Single Blastomeres Research Articles

Inhibition of Rho‑associated protein kinase increases the ratio of formation of blastocysts from single human blastomeres.

Y-27632 is a specific inhibitor of Rho-associated protein kinases (ROCKs), which are downstream effectors of Rho GTPase. The present study aimed to determine the effect of the specific ROCK inhibitor, Y-27632, on fresh human embryos and on single blastomeres obtained from discarded human embryos. A total of 784 poor-quality embryos were included, of which 526 were allocated to blastocyst culture directly and the remaining 258 were allocated to blastomere isolation. Embryos and single blastomeres were cultured either with, or without, Y-27632. Embryonic development was observed and recorded daily from day 5 onwards. Y-27632 did not affect the ratio of blastocyst formation or the quality of the human embryos. The duration of blastocyst formation was compared between the two groups in the embryo culture. On day 5, the blastocyst formation ratio in the experimental group was 11.4% (26/228), which was significantly (P=0.015) lower than the corresponding rate (19.7%; 44/223) in the control group. Survival analysis of the blastocyst formation duration showed that the median formation duration in the experimental group was significantly higher than that of the control group. The present study also obtained 1,192 blastomeres from 258 discarded day 3 embryos, and sibling blastomeres of similar sizes were equally allocated to experimental and control groups (n=596 in each). Treatment with Y-27632 increased the blastocyst formation ratio of human individual blastomeres, with 82 blastocysts of 596 blastomeres (13.8%), and 51 blastocysts of 596 blastomeres (8.6%) formed in the presence and absence of Y-27632, respectively (P=0.004). Compared with the control group, the mRNA and protein expression levels of E-cadherin in the blastocysts from blastomeres were enhanced by Y-27632 (P=0.022). In conclusion, the present study demonstrated that Y-27632 has different effects on the cleavage-stage of embryos and single blastomeres. Y-27632 increases the ratio of formation of blastocysts from single human blastomeres, but inhibits the direct formation of blastocysts from discarded human embryos.

Single-Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16-Cell Frog (Xenopus) Embryo.

We advance mass spectrometry from a cell population‐averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh‐resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE‐μESI‐HRMS enabled the identification of 500–800 nonredundant protein groups by measuring 20 ng, or <0.2% of the total protein content in single blastomeres that were isolated from the 16‐cell frog (Xenopus laevis) embryo, amounting to a total of 1709 protein groups identified between n=3 biological replicates. By quantifying ≈150 nonredundant protein groups between all blastomeres and replicate measurements, we found significant translational cell heterogeneity along multiple axes of the embryo at this very early stage of development when the transcriptional program of the embryo has yet to begin.

Single‐Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16‐Cell Frog (Xenopus) Embryo

AbstractWe advance mass spectrometry from a cell population‐averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh‐resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE‐μESI‐HRMS enabled the identification of 500–800 nonredundant protein groups by measuring 20 ng, or &lt;0.2% of the total protein content in single blastomeres that were isolated from the 16‐cell frog (Xenopus laevis) embryo, amounting to a total of 1709 protein groups identified between n=3 biological replicates. By quantifying ≈150 nonredundant protein groups between all blastomeres and replicate measurements, we found significant translational cell heterogeneity along multiple axes of the embryo at this very early stage of development when the transcriptional program of the embryo has yet to begin.

Is oocyte meiotic spindle morphology associated with embryo ploidy? A prospective cohort study

To explore whether an association exists between oocyte meiotic spindle morphology visualized by polarized light microscopy at the time of intracytoplasmic sperm injection and the ploidy of the resulting embryo. Prospective cohort study. Private IVF clinic. Patients undergoing preimplantation genetic screening/diagnosis (n = 113 patients). Oocyte meiotic spindles were assessed by polarized light microscopy and classified at the time of intracytoplasmic sperm injection as normal, dysmorphic, translucent, telophase, or no visible spindle. Single blastomere biopsy was performed on day 3 of culture for analysis by array comparative genomic hybridization. Spindle morphology and embryo ploidy association was evaluated by regression methods accounting for non-independence of data. The frequency of euploidy in embryos derived from oocytes with normal spindle morphology was significantly higher than all other spindle classifications combined (odds ratio [OR] 1.93, 95% confidence interval [CI] 1.33-2.79). Oocytes with translucent (OR 0.25, 95% CI 0.13-0.46) and no visible spindle morphology (OR 0.35, 95% CI 0.19-0.63) were significantly less likely to result in euploid embryos when compared with oocytes with normal spindle morphology. There was no significant difference between normal and dysmorphic spindle morphology (OR 0.73, 95% CI 0.49-1.08), whereas no telophase spindles resulted in euploid embryos (n = 11). Assessment of spindle morphology was found to be independently associated with embryo euploidy after controlling for embryo quality (OR 1.73, 95% CI 1.16-2.60). Oocyte spindle morphology is associated with the resulting embryo's ploidy. Oocytes with normal spindle morphology are significantly more likely to produce euploid embryos compared with oocytes with meiotic spindles that are translucent or not visible.

Cell-marking techniques for cell lineage tracing

A zygote, the only totipotent cell of the developing organism, transforms into a complex, multicellular entity with billions (for humans) of highly specialized cells and tissues. Most adult tissues are maintained by a combination of highly dynamic processes of senescence, apoptosis and rejuvenation with new cells constantly arising from dispersed depots of stem cells. Studying individual cell fates and their intertwined relations thus aids in understanding ontogenetic development as well as pathogenic processes in the body. Direct observations of developing embryos uncovered the fate of single blastomeres of ascidia and nematode. In both cases, research benefited from the simplicity of these objects, because, in ascidia, each blastomere has unique signatures naturally, and, in nematode, transparency of a worm’s 959-cell body allows every cell to be traced through development individually. In most cases, however, studying cellular lineages and identification of stem cells’ subpopulations are a true challenge for investigators. To trace the cell’s fate, novel methods were invented that introduce special tags into cells, the tags that would be inherited during cell divisions. Every descendent of a marked cell bears the same tag and can easily be distinguished from unrelated cellular neighbors. This review focuses on modern methods for cell tracing with dyes and genetic constructs encoding protein reporters that mark cell lineages. Special focus is on genome-integrated tags (genetic labeling), such as viral and cellular barcoding. One chapter of the review describes novel advancements in the field of CRISPR/Cas9-based cellular barcoding.

Retraction Note to: Aneuploidy analysis of non-pronuclear embryos from IVF with use of array CGH: a case report.

By using array comparative genomic hybridization (array CGH), to analyze the aneuploidy of the single blastomeres from non-pronuclear embryos on cleavage-stage in IVF cycle. Four non-pronuclear embryos were got from an IVF cycle, and the each single cell was biopsied from the four cleavage-stage embryos on the third day after the insemination which was investigated by using array CGH. After the biopsy, all the embryos continued to cleave, and lately entered the morula stage on the fifth day, just one embryo 3 was developed to early blastocyst stage on the sixth day. The four blastomere 24 chromosomes showed one X monomer and three normal XY diploids; the autosome chromosomes of blastomeres were abnormally gained or lost at different chromosome from four embryos, such as Embryo 1 : 49,X (−1, −5, −11, −19, −20, −21, −Y, +3, +6, +7, +8, +10, +13, +14, +16, +17, +18); Embryo 2 : 44,XY (−12, −15); Embryo 3: 47,XY (−3, −8, −9, −21, +7, +17, +18, +19, +20); Embryo 4 : 54,XY (+4, +7, +10, +12, +13, +16, +17, +22). With the use of the array CGH, the aneuploidy analysis could review the abnormal chromosomes of single blastomere from the non-pronuclear embryos, which can harbor the risk of abnormal sex chromosome and autosome chromosomes.

Does sperm DNA fragmentation affect the developmental potential and the incidence of apoptosis following blastomere biopsy?

Common methods employed in assisted reproduction technology (ART) include intracytoplasmic sperm injection (ICSI) with an unspecified level of sperm DNA fragmentation (SDF) and preimplantation genetic diagnosis (PGD). The aim of this study was to investigate the impact of SDF on human preimplantation embryo development and the incidence of apoptosis following a single blastomere biopsy. Using sperm chromatin dispersion (SCD) to assess SDF, a total of 20 processed semen samples were categorized into two groups; group I: SDF ≤30% and group II: SDF >30%. After ICSI, fertilization, cleavage, and embryo quality score were assessed. A single blastomere was biopsied from day 3 embryos and development was monitored on day 4. The frequency of apoptosis in biopsied embryos was assayed by TUNEL and the level of BCL-2, BAX, hsa-mir-15a, and hsa-mir-16-1 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). SCD was found to be negatively correlated with sperm motility and normal form spermatozoa (p < 0.05). The rate of fertilization, cleavage, and embryo quality score were not significantly different between the two groups (all p > 0.05). SDF >30% had no negative effect on potential development and did not increase the proportion of apoptotic cells and the level of apoptosis-related genes and microRNAs (miRNAs) in group II vs. group I (p > 0.05). It appears that at the levels assessed paternal genome damage had little if any negative effect on preimplantaton embryo development and apoptosis following single blastomere biopsy. This may reflect the selection of morphologically normal sperm for ICSI and the repair capacity of the oocyte.

Reproductive biology and medicine 2015--the year in review.

It would be an understatement to suggest that the discoveries in reproductive biology and medicine during 2015 were anything short of remarkable. And to rank these according to potential impact on our field would be a disservice to the groups that have persevered in tackling fundamental questions with the most sophisticated of techniques ensconced in the biomedical research enterprise of this decade. And so, with a measure of humility, a sense of awe, and the poetic license liberally taken by your EIC, a short summary of what might be considered a sentinel of what reproductive medicine may look like in the coming years is provided below. Progress in the area of germline stem cells continues to dominate a research landscape aiming to define the embryonic origins of primordial germ cells (PGCs) in molecular terms while at the same time nudging this line of research into the domain of treating infertility in men and women. That the horizon indeed holds the promise of making germline stem cells in vivo and in vitro is evidenced by the latest contributions from the laboratory of Surani and colleagues who have now mapped a molecular signature for the production of PGCs in humans (Tang et al., A Unique Gene Regulatory Network Resets the Human Germline Epigenome for Development. Cell. 2015 Jun 4;161(6):1453–67. doi: 10. 1016/j.cell.2015.04.053. http://www.ncbi.nlm.nih.gov/ pubmed/26046444). And the continuing quest for ways to obtain the best quality stem cells, which the field of regenerative medicine has been waiting for (as human ARTs sets the gold standards for quality embryo production), has taken an interesting twist of fate with a recent publication from the laboratory of Susan Fisher and her colleagues at UCSF (Zdavkovic et al., Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification. http://dev.biologists.org/lookup/ doi/10.1242/dev.122846). Taking single blastomeres from four 8-cell and one 12-cell embryos from a single individual lead to the derivation of ten ES cell lines, each of which displayed unique transcriptomic and protein heterogeneities, providing new insights into the allocation of cell lineages in the human embryo. Gene editing in human embryos captured the attention of many this past year. The power of CRISPR/Cas9 entered the arena of reproductive medicine with much fanfare as a result of the publication by Liang and colleagues proof-of-principle case using ART generated three PN zygotes (Liang et al., CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015 May;6(5):363–72. doi: 10.1007/ s13238-015-0153-5. Epub 2015 Apr 18. http://www.ncbi. nlm.nih.gov/pubmed/25894090). This work sparked renewed interest in prospects for genetic engineering but in tandem has brought the level of discourse to new heights in light of ethical and societal implications that are only now being subject to the degree of scrutiny they deserve. That RNAs of the many varieties now understood to have important physiological functions have been catching their fair share of attention at both the basic and clinical levels has come as no surprise. From the Kravetz and Diamond groups, this year came the exciting finding that certain spermassociated RNAs detected by next gen sequencing could be Capsule 2015 was a year of unprecedented discovery in the fields of reproductive biology and medicine. Here, we take note of ten publications that have drawn upon technological advances of the past decade to bring new ways of thinking about old problems into the realm of human ARTs and reproductive genetics.

Establishment of a novel HLA genotyping method for preimplantation genetic diagnonis using multiple displacement amplification-polymerase chain reaction-sequencing based technique

To establish a novel HLA genotyping method for preimplantation genetic diagnonis (PGD) using multiple displacement amplification-polymerase chain reaction-sequencing based technique (MDA-PCR-SBT). Peripheral blood samples and 76 1PN, 2PN, 3PN discarded embryos from 9 couples were collected. The alleles of HLA-A, B, DR loci were detected from the MDA product with the PCR-SBT method. The HLA genotypes of the parental peripheral blood samples were analyzed with the same protocol. The genotypes of specific HLA region were evaluated for distinguishing the segregation of haplotypes among the family members, and primary HLA matching was performed between the embryos. The 76 embryos were subjected to MDA and 74 (97.4%) were successfully amplified. For the 34 embryos from the single blastomere group, the amplification rate was 94.1%, and for the 40 embryos in the two blastomeres group, the rate was 100%. The dropout rates for DQ allele and DR allele were 1.3% and 0, respectively. The positive rate for MDA in the single blastomere group was 100%, with the dropout rates for DQ allele and DR allele being 1.5% and 0, respectively. The positive rate of MDA for the two blastomere group was 100%, with the dropout rates for both DQ and DR alleles being 0. The recombination rate of fetal HLA was 20.2% (30/148). Due to the improper classification and abnormal fertilized embryos, the proportion of matched embryos HLA was 20.3% (15/74),which was lower than the theoretical value of 25%. PGD with HLA matching can facilitate creation of a HLA-identical donor (saviour child) for umbilical cord blood or bone marrow stem cells for its affected sibling with a genetic disease. Therefore, preimplantation HLA matching may provide a tool for couples desiring to conceive a potential donor progeny for transplantation for its sibling with a life-threatening disorder.

Dynamic expression of chromatin modifiers during developmental transitions in mouse preimplantation embryos.

During mouse preimplantation development, major changes in cell fate are accompanied by extensive alterations of gene expression programs. Embryos first transition from a maternal to zygotic program and subsequently specify the pluripotent and the trophectodermal cell lineages. These processes are regulated by key transcription factors, likely in cooperation with chromatin modifiers that control histone and DNA methylation. To characterize the spatiotemporal expression of chromatin modifiers in relation to developmental transitions, we performed gene expression profiling of 156 genes in individual oocytes and single blastomeres of developing mouse embryos until the blastocyst stage. More than half of the chromatin modifiers displayed either maternal or zygotic expression. We also detected lineage-specific expression of several modifiers, including Ezh1, Prdm14, Scmh1 and Tet1 underscoring possible roles in cell fate decisions. Members of the SET-domain containing SMYD family showed differential gene expression during preimplantation development. We further observed co-expression of genes with opposing biochemical activities, such as histone methyltransferases and demethylases, suggesting the existence of a dynamic chromatin steady-state during preimplantation development.

Novel PRD-like homeodomain transcription factors and retrotransposon elements in early human development.

Transcriptional program that drives human preimplantation development is largely unknown. Here, by using single-cell RNA sequencing of 348 oocytes, zygotes and single blastomeres from 2- to 3-day-old embryos, we provide a detailed analysis of the human preimplantation transcriptome. By quantifying transcript far 5′-ends (TFEs), we include in our analysis transcripts that derive from alternative promoters. We show that 32 and 129 genes are transcribed during the transition from oocyte to four-cell stage and from four- to eight-cell stage, respectively. A number of identified transcripts originates from previously unannotated genes that include the PRD-like homeobox genes ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB and LEUTX. Employing de novo promoter motif extraction on sequences surrounding TFEs, we identify significantly enriched gene regulatory motifs that often overlap with Alu elements. Our high-resolution analysis of the human transcriptome during preimplantation development may have important implications on future studies of human pluripotent stem cells and cell reprograming.

Validation of next-generation sequencing for comprehensive chromosome screening of embryos

Massively parallel genome sequencing, also known as next-generation sequencing (NGS), is the latest approach for preimplantation genetic diagnosis. The purpose of this study was to determine whether NGS can accurately detect aneuploidy in human embryos. Low coverage genome sequencing was applied to trophectoderm biopsies of embryos at the blastocyst stage of development. Sensitivity and specificity of NGS was determined by comparison of results with a previously validated platform, array-comparative genomic hybridization (aCGH). In total, 156 samples (116 were blindly assessed) were tested: 40 samples were re-biopsies of blastocysts where the original biopsy specimen was previously tested for aCGH; four samples were re-biopsies of single blastomeres from embryos previously biopsied at the cleavage stage and tested using aCGH; 18 samples were single cells derived from well-characterized cell lines; 94 samples were whole-genome amplification products from embryo biopsies taken from previous preimplantation genetic screening cycles analysed using aCGH. Per embryo, NGS sensitivity was 100% (no false negatives), and 100% specificity (no false positives). Per chromosome, NGS concordance was 99.20%. With more improvement, NGS will allow the simultaneous diagnosis of single gene disorders and aneuploidy, and may have the potential to provide more detailed insight into other aspects of embryo viability.

Study on preimplantation genetic diagnosis and follow-up for Duchenne muscular dystrophy

Objective To carry out preimplantation genetic diagnosis (PGD) for Duchenne muscular dystrophy (DMD) carrier, so as to prevent the birth of affected infants with DMD. Methods One DMD gene carrier with a deletion of exon 10-30 received fertilization with intracytoplasmic sperm injection (ICSI). DMD gene and haplotype were tested after amplification of genome DNA in multiple displacement amplification (MDA), then healthy embryos were transferred to uterus according to the genetic results. Genetic testing was made in second trimester and after delivery, and also periodic follow-up was made for over 3 years. Results The second cycle of PGD was successful, and a total of 14 single blastomeres obtained from 7 embryos were used for genetic analysis. The success rate of MDA was 13/14, and the allele dropout rate was 18.75% (18/96). Three unaffected embryos were transferred, resulting in twin pregnancy. One healthy boy and one healthy girl were born in cesarean section at the pregnant week of 35. Genetic results on DNA from both amniotic fluid at 16 weeks of gestation and peripheral blood after birth were normal. During the 3-year follow-up, both 2 infants were normal in growth and development, motor function and dynamic monitor of serum creatine kinase (CK). Conclusions Preimplantation genetic diagnosis can help DMD gene carrier give birth to healthy infants, and these infants have normal development. DOI: 10.3969/j.issn.1672-6731.2015.06.008

Molecular asymmetry in the 8-cell stage Xenopus tropicalis embryo described by single blastomere transcript sequencing

Correct development of the vertebrate body plan requires the early definition of two asymmetric, perpendicular axes. The first axis is established during oocyte maturation, and the second is established by symmetry breaking shortly after fertilization. The physical processes generating the second asymmetric, or dorsal–ventral, axis are well understood, but the specific molecular determinants, presumed to be maternal gene products, are poorly characterized. Whilst enrichment of maternal mRNAs at the animal and vegetal poles in both the oocyte and the early embryo has been studied, little is known about the distribution of maternal mRNAs along either the dorsal–ventral or left–right axes during the early cleavage stages. Here we report an unbiased analysis of the distribution of maternal mRNA on all axes of the Xenopus tropicalis 8-cell stage embryo, based on sequencing of single blastomeres whose positions within the embryo are known. Analysis of pooled data from complete sets of blastomeres from four embryos has identified 908 mRNAs enriched in either the animal or vegetal blastomeres, of which 793 are not previously reported as enriched. In contrast, we find no evidence for asymmetric distribution along either the dorsal–ventral or left–right axes. We confirm that animal pole enrichment is on average distinctly lower than vegetal pole enrichment, and that considerable variation is found between reported enrichment levels in different studies. We use publicly available data to show that there is a significant association between genes with human disease annotation and enrichment at the animal pole. Mutations in the human ortholog of the most animally enriched novel gene, Slc35d1, are causative for Schneckenbecken dysplasia, and we show that a similar phenotype is produced by depletion of the orthologous protein in Xenopus embryos.

Comprehensive preimplantation genetic screening and sperm deoxyribonucleic acid fragmentation from three males carrying balanced chromosome rearrangements

To assess whether preimplantation genetic screening can successfully identify cytogenetically normal embryos in couples carrying balanced chromosome rearrangements in addition to increased sperm DNA fragmentation. Comprehensive preimplantation genetic screening was performed on three couples carrying chromosome rearrangements. Sperm DNA fragmentation was assessed for each patient. Academic center. One couple with the male partner carrying a chromosome 2 pericentric inversion and two couples with the male partners carrying a Robertsonian translocation (13:14 and 14:21, respectively). A single blastomere from each of the 18 cleavage-stage embryos obtained was analysed by metaphase comparative genomic hybridization. Single- and double-strand sperm DNA fragmentation was determined by the alkaline and neutral Comet assays. Single- and double-strand sperm DNA fragmentation values and incidence of chromosome imbalances in the blastomeres were analyzed. The obtained values of single-strand sperm DNA fragmentation were between 47% and 59%, and the double-strand spermDNA fragmentation values were between 43% and 54%. No euploid embryos were observed in the couple showing the highest single-strand sperm DNA fragmentation. However, euploid embryos were observed in the other two couples: embryo transfer was performed, and pregnancy was achieved by the couple showing the lowest sperm DNA fragmentation values. Preimplantation genetic screening enables the detection of euploid embryos in couples affected by balanced chromosome rearrangements and increased sperm DNA fragmentation. Even though sperm DNA fragmentation may potentially have clinical consequences on fertility, comprehensive preimplantation genetic screening allows for the identification and transfer of euploid embryos.

Blastocoel fluid from differentiated blastocysts harbors embryonic genomic material capable of a whole-genome deoxyribonucleic acid amplification and comprehensive chromosome microarray analysis

To obtain embryonic molecular karyotypes from genomic DNA (deoxyribonucleic acid) isolated from blastocoel fluid (BF) and to compare these karyotypes with the karyotypes from the remaining inner cell mass (ICM) and trophectoderm (TE) of the blastocyst. Prospective cohort study. Academic center and preimplantation genetics laboratory. Ninety-six donated cryopreserved embryos. Embryo biopsy, BF aspiration, DNA analysis using a comparative genomic hybridization microarray (aCGH). The aCGH of a single blastomere, BF-DNA, and ICM-TE. The BF-DNA samples resulted in a successful aCGH in 63% of cases. Discordance in karyotypes was found between the BF-DNA and the ICM-TE in 52% of cases. A total of 70% of aneusomic (mosaicism), cleavage-stage embryos differentiated into euploid blastocysts. Probabilities for diagnostic accuracy were calculated and demonstrated the following: sensitivity of 0.88 (95% confidence interval [CI]: 0.62-0.98); specificity of 0.55 (95% CI: 0.39-0.70); positive predictive value of 0.41 (95% CI: 0.25-0.60); negative predictive value of 0.92 (95% CI: 0.75-0.99). Genomic DNA from the BF can be amplified and characterized by comprehensive chromosome microarrays. The results demonstrated that aneusomic cleavage-stage embryos differentiated into euploid blastocysts, possibly using a mechanism that marginalizes aneuploid nuclei into the blastocoel cavity. In addition, owing to the high discordance between the karyotypes obtained from the BF-DNA and the ICM-TE, using BF-DNA for preimplantation genetic testing is not yet advised.

Embryo genome profiling by single-cell sequencing for preimplantation genetic diagnosis in a β-thalassemia family.

The embryonic genome, including genotypes and haplotypes, contains all the information for preimplantation genetic diagnosis, representing great potential for mendelian disorder carriers to conceive healthy babies. We developed a strategy to obtain the full embryonic genome for a β-thalassemia-carrier couple to have a healthy second baby. We carried out sequencing for single blastomere cells and the family trio and further developed the analysis pipeline, including recovery of the missing alleles, removal of the majority of errors, and phasing of the embryonic genome. The final accuracy for homozygous and heterozygous single-nucleotide polymorphisms reached 99.62% and 98.39%, respectively. The aneuploidies of embryos were detected as well. Based on the comprehensive embryonic genome, we effectively performed whole-genome mendelian disorder diagnosis and human leukocyte antigen matching tests. This retrospective study in a β-thalassemia family demonstrates a method for embryo genome recovery through single-cell sequencing, which permits detection of genetic variations in preimplantation genetic diagnosis. It shows the potential of single-cell sequencing technology in preimplantation genetic diagnosis clinical practices.

Cytoplasmic localization and asymmetric division in the early embryo of Caenorhabditis elegans.

During the initial cleavages of the Caenorhabditis elegans embryo, a series of rapid and invariant asymmetric cell divisions pattern the fate, size, and position of four somatic blastomeres and a single germline blastomere. These asymmetric divisions are orchestrated by a collection of maternally deposited factors that are initially symmetrically distributed in the newly fertilized embryo. Maturation of the sperm-derived centrosome in the posterior cytoplasm breaks this symmetry by triggering a dramatic and highly stereotyped partitioning of these maternal factors. A network of conserved cell polarity regulators, the PAR proteins, form distinct anterior and posterior domains at the cell cortex. From these domains, the PAR proteins direct the segregation of somatic and germline factors into opposing regions of the cytoplasm such that, upon cell division, they are preferentially inherited by the somatic blastomere or the germline blastomere, respectively. The segregation of these factors is controlled, at least in part, by a series of reaction-diffusion mechanisms that are asymmetrically deployed along the anterior/posterior axis. The characterization of these mechanisms has important implications for our understanding of how cells are polarized and how spatial organization is generated in the cytoplasm. For further resources related to this article, please visit the WIREs website.

In vitro Development of Chimeric Bovine Embryos Created by Combining a Single Blastomere of SCNT Embryos with an IVF Embryo

In this study, we produced chimeric embryos from a single blastomere of somatic cell nuclear transfer (SCNT) bovine embryo and in-vitro fertilized (IVF) bovine embryo, and examined the rate of blastocyst development and the contribution of the SCNT blastomere to the blastocyst in order to improve cloning efficiency. We produced SCNT embryos from bovine fibroblasts carrying a luciferase gene under the control of the β-actin promoter. At 96 hours post fusion, chimeric embryos were produced by transferring a single blastomere of a 16-cell SCNT embryo to the perivitelline space of an IVF embryo. At 4 days after production of the chimeric embryos, half of the embryos reached the blastocyst stage, which is the same as that of IVF embryos. Furthermore, luciferase activity in blastocysts from the chimeric embryos was detected in both the inner cell mass and

Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification.

Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active β-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines.