [162]. Couples considering to transfer mosaic embryos should be informed about potential pregnancy risks and outcomes; it should be explained them that these

transfers may be characterized by lower implantation and pregnancy rates, beyond a higher risk of genetic abnormalities and adverse pregnancy outcome.

It must be underlined a recently published case report: a blastocyst showing a 35% mosaicsim of monosomy 2 was transferred in a 39-year old woman with diminished ovarian reserve resulting in a clinical pregnancy. Amniocentesis revealed a mosaic trisomic karyotype: 46,XX(98)/47,XX + 2(2). This case demonstrates the need for a strict prenatal monitoring and diagnosis by early amniocentesis [169].

Preimplantation Genetic Diagnosis International Society (PGDIS) and Controversies in Preconception and Perinatal Genetic Diagnosis (CoGEN) have been suggested to always prefer the transfer of embryos with low level of mosaicism (20–40%), avoiding the transfer of those with potentially viable aneuplodies or intrauterine growth restriction [170,171].

Mosaicism rate is less than 1–2% in viable pregnancies suggesting the existence of a self-correction process that removes aneuploid cells from embryos after implantation [172]. Some models have been proposed to explain this phenomenon: one of them suggests cell death; the other a reduced proliferation of aneuploid cells when compared to euploid ones [173]. In 2019, a study by Popovic et al. [174], confirmed that, in human embryos, cell proliferation and death have different dynamics among euploid, aneuploid, or mosaic blastocysts. He used an extended in vitro embryo culture protocol to study the effect of mosaicsm on early preimplantation, up to 12 days post-fertilization. Blastocysts with high mosacism levels were more likely to be non-viable at this stage of development. This could confirm the self-correction model, since aneuploid cells could proliferate more slowly or undergo apoptosis, whilst euploid ones could proliferate faster to compensate. There is still no evidence, anyway, that could support this model.
