Human pluripotent stem cells provide an inexhaustible model to study human embryogenesis in vitro. Recent studies have provided diverse models to generate human blastoids by self-organization of different pluripotent stem cells or somatic reprogramming intermediates. However, whether blastoids can be generated from other cell types or whether they can recapitulate postimplantation development in vitro is unknown. Here, we develop a strategy to generate human blastoids from heterogeneous intermediates with epiblast, trophectoderm, and primitive endoderm signatures of the primed-to-naïve conversion process, which resemble natural blastocysts in morphological architecture, composition of cell lineages, transcriptome, and lineage differentiation potential. In addition, these blastoids reflect many features of human peri-implantation and pregastrulation development when further cultured in an in vitro 3D culture system. In summary, our study provides an alternative strategy to generate human blastoids and offers insights into human early embryogenesis by modeling peri- and postimplantation development in vitro.
Humans ; Pluripotent Stem Cells/metabolism* ; Embryo, Mammalian/metabolism* ; Cell Differentiation ; Blastocyst ; Cell Lineage ; Embryonic Development

Animals ; Blastocyst ; Embryo Loss ; HeLa Cells ; Humans ; Mice ; Nuclear Proteins ; deficiency ; genetics ; metabolism

Coordination of cell division and cell fate is crucial for the successful development of mammalian early embryos. Aurora kinases are evolutionarily conserved serine/threonine kinases and key regulators of mitosis. Aurora kinase B (AurkB) is ubiquitously expressed while Aurora kinase C (AurkC) is specifically expressed in gametes and preimplantation embryos. We found that increasing AurkC level in one blastomere of the 2-cell embryo accelerated cell division and decreasing AurkC level slowed down mitosis. Changing AurkB level had the opposite effect. The kinase domains of AurkB and AurkC were responsible for their different ability to phosphorylate Histone H3 Serine 10 (H3S10P) and regulate metaphase timing. Using an Oct4-photoactivatable GFP fusion protein (Oct4-paGFP) and fluorescence decay after photoactivation assay, we found that AurkB overexpression reduced Oct4 retention in the nucleus. Finally, we show that blastomeres with higher AurkC level elevated pluripotency gene expression, which were inclined to enter the inner cell mass lineage and subsequently contributed to the embryo proper. Collectively, our results are the first demonstration that the activity of mitotic kinases can influence cell fate decisions in mammalian preimplantation embryos and have important implications to assisted reproduction.
Animals ; Aurora Kinase B ; metabolism ; Aurora Kinase C ; metabolism ; Blastocyst ; metabolism ; Gene Expression Regulation, Developmental ; physiology ; Histones ; metabolism ; Mice ; Phosphorylation ; physiology

OBJECTIVETo correlate sperm nucleoprotein transition (SNT) with sperm morphology, DNA damage and embryo development, and assess its value for assisted reproductive technology (ART).

METHODSThe SNT of 437 infertile men underwent ART were assayed, and its correlation with sperm morphology, DNA damage, fertilization rate, normal fertilization rate, cleavage rate, available embryo rate, D3 high quality embryo rate, blastocyst formation rate and high quality blastocyst rate were analyzed.

RESULTSThe normal morphology rate of sperms, DNA damage, fertilization rate, normal fertilization rate, cleavage rate, embryo transfer rate (ETR), D3 high quality embryo rate, blastocyst formation rate (BFR) and high quality blastocyst in normal males (Group A, abnormal rate≤30%, 135 subjects) did not significantly differ from those with an abnormal rate between 30% and 60% (Group B, 170 subjects) (P>0.05). For those with an abnormal rate of above 60% (Group C, 132 subjects), the sperm normal morphology rate, DNA damage, normal fertilization rate, ETR, D3 high quality embryo rate, high quality blastocyst rate were significantly lower compared with Group A (P<0.01), while no significant difference was found in fertilization rate, cleavage rate and BFR between groups A and C (P>0.05).

CONCLUSIONSNT is related with sperm morphology rate, DNA damage and embryo development, and should be assessed before ART.

Adult ; Blastocyst ; metabolism ; DNA Damage ; Embryo Transfer ; Embryonic Development ; Female ; Fertilization in Vitro ; Humans ; Infertility, Male ; genetics ; metabolism ; Male ; Nucleoproteins ; genetics ; metabolism ; Spermatozoa ; metabolism

OBJECTIVETo establish a novel HLA genotyping method for preimplantation genetic diagnonis (PGD) using multiple displacement amplification-polymerase chain reaction-sequencing based technique (MDA-PCR-SBT).

METHODSPeripheral 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.

RESULTSThe 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%.

CONCLUSIONPGD 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.

Blastocyst ; cytology ; metabolism ; Female ; Genotype ; Genotyping Techniques ; methods ; HLA Antigens ; genetics ; HLA-DQ beta-Chains ; genetics ; HLA-DRB1 Chains ; genetics ; Humans ; Polymerase Chain Reaction ; methods ; Pregnancy ; Preimplantation Diagnosis ; methods ; Reproducibility of Results ; Sequence Analysis, DNA ; methods

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.
Animals ; Blastocyst/cytology ; Cloning, Organism/*veterinary ; Culture Media/metabolism ; Dogs/*embryology ; Embryo Culture Techniques/*veterinary ; *Embryonic Development ; Nuclear Transfer Techniques/*veterinary

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
Blastocyst ; CRISPR-Cas Systems ; Hemoglobins, Abnormal ; genetics ; metabolism ; Humans ; Zygote

OBJECTIVE: Under estrogen deficiency, blastocysts cannot initiate implantation and enter dormancy. Dormant blastocysts live longer in utero than normal blastocysts, and autophagy has been suggested as a mechanism underlying the sustained survival of dormant blastocysts during delayed implantation. Autophagy is a cellular degradation pathway and a central component of the integrated stress response. Reactive oxygen species (ROS) are produced within cells during normal metabolism, but their levels increase dramatically under stressful conditions. We investigated whether heightened autophagy in dormant blastocysts is associated with the increased oxidative stress under the unfavorable condition of delayed implantation. METHODS: To visualize ROS production, day 8 (short-term dormancy) and day 20 (long-term dormancy) dormant blastocysts were loaded with 1-microM 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA). To block autophagic activation, 3-methyladenine (3-MA) and wortmannin were used in vivo and in vitro, respectively. RESULTS: We observed that ROS production was not significantly affected by the status of dormancy; in other words, both dormant and activated blastocysts showed high levels of ROS. However, ROS production was higher in the dormant blastocysts of the long-term dormancy group than in those of the short-term group. The addition of wortmannin to dormant blastocysts in vitro and 3-MA injection in vivo significantly increased ROS production in the short-term dormant blastocysts. In the long-term dormant blastocysts, ROS levels were not significantly affected by the treatment of the autophagy inhibitor. CONCLUSION: During delayed implantation, heightened autophagy in dormant blastocysts may be operative as a potential mechanism to reduce oxidative stress. Further, ROS may be one of the potential causes of compromised developmental competence of long-term dormant blastocysts after implantation.
Animals ; Autophagy* ; Blastocyst* ; Estrogens ; Mental Competency ; Metabolism ; Mice* ; Oxidative Stress ; Reactive Oxygen Species*

The present study was conducted to develop an effective method for establishment of porcine parthenogenetic embryonic stem cells (ppESCs) from parthenogenetically activated oocyte-derived blastocysts. The addition of 10% fetal bovine serum (FBS) to the medium on the 3rd day of oocyte culturing improved the development of blastocysts, attachment of inner cell masses (ICMs) onto feeder cells, and formation of primitive ppESC colonies. ICM attachment was further enhanced by basic fibroblast growth factor, stem cell factor, and leukemia inhibitory factor. From these attached ICMs, seven ppESC lines were established. ppESC pluripotency was verified by strong enzymatic alkaline phosphatase activity and the expression of pluripotent markers OCT3/4, Nanog, and SSEA4. Moreover, the ppESCs were induced to form an embryoid body and teratoma. Differentiation into three germ layers (ectoderm, mesoderm, and endoderm) was confirmed by the expression of specific markers for the layers and histological analysis. In conclusion, data from the present study suggested that our modified culture conditions using FBS and cytokines are highly useful for improving the generation of pluripotent ppESCs.
Animals ; Blastocyst/*cytology ; Cell Culture Techniques/*veterinary ; *Cell Differentiation ; Cytokines/metabolism ; Embryonic Stem Cells/*cytology ; Parthenogenesis ; Pluripotent Stem Cells/*cytology ; Swine/*physiology

Insufficient epigenetic reprogramming of donor nuclei is believed to be one of the most important causes of low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Previous studies have shown that both the in vitro and in vivo development of mouse SCNT embryos could be increased significantly by treatment with various histone deacetylase inhibitors (HDACi), including Trichostatin A, Scriptaid, and m-carboxycinnamic acid bishydroxamide (CBHA), in which only the effect of CBHA has not yet been tested in other species. In this paper we examine the effect of CBHA treatment on the development of porcine SCNT embryos. We have discovered the optimum dosage and time for CBHA treatment: incubating SCNT embryos with 2 μmol/L CBHA for 24 h after activation could increase the blastocyst rate from 12.7% to 26.5%. Immunofluorescence results showed that the level of acetylation at histone 3 lysine 9 (AcH3K9), acetylation at histone 3 lysine 18 (AcH3K18), and acetylation at histone 4 lysine 16 (AcH4K16) was raised after CBHA treatment. Meanwhile, CBHA treatment improved the expression of development relating genes such as pou5f1, cdx2, and the imprinted genes like igf2. Despite these promising in vitro results and histone reprogramming, the full term development was not significantly increased after treatment. In conclusion, CBHA improves the in vitro development of pig SCNT embryos, increases the global histone acetylation and corrects the expression of some developmentally important genes at early stages. As in mouse SCNT, we have shown that nuclear epigenetic reprogramming in pig early SCNT embryos can be modified by CBHA treatment.
Acetylation ; Animals ; Blastocyst ; cytology ; Cell Nucleus ; metabolism ; Cinnamates ; pharmacology ; Embryo, Mammalian ; drug effects ; metabolism ; Embryonic Development ; drug effects ; Epigenesis, Genetic ; Female ; Gene Expression ; Histone Deacetylase Inhibitors ; pharmacology ; Histones ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; In Vitro Techniques ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Nuclear Transfer Techniques ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Swine