Regional and developmental characteristics of human embryo mosaicism revealed by single cell sequencing

Chromosomal mosaicism is common throughout human pre- and post-implantation development. However, the incidence and characteristics of mosaicism in human blastocyst remain unclear. Concerns and confusions still exist regarding the interpretation of chromosomal mosaicism on preimplantation genetic testing for aneuploidy (PGT-A) results and embryo development. Here, we aimed to estimate the genetic concordance between trophectoderm (TE), inner cell mass (ICM) and the corresponding human embryonic stem cells (hESCs), and to explore the characteristics of mosaicism in human blastocyst and hESCs on a single cell level. The single cell sequencing results of TE cells indicated that 65.71% of the blastocysts were mosaic (23 in 35 embryos), while the ICM sequencing results suggested that 60.00% of the blastocysts were mosaic (9 in 15 embryos). The incidence of mosaicism for the corresponding hESCs was 33.33% (2 in 6 embryos). No significant difference was observed between the mosaic rate of TE and that of ICM. However, the mosaic rate of the corresponding hESCs was significantly lower than that of TE and ICM cells, suggesting that the incidence of mosaicism may decline during embryonic development. Upon single cell sequencing, we found several “complementary” copy number variations (CNVs) that were usually not revealed in clinical PGT-A which used multi-cell DNA sequencing (or array analysis). This indicates the potential diagnostic risk of PGT-A based multi-cell analysis routinely in clinical practice. This study provided new insights into the characteristics, and considerable influences, of mosaicism on human embryo development, as well as the clinical risks of PGT-A based on multi-cell biopsies and bulk DNA assays.     

Tissue-specific placental mosaicism for autosomal trisomies in spontaneous human abortuses: mechanisms of formation and phenotypic effects]

The frequencies of autosomal trisomies in extraembryonic human tissues were estimated in the cases of different abnormalities of prenatal development, from the confined placental mosaicism (CPM) with either relatively normal embryogenesis or restricted intrauterine growth to spontaneous abortion. A tissue-specific compartmentalization was found to be characteristic of cell lines with trisomies for individual autosomes. Analysis of various phenotypical effects of chromosomal aberrations associated with mosaicism is necessarily required to understand the mechanisms and factors responsible for tissue chromosomal mosaicism. Based on analysis of the cell karyotype during prenatal diagnosing of chromosome aberrations in tissues of both extraembryonic and embryonic origin, in 1996, Wolstenholme proposed a model of CPM for individual chromosomes. According to the model, the distribution of cell lines with autosomal trisomies between extraembryonic tissues depends on the ratio between meiotic and mitotic mutations early in embryonic development. However, the model cannot be used to study tissue chromosomal mosaicism in spontaneous abortions, because little information is available on cell karyotype in embryonic tissues themselves after intrauterine fetal death. In this work, a model of tissue-specific chromosomal mosaicism was suggested based on the data on cell karyotype determined in extraembryonic tissues alone, which can be helpful in evaluating the contribution of tissue chromosomal differences into the etiology of early intrauterine death. Along with the experimental evidence, comparative analysis of the two models indicated that the meiotic chromosome nondisjunction plays the major role in trisomy formation and the resultant spontaneous arrest of embryonic development. Other factors responsible for tissue-specific distribution of chromosomal aberrations are also discussed. These are differences in cell proliferative activity, as well as changes in compartmentalization and migration of cells with abnormal karyotypes.     

Tissue-Specific Placental Mosaicism for Autosomal Trisomies in Human Spontaneous Abortuses: Mechanisms of Formation and Phenotypical Effects

The frequencies of autosomal trisomies in extraembryonic human tissues were estimated in the cases of different abnormalities of prenatal development, from the confined placental mosaicism (CPM) with either relatively normal embryogenesis or restricted intrauterine growth to spontaneous abortion. A tissue-specific compartmentalization was found to be characteristic of cell lines with trisomies for individual autosomes. Analysis of various phenotypical effects of chromosomal aberrations associated with mosaicism is necessarily required to understand the mechanisms and factors responsible for tissue chromosomal mosaicism. Based on analysis of the cell karyotype during prenatal diagnosing of chromosome aberrations in tissues of both extraembryonic and embryonic origin, in 1996, Wolstenholme proposed a model of CPM for individual chromosomes. According to the model, the distribution of cell lines with autosomal trisomies between extraembryonic tissues depends on the ratio between meiotic and mitotic mutations early in embryonic development. However, the model cannot be used to study tissue chromosomal mosaicism in spontaneous abortions, because little information is available on cell karyotype in embryonic tissues themselves after intrauterine fetal death. In this work, a model of tissue-specific chromosomal mosaicism was suggested based on the data on cell karyotype determined in extraembryonic tissues alone, which can be helpful in evaluating the contribution of tissue chromosomal differences into the etiology of early intrauterine death. Along with the experimental evidence, comparative analysis of the two models indicated that the meiotic chromosome nondisjunction plays the major role in trisomy formation and the resultant spontaneous arrest of embryonic development. Other factors responsible for tissue-specific distribution of chromosomal aberrations are also discussed. These are differences in cell proliferative activity, as well as changes in compartmentalization and migration of cells with abnormal karyotypes.     

Abnormal skin fibroblast cytogenetics in four dysmorphic patients with normal lymphocyte chromosomes.

Four patients with features suggestive of chromosome disorders but with normal lymphocyte karyotypes were found to have chromosome aberrations in skin fibroblast karyotypes. Although mosaicism for chromosome abnormalities in lymphocyte cultures is common, apparent restriction of mosaicism to one tissue is unusual. We suggest that after examination of lymphocyte karyotypes, certain patients warrant cytogenetic evaluation of a second tissue, usually cultured skin fibroblasts.     

Interphase cytogenetics on paraffin-embedded sections of ovary for detection of genomic constitution in a patient with Turner's syndrome and chromosomal mosaicism

The difficulty of detecting sex chromosome mosaicism cytogenetically hinders the finding of an acceptable explanation for phenotypic-genotypic discrepancy amongst those patients. Fluorescence in situ hybridization (FISH) permits the genomic identification of patients with mosaic karyotypes in interphase nuclei by utilising an X chromosome-specific DNA probe (interphase cytogenetics). We evaluated the efficiency of interphase cytogenetics in the detection of the genomic constitution of the ovary from a patient with Turner's syndrome having mosaicism (46,XX/45,X0) previously established by blood lymphocyte karyotyping. We used a biotin-labelled alphoid repetitive sequence, pBAMX5, specific for the centromeric region of the human X chromosome. Although examination of ovarian sections and blood lymphocytes by FISH showed the presence of both 46,XX and 45,X0 cell lines, the genomic constitution of the germ cells/oocytes in ovarian primordial follicles was shown to be normal (46,XX). Our results (1) show the high applicability of interphase cytogenetics on paraffin sections, (2) indicate the possibility of genomic screening of different tissues that are otherwise not amenable to routine cytogenetic investigation and (3) offer a reliable methodological approach to defining accurate by the percentage of abnormal karyotypes in mosaicism of different organs and non-dividing tissues.     

由21三体综合征的核型引起的思考

不同的医学遗传学教材上对21三体综合征的概念及核型说法不一, 作者在查阅了大量教材的基础上, 对21三体综合征的概念及核型等问题进行了认真的思考, 并提出了自己的观点, 认为21三体综合征的核型应该为两种类型: 即三体型和嵌合型, 而先天愚型的核型应该为3种类型: 即三体型、嵌合型和易位型。     

Mechanisms leading to uniparental disomy and their clinical consequences

Uniparental disomy (UPD) refers to the situation in which both copies of a chromosome pair have originated from one parent. In humans, it can result in clinical conditions by producing either homozygosity for recessive mutations or aberrant patterns of imprinting. Furthermore, UPD is frequently found in conjunction with mosaicism for a chromosomally abnormal cell line, which can also contribute to phenotypic abnormalities. Investigations into the mechanisms by which UPD may arise have helped to expand our general awareness of the impact of chromosomal abnormalities and chromosomal mosaicism in normal human development. Specifically, it appears that errors in the transmission of a chromosome from parent to gamete and during early somatic cell divisions are remarkably common but that embryo and cell selection during early embryogenesis help to ensure the presence of a numerically balanced chromosome complement in the developing fetus. UPD is also likely to occur within a portion of cells in all individuals simply as a consequence of somatic recombination occurring during mitotic cell divisions. This can be an important step in cancer development as well as a contributing factor to other late onset diseases. This review summarizes mechanisms by which UPD may arise and their associated clinical consequences.     

Experimental evidence for the origins of determinative development in the polyclad turbellarians

Cell-deletion experiments were carried out on the embryo of the polyclad turbellarian Hoploplana inquilina to examine further the nature of development in primitive spiralians. The polyclads are of particular interest because they provide a link between the regulative development of acoels and the determinative development of annelids and molluscs. Single blastomeres were deleted at the two- and four-cell stages by puncture through the eggshell membrane with tungsten needles. All deletions resulted in abnormal larvae with consistent characteristics representing half or three-quarter Müller's larvae. The number of larval eyes was a particularly useful character in revealing mosaicism. This study establishes the polyclad embryo as determinative, but with important cell interactions also occurring during early development, and provides evidence that mosaicism became associated with spiral cleavage in the quartet form during the evolution of the Turbellaria.     

Molecular analysis in true hermaphroditism: demonstration of low-level hidden mosaicism for Y-derived sequences in 46,XX cases

True hermaphroditism (TH) is an unusual form of sex reversal, characterized by the development of testicular and ovarian tissue in the same subject. Approximately 60% of the patients have a 46,XX karyotype, 33% are mosaics with a second cell line containing a Y chromosome, while the remaining 7% are 46,XY. Molecular analyses have demonstrated that SRY is present in only 10% of TH with a 46,XX karyotype; therefore, in the remaining 90%, mutations at unknown X-linked or autosomal sex determining loci have been proposed as factors responsible for testicular development. True hermaphroditism presents considerable genetic heterogeneity with several molecular anomalies leading to the dual gonadal development as SRY point mutations or SRY hidden gonadal mosaicism. In order to identify genetic defects associated with subjects with the disease, we performed molecular analyses of the SRY gene in DNA from blood leukocytes and gonadal tissue in 12 true hermaphrodites with different karyotypes. Our results using PCR and FISH analyses reveal the presence of hidden mosaicism for SRY or other Y sequences in some patients with XX true hermaphroditism and confirms that mosaicism for SRY limited to the gonads is an alternative mechanism for testicular development in 46,XX true hermaphrodites.     

Gonosomale Mosaike

With an incidence of approximately 0.2?% in newborns gonosomal chromosome aberrations are of major relevance for clinical genetics. They frequently occur as numerical and/or structural gonosomal mosaicism. The correlation between genotype and phenotype is poor most probably due to different levels of mosaicism in different tissues and they represent a great challenge especially in prenatal diagnostics, requiring genetic counseling by an experienced clinical geneticist. Postnatally, gonosomal mosaicism in females often leads to the clinical symptoms of Turner’s syndrome (especially short stature and infertility) with an potentially increased risk for gonadoblastoma if an XY cell line is present. In males with Klinefelter’s syndrome mosaicism is also frequent (up to 20?%). Mosaicism in 47,XYY and 47,XXX karyotypes is rarely reported perhaps due to the innocuous phenotype seldom being an indication for chromosome analysis.     

Somatic mosaicism in cases with small supernumerary marker chromosomes

Somatic mosaicism is something that is observed in everyday lives of cytogeneticists. Chromosome instability is one of the leading causes of large-scale genome variation analyzable since the correct human chromosome number was established in 1956. Somatic mosaicism is also a well-known fact to be present in cases with small supernumerary marker chromosomes (sSMC), i.e. karyotypes of 47,+mar/46. In this study, the data available in the literature were collected concerning the frequency mosaicism in different subgroups of patients with sSMC. Of 3124 cases with sSMC 1626 (52%) present with somatic mosaicism. Some groups like patients with Emanuel-, cat-eye- or i(18p)- syndrome only tend rarely to develop mosaicism, while in Pallister-Killian syndrome every patient is mosaic. In general, acrocentric and non-acrocentric derived sSMCs are differently susceptible to mosaicism; non-acrocentric derived ones are hereby the less stable ones. Even though, in the overwhelming majority of the cases, somatic mosaicism does not have any detectable clinical effects, there are rare cases with altered clinical outcomes due to mosaicism. This is extremely important for prenatal genetic counseling. Overall, as mosaicism is something to be considered in at least every second sSMC case, array-CGH studies cannot be offered as a screening test to reliably detect this kind of chromosomal aberration, as low level mosaic cases and cryptic mosaics are missed by that.     

Localized chromosomal mosaicism as a cause of dysmorphic development

Regional chromosomal mosaicism has been found in tissue from the clitoral mass of an infant presenting with ambiguous genitalia. Chromosome analysis of skin from the clitoral mass was interpreted as 46,XX/52,XX, +2, +7, +8, +12, +13, +20, whereas study of ovarian tissue and peripheral lymphocytes found each to have normal 46,XX karyotypes with no indication of mosaicism. We believe that somatic cell mutation led to a hyperdiploid clonal cell line resulting in maldevelopment of this patient's external genitalia. This observation, which to our knowledge has not been previously reported, indicates that localized chromosomal abnormalities in some cases may be etiologic of isolated congenital malformations.     

The fate of mosaic aneuploid embryos during mouse development

More than any other species, humans have difficulty reproducing. As recent studies show that human infertility is ever increasing, much efforts are needed towards the understanding of our low fecundity. While aneuploidy is the leading cause of spontaneous pregnancy loss in humans, we still know surprisingly little about the developmental consequences of chromosomal abnormalities. We have here used a mouse model that spontaneously incites chromosomal primary aneuploidy in female haploid oocytes and find that after fertilization, these primary aneuploid cells become cytological unstable, generating diverse karyotypic mosaic embryos. The mosaic aneuploid embryos can develop and implant into the female uterine tissue and initiate the gastrulation process (E6.5) but quickly degrade and succumb by E8.0. We find that loss of embryo viability due to chromosomal mosaicism is caused by the activation of a spatially and temporally controlled p53-independent apoptotic mechanism and does not result from a failure to progress through mitosis. We conclude that an initial state of primary aneuploidy within an embryo results in a rapid evolution of mosaicism and early embryonic death. This gestational loss due to aneuploid mosaicism could account for the large proportion of human pregnancy loss prior to clinical recognition.     

Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in?~?50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.     

The human genome puzzle - the role of copy number variation in somatic mosaicism

The discovery of copy number variations (CNV) in the human genome opened new perspectives in the study of the genetic causes of inherited disorders and the etiology of common diseases. Differently patterned instances of somatic mosaicism in CNV regions have been shown to be present in monozygotic twins and throughout different tissues within an individual. A single-cell-level investigation of CNV in different human cell types led us to uncover mitotically derived genomic mosaicism, which is stable in different cell types of one individual. A unique study of immortalized B-lymphoblastoid cell lines obtained with 20 year interval from the same two subjects shows that mitotic changes in CNV regions may happen early during embryonic development and seem to occur only once, as levels of mosaicism remained stable. This finding has the potential to change our concept of dynamic human genome variation. We propose that further genomic studies should focus on the single-cell level, to understand better the etiology and physiology of aging and diseases mediated by somatic variations.     

Aneuploidy in the human cleavage stage embryo

The cleavage stage embryo (days 1-3) stands out due to the high level of chromosomal anomalies, especially mosaicism that arises prior to global embryonic genome activation. Molecular cytogenetic studies show that an average of 60% of in vitro derived embryos have at least one aneuploid cell by the time they are 3 days old. However, comprehensive studies of the chromosome content of individual cells have revealed that 25% of these embryos have no aneuploid cells, a fact that sits well with the knowledge that at most 1 in 5 have the capacity to implant. The evidence is that extensive mosaicism, affecting several chromosomes, interferes with development to a greater extent than does uniform aneuploidy. Follow-up studies on embryos after pre-implantation genetic aneuploidy screening indicate that the frequency of meiotic errors varies according to the referral reason, with the highest frequency being recorded for the recurrent miscarriage category and the lowest in the repeated implantation failure group where younger women have a good response to ovarian stimulation. The exceptionally high incidence of pre- and post-zygotic chromosomal anomalies seen in early human embryos is thus the product of several mechanisms. Firstly, the error-prone cell cycle during the embryonic cleavage stage and secondly, parental susceptibility to meiotic and mitotic chromosomal instability together with their general genetic background.     

A case of trisomy 12 mosaicism with pituitary malformation and polycystic ovary syndrome

We report the case of a patient (followed from birth to 15 years) presenting with trisomy 12 mosaicism, and focus on the endocrine phenotype associating a pituitary malformation and ovarian abnormalities. We describe the dysmorphic features and their evolution, the growth retardation and ovarian symptoms. Complete growth hormone deficiency was confirmed on auxological data, stimulation test and was related to pituitary stalk interruption, diagnosed by magnetic resonance imaging. Effect of growth hormone treatment was satisfactory resulting in a normal adult height. She also presented premature thelarche associated with right ovarian hypertrophy (4 to 5 fold the volume of the left ovary) which remained constant until 15 years of age. Diagnosis of trisomy 12 mosaicism was made on skin and ovarian karyotypes. The possible relation between these endocine findings and some genes located on chromosome 12 involved in pituitary and ovarian development is discussed.     

Value of molecular-genetic examination of patients with the Shereshevsky-Terner syndrome and married couples with disorders of reproductive function]

The molecular-cytogenetic investigation gives a significant opportunity for diagnosis of gonosome mosaics. Due to the molecular-cytogenetic method additional chromosomes in karyotypes in 4 of 13 patients are determined. A significant spreading of mosaicism type 45, XO/46, X among women with early spontaneous abortions is revealed.     

Mosaicism in the mouse trophectoderm

The issue of mosaicism in the mouse trophectoderm is examined by reviewing two sets of evidence: one arguing for a mosaic, the other for a non-mosaic character. Evidence for mosaicism includes documented cellular contribution from the inner cell mass to the trophectoderm, and data that reveal the gradual pace of the allocation process that separates the inner cell mass and trophectoderm lineages. Evidence suggesting a non-mosaic character for the trophectoderm is based on the polarization process undergone by exterior cells in the eight-celled embryo, the heritability of the changes brought about by this process, and the formation of gap junctions between the resulting apolar, trophectoderm progenitor cells. Since inner-cell-mass cells are developmentally labile, spatially heterogeneous and translocate to the polar trophectoderm, it is concluded that the polar trophectoderm is a mosaic tissue.