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Mosaicism between trophectoderm and inner cell mass

      Defining the actual incidence and prevalence of mosaicism in human blastocysts still remains a difficult task. The small amount of evidence generated by animal and human studies does not support the existence of mechanisms involved in developmental arrest, clonal depletion, or aneuploidy rescue for abnormal cells in euploid/aneuploid embryos during preimplantation development. However, studies in humans are mainly descriptive and lack functional evidence. Understanding the biological mechanisms that beset preimplantation differentiation holds the potential to reveal the role of aneuploidies and gene dosage imbalances in cell fate decision, providing important clues on the origin and evolution of embryonic mosaicism. The evidence on human blastocysts suggests that a mosaic euploid/aneuploid configuration is detected in around 5% of embryos. This figure supports the extremely low level of mosaicism reported in natural and IVF pregnancies. Similarly, the clinical management of patterns consistent with the presence of mosaicism in a trophectoderm biopsy during preimplantation genetic diagnosis cycles (PGD-A) is still a controversial issue. Despite the facts that some contemporary comprehensive chromosomal screening platforms can detect mosaic samples in cell mixture models with variable accuracy and many reproductive genetics laboratories are now routinely including embryonic mosaicism on their genetic reports, a diagnosis of certainty for mosaicism in PGD-A cycles is conceptually impracticable. Indeed, several technical and biological sources of errors clearly exist when trying to estimate mosaicism from a single trophectoderm biopsy in PGD-A cycles and must be understood to adequately guide patients during clinical care.

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      The unbalanced transmission of chromosomes in human gametes and early preimplantation embryos causes aneuploidy, which is a major cause of infertility, pregnancy loss, and intellectual disability in humans (
      • Hassold T.
      • Hunt P.
      To err (meiotically) is human: the genesis of human aneuploidy.
      ). In the preimplantation and prenatal setting, chromosome abnormalities span a wide range of genomic imbalances, from polyploidy, to whole chromosome and large structural aneuploidies, down to submicroscopic deletions and duplications. Whole chromosome aneuploidies, monosomies and trisomies for the entire chromosomes, are the far more prevalent abnormalities and have been extensively investigated due to their high incidence in human conceptions and their clear association with clinical phenotypes and infertility. Undoubtedly, they represent the single most common form of aneuploidy and the primary cause for implantation failure in IVF cycles and miscarriages in human pregnancies. Another well-defined characteristic of human aneuploidies is their strict correlation with female age. As women age, oocytes become increasingly susceptible to chromosome segregation errors during the meiotic process. Extensive analysis of main autosomal trisomies in clinical pregnancies revealed that more than 90% had a meiotic origin. The majority are due to maternal errors, with >75% due to errors in meiosis I and <25% due to errors in meiosis II, while current estimates suggest that a minority (1%–2%) of the spermatozoa are afflicted (
      • Hassold T.
      • Hunt P.
      To err (meiotically) is human: the genesis of human aneuploidy.
      ,
      • Chiang T.
      • Schultz R.M.
      • Lampson M.A.
      Meiotic origins of maternal age-related aneuploidy.
      ). Similar findings were observed in blastocyst-stage human embryos as well, where most of the aneuploidies appear to be meiotic in origin (
      • Ottolini C.S.
      • Newnham L.J.
      • Capalbo A.
      • Natesan S.A.
      • Joshi H.A.
      • Cimadomo D.
      • et al.
      Genome-wide maps of recombination and chromosome segregation in human oocytes and embryos show selection for maternal recombination rates.
      ). The age-related processes that lead to the exponential increase in aneuploid conceptions are increasingly understood, and novel insights into the molecular mechanisms of chromosome segregation during female meiosis are being unraveled (
      • Ottolini C.S.
      • Newnham L.J.
      • Capalbo A.
      • Natesan S.A.
      • Joshi H.A.
      • Cimadomo D.
      • et al.
      Genome-wide maps of recombination and chromosome segregation in human oocytes and embryos show selection for maternal recombination rates.
      ). Importantly, the fallibility of female meiosis is a panethnic and central biomedical subject (
      • Franasiak J.M.
      • Olcha M.
      • Shastri S.
      • Molinaro T.A.
      • Congdon H.
      • Treff N.R.
      • et al.
      Embryonic aneuploidy does not differ among genetic ancestry according to continental origin as determined by ancestry informative markers.
      ) that has led to the introduction of several preimplantation/prenatal diagnostic programs worldwide to counteract the impact of aneuploidies in pregnancies, especially for women of advanced reproductive age. Indeed, there are no therapies available to counteract the age-related increase in aneuploidies. The only preventive interventions are fertility preservation (oocyte vitrification) at a young age and the adoption of diagnostic measures in the preimplantation (preimplantation genetic diagnosis-aneuploidy testing [PGD-A]) or prenatal period to prevent their complications.
      For what concerns the diagnosis of aneuploidies, it is important to outline that when the error occurs in the gametes, the embryo will show the extra or missing chromosome or chromosome region in all the cells. Embryos carrying a meiotic-derived chromosome abnormality are commonly referred to as uniform aneuploid. In this situation, where the chromosomal abnormality is uniformly present across all the cells of the preimplantation embryo, the evolution and implication for that conception can be predicted with enough accuracy. Indeed, whatever preimplantation or prenatal diagnostic approach is applied, the diagnosis will not be subjected to sample bias, meaning that the biopsy sample obtained from the fetus or its related tissues will be representative of the embryonic chromosomal constitution. The high relative contribution of numeric aneuploidies of meiotic origin in embryos and pregnancies resulted in the successful application of diagnostic programs either at the blastocyst and prenatal stage (
      • Vermeesch J.R.
      • Voet T.
      • Devriendt K.
      Prenatal and pre-implantation genetic diagnosis.
      ). PGD-A at the blastocyst stage has been proven to be an effective strategy to improve embryo selection, reducing miscarriage risk in IVF treatment (
      • Chen M.
      • Wei S.
      • Hu J.
      • Quan S.
      Can comprehensive chromosome screening technology improve IVF/ICSI outcomes? A meta-analysis.
      ).
      Apart from uniform aneuploidies originating because of meiotic errors in both gametes, postzygotic errors in chromosome segregation can also occur and contribute to human aneuploidies and may be associated with developmental arrest or congenital abnormalities (
      • McCoy R.C.
      • Demko Z.P.
      • Ryan A.
      • Banjevic M.
      • Hill M.
      • Sigurjonsson S.
      • et al.
      Evidence of selection against complex mitotic-origin aneuploidy during preimplantation development.
      ). Mitotic errors during the first cleavage divisions result in mosaicism within the preimplantation embryo and potentially in cell lines with different karyotypes. Although meiotic aneuploidies are uniformly present in all cells and can be accurately detected and managed in clinical diagnostic programs (
      • Hassold T.
      • Hunt P.
      To err (meiotically) is human: the genesis of human aneuploidy.
      ,
      • Cohen J.
      Sorting out chromosome errors.
      ), the embryonic fate and the clinical consequences of mosaic aneuploidies may depend on many variables. These include which chromosome is involved in the aneuploidy, when the error occurred during preimplantation development, what proportion of the embryo is aneuploid, and where abnormal cells are located within the embryo (
      • Johnson D.S.
      • Cinnioglu C.
      • Ross R.
      • Filby A.
      • Gemelos G.
      • Hill M.
      • et al.
      Comprehensive analysis of karyotypic mosaicism between trophectoderm and inner cell mass.
      ,
      • Wapner R.J.
      • Simpson J.L.
      • Golbus M.S.
      • Zachary J.M.
      • Ledbetter D.H.
      • Desnick R.J.
      • et al.
      Chorionic mosaicism: association with fetal loss but not with adverse perinatal outcome.
      ,
      • Wilkins-Haug L.
      • Roberts D.J.
      • Morton C.C.
      Confined placental mosaicism and intrauterine growth retardation: a case-control analysis of placentas at delivery.
      ). As a consequence, the clinical implication of a mosaic aneuploidy can be seen as unique for each event and is difficult to interpret in the absence of well-defined genotype/phenotype associations. In this regard, the incidence and prevalence of chromosomal mosaicism in human blastocysts and its diagnosis in PGD-A cycles have recently been the subject of extensive investigation and debate. In particular, issues related to segregation and spatial allocation of aneuploid cells in a mosaic preimplantation embryo have recently raised concerns about the applicability and the effectiveness of PGD-A programs. Indeed, preimplantation embryos are normally assessed for genetic content by taking a small biopsy and testing the chromosomal constitution. For blastocyst-stage embryos, 5–10 randomly selected cells of the trophectoderm (TE) are commonly used to infer the chromosomal configuration of the inner cell mass (ICM). While for uniform aneuploidies this does not represent a limitation, in the context of a mosaic diploid/aneuploid embryo, the biopsied TE cells might not be representative of the actual chromosomal constitution of the ICM, causing misdiagnosis of the embryo's karyotype.
      Despite the fact that chromosomal mosaicism is diagnosed in <2% of prenatal specimens and only a small proportion of them (≈10%) is then confirmed in the fetus (
      • Malvestiti F.
      • Agrati C.
      • Grimi B.
      • Pompilii E.
      • Izzi C.
      • Martinoni L.
      • et al.
      Interpreting mosaicism in chorionic villi: results of a monocentric series of 1001 mosaics in chorionic villi with follow-up amniocentesis.
      ), estimates of preimplantation-stage mosaicism frequency are still inconsistent (
      • Taylor T.H.
      • Gitlin S.A.
      • Patrick J.L.
      • Crain J.L.
      • Wilson J.M.
      • Griffin D.K.
      The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans.
      ). Different approaches for data reporting and clinical management of mosaicism in PGD-A cycles have also been proposed (
      • Capalbo A.
      • Ubaldi F.M.
      • Rienzi L.
      • Scott R.
      • Treff N.
      Detecting mosaicism in trophectoderm biopsies: current challenges and future possibilities.
      ,
      • Munne S.
      • Grifo J.
      • Wells D.
      Mosaicism: “Survival of the fittest” versus “no embryo left behind”.
      ,
      • Scott Jr., R.T.
      • Galliano D.
      The challenge of embryonic mosaicism in preimplantation genetic screening.
      ). Due to the poor knowledge about the mechanisms of mitotic error and subsequent evolution of abnormal cells in preimplantation development, mosaicism has been also advocated as a major biological limitation to the success of blastocysts PGD-A programs in general (
      • Mastenbroek S.
      • Repping S.
      Preimplantation genetic screening: back to the future.
      ). The main issues under debate are the TE representativeness of the ICM, the capability of contemporary comprehensive chromosome screening (CCS) technologies to quantify the ratio of normal/abnormal cells in a blastocyst biopsy, and how to attribute a clinical value to mosaic results.
      Therefore, understanding the incidence and prevalence of mosaicism in blastocysts is essential and an area of intense scrutiny, with the objective to improve the diagnostic approaches and the treatment outcomes during medically assisted reproduction. The scope of this review is to communicate recent findings on the role of aneuploidies on preimplantation embryo development, provide a critical evaluation of existing data on the incidence and prevalence of chromosome mosaicism at the blastocyst stage, and propose a guideline on how these data may be appropriately managed in the PGD-A clinical setting.

      The impact of aneuploidies on preimplantation embryo development and differentiation

      Currently, a large amount of research is being carried out to investigate whether aneuploidy affects preimplantation development itself. Up to the moment of embryonic genome activation (EGA) at the 4- to 8-cell stage in humans (
      • Braude P.
      • Bolton V.
      • Moore S.
      Human gene expression first occurs between the four- and eight-cell stages of preimplantation development.
      ), embryo development is under the control of maternally inherited mRNAs and proteins (subcortical maternal complex). Gene transcription is mostly inactive (
      • Petropoulos S.
      • Edsgard D.
      • Reinius B.
      • Deng Q.
      • Panula S.P.
      • Codeluppi S.
      • et al.
      Single-cell RNA-Seq reveals lineage and X chromosome dynamics in human preimplantation embryos.
      ). It has been proposed that these early cell divisions are at higher risk for mitotic errors leading to mosaicism in cleavage-stage embryos (
      • Taylor T.H.
      • Gitlin S.A.
      • Patrick J.L.
      • Crain J.L.
      • Wilson J.M.
      • Griffin D.K.
      The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans.
      ,
      • van Echten-Arends J.
      • Mastenbroek S.
      • Sikkema-Raddatz B.
      • Korevaar J.C.
      • Heineman M.J.
      • van der Veen F.
      • et al.
      Chromosomal mosaicism in human preimplantation embryos: a systematic review.
      ). Aneuploidies in blastomeres give rise to dosage imbalances in the expression of genes from the affected chromosomes (
      • Zamani Esteki M.
      • Dimitriadou E.
      • Mateiu L.
      • Melotte C.
      • Van der Aa N.
      • Kumar P.
      • et al.
      Concurrent whole-genome haplotyping and copy-number profiling of single cells.
      ), and the high progression failure occurring at the compaction stage during in vitro development has been explained by the negative effect of aneuploidies on cell differentiation when EGA takes place. A large body of research indeed suggests that mosaicism is lower in blastocysts than in cleavage-stage embryos (
      • Taylor T.H.
      • Gitlin S.A.
      • Patrick J.L.
      • Crain J.L.
      • Wilson J.M.
      • Griffin D.K.
      The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans.
      ,
      • van Echten-Arends J.
      • Mastenbroek S.
      • Sikkema-Raddatz B.
      • Korevaar J.C.
      • Heineman M.J.
      • van der Veen F.
      • et al.
      Chromosomal mosaicism in human preimplantation embryos: a systematic review.
      ). Some of the differences in the magnitude of aneuploidy between cleavage and blastocyst stage are likely to reflect the lower diagnostic reliability of single-blastomere analysis compared with the multicell TE samples. It is thus likely that mosaicism has been overestimated in cleavage-stage embryo studies. Despite this, the existence of mechanism(s) that “correct” or “prevent” aneuploidy (referred to as “self-correction”) during preimplantation development has been suggested (
      • Bazrgar M.
      • Gourabi H.
      • Valojerdi M.R.
      • Yazdi P.E.
      • Baharvand H.
      Self-correction of chromosomal abnormalities in human preimplantation embryos and embryonic stem cells.
      ,
      • Mertzanidou A.
      • Spits C.
      • Nguyen H.T.
      • Van de Velde H.
      • Sermon K.
      Evolution of aneuploidy up to day 4 of human preimplantation development.
      ). One of the fundamental questions for the basic understanding of mosaicism in embryos relates to the characterization of whether chromosome perturbations and associated gene dosage imbalances might contribute to embryonic arrest or, for those surviving to the blastocyst stage, whether the abnormal cells can be selected against by apoptosis or lower mitotic progression or “corrected” by a second mitotic error. Insights from the basic biology of preimplantation differentiation events might provide important clues on the existence of putative corrective mechanisms. Three models have been proposed: clonal depletion, preferential allocation, and self-correction. The first hypothesis involves a physiological selection against chromosomally abnormal cells due to their lower developmental competence. The second mechanism involves the selective allocation of aneuploid cells to the TE, whereas the self-correction model presumes the restoration to a normal disomic configuration through a second segregation error event correcting the aneuploidy. Unraveling these mechanisms will provide the basis for a better understanding of the incidence and prevalence of mosaicism in embryos and improve the clinical protocols for blastocyst-stage PGD-A.

       Aneuploidies and Embryo Progression Arrest

      Nearly 40% of human preimplantation embryos arrest around day 3–4, when the 8-cell stage to morula compaction normally occurs. A proportion of these embryos contain fragmented cells, suggestive of error-prone cleavage divisions associated with DNA damage (
      • Kort D.H.
      • Chia G.
      • Treff N.R.
      • Tanaka A.J.
      • Xing T.
      • Vensand L.B.
      • et al.
      Human embryos commonly form abnormal nuclei during development: a mechanism of DNA damage, embryonic aneuploidy, and developmental arrest.
      ). However, many arrested cleavage-stage embryos have normal morphology, and the majority are aneuploid. Aneuploidies, resulting in gene dosage imbalance for key genes, might explain, at least in part, this high developmental failure that is encountered between cleavage and blastocyst stage when EGA and important cellular differentiation events take place.
      Consistent with aneuploidies affecting preimplantation development, recent studies claimed a higher incidence of chaotic chromosome aneuploidies in biopsies taken from 8-cell embryos (day 3) compared with blastocysts (
      • Vega M.
      • Breborowicz A.
      • Moshier E.L.
      • McGovern P.G.
      • Keltz M.D.
      Blastulation rates decline in a linear fashion from euploid to aneuploid embryos with single versus multiple chromosomal errors.
      ) derived from putative mitotic events (
      • McCoy R.C.
      • Demko Z.P.
      • Ryan A.
      • Banjevic M.
      • Hill M.
      • Sigurjonsson S.
      • et al.
      Evidence of selection against complex mitotic-origin aneuploidy during preimplantation development.
      ). In particular, results from a survey of 385 cleavage-stage embryos found that survival to blastocyst formation correlates with the number of chromosomal abnormalities detected in blastomeres (
      • Vega M.
      • Breborowicz A.
      • Moshier E.L.
      • McGovern P.G.
      • Keltz M.D.
      Blastulation rates decline in a linear fashion from euploid to aneuploid embryos with single versus multiple chromosomal errors.
      ). In this study, euploid embryos were twice as likely to progress to blastocyst and three times as likely to progress to the fully expanded stage compared with aneuploid cleavage-stage embryos. For each extra chromosome pair affected, a linear reduction of 22% on the probability of the embryo progressing to the blastocyst stage was observed. Currently, these data should be considered in the light of the impact of blastomere removal on development and on the reduced accuracy associated with a day 3 single-blastomere biopsy (12% no result rate in this study). Furthermore, 22% of euploid embryos did not progress to the blastocyst stage, suggesting that mechanisms unrelated to chromosomal content are still important factors for preimplantation embryo arrest.
      In a separate study, McCoy and colleagues recently also reported a higher incidence of putative complex mitotic-origin aneuploidies in day 3 blastomere biopsies compared with TE samples, which they conclude are purged by selection before blastocyst formation (
      • McCoy R.C.
      • Demko Z.P.
      • Ryan A.
      • Banjevic M.
      • Hill M.
      • Sigurjonsson S.
      • et al.
      Evidence of selection against complex mitotic-origin aneuploidy during preimplantation development.
      ). They used a single nucleotide polymorphism (SNP) array–based chromosome analysis with a proprietary parental support algorithm to distinguish between the meiotic or mitotic origin of aneuploidies found in blastomeres and TE biopsies. Furthermore, they compared the two groups (blastomere and TE samples) by calculating the difference in percentage of noneuploidy rates, stratifying by the total number of affected chromosomes. The assumption was that this metric could reflect the proportion of embryos that were either lost or self-corrected between the two preimplantation stages. The higher rate of mitotic aneuploidies detected in blastomeres led to the conclusion that an early selection mechanism exists, resulting in the developmental arrest of embryos with complex aneuploidy of primarily mitotic origin. However, due to the design of the study, where blastomere and TE biopsy data were fully independent (derived from unrelated embryos), it was not possible to distinguish between embryonic arrest and self-correction. Additionally, since the rate of arrested embryos was not reported, correlation between aneuploidies and embryo progression failure was not possible either. Moreover, despite the publication of algorithm validation data, the authors express reasonable concerns on the limited ability of the algorithm to distinguish aneuploidies of meiotic or mitotic origin (
      • Bisignano A.
      • Wells D.
      • Harton G.
      • Munne S.
      PGD and aneuploidy screening for 24 chromosomes: advantages and disadvantages of competing platforms.
      ,
      • Handyside A.H.
      PGD and aneuploidy screening for 24 chromosomes by genome-wide SNP analysis: seeing the wood and the trees.
      ). Finally, some concerns remain about the impact of chromosome aneuploidies on embryo progression.
      Although some evidence suggests that aneuploidies and associated haploinsufficiency of dosage-sensitive genes are involved in preimplantation embryo arrest, other data sets are in favor of the opposite. In particular, when human blastocysts are assessed in PGD-A cycles, all range and spectra of aneuploidies are detected, including complex aneuploidies where multiple chromosomes are affected and alteration of dosage-sensitive genes has occurred. Thus, meiotic aneuploidies do not appear to be selected against during preimplantation development (
      • Capalbo A.
      • Rienzi L.
      • Cimadomo D.
      • Maggiulli R.
      • Elliott T.
      • Wright G.
      • et al.
      Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts.
      ,
      • Franasiak J.M.
      • Forman E.J.
      • Hong K.H.
      • Werner M.D.
      • Upham K.M.
      • Treff N.R.
      • et al.
      The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening.
      ). Similarly, aneuploidies do not cause major differences in the morphological and morphokinetic behavior of developing human blastocysts (
      • Capalbo A.
      • Rienzi L.
      • Cimadomo D.
      • Maggiulli R.
      • Elliott T.
      • Wright G.
      • et al.
      Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts.
      ,
      • Rienzi L.
      • Capalbo A.
      • Stoppa M.
      • Romano S.
      • Maggiulli R.
      • Albricci L.
      • et al.
      No evidence of association between blastocyst aneuploidy and morphokinetic assessment in a selected population of poor-prognosis patients: a longitudinal cohort study.
      ).
      An alternative explanation for the higher chromosome/mitotic error rate observed in cleavage-stage arrested embryos may be that a primary cellular and developmental defect occurring in blastomeres causes downstream defective mitosis, resulting in several chromosome segregation errors in daughter cells. In this view, chromosomal abnormality can be seen as a proxy variable or a by-product of distinct cellular defects that result in abnormal chromosomal segregation and mitotic arrest. Currently, we lack evidence on how these mechanisms may cause mitotic arrest in preimplantation embryos. Characterization of these mechanisms will provide the basis for understanding whether aneuploidies in blastomeres cause preimplantation embryo arrest (thus explaining the lower rate of mitotic aneuploidies observed at the blastocyst stage) or whether they are entirely permissive of preimplantation progression.

       Aneuploidies and Blastocyst Differentiation

      Preimplantation differentiation is an extraordinarily complex and unique process in human biology that culminates in the formation of a blastocyst. At the blastocyst stage, the human embryo is composed of the extraembryonic TE and primitive endoderm, which will form the placenta and yolk sac, respectively, and the embryonic epiblast, which forms the fetus (
      • Carbone L.
      • Chavez S.L.
      Mammalian pre-implantation chromosomal instability: species comparison, evolutionary considerations, and pathological correlations.
      ,
      • Bedzhov I.
      • Graham S.J.
      • Leung C.Y.
      • Zernicka-Goetz M.
      Developmental plasticity, cell fate specification and morphogenesis in the early mouse embryo.
      ). The specification of these three cell types is achieved through two “cell fate decisions.” In the first cell fate decision, two major waves of asymmetric cell divisions at the 8- to 16- and 16- to 32-cell transitions and a minor wave at the 32- to 64-cell transition generate outer and inner cells that differ in cellular properties, position within the embryo, and ensuing fate (
      • Morris S.A.
      • Teo R.T.
      • Li H.
      • Robson P.
      • Glover D.M.
      • Zernicka-Goetz M.
      Origin and formation of the first two distinct cell types of the inner cell mass in the mouse embryo.
      ,
      • Fleming T.P.
      A quantitative analysis of cell allocation to trophectoderm and inner cell mass in the mouse blastocyst.
      ). Outer cells differentiate into TE, while inner cells form the pluripotent ICM and will be further differentiated during the second cell fate decision. At the blastocyst stage, the ICM differentiates into the primitive endoderm, which predominantly gives rise to the yolk sac, and the pluripotent epiblast, which is the precursor of the future fetus. The correct specification and organization of these different cell types are essential for development of the embryo beyond implantation. The molecular mechanisms driving the targeted differentiation of a morphologically identical cluster of cells is a fundamental question of reproductive biology. Increased knowledge of the molecular events involved in cell fate decision in preimplantation embryos could potentially shed some light on mechanisms guiding preferential allocation and clonal depletion of abnormal cells in mosaic embryos.
      In the past few years, exciting experimental findings have started to unravel the mechanisms by which blastomeres incorporate information about their physical environment into the “internal” genetic imperatives that drive their differentiation. An integrative view is emerging where predeterminism and stochasticity are both contributing factors to cell fate determination in preimplantation development (
      • Zernicka-Goetz M.
      • Huang S.
      Stochasticity versus determinism in development: a false dichotomy?.
      ). Determinism entails that lineage segregation is programmed into the zygote at fertilization by the localization of specific molecular determinants. On the other hand, stochasticity sees the outcome of a particular process as unpredictable with certainty given the starting conditions. As a consequence, external inputs such as the position of a blastomere, the forces it is subject to, and the inherited genomic constitution play a significant role in lineage determination. This approach would essentially allow any of the blastomeres to produce all cell lineages. Indeed, position, signaling, and inherent cellular heterogeneities all influence cell fate decisions. Therefore, cellular heterogeneity provides biases that guide, but do not determine, cell fate decisions, thus allowing flexibility in cellular commitment throughout preimplantation development.
      These preimplantation cell fate decisions rely on a combination of factors including cell polarity, position, and cell-cell signaling and are influenced by the heterogeneity between early embryonic cells. The molecular genetic basis for the differentiation of these early cell types has been studied extensively, and several comprehensive reviews on this subject are available (
      • Bedzhov I.
      • Graham S.J.
      • Leung C.Y.
      • Zernicka-Goetz M.
      Developmental plasticity, cell fate specification and morphogenesis in the early mouse embryo.
      ,
      • Biggins J.S.
      • Royer C.
      • Watanabe T.
      • Srinivas S.
      Towards understanding the roles of position and geometry on cell fate decisions during preimplantation development.
      ,
      • Ajduk A.
      • Zernicka-Goetz M.
      Polarity and cell division orientation in the cleavage embryo: from worm to human.
      ). Recent studies have brought a refreshing new view on how TE and ICM are generated after the compaction stage, revealing that the Hippo pathway and its effectors YAP and TAZ (YAP/TAZ) are central to this process (
      • Hirate Y.
      • Hirahara S.
      • Inoue K.
      • Suzuki A.
      • Alarcon V.B.
      • Akimoto K.
      • et al.
      Polarity-dependent distribution of angiomotin localizes Hippo signaling in preimplantation embryos.
      ,
      • Leung C.Y.
      • Zernicka-Goetz M.
      Angiomotin prevents pluripotent lineage differentiation in mouse embryos via Hippo pathway-dependent and -independent mechanisms.
      ,
      • Nishioka N.
      • Inoue K.
      • Adachi K.
      • Kiyonari H.
      • Ota M.
      • Ralston A.
      • et al.
      The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass.
      ). These hypotheses provide a mechanism not only for sensing but also for responding to the localization of blastomeres within the embryo. Interestingly, depletion of genes (null mutant alleles) involved in the establishment and maintenance of polarity does not appear to compromise preimplantation development. Neither YAP nor TAZ alone are essential for preimplantation development (
      • Morin-Kensicki E.M.
      • Boone B.N.
      • Howell M.
      • Stonebraker J.R.
      • Teed J.
      • Alb J.G.
      • et al.
      Defects in yolk sac vasculogenesis, chorioallantoic fusion, and embryonic axis elongation in mice with targeted disruption of Yap65.
      ,
      • Makita R.
      • Uchijima Y.
      • Nishiyama K.
      • Amano T.
      • Chen Q.
      • Takeuchi T.
      • et al.
      Multiple renal cysts, urinary concentration defects, and pulmonary emphysematous changes in mice lacking TAZ.
      ), while homozygosity for the Yap(tm1Smil) allele (Yap-/-) caused developmental arrest around E8.5 of postimplantation mouse embryo development. As a result, mutant mouse embryos that have not correctly specified one of their early cell lineages can be morphologically indistinguishable from their wild-type littermates until the blastocyst stage. Yet their development does not progress beyond the peri-implantation period (
      • Chazaud C.
      • Rossant J.
      Disruption of early proximodistal patterning and AVE formation in Apc mutants.
      ,
      • Wicklow E.
      • Blij S.
      • Frum T.
      • Hirate Y.
      • Lang R.A.
      • Sasaki H.
      • et al.
      HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst.
      ,
      • Nichols J.
      • Zevnik B.
      • Anastassiadis K.
      • Niwa H.
      • Klewe-Nebenius D.
      • Chambers I.
      • et al.
      Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.
      ,
      • Mitsui K.
      • Tokuzawa Y.
      • Itoh H.
      • Segawa K.
      • Murakami M.
      • Takahashi K.
      • et al.
      The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.
      ,
      • Nishioka N.
      • Yamamoto S.
      • Kiyonari H.
      • Sato H.
      • Sawada A.
      • Ota M.
      • et al.
      Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos.
      ,
      • Kang M.
      • Piliszek A.
      • Artus J.
      • Hadjantonakis A.K.
      FGF4 is required for lineage restriction and salt-and-pepper distribution of primitive endoderm factors but not their initial expression in the mouse.
      ).
      Further evidence suggests that aneuploidies and haploinsufficiency may not be detrimental for embryo differentiation at the blastocyst stage. A biological hallmark of preimplantation development is the rapid mitotic progression in the absence of stringent checkpoint schemes. Soon after EGA, human embryos show short cell cycles during the precompaction divisions (
      • Wong C.C.
      • Loewke K.E.
      • Bossert N.L.
      • Behr B.
      • De Jonge C.J.
      • Baer T.M.
      • et al.
      Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage.
      ). A number of cell cycle drivers, including G1 phase-specific factors, are intensively activated after EGA, but checkpoint proteins are lacking (
      • Kiessling A.A.
      Timing is everything in the human embryo.
      ). Due to the absence of appropriate checkpoints at early cleavage stages, it is likely that human embryos do not experience strong negative selection of cells bearing aneuploidy, chromosome breakage, or segmental aberrations. Consequently, these abnormal cells are included in the developing embryo, causing genetic mosaicism to persist beyond early developmental stages without affecting cellular differentiation in a ploidy-dependent fashion.
      Consistent with this finding, a recent study by Bolton and colleagues concluded that there was no evidence for preferential allocation of aneuploid cells in mosaic mouse embryo models (
      • Bolton H.
      • Graham S.J.
      • Van der Aa N.
      • Kumar P.
      • Theunis K.
      • Fernandez Gallardo E.
      • et al.
      Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential.
      ). To investigate both aneuploid cells' fate and the developmental potential of mosaic embryos, Bolton and colleagues generated a mouse model with euploid-aneuploid chromosome mosaicism using the drug Reversine to inhibit SAC protein monopolar spindle 1-like 1 (Mps1) kinase during the 4- to 8-cell stage transition. Reversine-treated embryos formed blastocysts at a similar rate as controls by E4.5 but had significantly fewer cells in all their lineages. For the first time, time-lapse imaging enabled direct evaluation of cell fate in mosaic embryos, revealing no evidence of preferential allocation of abnormal cells to embryonic or extraembryonic lineages. This finding was in agreement with studies on human embryos (
      • Capalbo A.
      • Wright G.
      • Elliott T.
      • Ubaldi F.M.
      • Rienzi L.
      • Nagy Z.P.
      FISH reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts shows high accuracy of diagnosis and no major diagnostic impact of mosaicism at the blastocyst stage.
      ). Interestingly, blastomeres with a history of chromosome abnormalities become progressively depleted from the embryo as it develops. This depletion becomes apparent during blastocyst maturation, when abnormal ICM cells show increased apoptosis and abnormal TE cells exhibit limited proliferation. However, this effect is more evident after blastulation. These data suggest that clonal depletion of abnormal cells takes place as development progresses. A limitation of this experimental model is that, after treatment with Reversine, abnormal cells in the mosaic embryo show aneuploidies involving copy number alteration for multiple chromosomes in a single cell. Thus, further investigation is required to confirm these mechanisms in embryos where only one or a few chromosomes are aneuploid. Nevertheless, Bolton et al.'s study strengthened the concept that haploinsufficiency for a multitude of genes does not severely determine the cell allocation of aneuploid blastomeres, while it might result in their lower survival rate compared with normal cells, especially in the postimplantation period.
      In agreement with these findings, we have reported no sign for preferential allocation of abnormal cells in mosaic human blastocysts using fluorescence in situ hybridization (FISH) chromosome analysis at the single-cell level (
      • Capalbo A.
      • Wright G.
      • Elliott T.
      • Ubaldi F.M.
      • Rienzi L.
      • Nagy Z.P.
      FISH reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts shows high accuracy of diagnosis and no major diagnostic impact of mosaicism at the blastocyst stage.
      ). In this study, we examined the distribution and rate of abnormal cells in the ICM and three TE sections including the whole blastocyst. Results suggest that abnormal cells in mosaic diploid/aneuploid embryos were evenly distributed across the different blastocyst sections, suggesting no sign for preferential allocation toward one or the other cell lineage. Unfortunately, this study only provided a snapshot of the blastocyst stage, and we were unable to track the cell fate from the cleavage stage to define the existence of clonal depletion of abnormal cells or embryonic arrest of mosaic embryos. Hence, similar studies in human embryos are required and will be of significant value to further define the role of mitotic errors in a blastomere's fate decision.
      Finally, the self-correction model that suggests that abnormal chromosomes are fixed to a normal disomic configuration by a second mitotic error event correcting the original one is not supported by recent findings that show very low incidence of uniparental disomy at the blastocyst stage (
      • Gueye N.A.
      • Devkota B.
      • Taylor D.
      • Pfundt R.
      • Scott Jr., R.T.
      • Treff N.R.
      Uniparental disomy in the human blastocyst is exceedingly rare.
      ).
      In summary, although human embryo studies on this subject are still limited, it is likely that gene dosage imbalance for factors involved in early differentiation events has a minor impact on preimplantation progression and cell allocation to ICM or TE lineages. Mosaic embryos might still be able to progress through the preimplantation window without tangible effects on their morphological appearance and without depletion or preferential allocation of abnormal cells to one or the other lineage. However, understanding of human preimplantation development is limited due to ethical and legal restrictions on embryo research and scarcity of material. Studies in humans are mainly descriptive and lack functional evidence. Most of the information on embryo development is obtained from animal models and embryonic stem cell cultures and should be extrapolated with caution. New imaging and genetic technologies now provide the tools to investigate these mechanisms. Therefore, integrated genetic, embryological, and developmental biology studies will hopefully provide important data that will be able to answer these fundamental questions in the near future.

      The chromsome constitution of mosaic blastocysts

      Estimating the incidence and prevalence of mosaicism in blastocyst-stage human embryos remains a particularly difficult task. Methodological and biological limitations have restricted the level of evidence that has been generated thus far.
      First, methodological limitations in studying the human blastocyst at the single-cell level with comprehensive chromosome testing technologies (CCS) should be acknowledged. Only one validated method for reliable ICM isolation from human blastocysts has been reported in the literature (
      • Capalbo A.
      • Wright G.
      • Elliott T.
      • Ubaldi F.M.
      • Rienzi L.
      • Nagy Z.P.
      FISH reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts shows high accuracy of diagnosis and no major diagnostic impact of mosaicism at the blastocyst stage.
      ). However, downstream protocols for an effective ICM and TE disaggregation into single cells are missing. Moreover, access to whole human embryos for research purposes requires specific patient consent and the approval of regulatory and ethical committees. As a consequence, the availability of embryos is often limited to discarded blastocysts, such as abnormal development or aneuploid embryos. These starting conditions result in the generation and extrapolation of biased data that do not represent the general IVF population.
      Second, inherent technical limitations of CCS methods for assessment of mosaicism in a multicellular TE/ICM biopsy still persist. Identification of the presence of mosaicism relies on the bioinformatics tools that interpret an intermediate position of chromosome copy number falling between the disomy and the aneuploidy thresholds without additional molecular procedures. Fluctuation of copy number profiles can be explained by a multitude of different technical and biological factors (Fig. 1). Amplification bias can be introduced at the DNA enrichment step, resulting in a pattern of intermediate chromosome copy number similar to the presence of mosaicism. Additional biological sources of errors should be considered when evaluating mosaicism from TE biopsies. These include, for example, the presence of monosomies and trisomies in a polyploid or a contaminated sample. For most of the clinically available CCS technologies, the presence of an extra or missing chromosome in a triploid/tetraploid embryo or biopsy or in a contaminated sample will be interpreted as mosaicism due to the intermediate chromosome copy number profile (Fig. 1). Furthermore, a TE biopsy might contain reciprocal aneuploidies (a mixture of monosomy and trisomy of the same whole chromosome) at a ratio that causes an averaging of the signal, leading to a false-negative diagnosis of disomy. Finally, although less likely to happen in a heterogeneous TE sample, the presence of cells in S-phase may appear as a mosaic aneuploidy (
      • Van der Aa N.
      • Cheng J.
      • Mateiu L.
      • Zamani Esteki M.
      • Kumar P.
      • Dimitriadou E.
      • et al.
      Genome-wide copy number profiling of single cells in S-phase reveals DNA-replication domains.
      ).
      Figure thumbnail gr1
      Figure 1Technical and biological sources of errors when trying to estimate mosaicism from TE biopsies. This illustration highlights the most important technical and biological limitations that can be encountered when trying to incorporate a mosaicism diagnosis into blastocyst PGD cycles for aneuploidies. The predicted adverse clinical outcomes are also reported. Both amplification bias and the presence of contamination can generate intermediate copy number profiles resembling presence of mosaicism in TE biopsies. The presence of reciprocal aneuploidies in a single TE biopsy after a mitotic nondisjunction event can hide the presence of mosaicism resulting in the unknowing transfer of abnormal embryos. Polyploid embryos with an extra or missing chromosome can also be detected as mosaic embryos. A TE biopsy with a significant presence of S-phase cells can also result in a copy number profile consistent with mosaicism. PR/cycles = pregnancy rate per started IVF cycle; MR = miscarriage rate; CAP = chromosomally abnormal pregnancy.
      Several different DNA amplification protocols and CCS platforms for downstream quantitation are available on the market. Each methodology relies on significantly different bioinformatics paradigms that cannot be transferred across. None of the CCS methods has clinically standardized criteria to unequivocally distinguish between technical variation and genuine mosaicism when the copy number value falls between the disomy and aneuploidy thresholds. Thus, the reported estimates of mosaicism are all potentially impacted by technical factors and biological singularities of the samples (
      • Capalbo A.
      • Ubaldi F.M.
      • Rienzi L.
      • Scott R.
      • Treff N.
      Detecting mosaicism in trophectoderm biopsies: current challenges and future possibilities.
      ,
      • Treff N.R.
      • Franasiak J.M.
      Detection of segmental aneuploidy and mosaicism in the human preimplantation embryo: technical considerations and limitations.
      ). In a recent opinion paper, we proposed a model to strengthen the evidence of mosaicism when studying cleavage- and blastocyst-stage embryos. The aim of this model is to minimize the impact of technical variation in future studies and provide more reliable information about the real incidence and prevalence of mosaicism in preimplantation embryos (
      • Capalbo A.
      • Ubaldi F.M.
      • Rienzi L.
      • Scott R.
      • Treff N.
      Detecting mosaicism in trophectoderm biopsies: current challenges and future possibilities.
      ).
      Although caution is needed when interpreting mosaicism data, several studies have recently reported on the frequency and distribution of mosaicism in blastocysts using FISH or alternative contemporary CCS methods (Table 1). The most recent evidence suggests that about 5% of blastocysts are euploid/aneuploid mosaic, as collectively reported by four studies investigating multiple biopsies including whole blastocysts (
      • Johnson D.S.
      • Cinnioglu C.
      • Ross R.
      • Filby A.
      • Gemelos G.
      • Hill M.
      • et al.
      Comprehensive analysis of karyotypic mosaicism between trophectoderm and inner cell mass.
      ,
      • Capalbo A.
      • Wright G.
      • Elliott T.
      • Ubaldi F.M.
      • Rienzi L.
      • Nagy Z.P.
      FISH reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts shows high accuracy of diagnosis and no major diagnostic impact of mosaicism at the blastocyst stage.
      ,
      • Fragouli E.
      • Lenzi M.
      • Ross R.
      • Katz-Jaffe M.
      • Schoolcraft W.B.
      • Wells D.
      Comprehensive molecular cytogenetic analysis of the human blastocyst stage.
      ,
      • Northrop L.E.
      • Treff N.R.
      • Levy B.
      • Scott Jr., R.T.
      SNP microarray-based 24 chromosome aneuploidy screening demonstrates that cleavage-stage FISH poorly predicts aneuploidy in embryos that develop to morphologically normal blastocysts.
      ). Table 1 shows a summary of these studies, highlighting the procedural aspects and observed outcomes. Even though all studies present some sort of bias and none of them was blinded, no evidence of preferential allocation of aneuploid cells toward the TE was observed in human blastocysts. In high-grade mosaic blastocysts, abnormal cells appear to be spread across the whole embryo, increasing the likelihood of detection during clinical protocols of TE biopsy and CCS. On the other hand, low-grade mosaicism might go undetected as the abnormal cells appear to be confined to specific areas of the embryo (
      • Capalbo A.
      • Wright G.
      • Elliott T.
      • Ubaldi F.M.
      • Rienzi L.
      • Nagy Z.P.
      FISH reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts shows high accuracy of diagnosis and no major diagnostic impact of mosaicism at the blastocyst stage.
      ). Although additional studies are urgently required, diploid/aneuploid mosaicism appears to be a relatively uncommon feature of blastocyst-stage human embryos. Also, evidence that mosaicism is not frequently observed in spontaneous or IVF-derived pregnancies further supports these observations (
      • Huang A.
      • Adusumalli J.
      • Patel S.
      • Liem J.
      • Williams 3rd, J.
      • Pisarska M.D.
      Prevalence of chromosomal mosaicism in pregnancies from couples with infertility.
      ). Huang and colleagues reported extremely low rates of mosaicism in more than 5,300 chorionic villus sampling analyses from either spontaneous or IVF-derived conceptions: 1.22% and 1.32%, respectively, while true fetal mosaicism detection (not only confined to placental cells) was 0.3% and 0.44%, respectively (
      • Huang A.
      • Adusumalli J.
      • Patel S.
      • Liem J.
      • Williams 3rd, J.
      • Pisarska M.D.
      Prevalence of chromosomal mosaicism in pregnancies from couples with infertility.
      ). These data are sufficient to define the prevalence of mitotic errors in human reproduction as relatively limited and of potentially low impact for PGD-A programs.
      Table 1Summary table of methodology and main findings from relevant studies on the cytogenetic constitution of ICM and TE samples from disaggregated human blastocysts.
      Northrop et al. (2010)Fragouli et al. (2008)Capalbo et al. (2013)Johnson et al. (2010)Overall
      MethodFISH + SNP array reanalysisFISH + CGH reanalysisaCGH + FISH reanalysisSNP array with parental support
      No. of samples50107051181
      No. of patients244261771
      Mean female age35333631Range, 26–42
      Origin of embryosBlastocysts diagnosed as aneuploid by FISH-based blastomere analysisBlastocysts diagnosed as aneuploid by FISH-based blastomere analysisBlastocysts diagnosed as aneuploid by aCGH-based TE analysisDonated blastocysts
      TE-confined mosaicism (%)4/50 (8)0/10 (0)0/70 (0)0/51 (0)4/181 (2.2)
      ICM-confined mosaicism (%)1/50 (2)0/10 (0)0/70 (0)1/51 (2)2/181 (1.1)
      Mosaic blastocysts (both ICM and TE) (%)3/50 (6)0/10 (0)2/70 (3)0/51 (0)5/181 (2.8)
      Most frequent mosaic chromosomeschr16; chr22Not applicablechr16; chr17; chr22; chr21
      Fully euploid blastocyst (%)29/50 (58)4/10 (40)19/70 (70)41/51 (80)92/181 (51.7)
      Fully aneuploid blastocyst (%)13/50 (26)6/10 (60)49/70 (27)9/51 (18)77/181 (42.8)
      Note: Abnormal cells in euploid-aneuploid mosaic embryos are similarly distributed between ICM and TE, indicating no sign of preferential allocation or confinement of chromosomally abnormal cells. The fact that results were almost identical for samples from the TE and ICM indicates that data obtained from a clinical TE sample can generally be considered diagnostic of the ICM chromosomal complement. CGH = comparative genomic hybridization.
      Next we will need to understand how mosaic embryos behave in clinical terms and how we can manage the presence of copy number profiles consistent with mosaicism in TE biopsies during PGD-A clinical cycles. The reproductive potential of a mosaic euploid/aneuploid blastocyst will likely be inversely correlated with the abnormal-to-normal cells ratio. To determine the postimplantation fate of euploid/aneuploid mosaic embryos, Bolton and colleagues transferred embryos derived from their murine model of mosaicism to recipient mothers. This experiment showed that mosaic embryos that contain sufficient normal euploid cells (>50%) have full developmental potential, while when the proportion of normal blastomeres was decreased to one third, there was a partial rescue of embryonic lethality. In humans, both functional and prospective nonselection studies aimed at modeling the clinical fate and the reproductive potential of embryos showing intermediate copy number profiles are lacking. Prospective cohort studies in PGD-A cycles are also of poor value to determine the predictive value of patterns consistent with mosaicism as these are subjected to a patient and embryo selection bias.
      In conclusion, despite the fact that some contemporary CCS platforms can detect mosaic samples in cell mixture models with variable accuracy (
      • Greco E.
      • Minasi M.G.
      • Fiorentino F.
      Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts.
      ,
      • Mamas T.
      • Gordon A.
      • Brown A.
      • Harper J.
      • Sengupta S.
      Detection of aneuploidy by array comparative genomic hybridization using cell lines to mimic a mosaic trophectoderm biopsy.
      ,
      • Goodrich D.
      • Tao X.
      • Bohrer C.
      • Lonczak A.
      • Xing T.
      • Zimmerman R.
      • et al.
      A randomized and blinded comparison of qPCR and NGS-based detection of aneuploidy in a cell line mixture model of blastocyst biopsy mosaicism.
      ), a diagnosis of certainty for mosaicism in PGD-A cycles is conceptually impracticable. Many reproductive genetics laboratories are now routinely including embryonic mosaicism on their diagnostic reports. However, for the arguments discussed above, both biological and technological limitations exist and must be understood in order to adequately counsel patients during clinical care. Furthermore, it should be stressed that the chromosomal variations observed in a TE biopsy will not unequivocally represent the mosaicism rate present within the whole embryo. The existence of a sample bias has to be acknowledged in patients consent forms as a limitation for mosaicism detection in PGD-A cycles.
      In the light of the arguments discussed above, mosaicism should be recognized as a biological limitation for PGD-A programs as it is for prenatal diagnosis. In addition, it appears reasonable to avoid reporting mosaicism in PGD-A cycles until its impact on clinical treatment has been clarified. A more conservative approach for reporting this type of result may be therefore preferred until evidence showing correlation between intermediate copy number profiles and mosaicism in embryos is available (i.e., “analysis showing a pattern that is consistent with the potential presence of mosaicism”). Similarly, if future nonselection studies and randomized controlled trials show a lower reproductive potential for blastocyst with intermediate copy number profiles, clinically validated criteria should be introduced into clinical practice. In this case, on top of their genetic diagnosis, additional viability scores could be assigned to embryos. Nonetheless, extensive genetic counseling detailing all the specific limitations should be given to patients.

      Concluding remarks and future perspectives

      The small amount of evidence gathered from animal and human studies suggests the absence of mechanisms involved in embryo arrest, clonal depletion, or aneuploidy rescue for abnormal cells in euploid/aneuploid embryos during preimplantation development and blastocyst differentiation. Thus, it is likely that abnormal cells in mosaic diploid/aneuploid blastocysts are randomly allocated to the ICM and TE. However, our understanding of human preimplantation development is limited due to ethical and legal restrictions on embryo research and scarcity of materials. Studies in humans are mainly descriptive and lack functional evidence. Most of the information on embryo development is inferred from animal models and embryonic stem cell cultures, which should therefore be applied to the human model with caution. Understanding the biological mechanisms that beset preimplantation differentiation might reveal how aneuploidies and related gene dosage imbalances impact cell fate decisions and provide important clues on the biology of mosaicism in preimplantation embryos.
      The lack of correction for expected false positives and the use of nonstandardized criteria for classification of embryos as mosaic have likely resulted in an overestimation of mosaicism in IVF embryos. The little evidence available on humans suggests that mosaic euploid/aneuploid configuration is a relatively uncommon feature and should not impact the effectiveness of PGD-A programs appreciably. Further studies are needed to corroborate these findings and to better characterize at the single-cell level the incidence and prevalence of mosaicism at the blastocyst stage. Undoubtedly, novel CCS technologies able to distinguish between meiotic and mitotic errors in embryonic biopsies will deliver powerful research and clinical tools for the comprehensive analysis of aneuploidies in human blastocyst and consequent PGD-A protocols improvement.
      At the present, reports of mosaicism should be avoided in blastocyst PGD-A cycles due to the several technical and biological issues present when attempting this type of diagnosis and the lack of evidence from nonselection studies. The observation of intermediate chromosome copy number profiles would be better reported as a “pattern consistent with the presence of mosaicism,” and genetic counseling should be provided to guide patients' decisions.

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