Early cleavage of human embryos: an effective method for predicting successful IVF/ICSI outcome.
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BACKGROUND: The need for effective parameters for selecting the best embryos is paramount when a large number of them are available for transfer. Other studies have reported that transfer of pre-selected embryos, based on cleavage to the 2-cell stage at 25 h and 27 h post-insemination/intracytoplasmic sperm injection (ICSI), increases implantation and pregnancy rates. We investigated whether extending the time for selection of cleaved embryos to 29 h post-insemination/ICSI had a similar effect on pregnancy and implantation rates. METHODS: Cleavage to the 2-cell stage was assessed at 25, 27 and 29 h post-insemination/ICSI. Embryos that had cleaved at any of these time points were designated as 'early cleavage' (EC), while others were designated as 'non-early cleavage' (NEC). EC embryos were selected and preferentially transferred. RESULTS: EC occurred in 57% of the cycles (61% IVF; 51% ICSI). Significantly (P = 0.02) more clinical pregnancies occurred in the EC group (23/42, 55%) compared with the group that had no embryo undergoing first cleavage up to 29 h post-insemination/ICSI (8/32, 25%). The EC group of patients was significantly younger than the NEC. CONCLUSION: Transfer of selected embryos that reached the 2-cell stage between 25 and 29 h post-insemination/ICSI is a reliable prognostic tool for patients undergoing assisted reproduction techniques. (+info)
How many blastomeres of the 4-cell embryo contribute cells to the mouse body?
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The aim of this study was to estimate how many blastomeres of the 4-cell mouse embryo contribute cells to the embryo proper and finally to the animal. To this end, 4-cell embryos of pigmented and albino genotypes were disaggregated and single blastomeres (henceforth called '1/4' or 'quarter' blastomeres) were reaggregated in the following combinations: one 'pigmented' blastomere + three 'albino' blastomeres or vice versa (henceforth called '1+3') and two pigmented blastomeres + two albino blastomeres (henceforth called '2+2'). The aggregations were cultured in vitro and transferred as blastocysts either to the oviduct or uterus of pseudopregnant females. Recipients were allowed to litter naturally, or the foetuses were removed by Caesarian section and raised by lactating foster mothers. Chimaerism was assessed on the basis of coat (adults) or eye pigmentation (dead neonates). Among 28 '1+3' animals, there were 13 chimaeric and 15 non-chimaeric individuals. The pigmentation of non-chimaeras was always concordant with the genotype of the three 1/4 blastomeres and not with the genotype of the single blastomere in the given aggregation. These results make rather unlikely the possibility that the mouse is built of cells derived either from one or all four 1/4 blastomeres. Both two remaining options (2 or 3 1/4 blastomeres) are conceivable but the observed ratio of chimaeras to non-chimaeras among '1+3' animals (13:15) fits better the assumption of two 1/4 blastomeres contributing cells to the animal body. This assumption finds additional support in the observation that among '2+2' animals there were non-chimaeras (5 out of 7) and these would not have been expected should three 1/4 blastomeres contribute cells to the mouse body. (+info)
Allele-specific non-CpG methylation of the Nf1 gene during early mouse development.
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Recent reports of cytosine methylation occurring at CpA and CpT dinucleotides in murine ES cells as well as in Drosophila have renewed interest in methylation at sites other than CpGs. Our examination of the murine neurofibromatosis type 1 gene by sodium bisulfite genomic sequencing has revealed non-CpG methylation primarily in the oocyte and the maternally derived allele of the 2-cell embryo, with markedly lower levels found in sperm. Non-CpG methylation was not found in later stages of embryo development or in adult tissue. Our results suggest that maternal-specific de novo non-CpG methylation has occurred sometime between ovulation and formation of the 2-cell embryo, while during the same period the paternally derived allele has undergone site-specific active demethylation. Our data demonstrate both stage and parent-of-origin specific changes in methylation patterns within the neurofibromatosis type 1 coding region-involving cytosines located at both CpG and non-CpG dinucleotides. (+info)
Dynamic reprogramming of DNA methylation in the early mouse embryo.
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Dynamic epigenetic modification of the genome occurs during early development of the mouse. Active demethylation of the paternal genome occurs in the zygote, followed by passive demethylation during cleavage stages, and de novo methylation, which is thought to happen after implantation. We have investigated these processes by using indirect immunofluorescence with an antibody to 5-methyl cytosine. In contrast to previous work, we show that demethylation of the male pronucleus is completed within 4 h of fertilisation. This activity is intricately linked with and not separable from pronucleus formation. In conditions permissive for polyspermy, up to five male pronuclei underwent demethylation in the same oocyte. Paternal demethylation in fertilised oocytes deficient for MBD2, the only candidate demethylase, occurred normally. Passive loss of methylation occurred in a stepwise fashion up to the morulae stage without any evidence of spatial compartmentalisation. De novo methylation was observed specifically in the inner cell mass (ICM) but not in the trophectoderm of the blastocyst and hence may have an important role in early lineage specification. This is the first complete and detailed analysis of the epigenetic reprogramming cycle during preimplantation development. The three phases of methylation reprogramming may have roles in imprinting, the control of gene expression, and the establishment of nuclear totipotency. (+info)
The redistribution of Ca2+ stores with inositol 1,4,5-trisphosphate receptor to the cleavage furrow in a microtubule-dependent manner.
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We reported that microinjection of Ca2+ store-enriched microsome fractions from cultured CHO cells and mouse cerebella to dividing newt eggs induced extra-cleavage furrows via inositol 1,4,5-trisphosphate-induced Ca2+ release (Mitsuyama et al., 1999). Our observation strongly suggested that Ca2+ stores with inositol 1,4,5-trisphosphate receptor (IP3R) induce and position a cleavage furrow, as Ca2+-releasing machinery, and that such is itself a putative cleavage stimulus. For confirmation, we immunocytochemically examined mitotic CHO cells using antibodies against Ca2+ store-related proteins. We found that polar dominant Ca2+ stores with IP3R during metaphase were re-distributed to the future cleavage cortex just preceding the onset of furrowing, and that this redistributing IP3R was present on microtubule bundles. When a microsome fraction from sacro/endoplasmic reticulum Ca2+-ATPase (SERCA)-GFP stably expressing CHO cells was microinjected into dividing newt eggs and observed by confocal microscopy, the microinjected Ca2+ stores with IP3R moved linearly toward the next cleavage furrow and this movement was blocked by nocodazole, a microtubule-depolarizing agent, but not by cytochalasin B, an F-actin-depolarizing agent. These observations strongly suggest that Ca2+ stores with IP3R are transferred and accumulate to the cleavage furrow by microtubule-based motility, as a cleavage stimulus. (+info)
Effect of partial incision of the zona pellucida by piezo-micromanipulator for in vitro fertilization using frozen-thawed mouse spermatozoa on the developmental rate of embryos transferred at the 2-cell stage.
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Cryopreservation of mouse spermatozoa is widely used, although considerable strain differences in fertilization rates using frozen-thawed mouse spermatozoa have been described. The C57BL/6 mouse strain is a very widely used for establishment of transgenic mice, but the fertilization rate associated with the use of cryopreserved C57BL/6 spermatozoa is very low compared with rates for other inbred strains. We have recently solved this difficulty by in vitro fertilization (IVF) in combination with partial zona pellucida dissection (PZD). However, this technique requires culture of fertilized eggs with PZD in vitro up to morula or blastocyst stage before transfer into the uterus because blastomeres are lost after transfer into the oviduct because of the relatively large artificial slit in the zona pellucida. To overcome this problem, we performed a partial zona pellucida incision by using a piezo-micromanipulator (ZIP) for IVF with frozen-thawed mouse spermatozoa. The blunt end of the micropipette touched the surface of the zona pellucida of the oocytes, and piezo pulses were used to incise the zona pellucida while the pipette was moved along by the surface of zona pellucida. The length of the incision was pir/6 microm. When cumulus-free ZIP and PZD oocytes were inseminated with frozen-thawed genetically modified C57BL/6J spermatozoa, the fertilization rates of ZIP and PZD oocytes were 52% and 48%, respectively. After embryo transfer at the 2-cell stage, 18% and 2% of the transferred embryos with ZIP and PZD developed to term, respectively. This difference was significant (P < 0.05). When ZIP and PZD zygotes were cultured to blastocyst stage and subsequently transferred to uterine horns of recipient animals, the difference between ZIP and PZD zygotes for development rate to full term was not significant. Our results indicate that ZIP is an effective alternative technique for IVF using cryopreserved mouse spermatozoa and subsequent embryo transfer. (+info)
Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro.
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BACKGROUND: The absence of reliable markers for the identification of viable embryos for transfer at the early cleavage stage is likely to contribute to the generally low implantation rates and high incidence of multiple gestation in IVF treatment. In this study, we investigate the relationship between timing of first cleavage and the incidence of blastocyst formation in vitro. METHODS: Couples (n = 70) with at least one embryo remaining after transfer were included in the analyses. All embryos (n = 579) were examined for early cleavage at 25 h after insemination. Following embryo transfer, the remaining embryos (n = 426) were cultured until day 7 of development, and assessed for blastocyst formation. RESULTS: Eighty-five embryos (14.7%) cleaved to the 2-cell stage within 25 h of insemination; 26 of these were selected for transfer on day 2. Of the 59 embryos remaining in culture, 19 (32.2%) developed to the blastocyst stage; this was a significantly higher number than was observed in embryos (61/367; 16.6%) that failed to cleave within 25 h of insemination (P < 0.01). Within these two groups of embryos the proportion of hatched blastocysts was 11/59 (18.6%) and 26/367 (7.1%) respectively (P < 0.005). CONCLUSIONS: These findings indicate that early cleavage is indicative of increased developmental potential in human embryos and may be useful as an additional criterion in the selection of embryos for transfer. (+info)
Formation of a large Vasa-positive germ granule and its inheritance by germ cells in the enigmatic Chaetognaths.
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Chaetognaths (arrow worms) are abundant hermaphrodite marine organisms whose phylogenetic position amongst protostomes and deuterostomes is still debated. Ancient histological observations dating from a century ago described the presence in eggs of a large granule, presumed to be a germ plasm, and its probable inheritance in four primary germ cells (PGCs). Using videomicroscopy, electron microscopy and immunocytochemistry (labelling with anti-Vasa antibodies) we have followed the cycle of aggregation and dispersion of germ plasm and nuage material in eggs, embryos, PGCs and oocytes in several species of benthic (Spadella) and planctonic (Sagitta) chaetognaths. In these animals, germ cells and gametes can be observed in vivo throughout the 1-2 month life cycle. After describing internal fertilization in live animals we show that the single large (15 microm diameter) germ granule forms by a spiralling aggregation movement of small germ islands situated in the vegetal cortex at the time of first mitosis. We also demonstrate that the granule forms autonomously in unfertilized activated eggs or fertilized egg fragments. Once formed, the germ granule first associates with the cleavage furrow and is segregated into one of the first two blastomeres. The germ granule is then translocated from the cortex to the mitotic spindle during 3(rd) cleavage and remains in the single most-vegetal blastomere until the 32-cell stage. At the 64-cell stage the germ granule is partitioned as nuage material into two founder PGCs and further partitioned into four PGCs situated at the tip of the archenteron during gastrulation. These four PGCs migrate without dividing to reach the transverse septum, then proliferate and differentiate into oocytes and spermatocytes of two ovaries and two testes. We noted that germ plasm and nuage material were associated with mitochondria, the nucleus, the spindle and the centrosome during some stages of development and differentiation of the germ line. Finally, we demonstrate that a Vasa-like protein is present in the germ granule, in PGCs and in the electron-dense material associated with the germinal vesicle of oocytes. These features stress the conservation of cellular and molecular mechanisms involved in germ cell determination. (+info)