Comparison of the development of human embryos cultured in either an EmbryoScope or benchtop incubator.

PURPOSE: In this current study, our main goal was to establish that EmbryoScope incubation environment is comparable to standard incubation. METHODS: The development of sibling human zygotes was compared after culture in either a benchtop incubator (SI) or an EmbryoScope time-lapse incubator (ES). Between May 2015 to April 2016, a total of 581 normally fertilized 2PN, pronuclear-stage embryos, from 47 patients were allocated to culture in either a benchtop incubator (SI) or an EmbryoScope incubator (ES). RESULTS: The development of embryos to cleavage (up to day 3) and blastocyst stages (day 5/6) was compared between the two different incubators. The proportion of good quality embryos was higher in the ES group compared to the SI on day 2 (66.8 vs. 50.5%, P = 0.014) and on day 3 (75.1 vs. 56.0%, P = 0.006). Those differences were statistically significant. A higher proportion of embryos developed to good quality blastocysts when cultured in the EmbryoScope compared to the benchtop (49.4 vs. 42.0%, P = 0.24), but this was not significant. Finally, no significant differences were noted with the proportion of blastocysts chosen for cryopreservation on day 5/6 in the two incubators. CONCLUSIONS: The findings support the view that the EmbryoScope incubator supports at least equivalent in vitro development of human embryos compared to other standard incubation methods and may promote improved development during early cleavage stages.

Multiannual observations of acetone, methanol, and acetaldehyde in remote tropical atlantic air: implications for atmospheric OVOC budgets and oxidative capacity.

Oxygenated volatile organic compounds (OVOCs) in the atmosphere are precursors to peroxy acetyl nitrate (PAN), affect the tropospheric ozone budget, and in the remote marine environment represent a significant sink of the hydroxyl radical (OH). The sparse observational database for these compounds, particularly in the tropics, contributes to a high uncertainty in their emissions and atmospheric significance. Here, we show measurements of acetone, methanol, and acetaldehyde in the tropical remote marine boundary layer made between October 2006 and September 2011 at the Cape Verde Atmospheric Observatory (CVAO) (16.85° N, 24.87° W). Mean mixing ratios of acetone, methanol, and acetaldehyde were 546 ± 295 pptv, 742 ± 419 pptv, and 428 ± 190 pptv, respectively, averaged from approximately hourly values over this five-year period. The CAM-Chem global chemical transport model reproduced annual average acetone concentrations well (21% overestimation) but underestimated levels by a factor of 2 in autumn and overestimated concentrations in winter. Annual average concentrations of acetaldehyde were underestimated by a factor of 10, rising to a factor of 40 in summer, and methanol was underestimated on average by a factor of 2, peaking to over a factor of 4 in spring. The model predicted summer minima in acetaldehyde and acetone, which were not apparent in the observations. CAM-Chem was adapted to include a two-way sea-air flux parametrization based on seawater measurements made in the Atlantic Ocean, and the resultant fluxes suggest that the tropical Atlantic region is a net sink for acetone but a net source for methanol and acetaldehyde. Inclusion of the ocean fluxes resulted in good model simulations of monthly averaged methanol levels although still with a 3-fold underestimation in acetaldehyde. Wintertime acetone levels were better simulated, but the observed autumn levels were more severely underestimated than in the standard model. We suggest that the latter may be caused by underestimated terrestrial biogenic African primary and/or secondary OVOC sources by the model. The model underestimation of acetaldehyde concentrations all year round implies a consistent significant missing source, potentially from secondary chemistry of higher alkanes produced biogenically from plants or from the ocean. We estimate that low model bias in OVOC abundances in the remote tropical marine atmosphere may result in up to 8% underestimation of the global methane lifetime due to missing model OH reactivity. Underestimation of acetaldehyde concentrations is responsible for the bulk (∼70%) of this missing reactivity.

Redistribution of microtubules and pericentriolar material during the development of polarity in mouse blastomeres.

The distribution of microtubules and microtubule organizing centers (MTOCs) during the development of cell polarity in eight-cell mouse blastomeres was studied by immunofluorescence and immunoelectron microscopy using monoclonal anti-tubulin antibodies and an anti-pericentriolar material (PCM) serum. In early eight-cell blastomeres microtubules were found mainly around the nucleus and in the cell cortex, whereas PCM foci were observed dispersed in the cytoplasm. During the eight-cell stage, microtubules disappeared from the area adjacent to the zone of intercellular contact and accumulated in the apical part of the cell while their number decreased in the basal domain. The PCM also relocalized to the apical domain of the cell, but this occurred after the redistribution of the microtubules by a mechanism that involved the microtubule network. The possible roles of both MTOCs and microtubules in establishing cell polarity are discussed.

Anti-glomerular basement membrane antibody-mediated nephritis complicating transplantation in a patient with Alport's syndrome.

Loss of an allograft caused by anti-GBM antibody-mediated nephritis is a rare complication of renal transplantation in Alport's syndrome. We describe a patient in whom this occurred. He belongs to the subgroup of patients with hereditary nephritis and deafness with an abnormal Goodpasture antigen, and he developed a high level of circulating anti-GBM antibodies within 20 days of transplantation of a kidney with a presumably normal Goodpasture antigen. The antibody titer fell, only to rise again when he developed evidence of acute infection with CMV. Coincident with this second rise in antibody titer he developed an anti-GBM antibody-mediated crescentic nephritis with resultant loss of graft function and transplant nephrectomy. This case provides support for the hypothesis that the abnormality in the basement membrane in some patients with Alport's syndrome involves the Goodpasture antigen, and raises the possibility that viral infection may have triggered autoantibody production.

Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte.

The effect on the microtubule system of human oocytes of cooling to room temperature for either 10 or 30 minutes has been investigated. Changes in spindle organization were found in all oocytes cooled for 30 minutes compared with control oocytes kept at 37 degrees C throughout. These changes included reduction in spindle size, disorganization of microtubules within the spindle itself, and sometimes a complete lack of microtubules. In some oocytes, chromosome dispersal from the metaphase plate was associated with these changes. Cooling the oocyte to room temperature for only 10 minutes produced a similar pattern of disruption to spindle structure in many cases. The spindles in oocytes that were cooled for either 10 or 30 minutes and then allowed to recover at 37 degrees C for either 1 or 4 hours were found to resemble those in noncooled control oocytes in less than one half of the cases examined, although in only a few cases did the chromosomes remain dispersed. The significance of these findings for the handling of oocytes during gamete intrafallopian transfer and in vitro fertilization procedures is discussed in relation to the levels of aneuploidy detected in early human embryos.

Acid Tyrode's solution can stimulate parthenogenetic activation of human and mouse oocytes.

Fresh and aged (24 hours after ovulation) human oocytes and recently ovulated mouse oocytes may be activated by exposure to acidified Tyrode's solution. No activation of either type of human oocyte was observed after exposure to hyaluronidase or pronase, but significant numbers of fresh mouse oocytes were activated after exposure to pronase but not to chymotrypsin. The implications of these results for the manipulation of human and mouse eggs in vitro are discussed.

Are human spermatozoa separated on a Percoll density gradient safe for therapeutic use?

Motile morphologically normal human spermatozoa can be separated from semen by buoyant density centrifugation on Percoll (Pharmacia Fine Chemicals AB, Uppsala, Sweden) gradients. In this study, the authors have examined (1) the efficiency of washing procedures to remove contaminating Percoll particles from the separated spermatozoa, and (2) the potential of Percoll particles, which contain silica, to cause an inflammatory response when used for intrauterine insemination, or when introduced into the fallopian tube during gamete intrafallopian transfer (GIFT) procedures, as assessed by an intraperitoneal injection into mice. Although Percoll was phagocytosed at the injection site, and therefore cannot be presumed to be totally inert, no generalized inflammatory response was detected. A double spin and wash technique was found to remove most residual Percoll from the spermatozoa, as assessed by scanning electron microscopy. These results suggest that procedures involving the use of Percoll for the separation of human spermatozoa for in vitro fertilization, GIFT, or intrauterine insemination should include stringent washing protocols that will remove most, if not all, contaminating Percoll from the sample.

Phonotactic influences on short-term memory.

The impact of phonotactic probabilities on serial recall was investigated in a series of experiments. In Experiments 1A and 1B, 7 and 8 year olds were tested on their serial recall of monosyllabic words and of nonwords varying in phonotactic frequencies. A recall advantage to words over nonwords remained when stimuli were balanced for phonotactic probability, but nonword recall showed superior accuracy for high over low probability nonwords, as in Experiment 2. The nonword frequency effect appears to reflect the frequency of constituent syllables rather than biphones. Both lexicality and high phonotactic frequency led to increased proportions of full over partial recall of the memory stimuli. These findings indicate that decayed memory traces in phonological short-term memory can be reconstructed using either lexical or phonotactic knowledge.

Working memory deficits in children with low achievements in the national curriculum at 7 years of age.

BACKGROUND: Close links between children's capacities to store and manipulate information over brief periods have been found with achievements on standardised measures of vocabulary, language comprehension, reading, and mathematics. AIM: The study aimed to investigate whether working memory abilities are also associated with attainment levels in the national curriculum assessments at 7 years of age. SAMPLE: Eighty-three children aged 6 and 7 years attending local education authority schools participated in the study. METHODS: Working memory skills were assessed by a test battery designed to tap individual components of Baddeley and Hitch's (1974) working memory model. Children were assigned to normal and low achievement groups on the basis of their performance on national curriculum tasks and tests in the areas of English and mathematics. RESULTS: Children with low levels of curriculum attainment showed marked impairments on measures of central executive function and of visuo-spatial memory in particular. A single cut-off score derived from the test battery successfully identified the majority of the children failing to reach nationally expected levels of attainment. CONCLUSIONS: Complex working memory skills are closely linked with children's academic progress within the early years of school. The assessment of working memory skills may offer a valuable method for screening children likely to be at risk of poor scholastic progress.

Preimplantation genetic diagnosis of skin fragility-ectodermal dysplasia syndrome.

Skin fragility-ectodermal dysplasia syndrome is an autosomal recessive disorder caused by loss-of-function mutations in the desmosomal protein, plakophilin 1. Clinically, there may be considerable morbidity from extensive skin erosions and painful fissures on the palms and soles. In the absence of any specific treatment, prenatal diagnosis is an option for couples at reproductive risk of recurrence. In 2000, we developed and applied a single cell nested polymerase chain reaction protocol to test one couple for compound heterozygous plakophilin 1 gene mutations by preimplantation genetic diagnosis (PGD). Although pregnancy was established, an unrelated trisomy 22 led to a spontaneous abortion. However, eight embryos of known genetic status were cryopreserved at that stage, and we planned to undertake subsequent frozen embryo replacement cycles that might lead to the birth of an unaffected child in this family. Embryo cryopreservation was carried out in June 2000 using standard protocols in a three-step freezing procedure. Four embryos were thawed in March 2003, one of which was viable and was used in a frozen embryo replacement cycle, but pregnancy did not occur. The remaining four embryos were thawed in February 2004, two of which were viable (both carriers of the paternal mutation) and these were used in a second frozen embryo replacement cycle, and a singleton pregnancy was established. The child's plakophilin 1 genotype was assessed by direct nucleotide sequencing across the site of both potential mutations. Following two frozen embryo replacement cycles, and almost 4 years after the initial embryo biopsy and mutation analysis, a pregnancy was achieved that progressed to term with the birth of a healthy baby girl. Nucleotide sequencing of cord blood DNA, taken immediately after delivery, showed that the child was a heterozygous carrier of the paternal mutation but not of the maternal mutation. This case demonstrates the value of embryo cryopreservation, which can increase the number of embryo replacement procedures and hence the cumulative pregnancy rate per retrieval cycle. Moreover, this is the first report of successful full-term pregnancy and birth of a healthy baby following exclusion of a severe genodermatosis by PGD. The successful outcome of PGD in this case illustrates what is technically possible for couples at risk of recurrence of a severe inherited skin disease.

The development of visuo-spatial working memory.

Children's performance on tests of visuo-spatial working memory improves with age, although relatively little is known about why this happens. One explanation concerns the development of the ability to recode visually presented information into phonological form. This process appears to be used from around 8 years of age and is a major contributor to tasks in which stimuli can be verbally labelled. However, evidence suggests that phonological recoding cannot account for all of the age-related change in performance on visuo-spatial working memory tasks. In this review, four other mechanisms (knowledge, processing strategies, processing speed, and attentional capacity) are considered in terms of their contribution to children's visuo-spatial working memory development.

Microtubules influence compaction in preimplantation mouse embryos.

The role of microtubules during compaction of the 8-cell-stage mouse embryo was investigated using the drugs Taxol (which leads to a non-controlled polymerization of tubulin) and Nocodazole (which causes depolymerization of microtubules). Taxol inhibits compaction in most non-compacted embryos and reverses it in already compacted embryos. These effects were observed on both cell flattening (as judged by phase-contrast microscopy) and on cell surface polarization (as judged by scanning electron microscopy and the surface binding of fluorescent concanavalin A). In contrast Nocodazole does not inhibit cell flattening, but rather accelerates its completion. Nocodazole influences the detailed organization of the surface pole and appears to reduce the incidence of surface polarization but does not reverse polarity once established to a significant extent. We conclude that microtubules exercise a constraining role during compaction, influencing cell shape, cell organization and the time at which compaction takes place.

The effect of dimethylsulphoxide on the microtubular system of the mouse oocyte.

The effect of dimethylsulphoxide (DMSO) on the organization of the microtubular system of the mouse oocyte has been examined. Exposure to DMSO causes the immediate appearance of multiple, cold-resistant microtubular asters associated with the foci of pericentriolar material (PCM) normally present in the oocyte. More prolonged exposure to DMSO leads to progressive disassembly of the spindle, and as a result dispersal of the chromosomes and polar PCM foci occurs, and tubulin polymerization becomes confined to PCM-organized asters. Those astral microtubules located between the PCM foci and the cortex of the oocyte appear to be particularly stable, resulting in the development of lengthening radial bundles of microtubules between the PCM and the surface and the progressive movement of the PCM foci towards the centre of the cell. In contrast, after activation of the oocyte the microtubules generated in the presence of DMSO remain located in a cortical mesh. The effects of DMSO do not appear to be fully reversible in most oocytes. We discuss the implications of these results both for the cytoplasmic organization of the oocyte and zygote, and for the attempts at cryopreservation of human oocytes for therapeutic use in infertility programmes.

Maturation and polarization of the endocytotic system in outside blastomeres during mouse preimplantation development.

The maturation and distribution of the endocytotic apparatus in outside cells of cleavage-stage mouse embryos have been studied to determine the nature and sequence of changes associated with the differentiation of the polarized trophectoderm epithelium of the blastocyst. Various quantitative and qualitative techniques used at the light and electron microscopic levels have revealed an incremental pattern of endocytotic maturation and polarization. Oocytes, eggs and blastomeres within embryos up to the early 8-cell stage contain clusters of prelysosomal endocytotic vesicles (endosomes) distributed randomly in the cortical cytoplasm. During the 8-cell stage and continuing into the early 16-cell stage, endosomes become progressively localized in the apical cytoplasm beneath the microvillous pole. Endosome polarization is initiated prior to overt polarization of the surface membrane. Concomitant with endosome polarization, pinocytotic activity at the cell surface, revealed by horseradish peroxidase labelling, becomes segregated preferentially to the apical rather than the basolateral membrane. The final maturation phase occurs at the late 16-cell stage when secondary lysosomes, characterized by trimetaphosphatase reactivity, form and polarize in the basal cytoplasm.

The influence of cooling on the organization of the meiotic spindle of the mouse oocyte.

The effect of cooling on the organization of the microtubule system of the mouse oocyte has been investigated. Cooling to 25, 18 or 4 degrees C for varying periods of time resulted in a progressive disassembly of the spindle and the dispersal of the chromosomes. The extent of the changes observed was greater at lower temperatures and with longer periods of exposure. Transfer of oocytes from either 37 or 4 to 24 degrees C resulted in the rapid and transient appearance of polar asters and of multiple cytoplasmic asters associated with the pericentriolar material present at the spindle poles and in the cytocortex of the oocyte. This transient aster formation appeared to be driven by the elevated levels of free tubulin released during spindle disassembly. An apparent reversal of many of the changes induced by low temperature was observed in many but not all oocytes on their restoration to 37 degrees C for 1 h. These results have implications for our understanding of microtubule organization in the oocyte, and for the handling of oocytes during IVF and GIFT therapeutic procedures and during the cryopreservation of oocytes.

The influence of cooling on the properties of the zona pellucida of the mouse oocyte.

Mouse oocytes were exposed to a variety of cooling regimes prior to insemination in vitro. Exposure to 4 degrees C, but not to 25 degrees C, was associated with a reduced fertilization rate, but development to the blastocyst stage of those oocytes that fertilized was not consistently different from that of non-cooled controls. The reduced fertilization rate seems to result from an effect of cooling on the zona pellucida, since it was not observed if the zona was removed prior to insemination, and since cooling rendered the zona pellucida resistant to the action of chymotrypsin. Using chymotrypsin resistance as an assay, the nature of the cooling-induced effect on the zona was investigated. It is suggested that rapid cooling to 4 degrees C may promote release of cortical granules and a premature zona reaction.

Alternative routes for the establishment of surface polarity during compaction of the mouse embryo.

During the process of compaction, mouse 8-cell blastomeres flatten upon each other and polarize along an axis perpendicular to cell contacts. If the process of flattening is prevented, polarization can still occur, but does so in a lower proportion of cells than for control populations, and without the normal contact-directed orientation. We compared contact-directed and noncontact-directed processes to see if they involve common mechanisms. In nonflattened cells, surface polarization was favored in cells with nuclei located close to the cell surface, and the positions of surface poles and of nuclei tended to coincide. We present evidence that microtubules are involved in the development of microvillous poles associated with nuclei. In contrast it is known that polarization of microvilli occurs in the absence of microtubules if blastomeres are allowed to flatten. We conclude that surface polarization of mouse blastomeres can be accomplished by at least two alternative routes. One requires flattening but is independent of microtubules, and another can occur without flattening but involves a microtubule-mediated interaction between the nucleus and the cell cortex. It seems that both these pathways operate in the undisturbed embryo.

Cryoprotection of human oocytes: inappropriate exposure to DMSO reduces fertilization rates.

Human oocytes were exposed to the cryoprotectant dimethyl-sulphoxide (DMSO) at either 4 or 37 degrees C. Subsequent fertilization of these oocytes showed that exposure to DMSO at 37 degrees C was associated with a greatly reduced fertilization rate when compared to untreated control oocytes, whereas no such reduction was seen in oocytes exposed to DMSO at 4 degrees C. The significance of these results for the potential cryopreservation of human oocytes is discussed.

Reliability and accuracy of polymerase chain reaction amplification of two unique target sequences from biopsies of cleavage-stage and blastocyst-stage human embryos.

Human embryos have been biopsied at either the cleavage or the blastocyst stage of development. One to two blastomeres were removed from cleavage-stage embryos and 2-6 cells from blastocysts. The biopsy specimens were subjected to gene amplification by the polymerase chain reaction (PCR) and a comparison made of amplification efficiencies of two unique target sequences, one located within the beta-globin gene and containing the sickle-cell locus and the other a polymorphic dinucleotide repeat. When the cleavage-stage biopsy sample consisted of an intact blastomere with a clearly discernible nucleus, an amplification efficiency of 89% was achieved for each target locus. This was similar to that achieved with cleavage-stage biopsy samples consisting of two blastomeres or with blastocyst biopsy samples consisting of 2-3 trophectoderm cells. When biopsy samples consisted of four or more trophectoderm cells, both target loci were amplified in all samples tested. When the biopsy sample was heterozygous at the dinucleotide repeat locus and the biopsy consisted of one or more intact cells with a clearly discernible nucleus, both alleles were amplified in > 80% of biopsy samples. When four or more trophectoderm cells were used for the PCR, both alleles were amplified in all heterozygous samples. Target sequences were never amplified from biopsy samples which lysed prior to transfer into the reaction tube. Analysis of DNA fragments amplified from the dinucleotide repeat locus indicated that in most cases faithful amplification of biopsy DNA template had taken place.(ABSTRACT TRUNCATED AT 250 WORDS)