Electromagnetic radiation (EMR) has deleterious effects on sperm motility and viability, as well as oocyte membrane and organelle structure. The aim was to assess the effects of cell phone radiation on preimplantation embryo morphokinetics and blastocyst viability in mice. For superovulation, 20 female mice were treated with intraperitoneal (IP) injections of 10 IU pregnant mare’s serum gonadotropin (Folligon® PMSG), followed by 10 IU of human chorionic gonadotropin (hCG) after 48 h. The zygotes (n = 150) from the control group were incubated for 4 days. The experimental zygotes (n = 150) were exposed to a cell phone emitting EMR with a frequency range 900–1800 MHz for 30 min on day 1. Then, all embryos were cultured in the time-lapse system and annotated based on time points from the 2-cell stage (t2) to hatched blastocyst (tHDyz), as well as abnormal cleavage patterns. Blastocyst viability was assessed using Hoechst and propidium iodide staining. Significant increases (P < 0.05) were observed in the cleavage division time points of t2, t8, t10, and t12 of the experimental group compared with the controls. In terms of blastocyst formation parameters, a delay in embryo development was observed in the experimental group compared with the controls. Data analysis of the time intervals between the two groups showed a significant difference in the s3 time interval (P < 0.05). Also, the rates of fragmentation, reverse cleavage, vacuole formation, and embryo arrest were significantly higher in the experimental group (P < 0.05). Furthermore, the cell survival rate in the experimental group was lower than the control group (P < 0.05). Exposure to EMR has detrimental consequences for preimplantation embryo development in mice. These effects can manifest as defects in the cleavage stage and impaired blastocyst formation, leading to lower cell viability.

Current live-cell imaging techniques make possible the observation of live events and the acquisition of large datasets to characterize the different parameters of the visualized events. They provide new insights into the dynamics of biological processes with unprecedented spatial and temporal resolutions. Here we describe the implementation and application of a new tool called TrackAnalyzer, accessible from Fiji and ImageJ. Our tool allows running semi-automated single-particle tracking (SPT) and subsequent motion classification, as well as quantitative analysis of diffusion and intensity for selected tracks relying on the graphical user interface (GUI) for large sets of temporal images (X–Y–T or X–Y–C–T dimensions). TrackAnalyzer also allows 3D visualization of the results as overlays of either spots, cells or end-tracks over time, along with corresponding feature extraction and further classification according to user criteria. Our analysis workflow automates the following steps: (1) spot or cell detection and filtering, (2) construction of tracks, (3) track classification and analysis (diffusion and chemotaxis), and (4) detailed analysis and visualization of all the outputs along the pipeline. All these analyses are automated and can be run in batch mode for a set of similar acquisitions.

The aim of this study was to determine if there was an association between the presence of cytoplasmic strings (CS) and their characteristics, with blastocyst quality, development and clinical outcome in human blastocysts. This two-centre cohort study was performed between July 2017 and September 2018 and involved a total of 1152 blastocysts from 225 patients undergoing in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). All embryos were cultured in Embryoscope+ and were assessed for CS using time-lapse images. A single assessor examined all blastocysts and reviewed videos using the EmbyroViewer® Software. Blastocyst quality was assessed on day 5 of embryo development. The number of CS, location and duration of their activity was recorded on days 5/6. A positive association between the presence of CS in human blastocysts with blastocyst quality was identified. Blastocysts with a higher number of CS present, were of higher quality and were in the more advanced stages of development. Top quality blastocysts had CS activity present for longer, as well as having a higher number of vesicles present travelling along the CS. Blastocysts that had CS present, had a significantly higher live birth rate. This study has confirmed that a higher number of CS and vesicles in human blastocysts is associated with top quality blastocysts and is not a negative predictor of development. They had a higher number of CS present that appeared earlier in development and, although ceased activity sooner, had a longer duration of activity. Blastocysts with CS had a significant increase in live birth rate.

The aim of this study was to provide a more comprehensive understanding of 1PN intracytoplasmic sperm injection (ICSI) zygotes. To achieve this objective, we assessed whether all 1PN-derived embryos showed a similar morphokinetic pattern, and if the morphokinetic behaviour of 1PN-derived embryos was comparable with that of 2PN-derived embryos. In total, 149 1PN ICSI zygotes (study group) and 195 2PN ICSI zygotes (control group) were included in the study. Embryo development potential was evaluated in terms of blastocyst rate. Morphokinetic parameters, including the pronucleus diameter and kinetics of in vitro development, were also analyzed. Embryos derived from 1PN ICSI zygotes showed impaired development compared with 2PN-derived embryos, with blastocyst rates of 28.9% and 67.2%, respectively. The diameter of the pronucleus of 1PN zygotes was larger than that of 2PN zygotes. When compared with 2PN-derived embryos, those derived from 1PN zygotes had a visible pronucleus for a shorter time, in addition to a longer syngamy time and slower kinetic behaviour from two to nine cells. When 1PN-derived blastocysts and 2PN-derived blastocysts were compared, the developmental kinetics were similar in both groups, except for a delayed and longer duration of the compaction phase in 1PN-derived embryos. In conclusion, monopronucleated ICSI zygotes present differences in developmental capacity and morphokinetic behaviour compared with 2PN ICSI zygotes, showing particular morphokinetic parameters related to pronucleus formation. Only the 1PN ICSI-derived embryos that reached the blastocyst stage have similar morphokinetic development to blastocysts from 2PN zygotes.

Biocompatibility testing of new materials is often performed in vitro by measuring the growth rate of mammalian cancer cells in time-lapse images acquired by phase contrast microscopes. The growth rate is measured by tracking cell coverage, which requires an accurate automatic segmentation method. However, cancer cells have irregular shapes that change over time, the mottled background pattern is partially visible through the cells and the images contain artifacts such as halos. We developed a novel algorithm for cell segmentation that copes with the mentioned challenges. It is based on temporal differences of consecutive images and a combination of thresholding, blurring, and morphological operations. We tested the algorithm on images of four cell types acquired by two different microscopes, evaluated the precision of segmentation against manual segmentation performed by a human operator, and finally provided comparison with other freely available methods. We propose a new, fully automated method for measuring the cell growth rate based on fitting a coverage curve with the Verhulst population model. The algorithm is fast and shows accuracy comparable with manual segmentation. Most notably it can correctly separate live from dead cells.

The effects of cytoplasmic volumes on development and developmental kinetics of in vitro produced porcine embryos were investigated. During hand-made cloning (HMC), selected cytoplasts were separated into two groups according to their size in relation to the initial oocyte: ~75% or ~50%. Following two fusion steps and activation (day 0), reconstructed embryos were cultured in vitro for 6 days. Cleavage rates on day 2 as well as blastocyst rates and cell numbers on day 6 were recorded. Results showed that embryo development was no different for ~50% versus ~75% cytoplasm at first fusion. This result was used in the following experiments, where the effect of varying cytoplasm volume in second fusion to obtain a final cytoplasm volume of ~75% to ~200% was tested. The results showed that the lowest quality was obtained when the final cytoplasm volume was ~75% and the highest quality at ~200% of the original oocyte. Similar results were observed in parthenogenetic (PA) embryos activated with different cytoplasmic volumes. A common pattern for the developmental kinetics of HMC and PA embryos was observed: the smaller group tended to have a longer time for the first two cell cycles, but subsequently a shorter time to form morula and blastocyst. In conclusion, the developmental kinetics of in vitro produced embryos was affected by the cytoplasm volume of the initial oocyte, and this further accounted for the developmental ability of the reconstructed embryos.