Abnormal Cleavage Research Articles

Evaluation of human embryo development in in vitro fertilization- and intracytoplasmic sperm injection-fertilized oocytes: A time-lapse study.

ObjectiveWe investigated whether the insemination method (in vitro fertilization [IVF] or intracytoplasmic sperm injection [ICSI]) affected morphokinetic events and abnormal cleavage events in embryonic development.MethodsA total of 1,830 normal fertilized embryos were obtained from 272 IVF and ICSI cycles that underwent ovum retrieval culture using a time-lapse system (Embryoscope) from June 2013 to March 2015. All embryos were investigated by a detailed time-lapse analysis that measured the developmental events in the hours after IVF or ICSI insemination.ResultsNo significant differences were observed between the two groups regarding clinical outcomes (p>0.05). ICSI-derived embryos showed significantly faster morphokinetics than those derived from conventional IVF, from the time to pronuclear fading to the time to 6 cells (p<0.05). However, no significant differences were found from the time to 7 cells to the time to expanded blastocyst (p>0.05). There were no differences in abnormal cleavage events between the two groups (p>0.05); they showed the same rates of direct cleavage from 1 to 3 cells, 2 multinucleated cells, 2 uneven cells, and reverse cleavage.ConclusionThe morphokinetics of embryo development was found to vary between IVF- and ICSI-fertilized oocytes, at least until the 6-cell stage. However, these differences did not affect the clinical outcomes of the embryo. Additionally, no significant differences in abnormal cleavage events were found according to the fertilization method.

138 THE RELATIONSHIP BETWEEN THE NORMALITY OF FIRST CLEAVAGE, THE GENE EXPRESSION IN BLASTOMERES, AND THE ABILITY TO DEVELOP TO THE BLASTOCYST STAGE IN IVF-DERIVED BOVINE 2-CELL STAGE EMBRYOS

Early first and second cleaved embryos after IVF associated with even blastomeres without fragments or protrusions were found to be a potent criterion for the selection of embryos with high developmental competence (Sugimura et al. 2012 PLOS ONE 7, e36627). The aim of this study was to examine the relationship between an early normal first cleavage pattern, the transcript abundance, and their development to the blastocyst stage in each blastomere in 2-cell stage bovine embryos. The IVF-derived bovine embryos were cultured individually in well-of-the-well culture dishes in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL−1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2. The first embryonic cleavage was categorized as being either normal (occurring within 28 h after IVF with 2 even blastomeres without fragment or protrusion) or abnormal (2 uneven blastomeres, with/without fragment/protrusion and/or later than 28 h after IVF). Then, cleaved embryos were placed in 0.5% actinase-E in Ca- and Mg-free PBS and blastomeres were separated by pipetting (n = 85; 4 replicates). In each embryo, one blastomere was subjected to quantitative RT-PCR to analyse the expression of developmentally important genes. The remaining blastomere was subsequently cultured in an individually identifiable manner to verify their ability to develop to the blastocyst stage. Primers were designed for 12 target genes related to pluripotency, cell cycle, metabolism, pregnancy reorganization, placentation, and fetal growth (OCT4, ATP1A1, CCNB1, CDH1, COX1, CTNNB1, GLUT8, MNSOD-3, SOX2, DYNLL1, IGFBP3, and PMSB1) and a reference gene (PPIA). Transcript abundance of target genes in individual blastomeres was compared between embryos showing normal and abnormal cleavage. Values were normalized to the average values of the reference genes and all the means were compared by the Student t-test. Blastomeres resulted from normal cleavage developed to the blastocyst stage on Day 7 to 8 (Day 0 = IVF) at significantly higher rates than those resulted from abnormal cleavage (65.7% v. 37.5%, respectively, P &lt; 0.05). Transcript abundance of OCT4 was significantly higher in blastomeres associated with all abnormal cleavage than in those associated with normal cleavage (P &lt; 0.05). The expression of CCNB1, COX1, ATP1A1, GLUT8, and PMSB1 in blastomeres associated with normal cleavage and blastocyst development was higher than that in those of abnormal cleavage (P &lt; 0.05). However, the level of OCT4, CCNB1, COX1, ATP1A1, and PMSB1 was lower in blastomeres associated with normal cleavage but failure of blastocyst development than those in blastomeres showing abnormal cleavage (P &lt; 0.05). Our results reveal that significantly higher expression of CCNB1, COX1, ATP1A1, and PMSB1 in blastomeres at the 2-cell stage in bovine embryos with superior developmental competence compared with those showing abnormal cleavage and low competence. Research was supported by JSPS KAKENHI (26450388).

A Quantitative Assay of In Vivo Cleavage of Von Willebrand Factor By ADAMTS13 Reveals Excessive Cleavage in Type 2B Von Willebrand Disease and Diminished Cleavage in Thrombotic Thrombocytopenic Purpura

Von Willebrand Factor (VWF) is a large glycoprotein that plays a crucial role in hemostasis by enabling platelets to adhere to sites of vascular injury, particularly under conditions of high shear stress. The multimeric structure of VWF is central to its activity; VWF is most active in its ultra-large multimer form (ULVWF). The size of plasma VWF is regulated by the plasma metalloprotease ADAMTS13, which cleaves VWF at the Tyr1605-Met1606 peptide bond within the A2 domain. Abnormal VWF cleavage has been implicated in a wide range of thrombotic and bleeding disorders, including thrombotic thrombocytopenic purpura (TTP) and Type 2 von Willebrand disease (VWD). These two diseases represent extreme phenotypes of VWF proteolysis-the former with too little cleavage and the latter with too much cleavage. ADAMTS13 proteolysis of VWF is currently assessed by either multimer gel analysis or ADAMTS13 activity assays. High-resolution agarose gels give an estimate of the extent to which a particular band represents cleaved vs native VWF. This method, however, is not quantitative and cannot distinguish whether a particular cleavage product is produced by ADAMTS13 or by another protease. Alternatively, ADAMTS13 activity assays are indirect measures of VWF cleavage, used especially to diagnose TTP. These assays measure the extent to which a subject's ADAMTS13 can cleave an exogenous substrate, usually a small peptide, and do not determine the extent of endogenous VWF cleavage. Therefore, we developed a mass spectrometry-based method to directly quantify cleavage of the Tyr1605-Met1606 peptide bond in plasma VWF. Here, we report quantification of VWF cleavage in plasma from normal donors, and TTP and VWD patients.Methods: VWF was isolated from plasma by immunoprecipitation using polyclonal VWF antibody and then digested with trypsin. Tryptic peptides, including the ADAMTS13-cleaved peptide and a control peptide, were quantified by nano ultra-performance liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) using heavy isotope-labelled peptides as internal standards. The extent of VWF cleavage by ADAMTS13 was determined as the concentration ratio of ADAMTS13-cleaved peptide to a control peptide representative of total VWF, and expressed as percent cleavage.Results: Given that the plasma concentration of VWF is relatively low, we first optimized conditions for sample preparation and analysis. Using NanoLC-MS/MS with isotopic dilution, we quantified ADAMTS13 cleavage of VWF in plasma samples (10-100 µl) containing 100-500 ng of VWF. We tested the reproducibility of the measurement using pooled normal plasma. The quantification was highly reproducible, with a wide linear range. VWF cleavage in pooled normal plasma was 4.9% ± 0.3% (mean ± SD, n=6), indicating that one VWF monomer in 20 is cleaved, on average. Cleavage was much higher in plasma from type 2B VWD patients (n=5), ranging from 15-30% depending on mutations. In contrast, we observed only 2.1-3.2% (n=6) cleavage in plasma VWF from TTP patients.Summary: This is the first report of quantification of ADAMTS13-mediated VWF cleavage in plasma. In normal plasma, approximately 5% of VWF A2 domains are cleaved, whereas cleavage was reduced to approximately 60% of normal on average in 6 TTP patients, and increased up to 6 times normal in patients with Type 2B VWD. In TTP, this level of proteolysis would be expected to result in accumulation of ULVWF multimers, as only between 1 in 50 to 1 in 30 monomers were cleaved. In type 2B VWD, by contrast, proteolysis may play a much larger role in the lack of larger multimers than previously appreciated, as up to 1 in 3 monomers were cleaved. This method should also prove useful in conditions such as severe malaria, in which it has been suggested that VWF cleavage is impaired, but this is not necessarily accompanied by low ADAMTS13 activity as measured with a small peptide substrate. DisclosuresNo relevant conflicts of interest to declare.

Blastomere extrusion and abnormal cleavage behavior in human embryos under time-lapse monitoring: possible way of embryo “self-correction”?

Blastomere extrusion and abnormal cleavage behavior in human embryos under time-lapse monitoring: possible way of embryo “self-correction”?

Does in vitro maturation (IVM) have an impact on morphokinetic parameters in comparison with IVF?

Polycystic ovarian syndrome (PCOS) patients are at higher risk of ovarian hyper stimulation syndrome (OHSS) due to the high dose of gonadotropin in IVF. To avoid the possibility of OHSS, IVM techniques have been developed and employed in clinical settings. However, IVM delivers lower pregnancy rates and birth rates than IVF. The objective of this study was to assess whether IVM treatment affects embryo development events together with morphokinetic parameters compared with IVF, and to identify the morphokinetic parameters specific to embryos that were suitable for implantation in IVM cycles. Retrospective study. The subjects were 41 couples who underwent 50 cycles of IVM at our clinic, from May, 2013 to Nov., 2015. After the failure of IVM treatment, 12 couples underwent standard IVF treatment. We assessed 194 embryos derived from IVM-ICSI and 48 embryos derived from IVF-ICSI. Their treatment outcomes were analyzed with morphokinetic events (cell division and interval of cell cleavage) by Primovision (vitrolife Sweden), and rate of clinical outcome. Moreover in IVM cycles, these time points were also compared between transferred embryos that implanted or did not implant. IVM zygotes showed significantly lower good quality embryo on day3, blastocyst formation, and good blastocyst rates compared with IVF. There were no differences in time points of cell division (tPNa to t8), pregnancy and abortion rates between the two groups. However, there were significant differences in synchrony of the second cell cycles (t4-t3:S2) (S2:4.5±5.0h vs. 1.4±2.3h, P=0.0002), and the rate of direct cleavage of one to three, or there to five cells was significantly higher in the IVM group (90/194 46.4% vs. 9/48 18.8%, P=0.0016). In the IVM group, there were no differences in development time points between implanted and non-implanted embryos. IVM zygotes showed increased abnormal cleavage compared with IVF; however, there were no significant differences in pregnancy rate or abortion rate between IVM and IVF. We rejected these abnormal cleaved embryos for transfer, indicating that in order to improve the pregnancy rate it is important to exclude the abnormal cleaved embryo, such as direct cleavage, from embryo transfer in IVM treatment.

Effects of cold-shock triploidy induction treatment on cell cleavage, hatching rate, and survival of triploid larvae of spotted sand bass (Paralabrax maculatofasciatus)

Three experiments assessed the effects of cold-shock induced triploidy on (1) the incidence of abnormal cell cleavage during embryogenesis, (2) hatching success and the proportion of live yolk-sac larvae in normally cleaved versus abnormally cleaved embryos, and (3) survival of triploid larvae of spotted sand bass (Paralabrax maculatofasciatus). All spawns were obtained by hormonal induction (LHRHa). Triploidy was induced by cold shock shortly after fertilization. In experiment 1, the percentage of abnormal cell cleavage was significantly higher (P &lt; 0.05) in the cold-shocked group than in the control group, while the hatching rate and the proportion of live yolk-sac larvae were significantly lower (P &lt; 0.05). In experiment 2, when normally and abnormally cleaved embryos from the control and cold-shocked groups were incubated separately, normal cleaved embryos in the cold-shocked group showed a significantly higher hatching rate and proportion of live yolk-sac larvae than abnormally cleaved embryos in the same group. No difference was observed in the control group. In experiment 3, a significant decrease (P &lt; 0.05) in the triploidy count was observed throughout the larval period. Final survival was drastically lower for triploid larvae compared to cold-shocked diploids and untreated eggs. Low triploidy percentages at the end of the larval period would seriously hinder the development of triploid sand bass culture.

Abnormally cleaving embryos are able to produce live births: a time-lapse study.

This study investigated the prevalence of abnormally cleaved embryos and determined which types of abnormally cleaved embryos (1-3c, 2-4c, 3-5c, 4-6c), might be suitable for transfer based on live birth data. One hundred seventy-one women (whose transferred embryos were confirmed to be either fully implanted or fully unimplanted) provided 1256 embryos, which were analyzed. Of these embryos, 320 embryos were transferred, of these transferred embryos, 291 embryos were normal and 29 embryos were abnormal, which five embryos were not analyzed because each one was presented one abnormal cleavage type. These 24 embryos were divided into four groups. Inclusion criteria were as follows: women under 37 years of age undergoing first fresh in vitro fertilization (IVF) treatment with a basal antral follicle count of 5-15, body mass index (BMI) of 18-25 kg/m(2), number of retrieved oocytes between 5 and 20, and tubal factors as the cause of infertility. Time-lapse imaging analysis software was used to compare temporal parameters of normal cleavage and abnormal cleavage groups (there were four abnormal groups, based on the prevalence of abnormal cleavage embryos). Cleavage times were analyzed before the abnormal cleavage occurred, and time intervals were analyzed after the abnormal cleavage based upon the types of abnormal cleavage. In addition, the time intervals of t4-t3 and t8-t5 were also analyzed; corresponding time parameters were measured in the normal group as well. Implantation rate, clinical pregnancy rate, ongoing pregnancy rate, and live birth rate were also measured in the normally cleaved and abnormally cleaved embryos. The prevalence of abnormal cleavage was 15.92% (200/1256). T8-t5 was the most important parameter in the prediction of potential development (production of a live-born baby) of abnormally cleaving embryos. Abnormally cleaving embryos were able to produced live births with T8-t5 the best parameter to predict the developmental potential of abnormally cleaving embryos.

164 EFFECTS OF KINETICS AND MORPHOLOGY ON EARLY EMBRYONIC DEVELOPMENT IN BOVINE OPU-IVF EMBRYOS

In recent years, the use of ovum pick up (OPU) and IVF for embryo production has increased worldwide; however, the conception rate of embryo transfer is lower for OPU-IVF embryos than for in vivo-derived embryos. This study aimed to determine the efficacy of embryo selection by a 3-step observation method by using a micro-well culture dish (Dai Nippon Printing, Tokyo, Japan). In this study, 9 Holstein and 15 Japanese Black cows were used, and the OPU-IVF was conducted from October 2014 to May 2015. The collected cumulus-oocyte complexes (COC) were cultured for 22 h in 25 mM HEPES-buffered TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL–1 of FSH. Sperm (at a final concentration of 5 × 106 spermatozoa mL–1) were incubated with COC for 6 h. After insemination, presumptive zygotes were separated from cumulus cells and sperm by pipetting. Then, the presumptive zygotes were cultured for 9 days in CR1aa supplemented with 5% CS by using a micro-well culture dish. Kinetics and morphology were observed at 27, 31, and 55 h post-insemination (hpi). The presumptive zygotes were divided to 3 groups (more than 2 cells, 2 cells, and no cleavage) at 27 and 31 hpi. Then, embryos at the 2-cell stage at 31 hpi were divided into 2 groups: 2-cell with normal cleavage and 2-cell embryos with abnormal cleavage (unequal cleavage, 2-cell with fragments, and 2-cell with protrusion). Subsequently, embryos were classified as 8-cell and more than 8 cell, or less than 8 cell at 55 hpi. The blastocyst rate (BL%) was analysed at 7, 8, and 9 days post IVF. Embryos selected by the 3-step observation method were used for fresh embryo transfer. The data were analysed by chi-squared test. In total, 856 oocytes were collected by OPU and 633 oocytes were cultured, of which 39.7% (263/663) developed to the blastocyst stage. The BL% of 2-cell embryos (72.5%, 116/160) was significantly higher (P &lt; 0.01) than that of no cleavage (47.0%, 117/249) at 27 hpi. The BL% of 2-cell (65.4%, 206/315) and more than 2-cell (53.0%, 35/66) was significantly higher (P &lt; 0.01 and P &lt; 0.05) than that of embryos divided as no cleavage (25.9%, 22/85) at 31 hpi. The BL% was not significantly different between 2-cell with normal cleavage (68.5%, 172/251) and abnormal cleavage (53.1%, 34/64). The BL% of 8-cell and more than 8-cell stage (72.8%, 182/250) was significantly higher (P &lt; 0.01) than that of embryos with less than 8 cells (38.8%, 81/209) at 55 hpi. Overall, 2-cell embryos at 27 hpi, 2-cell embryos with normal cleavage at 31 hpi, and 8-cell and more than 8 cell at 55 hpi showed the highest BL% (82.1%, 78/91). The conception rate was higher for following the selected fresh embryo transfer that was 70.6% (12/17) than average of in vitro fertilization embryos transfer that was 40.0%. These results demonstrate that the 3-step observation method used in this study can be effectively applied for the selection of IVF embryos that have a strong ability to develop into blastocysts and high competence for conception.

179 RELATIONSHIP BETWEEN GENE EXPRESSION IN INDIVIDUAL BLASTOMERES OF 2-CELL STAGE BOVINE EMBRYOS AND THE NORMALITY OF FIRST CLEAVAGE

Previously early first and second cleavages after IVF associated with even blastomeres without fragments or protrusions were found to be a potent criterion for the selection of embryos with high development competence (Sugimura et al. 2012 PLOS One 7, e36627). The aim of this study was to examine the relationship between an early normal first cleavage pattern and the transcript abundance in each blastomere in 2-cell stage bovine embryos. IVF-derived bovine embryos were cultured individually in microwells culture dish in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL–1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2. First cleavage and cleavage patterns were categorised as being either normal (the first cleavage within 28 h after IVF with 2 even blastomeres without fragment or protrusion) or abnormal (2 uneven blastomeres, with/without fragment/protrusion and/or later than 28 h after IVF at the first cleavage). Next, cleaved embryos were placed in 0.5% actinase-E in Ca- and Mg-free PBS and blastomeres were separated by pipetting. Individual blastomeres (n = 71, 10 replicates) were analysed for gene expression by quantitative RT-PCR. Primers were designed for 19 target genes related to pluripotency, cell cycle, metabolism, pregnancy reorganization, placentation and fetal growth (NANOG, OCT4, PLAC8, ATP1A1, CCNB1801, CDH1, COX1, CTNNB1, G6PDH, Glut8, MNSOD-3end, SOX2, DYNLL1, IGF1R, IGF2, IGF2R, IGFBP2, IGFBP3, and PMSB1) and a reference gene (PPIA). Transcript abundance of target genes in both of individual blastomeres of cleaved embryos was examined in embryos that cleaved early with a normal cleavage pattern and in those that showed abnormal cleavage pattern. Values were normalised to the average values of the reference genes and means were compared by the student t-test. Transcript abundance of OCT4, ATP1A1, CCNB1801, CDH1, COX1, CTNNB1, MNSOD-3end, IGF2R, and IGFBP2 was significantly higher in blastomeres associated with all categorised abnormal blastomeres compared towith an early normal cleavage (P &lt; 0.05). Furthermore, the expression of PLAC8, IGF1R, and PMSB1 in embryos having 2 uneven blastomeres, Glut8 and SOX2 in 2 uneven blastomeres with fragment/protrusion was higher than that in normal cleavage (P &lt; 0.05). However, the level of G6PDH was lower in embryos having 2 uneven blastomeres than that in those showing normal cleavage (P &lt; 0.05). Our results reveal blastomere gene expression in bovine embryos at the first cleavage may correlated with oocyte developmental competence. This study was supported by JSPS KAKENHI (26450388).

Time-lapse deselection model for human day 3 in vitro fertilization embryos: the combination of qualitative and quantitative measures of embryo growth

To present a time-lapse deselection model involving both qualitative and quantitative parameters for assessing embryos on day 3. Retrospective cohort study and prospective validation. Private IVF center. A total of 270 embryos with known implantation data (KID) after day 3 transfer from 212 IVF/intracytoplasmic sperm injection (ICSI) cycles were retrospectively analyzed for building the proposed deselection model, followed by prospective validation using an additional 66 KID embryos. None. Morphological score on day 3, embryo morphokinetic parameters, abnormal cleavage patterns, and known implantation results. All included embryos were categorized either retrospectively or prospectively into 7 grades (A+, A, B, C, D, E, F). Qualitative deselection parameters included poor conventional day 3 morphology, abnormal cleavage patterns identified via time-lapse monitoring, and <8 cells at 68 hours postinsemination. Quantitative parameters included time from pronuclear fading (PNF) to 5-cell stage and duration of 3-cell stage. KID implantation rates of embryos graded from A+ to F were 52.9%, 36.1%, 25.0%, 13.8%, 15.6%, 3.1%, and 0 respectively (area under the curve [AUC] = 0.762; 95% confidence interval [CI], 0.701-0.824), and a similar pattern was seen in either IVF (AUC = 0.721; 95% CI, 0.622-0.821) or ICSI embryos (AUC = 0.790; 95% CI, 0.711-0.868). Preliminary prospective validation using 66 KID embryos also showed statistically significant prediction in Medicult (AUC = 0.750; 95% CI, 0.588-0.912) and Vitrolife G-Series (AUC = 0.820; 95% CI, 0.671-0.969) suites of culture media. The proposed model involving both qualitative and quantitative deselection effectively predicts day 3 embryo implantation potential and is applicable to all IVF embryos regardless of insemination method by using PNF as the reference starting time point.

Irregular cleavage of early preimplantation human embryos: characteristics of patients and pregnancy outcomes.

This is a retrospective analysis of the morphokinetics, prevalence, and implantation potential of embryos with irregular first and second cleavages as identified by time-lapse microscopy. The study included 253 women who underwent 387 assisted reproduction treatments with intracytoplasmic sperm injection (ICSI). Each patient was assigned to one of three groups based on embryo cleavage results. In group I, one to two embryos per cycle showed irregular cleavage; group II, at least three embryos with abnormal cleavage; and in group III (the control group), all embryos cleaved normally. The number of embryos that cleaved from 1 to ≥3 cells or from 2 to ≥5 cells for each patient was recorded. Their prevalence and association with women's characteristics and pregnancy outcome were evaluated. The prevalence of irregular cleavage was 15.6 % among 1772 ICSI embryos. In 101 cycles, 1-2 embryos per cycle showed irregular cleavage (group I). In 32 cycles, at least 3 embryos showed abnormal cleavage (group II). In 254 cycles, all embryos cleaved normally (group III). The average age of the women in group II was significantly lower in comparison with groups I and III (32.5 ± 4.2 vs. 35.1 ± 4.9 and 35.5 ± 5.1, respectively, p < 0.02). In comparison of groups I and II, the odds ratio for ≥3 embryos with irregular cleavage in women younger than 35 was 3.48 (95 % CI, 1.28 to 9.46). Embryos with irregular cleavage were transferred in 16 women. Three live births were achieved following the transfer of single blastocysts derived from embryos with irregular cleavage from two to five cells. Early embryos with irregular cleavage are significantly more prevalent in younger women. When these embryos develop to the blastocyst stage, they may have normal implantation potential, leading to the birth of healthy babies.

Advantages of a Time-Lapse Imaging System in Assisted Reproductive Technology

Abstract: Clinical use of a time-lapse system and the develpoment of continuous culture media have enabled the establishment of modern incubation systems which decrease stress on the embryo. Furthermore, the use of a time-lapse system allows the accumulation of successive images of embryonic development. Various embryo morphological kinetics are beginning to be analyzed by using the collected data. A time-lapse system is useful for the accurate evaluation of implantation ability and deciding which embryos should be transferred first. Also, a time-lapse system allows the elimination of embryos which shoud not be transferred because of abnormal cleavage, or a multinucleated blastomere after the 4-cell stage.

Does the transition from standard incubator to a time-lapse imaging chamber impact embryonic development and neonatal outcomes?

Time lapse imaging chambers offer a unique environment for uninterrupted culture while still allowing visualization of embryo development. The altered culture environment may influence cell division and progression to the blastocyst stage. Ability to identify abnormal cleavage patterns as well as specific kinetic data with time lapse systems may further aid in embryo selection and ultimately pregnancy outcomes. This study compares embryonic development, clinical and neonatal outcomes after transition from standard box type incubators to time lapse.

Clinical significance of intercellular contact at the four-cell stage of human embryos, and the use of abnormal cleavage patterns to identify embryos with low implantation potential: a time-lapse study

To investigate the clinical significance of intercellular contact point (ICCP) in four-cell stage human embryos and the effectiveness of morphology and abnormal cleavage patterns in identifying embryos with low implantation potential. Retrospective cohort study. Private IVF center. A total of 223 consecutive IVF and intracytoplasmic sperm injection treatment cycles, with all resulting embryos cultured in the Embryoscope, and a subset of 207 cycles analyzed for ICCP number where good-quality four-cell embryos were available on day 2 (n = 373 IVF and n = 392 intracytoplasmic sperm injection embryos). None. Morphologic score on day 3, embryo morphokinetic parameters, incidence of abnormal biological events, and known implantation results. Of 765 good-quality four-cell embryos, 89 (11.6%) failed to achieve six ICCPs; 166 of 765 (21.7%) initially had fewer than six ICCPs but were able to establish six ICCPs before subsequent division. Embryos with fewer than six ICCPs at the end of four-cell stage had a lower implantation rate (5.0% vs. 38.5%), with lower embryology performance in both conventional and morphokinetic assessments, compared with embryos achieving six ICCPs by the end of four-cell stage. Deselecting embryos with poor morphology, direct cleavage, reverse cleavage, and fewer than six ICCPs at the four-cell stage led to a significantly improved implantation rate (33.6% vs. 22.4%). Embryos with fewer than six ICCPs at the end of the four-cell stage show compromised subsequent development and reduced implantation potential. Deselection of embryos with poor morphology and abnormal cleavage revealed via time-lapse imaging could provide the basis of a qualitative algorithm for embryo selection.

Enhanced neuronal degradation of amyloid-β oligomers allows synapse regeneration.

Enhanced neuronal degradation of amyloid-β oligomers allows synapse regeneration.

Holoprosencephaly: a survey of the entity, with embryology and fetal imaging.

Structural malformations of the brain are an important cause of childhood mortality and morbidity, with the latter having long-term financial and psychosocial implications for the affected child and family. Holoprosencephaly (HPE) is a severe brain malformation characterized by abnormal cleavage of the prosencephalon in the 5th gestational week. Aprosencephaly and atelencephaly occur earlier because of failure in the formation of the prosencephalon and telencephalon, respectively. The HPE holoprosencephaly spectrum classically includes alobar, semilobar, and lobar forms, although there are no clear-cut defining features. The middle interhemispheric variant (MIH), also known as syntelencephaly, is classified as a variant of HPE holoprosencephaly with midline interhemispheric fusion. Other conditions sometimes included in the spectrum of HPE holoprosencephaly include septo-optic dysplasia (SOD); "minimal" HPE holoprosencephaly , which is associated with subtle craniofacial malformations and mild developmental delay; and microform HPE holoprosencephaly , which by definition excludes brain involvement. The focus of this article will be on the spectrum of findings visible in fetal manifestation of the HPE holoprosencephaly spectrum. Brain embryology; the imaging characteristics, epidemiology, and embryology of HPE; and the more common associated anomalies, particularly those of the face ("the face predicts the brain") are reviewed. Recognition of these anomalies is important for accurate parental counseling, since the prognosis is poor but not invariably lethal; children with the milder forms may live well into their teens with severe developmental delays, endocrine dysfunction, and disrupted homeostasis. Available data on outcome in surviving children are summarized. Illustrative fetal ultrasonographic and magnetic resonance images are presented with clinical, autopsy, and postnatal imaging correlation.

Abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage.

Human embryos resulting from abnormal early cleavage can result in aneuploidy and failure to develop normally to the blastocyst stage. The nature of paternal influence on early embryo development has not been directly demonstrated although many studies have suggested effects from spermatozoal chromatin packaging, DNA damage, centriolar and mitotic spindle integrity, and plasma membrane integrity. The goal of this study was to determine whether early developmental events were affected by oxidative damage to the fertilizing sperm. Survival analysis was used to compare patterns of blastocyst formation based on P2 duration. Kaplan-Meier survival curves demonstrate that relatively few embryos with short (<1 hr) P2 times reached blastocysts, and the two curves diverged beginning on day 4, with nearly all of the embryos with longer P2 times reaching blastocysts by day 6 (p < .01). We determined that duration of the 2nd to 3rd mitoses were sensitive periods in the presence of spermatozoal oxidative stress. Embryos that displayed either too long or too short cytokineses demonstrated an increased failure to reach blastocyst stage and therefore survive for further development. Although paternal-derived gene expression occurs later in development, this study suggests a specific role in early mitosis that is highly influenced by paternal factors.

Exploring variations in the first and second cleavage stages of developing embryos using time-lapse imaging technique

ObjectiveThis study explores the relationship between abnormal cleavage such as direct cleavage from 1-3 cells and 2-5 cells, of developing embryos and its correlation to blastocyst formation.DesignA clinical retrospective study.Materials and MethodsEmbryo development was recorded and analyzed using Embryoscope, Unisense Fertilitech, Denmark. All morphokinetic observations were made with special attention to the time at which the pronuclei disappears, first and second cleavage times. 34 couples (maternal age≤38) underwent ICSI (n=263), of which 45 cycles led to an embryo transfer between December 2012 and September 2013. All transfers were frozen cycles and embryos were transferred at either cleavage or blastocyst stages.ResultsTabled 1TABLE 1.Blastocysts(MEAN±SD)p-valueBlastocysts Quality†(MEAN±SD)p-valueBlastocystsNot BlastocystsGoodpoorThe number of Embryos90/194104/194–28/9062/90–Time of the pronuclei disappearance(h)24.5±2.828.0±4.8p<0.00123.6±1.924.9±3.0p<0.05Time of the first cleavage(h)27.3±2.930.9±5.0p<0.00126.2±2.027.8±3.1p<0.01Time of the second cleavege(h)38.9±4.342.2±8.4p<0.0137.1±2.739.7±4.6p<0.01The 2cell at the first cleavege(%)96.7%59.6%p<0.001†Good Quality : more than G3BB(Gardner and Shoolcraft) Open table in a new tab ConclusionThese results indicated that good quality blastocysts developed slightly faster than others; thereby supporting the fact that the divisions must take place during a narrow window to maintain the viability of the embryo. Focusing on the first cleavage times, our study concludes that embryos divided from one to two cells had approximately 35% increased chance of successfully growing into a blastocyst than those that cleaved from one to 3 cells, therefore recognizing it as an imperative marker. Lastly, our results show that abnormally dividing embryos can potentially result in a positive pregnancy suggesting that if the embryo does develop into blastocyst after the 2-5 cells, there may be a chance to applying transfer. ObjectiveThis study explores the relationship between abnormal cleavage such as direct cleavage from 1-3 cells and 2-5 cells, of developing embryos and its correlation to blastocyst formation. This study explores the relationship between abnormal cleavage such as direct cleavage from 1-3 cells and 2-5 cells, of developing embryos and its correlation to blastocyst formation. DesignA clinical retrospective study. A clinical retrospective study. Materials and MethodsEmbryo development was recorded and analyzed using Embryoscope, Unisense Fertilitech, Denmark. All morphokinetic observations were made with special attention to the time at which the pronuclei disappears, first and second cleavage times. 34 couples (maternal age≤38) underwent ICSI (n=263), of which 45 cycles led to an embryo transfer between December 2012 and September 2013. All transfers were frozen cycles and embryos were transferred at either cleavage or blastocyst stages. Embryo development was recorded and analyzed using Embryoscope, Unisense Fertilitech, Denmark. All morphokinetic observations were made with special attention to the time at which the pronuclei disappears, first and second cleavage times. 34 couples (maternal age≤38) underwent ICSI (n=263), of which 45 cycles led to an embryo transfer between December 2012 and September 2013. All transfers were frozen cycles and embryos were transferred at either cleavage or blastocyst stages. ResultsTabled 1TABLE 1.Blastocysts(MEAN±SD)p-valueBlastocysts Quality†(MEAN±SD)p-valueBlastocystsNot BlastocystsGoodpoorThe number of Embryos90/194104/194–28/9062/90–Time of the pronuclei disappearance(h)24.5±2.828.0±4.8p<0.00123.6±1.924.9±3.0p<0.05Time of the first cleavage(h)27.3±2.930.9±5.0p<0.00126.2±2.027.8±3.1p<0.01Time of the second cleavege(h)38.9±4.342.2±8.4p<0.0137.1±2.739.7±4.6p<0.01The 2cell at the first cleavege(%)96.7%59.6%p<0.001†Good Quality : more than G3BB(Gardner and Shoolcraft) Open table in a new tab †Good Quality : more than G3BB(Gardner and Shoolcraft) ConclusionThese results indicated that good quality blastocysts developed slightly faster than others; thereby supporting the fact that the divisions must take place during a narrow window to maintain the viability of the embryo. Focusing on the first cleavage times, our study concludes that embryos divided from one to two cells had approximately 35% increased chance of successfully growing into a blastocyst than those that cleaved from one to 3 cells, therefore recognizing it as an imperative marker. Lastly, our results show that abnormally dividing embryos can potentially result in a positive pregnancy suggesting that if the embryo does develop into blastocyst after the 2-5 cells, there may be a chance to applying transfer. These results indicated that good quality blastocysts developed slightly faster than others; thereby supporting the fact that the divisions must take place during a narrow window to maintain the viability of the embryo. Focusing on the first cleavage times, our study concludes that embryos divided from one to two cells had approximately 35% increased chance of successfully growing into a blastocyst than those that cleaved from one to 3 cells, therefore recognizing it as an imperative marker. Lastly, our results show that abnormally dividing embryos can potentially result in a positive pregnancy suggesting that if the embryo does develop into blastocyst after the 2-5 cells, there may be a chance to applying transfer.

Abnormal cleavage of filamin a and cytoplasmic retention of androgen receptor in testicular biopsies in men with klinefelter syndrome (KS): an explanation for androgen resistance in KS

Abnormal cleavage of filamin a and cytoplasmic retention of androgen receptor in testicular biopsies in men with klinefelter syndrome (KS): an explanation for androgen resistance in KS

Cleavage and reduced CD36 ectodomain density on heart and spleen macrophages in the Spontaneously Hypertensive Rat

Metabolic disease is accompanied by a range of cellular defects (“comorbidities”) whose origin is uncertain. To investigate this pathophysiological phenomenon we used the Spontaneously Hypertensive Rat (SHR), which besides an elevated arterial blood pressure also has many other comorbidities, including a defective glucose and lipid metabolism. We have shown that this model of metabolic disease has elevated plasma matrix metalloproteinase (MMP) activity, which cleaves the extracellular domain of membrane receptors. We hypothesize here that the increased MMP activity also leads to abnormal cleavage of the scavenger receptor and fatty acid transporter CD36. To test this idea, chronic pharmaceutical MMP inhibition (CGS27023A) of the SHR and its normotensive control, the Wistar Kyoto Rat (WKY), was used to determine if inhibition of MMP activity serves to maintain CD36 receptor density and function. Surface density of CD36 on macrophages from the heart, spleen, and liver was determined in WKY, SHR, CGS-treated WKY (CGS WKY), and CGS-treated SHR (CGS SHR) by immunohistochemistry with an antibody against the CD36 ectodomain. The extracellular CD36 density was lower in SHR heart and spleen macrophages compared to that in the WKY. MMP inhibition by CGS served to restore the reduced CD36 density on SHR cardiac and splanchnic macrophages to levels of the WKY. To examine CD36 function, culture assays with murine macrophages (RAW 264.7) after incubation in fresh WKY or SHR plasma were used to test for adhesion of light-weight donor red blood cell (RBC) by CD36. This form of RBC adhesion to macrophages was reduced after incubation in SHR compared WKY plasma. Analysis of the supernatant macrophage media by Western blot shows a higher level of CD36 extracellular protein fragments following exposure to SHR plasma compared to WKY. MMP inhibition in the SHR plasma compared to untreated plasma, served to increase the RBC adhesion to macrophages and decrease the number of receptor fragments in the macrophage media. In conclusion, these studies bring to light that plasma in the SHR model of metabolic disease has an unchecked MMP degrading activity which causes cleavage of a variety of membrane receptors, including CD36, which attenuates several cellular functions typical for the metabolic disease, including RBC adhesion to the scavenger receptor CD36. In addition to other cell dysfunctions chronic MMP inhibition restores CD36 in the SHR.