Preimplantation Embryogenesis Research Articles

Quantitative and qualitative changes of mitochondria in human preimplantation embryos

The oxygen consumption rates (OCRs) in mice and cattle have been reported to change during preimplantation embryogenesis. On the other hand, mitochondrial DNA (mtDNA) copy number has been shown to be unchanged in mice and changed in cattle and pigs. The interactions between mitochondrial functions and mtDNA copy numbers in human embryos during preimplantation development remain obscure. Sixteen oocytes and 100 embryos were used to assess mtDNA copy numbers and OCR. Three oocytes and 12 embryos were used to determine cytochrome c oxidase activity. All specimens were obtained between July 2004 and November 2014, and donated from couples after they had given informed consent. Mature oocytes and embryos at 2-14-cell, morula, and blastocyst stages were used to assess their OCR in the presence or absence of mitotoxins. The mtDNA copy number was determined using the samples after analysis of OCR. The relationships between developmental stages and OCR, and developmental stages and mtDNA copy number were analyzed. Furthermore, cytochrome c oxidase activity was determined in oocytes and 4-cell to blastocyst stage embryos. The structure of inner mitochondrial membranes and their respiratory function developed with embryonic growth and the mtDNA copy numbers decreased transiently compared with those of oocytes. The undifferentiated state of inner cell mass cells appears to be associated with a low OCR. On the other hand, the mtDNA copy numbers increased and aerobic metabolism of mitochondria increased in trophectoderm cells. The mitochondrial respiratory function of human embryos developed along with embryonic growth although the copy numbers of mtDNA decreased transiently before blastulation. OCRs increased toward the morula stage ahead of an increase of mtDNA at the time of blastulation. Data regarding changes in mitochondrial function and mtDNA copy number during preimplantation development of human embryos will be useful for the development of ideal culture media.

Exploring timing activation of functional pathway based on differential co-expression analysis in preimplantation embryogenesis.

Recent genome-wide omics studies have confirmed the early embryogenesis strictly dependent on the rigorous spatiotemporal activation and multilevel regulation. However, the full effect of functional pathway was not considered. To obtain complete understanding of the gene activation during early development, we performed systematic comparisons based on differential co-expression analysis for bovine preimplantation embryo development (PED). The results confirmed that the functional pathways actively transcribes as early as the 2-cell and 4-cell waves, which Basal transcription factor, Endocytosis and Spliceosome pathway can represent first signs of embryonic activity. Endocytosis act as one of master activators for uncovering a series of successive waves of maternal pioneer signal regulator with the help of Spliceosome complex. Furthermore, the results showed that pattern recognition receptors began to perform its essential function at 4-cell stage, which might be needed to coordinate the later major activation. And finally, our work presented a probable dynamic landscape of key functional pathways for embryogenesis. A clearer understanding of early embryo development will be helpful for Assisted Reproductive Technology (ART) and Regenerative Medicine (RM).

Multi-center study: innovative control of ambient air quality in multiple IVF laboratories is associated with statistically significant improvements in clinical outcomes - analysis of 5319 cycles

Successful preimplantation embryogenesis and the reproductive potential of the human embryo are critically dependent upon a number of variables including the changing organic chemistry of the ambient air (AA) within the IVF laboratory. AA contains dynamic levels of embryotoxic volatile organic compounds (VOCs) and viable particulates (VPs), which play a critical role in preimplantation toxicology and in the influence of AA on epigenetic processes. This report represents the largest cohort study evaluating the impact of comprehensive remediation of airborne pathogens on measures of embryogenesis and patient outcomes in multiple IVF programs using a proprietary air purification system (APS).

Inhibition of phosphorylated Ser473-Akt from translocating into the nucleus contributes to 2-cell arrest and defective zygotic genome activation in mouse preimplantation embryogenesis.

Phosphorylated Ser473-Akt (p-Ser473-Akt) is extensively studied as a correlate for the activity of Akt, which plays an important role in mouse oogenesis and preimplantation embryogenesis. However, little progress has been made about its effect on the mouse zygotic genome activation (ZGA) of 2-cell stage in mouse preimplantation embryos. In this study, we confirmed its localization in the pronuclei of 1-cell embryos and found that p-Ser473-Akt acquired prominent nucleus localization in 2-cell embryos physiologically. Akt specific inhibitors API-2 and MK2206 could inhibit the development of mouse preimplantation embryos in vitro, and induce 2-cell arrest at certain concentrations. 2-cell embryos exposed to 2.0 μmol/L API-2 or 30 μmol/L MK2206 displayed attenuated immunofluorescence intensity of p-Ser473-Akt in the nucleus. Simultaneously, qRT-PCR results revealed that 2.0 μmol/L API-2 treatment significantly downregulated the mRNA pattern of MuERV-L and eIF-1A, two marker genes of ZGA, suggesting a defect in ZGA compared with that of control group. Collectively, our work demonstrated the nuclear localization of p-Ser473-Akt during major ZGA, and Akt specific inhibitors API-2 and MK2206 which led to 2-cell arrest inhibited p-Ser473-Akt from translocating into the nucleus of 2-cell embryos with defective ZGA as well, implying p-Ser473-Akt may be a potential player in the major ZGA of 2-cell mouse embryos.

Unequal distribution of 16S mtrRNA at the 2-cell stage regulates cell lineage allocations in mouse embryos

Cell lineage determination during early embryogenesis has profound effects on adult animal development. Pre-patterning of embryos, such as that of Drosophila and Caenorhabditis elegans, is driven by asymmetrically localized maternal or zygotic factors, including mRNA species and RNA binding proteins. However, it is not clear how mammalian early embryogenesis is regulated and what the early cell fate determinants are. Here we show that, in mouse, mitochondrial ribosomal RNAs (mtrRNAs) are differentially distributed between 2-cell sister blastomeres. This distribution pattern is not related to the overall quantity or activity of mitochondria which appears equal between 2-cell sister blastomeres. Like in lower species, 16S mtrRNA is found to localize in the cytoplasm outside of mitochondria in mouse 2-cell embryos. Alterations of 16S mtrRNA levels in one of the 2-cell sister blastomere via microinjection of either sense or anti-sense RNAs drive its progeny into different cell lineages in blastocyst. These results indicate that mtrRNAs are differentially distributed among embryonic cells at the beginning of embryogenesis in mouse and they are functionally involved in the regulation of cell lineage allocations in blastocyst, suggesting an underlying molecular mechanism that regulates pre-implantation embryogenesis in mouse.

WNT4-like protein is a cortical granule component in mouse oocytes and functions in regulating preimplantation embryogenesis

Mammalian cortical granules (CG) are membrane-bound organelles located in the cortex of the unfertilized oocytes. Upon fertilization, CG undergo exocytosis to function in blocking polyspermy. While cortical granules are important in fertilization, their exact biochemical composition and reproductive function have not been fully defined. In the present study, a 66 kDa wingless-type MMTV integration site family, member 4 (WNT4)-like protein, with mouse CG origin was identified. Oocytes that were double labeled with lectin Lens culinaris agglutinin (LCA) and WNT4 antibody showed colocalization of the WNT4 molecules and cortical granules. The disappearance of WNT4 molecules in the artificially activated oocytes that were devoid of cortical granules confirmed their granule origin. Following fertilization, WNT4 remained associated with zygotes and blastomeres of 2-cell and 8-cell embryos; however the amount of protein present was reduced more than 2-fold as embryos developed. Prior to implantation, WNT4 appeared to be detectable only in the trophoblast cells. Our functional study revealed that WNT4 molecules were involved in regulating zygotic cleavage and early embryogenesis. To our knowledge, this is the first study demonstrating mammalian cortical granules contain signaling molecules that are involved in the regulation of the first phase of embryonic development.

Maternal-zygotic knockout reveals a critical role of Cdx2 in the morula to blastocyst transition

The first lineage segregation in the mouse embryo generates the inner cell mass (ICM), which gives rise to the pluripotent epiblast and therefore the future embryo, and the trophectoderm (TE), which will build the placenta. The TE lineage depends on the transcription factor Cdx2. However, when Cdx2 first starts to act remains unclear. Embryos with zygotic deletion of Cdx2 develop normally until the late blastocyst stage leading to the conclusion that Cdx2 is important for the maintenance but not specification of the TE. In contrast, down-regulation of Cdx2 transcripts from the early embryo stage results in defects in TE specification before the blastocyst stage. Here, to unambiguously address at which developmental stage Cdx2 becomes first required, we genetically deleted Cdx2 from the oocyte stage using a Zp3-Cre/loxP strategy. Careful assessment of a large cohort of Cdx2 maternal-zygotic null embryos, all individually filmed, examined and genotyped, reveals an earlier lethal phenotype than observed in Cdx2 zygotic null embryos that develop until the late blastocyst stage. The developmental failure of Cdx2 maternal-zygotic null embryos is associated with cell death and failure of TE specification, starting at the morula stage. These results indicate that Cdx2 is important for the correct specification of TE from the morula stage onwards and that both maternal and zygotic pools of Cdx2 are required for correct pre-implantation embryogenesis.

Statistically significant improvements in clinical outcomes using engineered molecular media and genomically modeled ultraviolet light for comprehensive control of ambient air (AA) quality

Successful preimplantation embryogenesis is critically dependent upon the culture environment and changing organic chemistry of the AA within the IVF laboratory. AA contains dynamic levels of embryotoxic viable particulates and volatile organic compounds which play a critical role in preimplantation toxicology. Our retrospective analysis examined the impact of comprehensive control of airborne pathogens on measures of embryogenesis in multiple IVF programs using a proprietary air purification system (Aire∼IVF®) designed specifically to protect the human embryo.

The Mmachc gene is required for pre-implantation embryogenesis in the mouse

Patients with mutations in MMACHC have the autosomal recessive disease of cobalamin metabolism known as cblC. These patients are unable to convert cobalamin into the two active forms, methylcobalamin and adenosylcobalamin and consequently have elevated homocysteine and methylmalonic acid in blood and urine. In addition, some cblC patients have structural abnormalities, including congenital heart defects. MMACHC is conserved in the mouse and shows tissue and stage-specific expression pattern in midgestation stage embryos. To create a mouse model of cblC we generated a line of mice with a gene-trap insertion in intron 1 of the Mmachc gene, (MmachcGt(AZ0348)Wtsi). Heterozygous mice show a 50% reduction of MMACHC protein, and have significantly higher levels of homocysteine and methylmalonic acid in their blood. The MmachcGt allele was inherited with a transmission ratio distortion in matings with heterozygous animals. Furthermore, homozygous MmachcGt embryos were not found after embryonic day 3.5 and these embryos were unable to generate giant cells in outgrowth assays. Our findings confirm that cblC is modeled in mice with reduced levels of Mmachc and suggest an early requirement for Mmachc in mouse development.

DNA methylation and its role in the trophoblast cell lineage.

DNA methylation functions as cellular memory beyond generations of cells and is involved in many biological processes. Because of its relatively stable nature compared with the transcriptome, the DNA methylation profile of cells can also be used to evaluate developmental similarity and cellular phenotypes. Recent insights into 5-hydroxymethylcytosine have started to reshape our view of the epigenetic regulation of mammalian development. Both global DNA methylation and hydroxymethylation levels change dynamically during preimplantation embryogenesis. It is known that DNA methylation plays an essential role in embryonic cell fate restriction, whereas its role in trophoblast development requires further research. Two distinct blastocyst-derived stem cell lines, embryonic stem (ES) cells and trophoblast stem (TS) cells, are used to study the epigenetic mechanisms underlying cell lineage maintenance and the regulation of cell differentiation. Such studies will allow us to understand the details of the epigenetic landscape of trophoblast development, which should offer valuable information for managing pregnancy-related diseases in humans.

First births with a simplified culture system for clinical IVF and embryo transfer

This study reports the outcome results from a pilot clinical trial using a simplified laboratory method for human IVF. This system reproducibly generates de novo the atmospheric and culture conditions that support normal fertilization and preimplantation embryogenesis to the hatched blastocyst stage without the need for specialized medical-grade gases or equipment. Development from insemination to the hatched blastocyst stage occurs undisturbed in a completely closed system that enables timed performance assessments for embryo selection in situ that, in this study, involved single-embryo transfers on day 3. With the simplified culture system, 8/23 embryos implanted, one miscarried at 8weeks of gestation and seven healthy babies have been born. The methodology and results are discussed with regard to how this simplified system can be adopted worldwide to meet the growing need for accessible and affordable IVF.A common notion concerning the demographics of infertility is that it is largely a phenomenon associated with developed countries, where infertility treatments are commonplace. In fact, most infertile couples reside in developing/low-resource countries where infertility diagnosis and treatment is nonexistent, inaccessible or unaffordable by the vast majority of young men and women in need. The irony of this situation is that bilateral tubal occlusions, for which IVF was originally indicated and is the most effective treatment, is by far the most common cause of their infertility. We have addressed one aspect of this issue, the IVF laboratory, as part of a wider effort by the Walking Egg Project to design and establish small, dedicated centres in developing countries to provide assisted reproduction technologies that are affordable and accessible to a wider proportion of the population in need. The methods for conventional IVF designed to addresses tubal obstructions are relatively simple and free of complex instrumentation and the highly developed infrastructure common to high-resource centres. This simplified IVF system self-generates culture conditions in a closed system. After prolonged preclinical testing, a pilot clinical study was initiated in 2012 in Genk, Belgium. The findings suggest that a significant first step has been achieved in the effort to bring advanced assisted reproduction to developed countries using a low-resource but highly effective IVF system capable of bringing modern reproductive medicine to infertile couples in low-resource societies.

Induction of a Human Pluripotent State with Distinct Regulatory Circuitry that Resembles Preimplantation Epiblast

Human embryonic stem cells (hESCs) are derived from the inner cell mass of the blastocyst. Despite sharing the common property of pluripotency, hESCs are notably distinct from epiblast cells of the preimplantation blastocyst. Here we use a combination of three small-molecule inhibitors to sustain hESCs in a LIF signaling-dependent hESC state (3iL hESCs) with elevated expression of NANOG and epiblast-enriched genes such as KLF4, DPPA3, and TBX3. Genome-wide transcriptome analysis confirms that the expression signature of 3iL hESCs shares similarities with native preimplantation epiblast cells. We also show that 3iL hESCs have a distinct epigenetic landscape, characterized by derepression of preimplantation epiblast genes. Using genome-wide binding profiles of NANOG and OCT4, we identify enhancers that contribute to rewiring of the regulatory circuitry. In summary, our study identifies a distinct hESC state with defined regulatory circuitry that will facilitate future analysis of human preimplantation embryogenesis and pluripotency.

Peri-implantational in vivo and in vitro embryo-trophoblast development after perigestational alcohol exposure in the CD-1 mouse

Long-term pregestational ethanol exposure induced altered fertilization and preimplantation embryogenesis. We evaluated preimplantational embryo-trophoblast differentiation, growth and invasiveness after perigestational ethanol 10% ingestion for 15 days preceding and up to day 4 (treated females [TF]: TF-D4 group) or 5 (TF-D5) of CD-1 gestation (control females [CF] with water). In TF-D4, expanded and hatched blastocyst numbers were significantly reduced (p < 0.05) versus CF-D4. Abnormal embryos and percentage of pyknotic nuclei were increased, and early blastocyst growth (nuclear number/embryo) and mitotic index was reduced (p < 0.05) versus CF-D4. On day 5 of gestation, TF-D5 presented significantly reduced total embryos and advanced embryo type 3 number versus CF-D5 (p < 0.05). During in vitro development, up to 72-hour culture, TF-D5 had reduced embryo type 1 (the least developed) and 3 percentages (p < 0.05) versus controls, whereas embryo type 2 percentage increased (p < 0.05) versus CF-D5. Embryo-trophoblast growth was studied during culture by morphometry. Embryo size ranges were classified as small, medium and large embryos. At 48-hour culture, small and medium embryos of TF had significantly increased mean area versus CF (p < 0.05), whereas large embryos had reduced mean area at 24-hour culture. Perigestational alcohol exposure up to days 4–5 induced embryo differentiation retardation, abnormal blastocyst growth and alterations of embryo-trophoblast growth and expansion during implantation, suggesting impaired regulation of trophoblast invasion and a relation with early pregnancy loss after mouse perigestational alcohol consumption.

Epigenetic Re-Programming during Mammalian Preimplantation Embryogenesis and PGC Development

Epigenetic Re-Programming during Mammalian Preimplantation Embryogenesis and PGC Development

Epigenetic Re-Programming during Mammalian Preimplantation Embryogenesis and PGC Development

Epigenetic programming and reprogramming, by means of DNA methylation and histone modifications etc., control the mammalian development to a large extent. They are also artificially altered for cell fate conversion and regeneration. Though epigenetic modifications change with slow dynamics during somatic cell lineage differentiation, they undergo a genome-wide dramatic change with extensive DNA de-methylation and histone modification during two specific time windows, the early embryogenesis and the Primordial Germ Cell (PGC) development stage. Here we reviewed these global epigenetic reprogramming occurred during normal development, mainly focusing on DNA methylation, histone modification and X-chromosome inactivation. Epigenetic reprogramming participates in many key biological processes such as genomic imprinting, X-chromosome inactivation, gene expression regulation and tumorigenesis and genome stability. Understanding the mechanisms of epigenetic reprogramming during the early embryogenesis and PGC formation would facilitate our knowledge of the developmental process and disease progress.

G9a co-localized with histone H3 lysine 9 monomethylation but not dimethylation in a nuclear membrane-dependent manner during mouse preimplantation embryo development

Histone H3 lysine 9 (H3K9) methylation plays an important role in the regulation of preimplantation embryo development. G9a has been reported to be a major H3K9mono (m1)/dimethylation(m2) methyltransferase and to contain nuclear localization signals. This study was performed to investigate the correlation between H3K9 methylation level and G9a localization when the nuclear membrane undergoes periodic reconstruction in the cell cycle during preimplantation embryo development. The fluorescence intensity was examined via immunofluorescence. The mRNA expression of G9awas determined using real-time reverse transcriptase (RT)-PCR. Eight-cell embryos were cultured in KSOM supplemented with nocodazole (0.5μM) for 12h. In this study, it was observed that the fluorescence intensity of H3K9m2 and G9a began to increase significantly from the 4-cell stage and reached the peak at the morula stage (p < 0.001), but the fluorescence intensity declined to 4-cell-stage levels when it reached the blastula stage. We observed a similar pattern when we examined G9a mRNA expression. Once the nuclear membrane disintegrated, G9a and H3K9m1 were not detectable by immunofluorescence; when it was reconstructed, G9a and H3K9m1 had relocated to the cell nucleus. However, no significant change was observed in the H3K9m2 localization or in the G9a mRNA level (p > 0.05) during the whole process. JHDM2A was consistently localized in the cytoplasm irrespective of the presence or absence of a nuclear membrane. These results indicate dynamic changes in the expression level of H3K9m2 and G9a as preimplantation embryogenesis progresses. G9a co-localized with H3K9 m1 in a nuclear membrane-dependent manner during mouse preimplantation embryo development.

Cell volume regulation in mammalian oocytes and preimplantation embryos

The earliest stages of preimplantation embryos are particularly sensitive to increased osmolarity, even within the physiological range. This sensitivity contributed to persistent developmental arrest, even when embryos were cultured in vitro in older, conditioned culture media, and seems to arise when embryos at the 1- and 2-cell stages accumulate inorganic ions used for cell volume homeostasis at too high a level, through activation of coupled Na(+)/H(+) and HCO(3)-/Cl(-) exchange. Such accumulation of inorganic ions can be disruptive since, above a certain level, the increased ionic strength disrupts cellular biochemistry and macromolecular functions and alters membrane potential. To counter this, embryos have evolved mechanisms of cell volume regulation that are unique to early preimplantation embryogenesis. The primary role of these is glycine accumulation via the GLYT1 transporter, with a secondary contribution by betaine accumulation via the SIT1 transporter. Independent cell-volume regulation first arises in the oocyte only after ovulation is triggered, when the strong oocyte-zona pellucida adhesion present in germinal vesicle stage oocytes in the ovarian follicle is released and GLYT1 becomes activated to begin accumulating glycine. Open questions still remain regarding how these processes are regulated.

Rapid Mitochondrial DNA Segregation in Primate Preimplantation Embryos Precedes Somatic and Germline Bottleneck

The timing and mechanisms of mitochondrial DNA (mtDNA) segregation and transmission in mammals are poorly understood. Genetic bottleneck in female germ cells has been proposed as the main phenomenon responsible for rapid intergenerational segregation of heteroplasmic mtDNA. We demonstrate here that mtDNA segregation occurs during primate preimplantation embryogenesis resulting in partitioning of mtDNA variants between daughter blastomeres. A substantial shift toward homoplasmy occurred in fetuses and embryonic stem cells (ESCs) derived from these heteroplasmic embryos. We also observed a wide range of heteroplasmic mtDNA variants distributed in individual oocytes recovered from these fetuses. Thus, we present here evidence for a previously unknown mtDNA segregation and bottleneck during preimplantation embryo development, suggesting that return to the homoplasmic condition can occur during development of an individual organism from the zygote to birth, without a passage through the germline.

Prospective, multi-center, double-blind, randomized clinical trial evaluating the use of hyaluronan bound sperm (HBS) in ICSI: statistically significant improvement in clinical outcomes

Oligozoospermic men often demonstrate seminal populations with increased chromosomal aberrations and compromised DNA integrity. The selection of sperm for ICSI is critical and the pathogenesis associated with male factor infertility can exert an adverse impact on preimplantation embryogenesis. The association between HBS, developmental and nuclear competence has led to the current study examining the use of H in sperm selection for ICSI.

Increased clinical pregnancy rates (CPR) and statistically significant decrease in loss rates using hyaluronan in sperm selection: prospective, multi-center, double-blind, randomized clinical trial

The in vitro selection of sperm for ICSI influences the paternal contribution to preimplantation embryogenesis. Hyaluronan (H), found within the cumulus matrix, may play a role in the selection of competent sperm during in vivo fertilization. H-bound sperm (HBS) exhibit increased chromosomal integrity and developmental maturity. The relationship between HBS and enhanced competence led to the current clinical trial examining the use of H in sperm selection for ICSI.