8-cell Human Embryos Research Articles

Microarray evidence that 8-cell human embryos express some hormone family members including oxytocin.

This study is to discover hormone pathways active in early cleaving human embryos. A list of 152 hormones and receptors were compiled to query the microarray database of mRNAs in 8-cell human embryos, two lines of human embryonic stem cells plus human fibroblasts before and after induced pluripotency. Over half of the 152 hormones and receptors were silent on the arrays of all cell types, and more were detected at high or moderate levels on the 8-cell arrays than on the pluripotent cell or fibroblast arrays. Eight hormone family genes were uniquely detected at least 22-fold higher on the 8-cell arrays than the stem cell arrays: AVPI1, CCK, CORT, FSTL4, GIP, GPHA2, OXT, and PPY suggesting novel roles for these proteins in early development. Oxytocin was detected by pilot immunoassay in culture media collected from Day 3 embryos. Robust detection of CRHR1 and EPOR suggests the 8-cell embryo may be responsive to maternal CRH and EPO. The over-expression of POMC and GHITM suggests POMP peptide products may have undiscovered roles in early development and GHITM may contribute to mitochondrial remodeling. Under-detected on the 8-cell arrays at least tenfold were two key enzymes in steroid biosynthesis, DHCR24 and FDPS. The 8-cell human embryo may be secreting oxytocin, which could stimulate its own progress down the fallopian tube as well as play a role in early neural precursor development. The 8-cell embryo does not synthesize reproductive steroid hormones. As previously reported for growth factor families, the early embryo over-expresses more hormones than hormone receptors.

Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor.

Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.

8C-like cells capture the human zygotic genome activation program invitro.

SummaryThe activation of the embryonic genome marks the first major wave of transcription in the developing organism. Zygotic genome activation (ZGA) in mouse 2-cell embryos and 8-cell embryos in humans is crucial for development. Here, we report the discovery of human 8-cell-like cells (8CLCs) among naive embryonic stem cells, which transcriptionally resemble the 8-cell human embryo. They express ZGA markers, including ZSCAN4 and LEUTX, and transposable elements, such as HERVL and MLT2A1. 8CLCs show reduced SOX2 levels and can be identified using TPRX1 and H3.Y marker proteins in vitro. Overexpression of the transcription factor DUX4 and spliceosome inhibition increase human ZGA-like transcription. Excitingly, the 8CLC markers TPRX1 and H3.Y are also expressed in ZGA-stage 8-cell human embryos and may thus be relevant in vivo. 8CLCs provide a unique opportunity to characterize human ZGA-like transcription and might provide critical insights into early events in embryogenesis in humans.

Effects of three-area laser-assisted zona thinning in 8-cell human embryos on pregnancy outcomes in vitro fertilization.

ObjectiveThis study conducted a preliminary examination of the effects of three-area laser-assisted zona thinning (LAZT) during the cleavage stage of embryo development on the hatching process in human in vitro fertilization-embryo transfer (IVF-ET) with subjects of advanced female age or frozen-thawed (FT) embryos.MethodsEight-cell stage embryos were treated with LAZT in three areas of the zona pellucida at 120° intervals. The control group was embryos without LAZT. Of the 72 consecutive fresh cycles and the 28 FT embryo transfer cycles, the patients in 55 fresh cycles and 17 FT cycles declined LAZT, and those cycles were defined as the control group.ResultsIn the fresh cycles, the pregnancy rates were similar in the LAZT and control groups. However, in the FT cycles, the pregnancy rate was significantly higher in the LAZT group than in the control group (45.5% in the LAZT group vs. 23.5% in the control group, p<0.05).ConclusionThese results show that multi-area LAZT resulted in significantly improved pregnancy outcomes in human 8-cell embryos compared to controls.

Cleavage of Human Embryos: Options and Diversity

In order to estimate the diversity of embryo cleavage relatives to embryo progress (blastocyst formation), time-lapse imaging data of preimplantation human embryo development were used. This retrospective study is focused on the topographic features and time parameters of the cleavages, with particular emphasis on the lengths of cleavage cycles and the genealogy of blastomeres in 2- to 8-cell human embryos. We have found that all 4-cell human embryos have four developmental variants that are based on the sequence of appearance and orientation of cleavage planes during embryo cleavage from 2 to 4 blastomeres. Each variant of cleavage shows a strong correlation with further developmental dynamics of the embryos (different cleavage cycle characteristics as well as lengths of blastomere cycles). An analysis of the sequence of human blastomere divisions allowed us to postulate that the effects of zygotic determinants are eliminated as a result of cleavage, and that, thereafter, blastomeres acquire the ability of own syntheses, regulation, polarization, formation of functional contacts, and, finally, of specific differentiation. This data on the early development of human embryos obtained using noninvasive methods complements and extend our understanding of the embryogenesis of eutherian mammals and may be applied in the practice of reproductive technologies.

Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells.

Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that it is an independent circadian rhythm-competent equivalence group poised to signal its environment, defend against maternal immune rejection, and begin the rapid commitment events of early embryogenesis.

Functional Genomics of 5- to 8-Cell Stage Human Embryos by Blastomere Single-Cell cDNA Analysis

Blastomere fate and embryonic genome activation (EGA) during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM) (n = 120), stemness (n = 190) and Trophectoderm (TE) (n = 45), were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1), stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT), and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR). The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented.

Defining cell fate and embryonic genome activation by global single-cell cDNA analysis of blastomeres from 5 to 8-cell human embryos

Defining cell fate and embryonic genome activation by global single-cell cDNA analysis of blastomeres from 5 to 8-cell human embryos

Totipotent 8Cell human embryos may be naturally susceptible to aneuploidy

OBJECTIVE: To compare gene expression in totipotent 8Cell human embryos with pluripotent human embryonic stem (hES) cells. DESIGN: Expression patterns of 74 genes involved in pluripotency in hES cells were compared by whole human genome microarray analyses of mRNAs in two pools of 8-Cell human embryos, two lines of pluripotent hES cells, and fibroblasts before and after induced pluripotency (iPS cells). MATERIALS AND METHODS: The 8Cell microarray studies were reviewed and approved by institutional review boards in Boston, MA and Athens, Greece. Two pools of 5 normal appearing 8Cell embryos were subjected to RNA extraction and hybridization to Agilent whole human genome microarrays and compared with two lines of hES cells for 74 pluripotency genes. Statistical analyses indicated 7-fold higher or lower levels of fluorescence were potentially biologically relevant. RESULTS: Thirty pluripotency genes were over- or under-expressed by the 8Cell embryos, including the six transcription factors: KLF4, LIN28, MYC, NANOG, POU5F1, and SOX2, shown to transform normal human fibroblasts to iPS cells. Of the six, only MYC and KLF4 were over-detected on the 8-Cell arrays, along with DPPA5, ELOF1, IMP4, TCL1A, ZNF738. LEFTY1 and -2 were surprisingly quiet in the 8-cell embryos, as was DNMT3B, GAL, PODXL, TDGF1 and TERF1. CONCLUSION: Taken together, the findings indicate the totipotent embryo cells may be naturally susceptible to aneuploidy. Since most cells in pre-blastocyst embryos will give rise to placenta, only the few cells giving rise to the pluripotent inner cell mass (ICM) must be faithfully euploid. The microarrays emphasize striking differences in gene expression profiles between totipotent 8-Cell human embryos and lines of human embryonic stem cells cultured from ICMs, suggesting checkpoints to ensure accurate chromosomal allocation to daughter cells may be established in the ICM.

Genome-wide microarray evidence that 8-cell human blastomeres over-express cell cycle drivers and under-express checkpoints

PurposeTo understand cell cycle controls in the 8-Cell human blastomere.MethodsData from whole human genome (43,377 elements) microarray analyses of RNAs from normal 8-Cell human embryos were compiled with published microarrays of RNAs from human fibroblasts, before and after induced pluripotency, and embryonic stem cells. A sub database of 3,803 genes identified by high throughput RNA knock-down studies, plus genes that oscillate in human cells, was analyzed.ResultsThirty-five genes over-detected at least 7-fold specifically on the 8-Cell arrays were enriched for cell cycle drivers and for proteins that stabilize chromosome cohesion and spindle attachment and limit DNA and centrosome replication to once per cycle.ConclusionsThese results indicate that 8-cell human blastomere cleavage is guided by cyclic over-expression of key proteins, rather than canonical checkpoints, leading to rapidly increasing gene copy number and a susceptibility to chromosome and cytokinesis mishaps, well-noted characteristics of preimplantation human embryos.Electronic supplementary materialThe online version of this article (doi:10.1007/s10815-010-9407-6) contains supplementary material, which is available to authorized users.

The cell cycle of the 8-cell human embryo is uniquely controlled by a novel gene set

OBJECTIVE: To gain a better understanding of cell cycle control in early human embryos. DESIGN: High throughput strategies to correlate cell morphology with RNA-intereference of gene expression in human cells have been reported (1). Such experiments are not feasible with 8-cell human embryos, so we have compared microarray datasets to gain a better understanding of cell cycle control in early human embryos.The 8-cell embryo microarray data were combined with published data for human fibroblasts, before and after induced pluripotency, and two lines of human embryonic stem cells(2). The combined dataset was then queried for comparison with the 1352 putative cell cycle genes in the U2OS cell cycle. MATERIALS AND METHODS: Ten embryos were flash-frozen at the 8-cell stage for RNA extraction, amplification, and whole-genome microarray hybridization (1). The study was approved by ethics research committees of the University of Athens and Bedford Research Foundation. RESULTS: The U2OS dataset matched to 2148 gene elements in the 8C/fibro/hES database, 24% of which had no known function. Ninety U2OS cell cycle genes were silent and 166 U2OS cell cycle genes were overexpressed on all 8C/fibro/hES arrayst. Forty-three genes were under-expressed in the 8-cells. In contrast, 193 genes were over-expressed in the 8-cells: 47 CDD category, 101 G0/1, 27 S, 16 G2/M categories. Among the over-expressed were genes previously reported, such as: PCNA, MYC, CCNE1, CCNA2, PLK1, CDC2. Unanticipated new genes included FTL, ferritin light chain; MAGOH, “grandchildless” gene in Drosophila; KCNH5, potassium channel; ALOX15, arachidonate lipoxygenase; and MAGEB2, melanoma antigen. CONCLUSIONS: Cell cycle genes were more often over- expressed by 8-cell human embryos, which is under unique cell cycle control, possibly by circadian oscillation. (1)Mukerji, PNAS, 2006; (2)Kiessling, et al J Repro Genetics, 2009

Evidence that human blastomere cleavage is under unique cell cycle control

PurposeTo understand the molecular pathways that control early human embryo development.MethodsImproved methods of linear amplification of mRNAs and whole human genome microarray analyses were utilized to characterize gene expression in normal appearing 8-Cell human embryos, in comparison with published microarrays of human fibroblasts and pluripotent stem cells.ResultsMany genes involved in circadian rhythm and cell division were over-expressed in the 8-Cells. The cell cycle checkpoints, RB and WEE1, were silent on the 8-Cell arrays, whereas the recently described tumor suppressor, UHRF2, was up-regulated >10-fold, and the proto-oncogene, MYC, and the core element of circadian rhythm, CLOCK, were elevated up to >50-fold on the 8-Cell arrays.ConclusionsThe canonical G1 and G2 cell cycle checkpoints are not active in totipotent human blastomeres, perhaps replaced by UHRF2, MYC, and intracellular circadian pathways, which may play important roles in early human development.Electronic supplementary materialThe online version of this article (doi:10.1007/s10815-009-9306-x) contains supplementary material, which is available to authorized users.

Re-analysis by fluorescencein situ hybridisation of spare embryos cultured until Day 5 after preimplantation genetic diagnosis for a 47, XYY infertile patient demonstrates a high incidence of diploid mosaic embryos: a case report

Mosaicism in 4-8-cell human embryos analysed by fluorescence in situ hybridisation (FISH) has been widely reported, but few studies have addressed the incidence of mosaicism in more advanced embryonic stages. In the present study we analysed spare human embryos in a case of preimplantation genetic diagnosis (PGD) for increased risk of aneuploidy because of an infertile 47,XYY man. After replacement of two embryos typed as 1818XX at PGD, six spare embryos (not frozen because of their low quality) were re-analysed on Day 5 for PGD confirmation. Out of five embryos typed as 1818XY at PGD, four were diploid mosaic (DM) and one was normal in all cells. The sixth embryo, typed as 18XYY/1818181818X at PGD, was a DM. In spite of the bias of our small series of morphologically low-quality embryos, the surprisingly high proportion of mosaics (which confirms previous findings) questions the validity of PGD, but supports the strategy of transferring only the embryos where two blastomeres gave normal and concordant results at PGD. More data are required to understand the clinical significance of early diploid mosaicism (and its impact on implantation rate) and to determine whether some diploid mosaic embryos might be considered safe for transfer.

Micromanipulation of gametes and embryos: Biopsy of a blastomere from an 8-cell human embryo.

The preimplantation diagnosis of genetic disease in preimplantation human embryos was introduced several years ago. It involves removing one or more cells from a cleaving embryo or a blastocyst, and then assessing the isolated cells for evidence of chromosomal anomalies or genetic disease, usually by means of fluorescence in-situ hybridization (FISH), the polymerase chain reaction (PCR) and variants of it. The whole diagnostic procedure can be completed within one day, and correctly diagnosed embryos then replaced in the mother’s uterus. The number of correctly-diagnosed children is now accumulating as more clinics introduce this procedure into their practice. Details of PCR and FISH can be found in various publications (e.g. Delhanty and Handyside,1995). One form of operation is to remove a single blastomere from an 8-cell embryo, and this is the procedure shown in this video. This procedure is demonstrated on two approximately 8-cell human embryos growing in vitro. A series of steps are involved in the process. The initial step is to make a hole in the zona pellucida, and so permit the passage of pipettes through this investment. Access is thus gained to the enclosed embryo. Two examples are shown on this video. The first example illustrates a standard operation, from the drilling of the zona pellucida to the gentle loosening and withdrawal of a single blastomere. The second example is concerned with the problems arising when the first blastomere is damaged during the procedure, and it becomes essential to biopsy a second blastomere.

Chromosome mosaicism in human embryos.

In the human, mosaicism may occur before implantation; but, to determine when it first occurs, it is necessary to study the chromosomal complement of all blastomeres. Full karyotypes of blastomeres from 2- to 8-cell human embryos by conventional karyotyping of metaphase spreads are difficult to obtain. The aim of this study was to assess the stage at which mosaicism occurred in preimplantation human embryos through use of fluorescence in situ hybridization (FISH) with multiple probes. All or most blastomeres from 2- to 12-cell human embryos were analyzed by FISH using probes for gonosomes and chromosome 18. FISH was performed on blastomeres from 117 morphologically normal monospermic embryos that were not transferred after preimplantation diagnosis because of their risk of carrying X-linked disease; 20 (17.1%) of these embryos were mosaic. Another group of 163 arrested or morphologically abnormal monospermic embryos were also analyzed by FISH; 47 (28.8%) of these embryos were mosaic. In addition, 37 dispermic embryos were analyzed, and 28 (75.7%) of these were found to be mosaic. Mosaicism first occurred at the second cleavage division when the monospermic embryo was mostly diploid and at the first cleavage division when the embryo was mostly haploid, polyploid, or dispermic.

Ultrarapid freezing of early cleavage stage human embryos and eight-cell mouse embryos

Early cleavage stage human embryos and 8-cell mouse embryos were snap-frozen after a brief exposure to high concentrations of dimethyl sulfoxide (DMSO; 2 or 3.5 M) and 0.25 M sucrose and thawed in a warm water bath. Eleven of 12 3- to 8-cell human embryos survived freezing and thawing with more than 50% of their original blastomeres intact. However, pregnancy was not initiated when the 11 embryos were transferred to six patients. It was shown that continuation of embryonic development in vitro and in vivo was significantly better when 8-cell mouse embryos were snap-frozen in 3.5 M DMSO than in 2 M DMSO. When frozen in 3.5 M DMSO, 78% of 8-cell embryos survived on thawing, 84% developed to blastocysts in vitro, 63% implanted, and 42% developed to fetuses. Ultrarapid freezing is a quick and inexpensive method for mouse embryo cryobanking, but further studies are required to confirm the viability of frozen human embryos.

Cryopreservation of human embryos.

Studies on the cryopreservation of ninety-seven 4-cell and 8-cell human embryos indicate that morphologic survival can be achieved by means of two different cryoprotectants and two different freezing procedures. To date, pregnancies can be achieved after freezing and thawing of 8-cell human embryos cooled at 0.3 degree C per minute to -80 degrees C in the presence of 1.5 molar dimethyl sulfoxide (DMSO) and thawed at +8 degrees C per minute from -80 degrees C to +4 degrees C. By this procedure, 27 of 47 (57%) embryos frozen survived with 50% or more of their blastomeres intact. The transfer of these 27 embryos to 22 patients resulted in five pregnancies (22%). Morphologic survival of 4-cell and 8-cell human embryos after freezing and thawing is not affected by slight irregularities in blastomere size or the presence of small cytoplasmic fragments. Light and electron microscopic examination of fixed specimens indicates a good correlation between the appearance of frozen-thawed embryos at the dissecting microscope level and the extent of cryoinjury.

Chromosome abnormalities in human embryos after in vitro fertilization.

In vitro fertilization and embryo transfer is now an established treatment for certain forms of human infertility. High success rates have been reported following oocyte recovery and fertilization in vitro, the only major remaining difficulty being the high failure rate (80%) of implantation after replacement of the embryo into the mother. One possible reason for this failure of implantation is that some embryos may carry a lethal chromosome abnormality which does not allow development beyond the preimplantation stage. The need for information on the chromosomal status of early embryos fertilized and developed in vitro is recognized but two reported attempts to examine them have met with technical problems. Here we describe a method for examining chromosomes in 8-cell human embryos. Although we have made complete chromosome analyses for only three oocytes, our findings are surprising in that two of the three embryos were chromosomally abnormal. Data on the DNA content of the nuclei in a further eight cases showed that approximately 20% overall may be haploid.