Embryo morphokinetics is potentially associated with clinical outcomes of single-embryo transfers in preimplantation genetic testing for aneuploidy cycles

Research questionAre the morphokinetics of euploid blastocysts evaluated by a generally applicable algorithm associated with the clinical outcomes of single-embryo transfer (SET)?DesignTime-lapse microscopy was used to compare morphokinetic variables between expanded blastocysts derived from preimplantation genetic testing for aneuploidy cycles using high-resolution next-generation sequencing (hr-NGS). The clinical efficacy of the morphokinetic algorithm KIDScore D5 was evaluated after euploid SET.ResultsCompared with euploid blastocysts, low-level mosaic blastocysts presented comparable morphokinetic and morphological features. However, high-level mosaic blastocysts exhibited significant delays in t5 (median 51.9 h post insemination (hpi), P = 0.034) (where t is the time for the embryo to reach the specific stage in hours after ICSI or conventional IVF) and t8 (median 58.6 hpi, P = 0.032) accompanied by a prolonged time period for the third cell cycle (median 14.7 h, P = 0.012). A significantly higher incidence (P = 0.011) of multinucleation indicated a susceptibility of high-level mosaic blastocysts to mitotic errors. Only a delay in the time for the embryo to reach the full blastocyst stage (median 106.0 hpi, P = 0.039) was revealed in aneuploid blastocysts, reflecting the reduced formation of good-quality blastocysts (42.6% versus 65.7%, P < 0.001). Euploid blastocysts with specific morphokinetic characteristics were graded using the KIDScore D5 algorithm. Grade C embryos achieved significantly lower rates of clinical pregnancy, implantation and ongoing pregnancy (25%, 25% and 10%, respectively) compared with the grade A (76.2%, 79.4% and 68.3%, respectively) or grade B (62.5%, 66.7% and 62.5%, respectively) embryos (P = 0.0171 to <0.0001).ConclusionsAlthough morphokinetic features appear dissimilar in embryos with different diploid–aneuploid mosaic levels, predicting chromosomal abnormalities using morphokinetics alone is still insufficient. When combined with hr-NGS, use of the generally applicable KIDScore D5 algorithm has the potential to discriminate euploid blastocysts with different developmental competence.     

Automated segmentation and tracking of non-rigid objects in time-lapse microscopy videos of polymorphonuclear neutrophils

Time-lapse microscopy is an important technique to study the dynamics of various biological processes. The labor-intensive manual analysis of microscopy videos is increasingly replaced by automated segmentation and tracking methods. These methods are often limited to certain cell morphologies and/or cell stainings. In this paper, we present an automated segmentation and tracking framework that does not have these restrictions. In particular, our framework handles highly variable cell shapes and does not rely on any cell stainings. Our segmentation approach is based on a combination of spatial and temporal image variations to detect moving cells in microscopy videos. This method yields a sensitivity of 99% and a precision of 95% in object detection. The tracking of cells consists of different steps, starting from single-cell tracking based on a nearest-neighbor-approach, detection of cell–cell interactions and splitting of cell clusters, and finally combining tracklets using methods from graph theory. The segmentation and tracking framework was applied to synthetic as well as experimental datasets with varying cell densities implying different numbers of cell–cell interactions. We established a validation framework to measure the performance of our tracking technique. The cell tracking accuracy was found to be >99% for all datasets indicating a high accuracy for connecting the detected cells between different time points.     

The type of GnRH analogue used during controlled ovarian stimulation influences early embryo developmental kinetics: a time-lapse study

Objective

To explore if the GnRH analogue used for controlled ovarian stimulation (COS) and the ovulation triggering factor (GnRH agonist + hCG triggering versus GnRH antagonist + GnRH agonist triggering) affect embryo development and kinetics.

Study design

In a retrospective cohort study in the Instituto Valenciano de Infertilidad (IVI) Alicante and the Instituto Universitario-IVI Valencia, Spain, 2817 embryos deriving from 400 couples undergoing oocyte donation were analysed. After controlled ovarian stimulation and IVF/intracytoplamic sperm injection, the timing of embryonic cleavages was assessed by a video time-lapse system. The results were analysed using Student's t test for comparison of timings (hours) and Chi-squared test for comparison of proportions. A p-value < 0.05 was considered to be statistically significant.

Results

Embryos from cycles co-treated with GnRH antagonist + GnRH agonist (n = 2101) cleaved faster than embryos deriving from patients co-treated with GnRH agonist + hCG (n = 716): these differences were significant at the first stages of development but they disappeared as long as the embryo developed. Assessing embryo quality in terms of morphokinetic characteristics, we did not find significant differences between the two groups.

Conclusion(s)

By adopting a time-lapse video system, we can suggest that the type of protocol used for controlled ovarian stimulation influences embryo kinetics of development but these variations are not reflected in embryo quality.     

In vitro culture of human dental pulp cells: some aspects of cells emerging early from the explant

Cells emerging from a dental pulp explant were studied in order to elucidate the origin of precursor cells implicated in the formation of reparative dentin in vivo. Such cells observed at the very early stage of culture (days 5 – 10) were different from fibroblast-like cells obtained after one subculture. These early cells were round or elongated, with thin spinous processes, highly mobile, and contained numerous lipid vesicles. Incorporation of 1-[¹⁴C]palmitic acid did not show an increased incorporation of radiolabeled total lipids or triglycerides into these early cells compared to cells after four subcultures or control skin fibroblasts, suggesting that the lipids in vesicles were not synthesized by the cells. Since these cells also show a high level of fluid phase uptake via macropino-cytosis, it is suggested that these lipids originate from macropinocytosis. Between days 10 and 20, these cells spontaneously start conversion into cells that have a fibroblast-like morphology, are less mobile, and lack lipid vesicles. Their morphology, movement, and macropinocytosis suggest that these cells, which migrate from the pulp explants, are mesenchymal cells related to mononuclear phagocytes/ histiocytes. Received: 4 March 1997 / Accepted: 20 June 1997     

A system for interactive film analysis.

An interactive, minicomputer system has been constructed for analyzing dynamic phenomena recorded on movie film in a developmental biology laboratory. The minicomputer interfaces a stop-motion, variable speed projector, a digitizing pen, and real-time graphics display equipment. An analyst uses the pen to digitize features in a film, e.g. by following a cell. A computer-generated animation portraying all data entered is superimposed on the film image and synchronized with it. Noteworthy system features include: image overlays on a large screen, data entry with the projector running, large data capacity, computer control of the projector, and convenient data entry tools.     

The amyloid precursor protein of Alzheimer's disease is found on the surface of static but not actively motile portions of neurites

We have previously found that the amyloid precursor protein (APP) of Alzheimer's disease is present on the surface of rat cortical neurons in culture, in a segmental pattern which first becomes evident after 24 hours and is fully developed by five days. As APP has previously been reported to have a short half-life in neuronal cell lines, and has been shown to contain binding sites for various extracellular matrix components within its extracellular domain, we hypothesized that APP would be associated with portions of neurites undergoing rapid structural change, such as growth cones. To test this hypothesis, we observed selected neurons by video time-lapse differential interference microscopy on 24-hour-old primary rat neuronal cultures for up to 45 minutes, followed by fixation and immunocytochemistry to ascertain surface APP distribution on those same neurons. In contrast to our predictions, surface APP was not found on active portions of neurites, even if the activity produced no net translational movement. This result indicates that surface APP is actually associated with stable portions of neurites, a conclusion that tallies with other recent results showing that neuronal surface APP has a longer half-life than general cellular APP, and is associated with markers of adhesion patches, which themselves are relatively stable structures.     

Calphostin C as a rapid and strong inducer of apoptosis in human coronary artery smooth muscle cells

Vascular smooth muscle cells (VSMCs) play a major role in the development of atherosclerotic and restenotic lesions. The apoptotic process has been implicated in the development of this pathology. In this study, we characterized the induction of apoptosis by calphostin C (CC), a protein kinase C (PKC) inhibitor, in primary human coronary artery smooth muscle cells in the presence and absence of insulin-like growth factor-I (IGF-I). Additionally, we investigated the signal transduction pathways important for IGF-I mediated protection. Calphostin C induced apoptosis, as measured by terminal deoxy-UTP nick-end labeling (TUNEL), in a time- and dose-dependent manner, approaching 20% within 6 h of 50 nM calphostin C treatment. The amount of apoptosis increased to 44.58+/-8.08%, 47.54+/-1.66% and 78.1+/-11.9% after 8, 10 and 12 h of treatment, respectively (p<0.01 vs. control). IGF-I offered significant protection (p<0.05) at 8 and 10 h of treatment (60.6% and 52.5% protection, respectively). DNA ELISA confirmed the apoptotic effect of calphostin C and the protective effect of IGF-I. After 6 h of calphostin C treatment, DNA ELISA revealed 11.20+/-1.53 fold greater apoptosis as compared to baseline values. IGF-I treatment offered a level of protection of 46.6% as measured by DNA ELISA (p=0.06). Apoptosis was further qualitatively confirmed by time-lapse video microscopy and scanning electron microscopy. Interestingly, inhibitors of phosphatidylinositol-3-kinase (PI-3-K), p38 and extracellular regulated kinase (ERK) activation significantly (p<0.05 vs. calphostin C only treatment) increased apoptosis when used in conjunction with calphostin C. Inhibitors of phospatidylinositol-3-kinase and ERK activation reversed IGF-I protection. However, the p38 inhibitor SB203580 failed to reverse IGF-I protection. This study characterized an apoptotic system for human coronary artery smooth muscle cells offering a rapid and strong induction of programmed cell death (PCD) that remains responsive to the survival effects of IGF-I. Studies utilizing this system may prove useful in understanding the apoptotic response of VSMCs in the arterial wall.     

Prolonged blastomere movement induced by the delay of pronuclear fading and first cell division adversely affects pregnancy outcomes after fresh embryo transfer on Day 2: a time-lapse study

Research question

What is the incidence, origin and clinical significance of blastomere movement after the first cell division in the human embryo?

A Bayesian filtering approach to incorporate 2D/3D time-lapse confocal images for tracking angiogenic sprouting cells interacting with the gel matrix

We present a new approach to incorporating information from heterogeneous images of migrating cells in 3D gel. We study 3D angiogenic sprouting, where cells burrow into the gel matrix, communicate with other cells and create vascular networks. We combine time-lapse fluorescent images of stained cell nuclei and transmitted light images of the background gel to track cell trajectories. The nuclei images are sampled less frequently due to photo toxicity. Hence, 3D cell tracking can be performed more reliably when 2D sprout profiles, extracted from gel matrix images, are effectively incorporated. We employ a Bayesian filtering approach to optimally combine the two heterogeneous images with different sampling rates. We construct stochastic models to predict cell locations and sprout profiles and condition the likelihood of nuclei location by the sprout profile. The conditional distribution is non-Gaussian and the cell dynamics is non-linear. To jointly update cell and sprout estimates, we use a Rao–Blackwell particle filter. Simulation and experimental results show accurate tracking of multiple cells along with sprout formation, demonstrating synergistic effects of incorporating the two types of images.