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Embryo morphokinetic analysis through time-lapse embryo imaging is envisioned as a method to improve selection of developmentally competent embryos. Morphokinetic analysis could be utilized to evaluate the effects of experimental manipulation on pre-implantation embryo development. The objectives of this study were to establish a normative morphokinetic database for in vitro fertilized rhesus macaque embryos and to assess the impact of atypical initial cleavage patterns on subsequent embryo development and formation of embryo outgrowths. The cleavage pattern and the timing of embryo developmental events were annotated retrospectively for unmanipulated in vitro fertilized rhesus macaque blastocysts produced over four breeding seasons. Approximately 50% of the blastocysts analyzed had an abnormal early cleavage event. The time to the initiation of embryo compaction and the time to completion of hatching was significantly delayed in blastocysts with an abnormal early cleavage event compared to blastocysts that had cleaved normally. Embryo hatching, attachment to an extracellular matrix, and growth during the implantation stage in vitro was not impacted by the initial cleavage pattern. These data establish normative morphokinetic parameters for in vitro fertilized rhesus macaque embryos and suggest that cleavage anomalies may not impact embryo implantation rates following embryo transfer., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aim: To explore the effect of embryo selection using the time-lapse monitoring (TLM) system compared with conventional morphological selection (CMS) on in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcomes., Methods: We searched PubMed, Ovid-Embase, and The Cochrane Library for the following studies: At Comparison 1, embryo selection using TLM images in a TLM incubator based on morphology versus embryo selection using CMS in a conventional incubator based on morphology; at Comparison 2, embryo selection using TLM based on morphokinetics versus embryo selection using CMS based on morphology. The primary outcomes were the live birth rate (LBR), ongoing pregnancy rate (OPR), clinical pregnancy rate (CPR), and implantation rate (IR), and the secondary outcome was the miscarriage rate (MR)., Results: A total of 14 randomized control trials (RCTs) were included. Both based on morphology, TLM incubators increased the IR (risk ratio [RR]: 1.10; 95% confidence interval [CI]: 1.01, 1.18; I 2  = 0%, moderate-quality evidence) compared to conventional incubators. Low- to moderate-quality evidence suggests that TLM incubators did not improve LBR, OPR, CPR, and MR compared to conventional incubators. In addition, low- to moderate-quality evidence indicates that embryo selection using TLM based on morphokinetics did not improve LBR, OPR, CPR, IR, or MR compared to CMS based on morphology., Conclusions: Low- to moderate-quality evidence suggests that neither TLM incubators nor embryo selection using TLM based on morphokinetics improved clinical outcomes (LBR, OPR, CPR, and MR) compared with CMS based on morphology. TLM is still an investigational procedure for IVF/ICSI practice., (© 2023 Japan Society of Obstetrics and Gynecology.)

Objective: This study aimed to investigate the accuracy of convolutional neural network models in the assessment of embryos using time-lapse monitoring., Data Sources: A systematic search was conducted in PubMed and Web of Science databases from January 2016 to December 2022. The search strategy was carried out by using key words and MeSH (Medical Subject Headings) terms., Study Eligibility Criteria: Studies were included if they reported the accuracy of convolutional neural network models for embryo evaluation using time-lapse monitoring. The review was registered with PROSPERO (International Prospective Register of Systematic Reviews; identification number CRD42021275916)., Methods: Two reviewer authors independently screened results using the Covidence systematic review software. The full-text articles were reviewed when studies met the inclusion criteria or in any uncertainty. Nonconsensus was resolved by a third reviewer. Risk of bias and applicability were evaluated using the QUADAS-2 tool and the modified Joanna Briggs Institute or JBI checklist., Results: Following a systematic search of the literature, 22 studies were identified as eligible for inclusion. All studies were retrospective. A total of 522,516 images of 222,998 embryos were analyzed. Three main outcomes were evaluated: successful in vitro fertilization, blastocyst stage classification, and blastocyst quality. Most studies reported >80% accuracy, and embryologists were outperformed in some. Ten studies had a high risk of bias, mostly because of patient bias., Conclusion: The application of artificial intelligence in time-lapse monitoring has the potential to provide more efficient, accurate, and objective embryo evaluation. Models that examined blastocyst stage classification showed the best predictions. Models that predicted live birth had a low risk of bias, used the largest databases, and had external validation, which heightens their relevance to clinical application. Our systematic review is limited by the high heterogeneity among the included studies. Researchers should share databases and standardize reporting., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Detecting and tracking multiple moving objects in a video is a challenging task. For living cells, the task becomes even more arduous as cells change their morphology over time, can partially overlap, and mitosis leads to new cells. Differently from fluorescence microscopy, label-free techniques can be easily applied to almost all cell lines, reducing sample preparation complexity and phototoxicity. In this study, we present ALFI, a dataset of images and annotations for label-free microscopy, made publicly available to the scientific community, that notably extends the current panorama of expertly labeled data for detection and tracking of cultured living nontransformed and cancer human cells. It consists of 29 time-lapse image sequences from HeLa, U2OS, and hTERT RPE-1 cells under different experimental conditions, acquired by differential interference contrast microscopy, for a total of 237.9 hours. It contains various annotations (pixel-wise segmentation masks, object-wise bounding boxes, tracking information). The dataset is useful for testing and comparing methods for identifying interphase and mitotic events and reconstructing their lineage, and for discriminating different cellular phenotypes., (© 2023. Springer Nature Limited.)

Motivation: Reliable label-free methods are needed for detecting and profiling apoptotic events in time-lapse cell-cell interaction assays. Prior studies relied on fluorescent markers of apoptosis, e.g. Annexin-V, that provide an inconsistent and late indication of apoptotic onset for human melanoma cells. Our motivation is to improve the detection of apoptosis by directly detecting apoptotic bodies in a label-free manner., Results: Our trained ResNet50 network identified nanowells containing apoptotic bodies with 92% accuracy and predicted the onset of apoptosis with an error of one frame (5 min/frame). Our apoptotic body segmentation yielded an IoU accuracy of 75%, allowing associative identification of apoptotic cells. Our method detected apoptosis events, 70% of which were not detected by Annexin-V staining., Availability and Implementation: Open-source code and sample data provided at https://github.com/kwu14victor/ApoBDproject., (© The Author(s) 2023. Published by Oxford University Press.)

Manual dish preparation for IVF in human fertility clinics or animal laboratories heavily relies on embryologists' experience, which can lead to occupational illness due to long-term and monotonous operation. Therefore, introducing an automated technique to replace traditional methods is crucial for improving working efficiency and reducing work burden for embryologists. In the current study in the mouse, both manual and automated methods were used to prepare IVF or embryo culture dishes. A one-way analysis of variance was conducted to compare several factors, including preparation time, qualified rates, media osmolality of dishes, fertilization rates, and embryonic development to assess the efficiency and potential of automated preparation. The results showed that automation system significantly reduced the required time and increased the efficiencies and qualified rates of dish preparation, especially for embryo culture dishes, without significantly altering medium osmolalities. There were no significant differences between two preparations in fertilization rates and embryo development in mice. Thus, automated dish preparation can improve working efficiency and qualified rates while maintaining fertilization rates and subsequent embryonic development without compromising osmolality stability of medium. It presents a superior alternative to manual preparation, reducing the workload of embryologists and facilitating the standardization of operational procedures., (© 2023. Springer Nature Limited.)

Time-lapse imaging (TLI) allows continuous monitoring of embryo development without disturbing culture conditions by removing embryos from the incubator. The study of embryo kinetics using TLI has given rise to some new markers for embryo selection that are able to document and evaluate embryo morphology and the timing of developmental events through continuous live image tracking. Time-lapse imaging has emerged as a powerful tool for creating predictive models of in vitro fertilization outcomes. Fourty-seven articles were included in the present review to investigate the current situation of TLI in in vitro fertilization laboratories. Morphokinetics of the embryo during its in vitro development have been described using parameters indicative of the different events capable of predicting the ability to reach blastocyst stage, implantation and pregnancy rates, live birth outcome, and embryo ploidy., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Purpose: Maintaining a stable pH at optimal level in human embryo culture media is crucial for embryo development but poses a challenge for all IVF laboratories. We validate analytically reliable conditions for pH measurement that are as close as possible to the embryo microenvironment during IVF., Methods: This was a multicentric study. A Siemens EPOC portable blood gas analyzer was used. The analytical validation was carried out under the culture medium (Global Total HSA®) conditions of use (microdroplets, under oil overlay, in a IVF incubator with (EmbryoScope®) or without a time lapse system (K system G210+®) and using IVF dishes. The validation included repeatability ("within-run" precision), total precision (between-day precision), trueness based on inter-laboratory comparison, inaccuracy based on external quality assessment and comparison to the reference technique. We also assessed the pre-analytical medium incubation time required to obtain a target value., Results: A measurement after an incubation period of 24 to 48 h is more representative of the pH to which the embryo will be exposed throughout the culture. The "within-run" and "between-day" precision show very low coefficients of variation (CV%): 0.17 to 0.22% and 0.13 to 0.34%, respectively, with IVF culture media. Trueness (% bias) range from - 0.07 to - 0.03%. We demonstrate good correlation between EPOC and reference pH electrode with an overestimation of 0.03 pH units of EPOC., Conclusion: Our method demonstrates good analytical performance for IVF laboratories wishing to implement a robust quality assurance system to monitor pH in embryo culture media. Compliance with stringent pre-analytical and analytical conditions is essential., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Time-lapse microscopy is the only method that can directly capture the dynamics and heterogeneity of fundamental cellular processes at the single-cell level with high temporal resolution. Successful application of single-cell time-lapse microscopy requires automated segmentation and tracking of hundreds of individual cells over several time points. However, segmentation and tracking of single cells remain challenging for the analysis of time-lapse microscopy images, in particular for widely available and non-toxic imaging modalities such as phase-contrast imaging. This work presents a versatile and trainable deep-learning model, termed DeepSea, that allows for both segmentation and tracking of single cells in sequences of phase-contrast live microscopy images with higher precision than existing models. We showcase the application of DeepSea by analyzing cell size regulation in embryonic stem cells., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

The success of an assisted reproduction cycle should be the achievement of a healthy singleton live birth following the replacement of one embryo. Therefore, one of the most critical points for embryologists has been the selection criteria and how to choose the best embryo to transfer with high implantation potential. In this vein, morphological evaluation has been historically the method applied. However, this practice relies on a limited number of single observations and is associated with high operator variability. Recently, a major innovation in embryo culture has been the introduction of a new type of incubator with integrated time-lapse monitoring, which enables the embryologist to analyze the dynamic events of embryo development, from fertilization to blastocyst formation. This novel practice is quickly growing and has been implemented in many IVF clinics worldwide. Therefore, the main aim of this review is to illustrate the benefits of time-lapse technology in a modern embryology laboratory. In particular, we discuss the blastocyst collapse(s) event and morphometric blastocyst assessment and analyse their association with embryo viability and implantation potential.

Research Question: Do morphokinetic parameters vary between male and female preimplantation embryos?, Design: This was a retrospective cohort study of 175 cycles between March 2018 and June 2021 at two reproductive centres. It included time-lapse data from 92 female and 83 male preimplantation embryos exclusively issued from fresh oocyte donation and undergoing intracytoplasmic sperm injection (ICSI). Only fresh elective single-embryo transfers on day 5 were assessed, and the sex of the embryo was confirmed at birth. The morphokinetic parameters analysed were measured in hours post-insemination (hpi). A two-tailed Student's t-test was used to compare the morphokinetics between embryo sexes and a value of P < 0.05 was considered statistically significant., Results: Following strict inclusion criteria to avoid poor-quality preimplantation embryos, no significant differences were found in morphokinetic parameters when comparing cycles that resulted in female versus male live births for the following: time to pronuclear fading (22.1 ± 2.4 versus 22.4 ± 2.9 hpi; P = 0.52); time to the 2-cell stage (24.6 ± 2.5 versus 25.0 ± 2.5 hpi; P = 0.34); time to the 3-cell stage (35.3 ± 3.3 versus 35.8 ± 3.1 hpi; P = 0.28); time to the 4-cell stage (36.3 ± 3.4 versus 36.9 ± 3.7 hpi; P = 0.20); time to the 5-cell stage (47.9 ± 4.6 versus 48.0 ± 4.8 hpi; P = 0.88); time to the 8-cell stage (54.0 ± 6.5 versus 54.1 ± 6.5 hpi; P = 0.91); time to the start of blastulation (86.3 ± 14.6 versus 85.7 ± 15.5 hpi; P = 0.78); and time to the full blastocyst stage (93.0 ± 16.9 versus 93.2 ± 17.2 hpi; P = 0.94)., Conclusions: There are no significant differences in morphokinetics between male and female preimplantation embryos., (Copyright © 2023 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Since the first pioneering studies on time-lapse systems (TLSs) for embryo incubation, many things have changed. Two main factors influence the development of modern time-lapse incubators for human in-vitro fertilization (IVF): 1) the switch from traditional cell culture incubators to benchtops incubators, more suitable for human IVF; and 2) the improvement of imaging technology. Another major factor for the increase in the utilization of TLSs in IVF labs over the last decade was the advances in computer/wireless and smartphone/tablet technology, which allowed patients to see the footage of their growing embryos. Hence, more user-friendly features have allowed their introduction and routine use in IVF labs while image-capturing software has enabled storage and providing additional information to the patients concerning the development of their embryos. This review aims to describe the history and the different TLSs available in the market, to summarize the research and clinical results obtained by using this technology, and to reflect on how this technology is changing the modern IVF laboratory. The current limitations of TLSs will be also reviewed.

Background: Time-lapse monitoring is increasingly used in fertility laboratories to culture and select embryos for transfer. This method is offered to couples with the promise of improving pregnancy chances, even though there is currently insufficient evidence for superior clinical results. We aimed to evaluate whether a potential improvement by time-lapse monitoring is caused by the time-lapse-based embryo selection method itself or the uninterrupted culture environment that is part of the system., Methods: In this three-armed, multicentre, double-blind, randomised controlled trial, couples undergoing in-vitro fertilisation or intracytoplasmic sperm injection were recruited from 15 fertility clinics in the Netherlands and randomly assigned using a web-based, computerised randomisation service to one of three groups. Couples and physicians were masked to treatment group, but embryologists and laboratory technicians could not be. The time-lapse early embryo viability assessment (EEVA; TLE) group received embryo selection based on the EEVA time-lapse selection method and uninterrupted culture. The time-lapse routine (TLR) group received routine embryo selection and uninterrupted culture. The control group received routine embryo selection and interrupted culture. The co-primary endpoints were the cumulative ongoing pregnancy rate within 12 months in all women and the ongoing pregnancy rate after fresh single embryo transfer in a good prognosis population. Analysis was by intention to treat. This trial is registered on the ICTRP Search Portal, NTR5423, and is closed to new participants., Findings: 1731 couples were randomly assigned between June 15, 2017, and March 31, 2020 (577 to the TLE group, 579 to the TLR group, and 575 to the control group). The 12-month cumulative ongoing pregnancy rate did not differ significantly between the three groups: 50·8% (293 of 577) in the TLE group, 50·9% (295 of 579) in the TLR group, and 49·4% (284 of 575) in the control group (p=0·85). The ongoing pregnancy rates after fresh single embryo transfer in a good prognosis population were 38·2% (125 of 327) in the TLE group, 36·8% (119 of 323) in the TLR group, and 37·8% (123 of 325) in the control group (p=0·90). Ten serious adverse events were reported (five TLE, four TLR, and one in the control group), which were not related to study procedures., Interpretation: Neither time-lapse-based embryo selection using the EEVA test nor uninterrupted culture conditions in a time-lapse incubator improved clinical outcomes compared with routine methods. Widespread application of time-lapse monitoring for fertility treatments with the promise of improved results should be questioned., Funding: Health Care Efficiency Research programme from Netherlands Organisation for Health Research and Development and Merck., Competing Interests: Declaration of interests DCK received the Fertility Society of Australia exchange award. MHJMC reports an unrestricted grant for implementing Value Based Healthcare paid to their institution and a personal speakers fee from Merck (Netherlands). FB reports a research support grant from Merck (Netherlands), Health Care Efficiency Research program grant from the Netherlands Organisation for Health Research and Development, speaker fees and scientific dinner symposium from Besins Healthcare Monaco, and is a member of the advisory board for Merck (Netherlands) and Ferring (Netherlands). CHdK reports a donation from Merck (Netherlands) for the European Society of Human Reproduction and Embryology annual meeting 2022 in Milan. MvW is coordinating editor of the Cochrane Gynecology and Fertility Group. CBL is Editor-In-Chief for Human Reproduction. All other authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Bacterial growth can be studied at the single cell level through time-lapse microscopy imaging. Technical advances in microscopy lead to increasing image quality, which in turn allows to visualize larger areas of growth, containing more and more cells. In this context, the use of automated computational tools becomes essential. In this paper, we present STrack, a tool that allows to track cells in time-lapse images in a fast and efficient way. We compared it to 3 recently published tracking tools on images ranging over 6 different bacterial strains with various morphologies. STrack showed to be the most consistent tracking tool, returning more than 80% of correct cell lineages on average, in comparison to manually annotated ground-truth. The python implementation of STrack, a docker structure, and a tutorial on how to download and use the tool can be found on the following github page: https://github.com/Helena-todd/STrack. IMPORTANCE Automated image analysis of growing prokaryotic cell populations becomes indispensable with larger data sets, such as derived by time-lapse microscopy. The tracking of the same individual cells and their daughter lineages is cumbersome and prone to errors in image alignment or poor resolution. Here, we present a simplified but highly effective tool for non-specialists to engage in cell tracking. The tool can be downloaded and run as a contained script-structure requiring minimal user input. Run times are fast, in comparison to other equivalent tools, and outputs consist of cell tables that can be subsequently used for lineage analysis, for which we offer examples. By providing open code, training data sets, as well as simplified script execution, we aimed to facilitate wide usage and further tool development for image analysis.

Over the years, the utilization of in vitro fertilization (IVF) in bovine embryo production has increased globally to accelerate the selection of cows with high genetic values. The selection of embryos with high implantation potential is a critical factor in establishing pregnancy. Time-lapse monitoring (TLM) has emerged as a new technique that allows frequent and non-invasive imaging of developing embryos. TLM is considered to have several advantages over the conventional morphological evaluation of embryos, which has been widely used in bovine embryo production. Establishing a novel embryo selection algorithm specifically for bovine IVF embryos is a critical challenge, but information on the association between morphokinetic data obtained using TLM and the implantation potential of embryos is still limited. This review outlines the potential application of TLM technology to improve the fertility of bovine IVF embryos, focusing on the results of human and bovine TLM studies that can be applied to select bovine embryos with high implantation potential. First, the progress of the TLM technology in bovine embryo production is summarized. The association between kinetic and morphological parameters and the developmental and implantation potential of human and bovine embryos is outlined. Finally, the benefits of evaluating blastocyst collapse and re-expansion as indicators of bovine embryo viability and the possible application of TLM to detect chromosomal abnormalities and determine embryo sex will be discussed.

Research Question: Can time-lapse parameters and the transcriptional profile of cumulus cells be used to achieve a more stringent and non-invasive method of embryo assessment and to identify possible factors affecting the embryo's ability to implant in repeated implantation failure (RIF) patients?, Design: A total of 190 embryos from 18 oocyte donors and 145 embryos from 15 RIF patients were evaluated based on time-lapse parameters. Three morphokinetic parameters including T5 (time to reach five cells), T3 (time to reach three cells) and CC2 (time to two to three cells) were recorded for all embryos. Embryos that had all three parameters in the normal range were graded as high quality and comparison between these parameters were compared in high-quality embryos between two groups. The transcriptional profile of cumulus cells related to high-quality embryos of both groups were analysed by RNA sequencing and compared. Finally, the possible relationship between differentially expressed genes and time-lapse parameters was examined., Results: T5 was significantly lower in the RIF group than the donor group (P = 0.011). The cumulus cell transcriptome analysis showed 193 genes were down-regulated and 222 genes up-regulated. The mammalian target of rapamycin and the transforming growth factor beta pathways were significantly increased in the RIF group compared to the donor group (P = 0.007 and 0.01, respectively). Vitamin B12 and fatty acid beta-oxidation pathways were also significantly reduced in the RIF group compared to the donor group (P = 0.006 and 0.01, respectively)., Conclusions: Differences in the transcriptomic profiles of cumulus cells and some morphokinetic parameters may be one of the main factors contributing to unexplained RIF., (Copyright © 2022 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

The goal for the present study was to investigate whether previous infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may compromise embryo morphokinetics and implantation. For that, a historical cohort study was performed in a private university-affiliated in vitro fertilization center. The study included 1628 embryos from 88 patients undergoing intracytoplasmic sperm injection (ICSI) cycles. Patients were age-matched in a 1:3 ratio to either a coronavirus disease (COVID) group, including patients with a positive SARS-CoV-2 immunoglobulin test (n = 22 patients, 386 embryos), or a control group, including patients with a negative SARS-CoV-2 immunoglobulin test (n = 66, 1242 embryos). The effect of previous infection with SARS-CoV-2 on morphokinetic events and ICSI outcomes was evaluated. Embryos derived from patients in the COVID group presented longer time to pronuclei appearance and fading, time to form two, three, four and five cells, and time to blastulation. The durations of the third cell cycle and to time to complete synchronous divisions were also significantly increased in the COVID group compared with the control group, whereas known implantation diagnosis score Day 5 ranked significantly lower in the COVID group. No differences were observed between the COVID and control groups on clinical outcomes. In conclusion, patients planning parenthood, who have recovered from COVID-19 infection, must be aware of a possible effect of the infection on embryo development potential., (© 2022 Wiley Periodicals LLC.)

Detailed and quantitative analyses of the cellular events underlying the formation of specific organs or tissues is essential to understand the general mechanisms of morphogenesis and pattern formation. Observation of live tissues or whole-mount fixed specimens has emerged as the method of choice for identifying and quantifying specific cellular and tissular structures within the organism. In both cases, cell and subcellular structure identification and good quality image acquisition for these analyses are essential. Many markers for live imaging and fixed tissue are now available for detecting cell membranes, subcellular structures, and extracellular structures like the extracellular matrix (ECM). Combination of live imaging and analysis of fixed tissue is ideal to obtain a general and detailed picture of the events underlying embryonic development. By applying morphometric methods to both approaches, we can, in addition, obtain a quantitative evaluation of the specific parameters under investigation in morphogenetic and cell biological studies. In this chapter, we focus on the development of the tracheal system of Drosophila melanogaster, which provides an ideal paradigm to understand the formation of branched tubular organs. We describe the most used methods of imaging and morphometric analysis in tubulogenesis using mainly (but not exclusively) examples from embryonic development. We cover embryo preparation for fixed and live analysis of tubulogenesis, together with methods to visualize larval tracheal terminal cell branching and lumen formation. Finally, we describe morphometric analysis and quantification methods using fluorescent images of tracheal cells., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Time-lapse microscopy plays critical roles in the studies of cellular dynamics. However, setting up a time-lapse movie experiments is not only laborious but also with low output, mainly due to the cell-losing problem (i.e., cells moving out of limited field of view), especially in a long-time recording. To overcome this issue, we have designed a cost-efficient way that enables cell patterning on the imaging surfaces without any physical boundaries. Using mouse embryonic stem cells as an example system, we have demonstrated that our boundary-free patterned surface solves the cell-losing problem without disturbing their cellular phenotype. Statistically, the presented system increases the effective-throughput of time-lapse microscopy experiments by an order of magnitude., Competing Interests: The authors have declared that no competing interests exist.

The primary purpose of this time-lapse data analysis was to identify the association between the nucleation status of a Day 2 preimplantation embryo and live births following in vitro fertilization (IVF). The retrospective data analysis was based on 2769 transferred embryos from 1966 treatment cycles and utilised only Known Implantation Data (KID) for live births. Nucleation errors (NE) such as micronucleation, binucleation, multinucleation and minor error groups, were annotated in the time-lapse images which were taken every 15 minutes for a minimum of 44 hours post insemination. Further, factors that may impact NE and the relationship of early morphological attributes and morphokinetic variables with NE occurrence were explored. The frequency of NE among the transferred embryos was 23.8%. The reversibility of NE evidenced by their presence at the two-cell stage, but absence at the four-cell stage was 89.6%. Embryos exhibiting nucleation errors at the two-cell stage had significantly lower live birth rates compared to embryos with no nucleation errors, constituting a significant predictor. A Generalized Additive Mixed Model was used to control for confounders and for controlling clustering effects from dual embryo transfers. Increased incidences of NE were observed with increasing age, with delayed occurrence of cell divisions and in oocytes inseminated with surgically retrieved spermatozoa. NE assessment and their impact on live birth provides valuable markers for early preimplantation embryo selection. In addition, the high incidence of reversibility of NE and their possible impact on live birth suggest that incorporating two-cell nuclear status annotations in embryo selection, alongside morphology and morphokinetics, is of value., Competing Interests: Co-author B.M. Petersen is the owner of BMP Analytics, an independent consultancy company. B.M.P has acted as freelance consultant for Vitrolife, as well as other enterprises within IVF and applied science in general. The corresponding author S.S, J.W.H and A.S are employees of Klinikk Hausken. M.M.R and R.S are employed at the Norwegian Research Centre on Women’s Health, Oslo University Hospital.The authors have declared no conflicting interests exists regarding the research presented in this manuscript. There are no pending patents, products in development or marketed products involved in this research. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

Conventional embryo assessment is performed by removing embryos from incubators at least once a day. However, it is static and limited to specific time points, reducing the amount of information that could potentially be obtained. Fortunately, the time-lapse system is a powerful technology that enables to observe embryo development progression by image acquisition at recurrent time intervals, without interfering in the culture conditions. There are numerous studies that used time-lapse incubators, focusing on embryo kinetics, patient characteristics and clinical outcomes. This review aims to find agreements in the literature concerning embryo kinetics and patient characteristics: age, body mass index, smoking habit, polycystic ovary syndrome and endometriosis; as well as culture conditions that involved culture media and oxygen concentration. Furthermore, they showed differences according to ploidy status, direct/reverse cleavage, gender and the potential association between embryo collapse and clinical outcomes.

Fertilization underpins the vital transition from gametic meiosis to embryonic mitosis. For decades, in human IVF, microscopic observation at a single time point has limited our appreciation of the morphokinetic complexity of this process. More recently, the introduction of time lapse technology-also enhanced by combination with artificial intelligence-has revealed the finest morphokinetic details of the beginning of human development. Overall, a picture has finally emerged in which the precise timing, morphology and geometry of several fertilization events offer clues to predict the fate of the embryo-a key aspect of assisted reproduction. In this scenario, correct unfolding of intra- and interpronuclear rearrangements emerge as a crucial factor to create a platform able to preserve genetic and cellular integrity at the first mitotic cleavage., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Research Question: What is the reliability of Geri® Assess 2.0 software time-lapse technology for annotating kinetic events and identifying abnormal phenotypes in preimplantation human embryos?, Design: Embryos were annotated using Assess 2.0 for the appearance and fading of pronuclei, and for progression to the 2-, 3-, 4-, 5- and 6-cell stages and to three blastocyst stages. Identification of reverse cleavage and direct cleavage phenotypes was also recorded. Manual annotation was undertaken after these events in a blinded fashion. Embryo scores were compared between Assess 2.0 and manual annotation., Results: A total of 513 oocytes from 34 women were included. Detection rates for Assess 2.0 versus manual annotation among the 10 kinetic events and including direct cleavage and reverse cleavage ranged between 0% and 94.4%. The percentage of discordant pairs was significantly different for all 12 events analysed (P-value range 0.036 to <0.0001). The sensitivity of Assess 2.0 ranged from 68.2% to 94.4% and specificity ranged from 63.8% to 97.3%. Assess 2.0 called for verification by the embryologist for at least one event in 55.2% of oocytes assessed. Of the 297 embryos scored by manual annotation, Assess 2.0 assigned the same score for only 125 (42.1%), although after manual corrections, concordance with manual annotation scores was raised to 66.0%., Conclusions: The results reveal striking differences between Assess 2.0 and manual annotation for kinetic annotations. Failure of Assess 2.0 to detect direct cleavage events and the low detection rate of reverse cleavage are further limitations. These collective findings highlight the importance of validating time-lapse annotation software before clinical implementation. Manual verification of Assess 2.0 outputs remains essential for accurate data interpretation., (Copyright © 2022 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Background: Embryo morphology is a predictive marker for implantation success and ultimately live births. Viability evaluation and quality grading are commonly used to select the embryo with the highest implantation potential. However, the traditional method of manual embryo assessment is time-consuming and highly susceptible to inter- and intra-observer variability. Automation of this process results in more objective and accurate predictions., Method: In this paper, we propose a novel methodology based on deep learning to automatically evaluate the morphological appearance of human embryos from time-lapse imaging. A supervised contrastive learning framework is implemented to predict embryo viability at day 4 and day 5, and an inductive transfer approach is applied to classify embryo quality at both times., Results: Results showed that both methods outperformed conventional approaches and improved state-of-the-art embryology results for an independent test set. The viability result achieved an accuracy of 0.8103 and 0.9330 and the quality results reached values of 0.7500 and 0.8001 for day 4 and day 5, respectively. Furthermore, qualitative results kept consistency with the clinical interpretation., Conclusions: The proposed methods are up to date with the artificial intelligence literature and have been proven to be promising. Furthermore, our findings represent a breakthrough in the field of embryology in that they study the possibilities of embryo selection at day 4. Moreover, the grad-CAMs findings are directly in line with embryologists' decisions. Finally, our results demonstrated excellent potential for the inclusion of the models in clinical practice., Competing Interests: Declaration of Competing Interest Authors declare that they have no conflict of interest., (Copyright © 2022. Published by Elsevier B.V.)

Significance: Quantitative differential phase contrast (qDPC) microscopy enhances phase contrast by asymmetric illumination using partially coherent light and multiple intensity measurements. However, for live cell imaging, motion artifacts and image acquisition time are important issues. For live cell imaging, a large number of intensity measurements can limit the imaging quality and speed. The minimum number of intensity measurements in qDPC can greatly enhance performance for live imaging., Aim: To obtain high-contrast, isotropic qDPC images with two intensity measurements and perform time-lapse imaging of biological samples., Approach: Based on the color-coded design, a dual-color linear-gradient pupil is proposed to achieve isotropic phase contrast response with two intensity measurements. In our method, the purpose of designing a dual-color coded pupil is twofold: first, to obtain a linear amplitude gradient for asymmetric illumination, which is required to get a circular symmetry of transfer function, and second, to reduce the required number of frames for phase retrieval., Results: To demonstrate the imaging performance of our system, standard microlens arrays were used as samples. We performed time-lapse quantitative phase imaging of rat astrocytes under a low-oxygen environment. Detailed morphology and dynamic changes such as the apoptosis process and migration of cells were observed., Conclusions: It is shown that dual-color linear-gradient pupils in qDPC can outperform half-circle and vortex pupils, and isotropic phase transfer function can be achieved with only two-axis measurements. The reduced number of frames helps in achieving faster imaging speed as compared to the typical qDPC system. The imaging performance of our system is evaluated by time-lapse imaging of rat astrocytes. Different morphological changes in cells during their life cycle were observed in terms of quantitative phase change values.

Mitosis detection plays an important role in the analysis of cell status and behavior and is therefore widely utilized in many biological research and medical applications. In this article, we propose a deep reinforcement learning-based progressive sequence saliency discovery network (PSSD)for mitosis detection in time-lapse phase contrast microscopy images. By discovering the salient frames when cell state changes in the sequence, PSSD can more effectively model the mitosis process for mitosis detection. We formulate the discovery of salient frames as a Markov Decision Process (MDP)that progressively adjusts the selection positions of salient frames in the sequence, and further leverage deep reinforcement learning to learn the policy in the salient frame discovery process. The proposed method consists of two parts: 1)the saliency discovery module that selects the salient frames from the input cell image sequence by progressively adjusting the selection positions of salient frames; 2)the mitosis identification module that takes a sequence of salient frames and performs temporal information fusion for mitotic sequence classification. Since the policy network of the saliency discovery module is trained under the guidance of the mitosis identification module, PSSD can comprehensively explore the salient frames that are beneficial for mitosis detection. To our knowledge, this is the first work to implement deep reinforcement learning to the mitosis detection problem. In the experiment, we evaluate the proposed method on the largest mitosis detection dataset, C2C12-16. Experiment results show that compared with the state-of-the-arts, the proposed method can achieve significant improvement for both mitosis identification and temporal localization on C2C12-16.

Elucidating phenotypic heterogeneity in clonal bacterial populations is important for both the fundamental understanding of bacterial behavior and the synthetic engineering of bacteria in biotechnology. In this study, we present and validate a high-throughput and high-resolution time-lapse fluorescence microscopy-based strategy to easily and systematically screen for heterogeneously expressed genes in the Bacillus subtilis model bacterium. This screen allows detection of expression patterns at high spatial and temporal resolution, which often escape detection by other approaches, and can readily be extrapolated to other bacteria. A proof-of-concept screening in B. subtilis revealed both recognized and yet unrecognized heterogeneously expressed genes, thereby validating the approach. IMPORTANCE Differential gene expression among isogenic siblings often leads to phenotypic heterogeneity and the emergence of complex social behavior and functional capacities within clonal bacterial populations. Despite the importance of such features for both the fundamental understanding and synthetic engineering of bacterial behavior, approaches to systematically map such population heterogeneity are scarce. In this context, we have elaborated a new time-lapse fluorescence microscopy-based strategy to easily and systematically screen for such heterogeneously expressed genes in bacteria with high resolution and throughput. A proof-of-concept screening in the Bacillus subtilis model bacterium revealed both recognized and yet unrecognized heterogeneously expressed genes, thereby validating our approach.

Cell tracking is one of the most critical tools for time-lapse image analysis to observe cell behavior and cell lineages over a long period of time. However, the accompanying graphical user interfaces are often difficult to use and do not incorporate seamless manual correction, data analysis tools, or simple training set design tools if it is machine learning based. In this paper, we introduce our cell tracking software "LIM Tracker". This software has a conventional tracking function consisting of recognition processing and link processing, a sequential search-type tracking function based on pattern matching, and a manual tracking function. LIM Tracker enables the seamless use of these functions. In addition, the system incorporates a highly interactive and interlocking data visualization method, which displays analysis result in real time, making it possible to flexibly correct the data and reduce the burden of tracking work. Moreover, recognition functions with deep learning (DL) are also available, which can be used for a wide range of targets including stain-free images. LIM Tracker allows researchers to track living objects with good usability and high versatility for various targets. We present a tracking case study based on fluorescence microscopy images (NRK-52E/EKAREV-NLS cells or MCF-10A/H2B-iRFP-P2A-mScarlet-I-hGem-P2A-PIP-NLS-mNeonGreen cells) and phase contrast microscopy images (Glioblastoma-astrocytoma U373 cells). LIM Tracker is implemented as a plugin for ImageJ/Fiji. The software can be downloaded from https://github.com/LIMT34/LIM-Tracker ., (© 2022. The Author(s).)

Assessing and selecting the most viable embryos for transfer is an essential part of in vitro fertilization (IVF). In recent years, several approaches have been made to improve and automate the procedure using artificial intelligence (AI) and deep learning. Based on images of embryos with known implantation data (KID), AI models have been trained to automatically score embryos related to their chance of achieving a successful implantation. However, as of now, only limited research has been conducted to evaluate how embryo selection models generalize to new clinics and how they perform in subgroup analyses across various conditions. In this paper, we investigate how a deep learning-based embryo selection model using only time-lapse image sequences performs across different patient ages and clinical conditions, and how it correlates with traditional morphokinetic parameters. The model was trained and evaluated based on a large dataset from 18 IVF centers consisting of 115,832 embryos, of which 14,644 embryos were transferred KID embryos. In an independent test set, the AI model sorted KID embryos with an area under the curve (AUC) of a receiver operating characteristic curve of 0.67 and all embryos with an AUC of 0.95. A clinic hold-out test showed that the model generalized to new clinics with an AUC range of 0.60-0.75 for KID embryos. Across different subgroups of age, insemination method, incubation time, and transfer protocol, the AUC ranged between 0.63 and 0.69. Furthermore, model predictions correlated positively with blastocyst grading and negatively with direct cleavages. The fully automated iDAScore v1.0 model was shown to perform at least as good as a state-of-the-art manual embryo selection model. Moreover, full automatization of embryo scoring implies fewer manual evaluations and eliminates biases due to inter- and intraobserver variation., Competing Interests: This study was supported by Vitrolife, the employer of J.B., J.R., J.T.L, M.F.K. All authors participated in the study design, data collection and analysis and preparation of the manuscript. The decision to publish was taken by J.B. Vitrolife produces and markets iDAScore. The study was also supported by Harrison.AI, the employer of D.T. He participated in the study design, data collection and analysis. D.T. has a patent related the current study. J.B. and J.R. are Vitrolife A/B shareholders. This does not alter our adherence to PLOS ONE policies on sharing data and materials, as detailed online in our guide for authors.

With self-supervised learning, both labeled and unlabeled data can be used for representation learning and model pretraining. This is particularly relevant when automating the selection of a patient's fertilized eggs (embryos) during a fertility treatment, in which only the embryos that were transferred to the female uterus may have labels of pregnancy. In this paper, we apply a self-supervised video alignment method known as temporal cycle-consistency (TCC) on 38176 time-lapse videos of developing embryos, of which 14550 were labeled. We show how TCC can be used to extract temporal similarities between embryo videos and use these for predicting pregnancy likelihood. Our temporal similarity method outperforms the time alignment measurement (TAM) with an area under the receiver operating characteristic (AUC) of 0.64 vs. 0.56. Compared to existing embryo evaluation models, it places in between a pure temporal and a spatio-temporal model that both require manual annotations. Furthermore, we use TCC for transfer learning in a semi-supervised fashion and show significant performance improvements compared to standard supervised learning, when only a small subset of the dataset is labeled. Specifically, two variants of transfer learning both achieve an AUC of 0.66 compared to 0.63 for supervised learning when 16% of the dataset is labeled.

The aim of this study was to investigate the use of time-lapse morphokinetic parameters and cumulus cells transcriptomic profile to achieve a more accurate and non-invasive method in embryo evaluation. Two hundred embryos from 20 couples were evaluated based on morphokinetic characteristics using time-lapse. Embryos were divided into the high-quality, moderate-quality, and bad-quality groups. Non-fertilized oocytes were considered as the fourth group. T5 (time to five cells), S2 (time from three to four cells), and CC2 (time from two to three cells) were recorded. Also, the cumulus cells of the respective oocytes were divided into high-quality, moderate-quality, bad-quality, and non-fertilized groups based on the grading of the embryos. Then their transcriptomic profiles were analyzed by RNA-sequencing. Finally, the correlation between differentially expressed genes and embryo time-lapse parameters was investigated. T5 was the only timing that showed a statistically significant difference between high-quality group and other groups. RNA-sequencing results showed that 37 genes were downregulated and 106 genes were upregulated in moderate, bad-quality, and non-fertilized groups compared to high-quality group (q value < 0.05). These genes were involved in the main biological processes such as cell cycle, DNA repair, cell signaling and communication, transcription, and cell metabolism. Embryos graded in different groups showed different transcriptomic profiles in the related cumulus cells. Therefore, it seems that embryo selection using the combination of cytokinetics and cumulus cells gene expression can improve the accuracy of the embryo selection and pregnancy rate in ART clinics., (© 2021. Society for Reproductive Investigation.)

Inhalation of mineral fibres is associated with the onset of an inflammatory activity in the lungs and the pleura responsible for the development of fatal malignancies. It is known that cell damage is a necessary step for triggering the inflammatory response. However, the mechanisms by which mineral fibres exert cytotoxic activity are not fully understood. In this work, the kinetics of the early cytotoxicity mechanisms of three mineral fibres (i.e., chrysotile, crocidolite and fibrous erionite) classified as carcinogenic by the International Agency for Research on Cancer, was determined for the first time in a comparative manner using time-lapse video microscopy coupled with in vitro assays. All tests were performed using the THP-1 cell line, differentiated into M0 macrophages (M0-THP-1) and exposed for short times (8 h) to 25 μg/mL aliquots of chrysotile, crocidolite and fibrous erionite. The toxic action of fibrous erionite on M0-THP-1 cells is manifested since the early steps (2 h) of the experiment while the cytotoxicity of crocidolite and chrysotile gradually increases during the time span of the experiment. Chrysotile and crocidolite prompt cell death mainly via apoptosis, while erionite exposure is also probably associated to a necrotic-like effect. The potential mechanisms underlying these different toxicity behaviours are discussed in the light of the different morphological, and chemical-physical properties of the three fibres., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Fluorescent carbon dots (CDs) are potential tools for the labeling of cells with many advantages such as photostability, multicolor emission, small size, rapid uptake, biocompatibility, and easy preparation. Affinity towards organelles can be influenced by the surface properties of CDs which affect the interaction with the cell and cytoplasmic distribution. Organelle targeting by carbon dots is promising for anticancer treatment; thus, intracellular trafficking and cytotoxicity of cationic CDs was investigated. Based on our previous study, we used quaternized carbon dots (QCDs) for treatment and monitoring the behavior of two human cancer cell MCF-7 and HeLa lines. We found similarities between human cancer cells and mouse fibroblasts in the case of QCDs uptake. Time lapse microscopy of QCDs-labeled MCF-7 cells showed that cells are dying during the first two hours, faster at lower doses than at higher ones. QCDs at a concentration of 100 µg/mL entered into the nucleus before cellular death; however, at a dose of 200 µg/mL, blebbing of the cellular membrane occurred, with a subsequent penetration of QCDs into the nuclear area. In the case of HeLa cells, the dose-depended effect did not happen; however, the labeled cells were also dying in mitosis and genotoxicity occurred nearly at all doses. Moreover, contrasted intracellular compartments, probably mitochondria, were obvious after 24 h incubation with 100 µg/mL of QCDs. The levels of reactive oxygen species (ROS) slightly increased after 24 h, depending on the concentration, thus the genotoxicity was likely evoked by the nanomaterial. A decrease in viability did not reach IC 50 as the DNA damage was probably partly repaired in the prolonged G0/G1 phase of the cell cycle. Thus, the defects in the G2/M phase may have allowed a damaged cell to enter mitosis and undergo apoptosis. The anticancer effect in both cell lines was manifested mainly through genotoxicity.

Improvements in microscopy software and hardware have dramatically increased the pace of image acquisition, making analysis a major bottleneck in generating quantitative, single-cell data. Although tools for segmenting and tracking bacteria within time-lapse images exist, most require human input, are specialized to the experimental set up, or lack accuracy. Here, we introduce DeLTA 2.0, a purely Python workflow that can rapidly and accurately analyze images of single cells on two-dimensional surfaces to quantify gene expression and cell growth. The algorithm uses deep convolutional neural networks to extract single-cell information from time-lapse images, requiring no human input after training. DeLTA 2.0 retains all the functionality of the original version, which was optimized for bacteria growing in the mother machine microfluidic device, but extends results to two-dimensional growth environments. Two-dimensional environments represent an important class of data because they are more straightforward to implement experimentally, they offer the potential for studies using co-cultures of cells, and they can be used to quantify spatial effects and multi-generational phenomena. However, segmentation and tracking are significantly more challenging tasks in two-dimensions due to exponential increases in the number of cells. To showcase this new functionality, we analyze mixed populations of antibiotic resistant and susceptible cells, and also track pole age and growth rate across generations. In addition to the two-dimensional capabilities, we also introduce several major improvements to the code that increase accessibility, including the ability to accept many standard microscopy file formats as inputs and the introduction of a Google Colab notebook so users can try the software without installing the code on their local machine. DeLTA 2.0 is rapid, with run times of less than 10 minutes for complete movies with hundreds of cells, and is highly accurate, with error rates around 1%, making it a powerful tool for analyzing time-lapse microscopy data., Competing Interests: The authors have declared that no competing interests exist.

Transcription factors (TFs) regulate cell fates, and their expression must be tightly regulated. Autoregulation is assumed to regulate many TFs' own expression to control cell fates. Here, we manipulate and quantify the (auto)regulation of PU.1, a TF controlling hematopoietic stem and progenitor cells (HSPCs), and correlate it to their future fates. We generate transgenic mice allowing both inducible activation of PU.1 and noninvasive quantification of endogenous PU.1 protein expression. The quantified HSPC PU.1 dynamics show that PU.1 up-regulation occurs as a consequence of hematopoietic differentiation independently of direct fast autoregulation. In contrast, inflammatory signaling induces fast PU.1 up-regulation, which does not require PU.1 expression or its binding to its own autoregulatory enhancer. However, the increased PU.1 levels induced by inflammatory signaling cannot be sustained via autoregulation after removal of the signaling stimulus. We conclude that PU.1 overexpression induces HSC differentiation before PU.1 up-regulation, only later generating cell types with intrinsically higher PU.1., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Ahmed et al.)

Purpose: To study the morphometric and morphokinetic profiles of pronuclei (PN) between male and female human zygotes., Method(s): This retrospective cohort study included 94 consecutive autologous single day 5 transfer cycles leading to a singleton live birth. All oocytes were placed in the EmbryoScope + incubator post-sperm injection with all annotations performed retrospectively by one embryologist (L-SO). Timing parameters included 2nd polar body extrusion (tPB2), sperm-originated PN (tSPNa) or oocyte-originated PN (tOPNa) appearance, and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2), and 0 h before PNF (stage 3), measuring PN area (um 2 ), PN juxtaposition, and nucleolar precursor bodies (NPB) arrangement., Results: Male zygotes had longer time intervals of tPB2&#95;tSPNa than female zygotes (4.8 ± 0.2 vs 4.2 ± 0.1 h, OR = 1.442, 95% CI 1.009-2.061, p = 0.044). SPN increased in size from stage 1 through 2 to 3 (435.3 ± 7.2, 506.7 ± 8.0, and 556.3 ± 8.9 um 2 , p = 0.000) and OPN did similarly (399.0 ± 6.1, 464.3 ± 6.7, and 513.8 ± 6.5 um 2 , p = 0.000), with SPN being significantly larger than OPN at each stage (p < 0.05 respectively). More male than female zygotes reached central PN juxtaposition at stage 1 (76.7% vs 51.0%, p = 0.010), stage 2 (97.7% vs 86.3%, p = 0.048), and stage 3 (97.7% vs 86.3%, p = 0.048). More OPN showed aligned NPBs than in SPN at stage 1 only (44.7% vs 28.7%, p = 0.023)., Conclusion(s): Embryos with different sexes display different morphokinetic and morphometric features at the zygotic stage. Embryo selection using such parameters may lead to unbalanced sex ratio in resulting offspring., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: The few studies that examined the effect of male and/or female features on early embryo development, notably using the time-lapse system (TL), reported conflicting results. This can be explained by the small number of studies using an adapted model., Methods: We used two original designs to study the female and male effects on embryo development: (1) based on embryos from donor oocytes (TL-DO), and (2) from donor sperm (TL-DS). Firstly, we analyzed the female and male similarities using an ad hoc intraclass correlation coefficient (ICC), then we completed the analysis with a multivariable model to assess the association between both male and female factors, and early embryo kinetics. A total of 572 mature oocytes (TL-DO: 293; TL-DS: 279), fertilized by intracytoplasmic sperm injection (ICSI) and incubated in a TL (Embryoscope®) were included from March 2013 to April 2019; 429 fertilized oocytes (TL-DO: 212; TL-DS: 217) were assessed. The timings of the first 48 h have been analyzed., Results: The similarities in the timings thought to be related to the female component were significant: (ICC in both DO-DS designs respectively: tPB2: 9-18%; tPNa: 16-21%; tPNf: 40-26%; t2: 38-24%; t3: 15-20%; t4: 21-32%). Comparatively, those related to male were lower. Surprisingly after multivariable analyses, no intrinsic female factors were clearly identified. However, in TL-DO design, oligozoospermia was associated with a tendency to longer timings, notably for tPB2 (p = 0.026)., Conclusion: This study quantifies the role of the oocyte in the first embryo cleavages, but without identified specific female factors. However, it also highlights that sperm may have an early embryonic effect., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

One obvious feature of life is that it is highly dynamic. The dynamics can be captured by movies that are made by acquiring images at regular time intervals, a method that is also known as time-lapse imaging. Looking at movies is a great way to learn more about the dynamics in cells, tissue, and organisms. However, science is different from Netflix, in that it aims for a quantitative understanding of the dynamics. The quantification is important for the comparison of dynamics and to study effects of perturbations. Here, we provide detailed processing and analysis methods that we commonly use to analyze and visualize our time-lapse imaging data. All methods use freely available open-source software and use example data that is available from an online data repository. The step-by-step guides together with example data allow for fully reproducible workflows that can be modified and adjusted to visualize and quantify other data from time-lapse imaging experiments., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Identification of cell phenotypic states within heterogeneous populations, along with elucidation of their switching dynamics, is a central challenge in modern biology. Conventional single-cell analysis methods typically provide only indirect, static phenotypic readouts. Transmitted light images, on the other hand, provide direct morphological readouts and can be acquired over time to provide a rich data source for dynamic cell phenotypic state identification. Here, we describe an end-to-end deep learning platform, UPSIDE (Unsupervised Phenotypic State IDEntification), for discovering cell states and their dynamics from transmitted light movies. UPSIDE uses the variational auto-encoder architecture to learn latent cell representations, which are then clustered for state identification, decoded for feature interpretation, and linked across movie frames for transition rate inference. Using UPSIDE, we identified distinct blood cell types in a heterogeneous dataset. We then analyzed movies of patient-derived acute myeloid leukemia cells, from which we identified stem-cell associated morphological states as well as the transition rates to and from these states. UPSIDE opens up the use of transmitted light movies for systematic exploration of cell state heterogeneity and dynamics in biology and medicine., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: P.S.B. receives research funding from AbbVie, Bristol-Myers Squibb, Cardiff Oncology, Pfizer, SecuraBio, Glycomimetics, Invivoscribe, JW Pharmaceutical, Novartis; and is an advisory board member with CVS Caremark.

Cdc42 is a key factor in directed cell migration and accumulates at the leading edge of migrating cells. However, what kind of proteins control Cdc42 and when is unclear. After mechanical wounding, protein kinase C α (PKCα), a conventional PKC isozyme, begins to accumulate at the edges of cells adjacent to the wounded cells (WCs). In this study, we hypothesized that PKCα may be implicated in directed cell migration at an early stage before Cdc42 controls the migration. We focused on the spatiotemporal distribution of PKCα, Cdc42, and Rac1 before cell migration. After wounding, at the edges of cells adjacent to the WCs, PKCα accumulation, Cdc42 accumulation, Rac1 accumulation, and filopodia formation occurred in that order. The PKCα inhibitor suppressed Cdc42 accumulation at the cell edges. These results suggest that inhibition of PKCα activity inhibits cell migration. In addition, it is not Cdc42 but PKCα that may decide the direction of cell migration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The purpose of this study was to determine if morphometric parameters that can be measured quantitatively using a time-lapse embryo incubator are associated with aneuploidy. Embryos cultured in a time-lapse incubator and assessed with preimplantation genetic testing for aneuploidy (PGT-A) were analyzed retrospectively. Morphokinetic analysis included timing of cell divisions. Quantitative morphometric measurements included the distance between the second and first polar body, zona pellucida thickness at the pronuclear stage and at the 2-cell stage, and blastomere area at the 2- and 4-cell stages. Symmetry at the 2-cell stage was determined by percent difference between blastomeres; symmetry at the 4-cell stage was the percent difference between the smallest and largest blastomeres. Maternal age, blastocyst grade and day of biopsy were recorded. Euploid embryo characteristics were compared to aneuploid embryos. A receiver operating characteristic (ROC) curve was used to evaluate cell symmetry as a predictor of aneuploidy. Embryos (n = 182) from 21 patients (age 22-43; median = 34) were analyzed. Of the 182 embryos, 45% were euploid. Euploid and aneuploid embryos had similar morphokinetics and morphometry across many measures. As expected, age and blastocyst grade were associated with embryo ploidy. It was notable that, additionally, symmetry at the 4-cell stage (27% vs 31%, p = 0.01) was also associated with embryo ploidy. The optimized cutoff from the ROC curve to predict aneuploidy was determined to be 21%. Embryos with > 21% asymmetry at the 4-cell stage had high rates of aneuploidy while morphokinetic parameters were similar. In conclusion, this suggests that embryo selection models using time-lapse parameters would improve if they incorporate cleavage-stage morphometrics., (© 2021. Society for Reproductive Investigation.)

Objective: To assess the relationship between maternal body mass index (BMI) and embryo morphokinetics on time-lapse microscopy (TLM)., Design: Retrospective cohort study., Methods: All IVF cycles between June 2015 and April 2017 were reviewed. Female BMI prior to egg retrieval was collected through chart review. BMI (kg/m 2 ) classification included underweight (< 18.5), normal weight (18.5-25), overweight (25-30), and obese (≥ 30). Embryos' morphokinetic parameters were assessed with TLM and included time to syngamy, 2-cell, 3-cell, 4-cell, and 8-cell. A generalized linear mixed model was used to control for potential confounders and multiple embryos resulting from a single IVF cycle., Results: A total of 2150 embryos from 589 IVF cycles were reviewed and included in the analysis. Classification based on BMI was as follows: underweight (N = 56), normal weight (N = 1252), overweight (N = 502), and obese (N = 340). After adjusting for race and use of intracytoplasmic sperm injection, the mean time to the 8-cell stage in the underweight group was 4.3 (95% CI: - 8.31, - 0.21) h less than in the normal weight group (P = 0.025) and 4.6 (95% CI: - 8.8, - 0.21) h less than in the obese group (p = 0.022). No significant difference was noted between race and TLM after controlling for possible confounders., Conclusions: Embryos from underweight women were demonstrated to have a faster time to the 8-cell stage than normal weight or obese women. No significant difference was noted for race. This study demonstrates that weight can be a factor contributing to embryo development as observed with TLM., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Stony coral exoskeletons build the foundation for the most biologically diverse marine ecosystems on Earth, coral reefs, which face major threats due to many anthropogenic-related stressors. Therefore, understanding coral biomineralization mechanisms is crucial for coral reef management in the coming decades and for using coral skeletons in geochemical studies. This study combines in-vivo imaging with cryo-electron microscopy and cryo-elemental mapping to gain novel insights into the biological microenvironment and the ion pathways that facilitate biomineralization in primary polyps of the stony coral Stylophora pistillata. We document increased tissue permeability in the primary polyp and a highly dispersed cell packing in the tissue directly responsible for producing the coral skeleton. This tissue arrangement may facilitate the intimate involvement of seawater at the mineralization site, also documented here. We further observe an extensive filopodial network containing carbon-rich vesicles extruding from some of the calicoblastic cells. Single-cell RNA-Sequencing data interrogation supports these morphological observations by showing higher expression of genes involved in filopodia and vesicle structure and function in the calicoblastic cells. These observations provide a new conceptual framework for resolving the ion pathway from the external seawater to the tissue-mineral interface in stony coral biomineralization processes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Peripheral myelin protein 2 (P2) is a fatty acid-binding protein expressed in vertebrate peripheral nervous system myelin, as well as in human astrocytes. Suggested functions of P2 include membrane stacking and lipid transport. Mutations in the PMP2 gene, encoding P2, are associated with Charcot-Marie-Tooth disease (CMT). Recent studies have revealed three novel PMP2 mutations in CMT patients. To shed light on the structure and function of these P2 variants, we used X-ray and neutron crystallography, small-angle X-ray scattering, circular dichroism spectroscopy, computer simulations and lipid binding assays. The crystal and solution structures of the I50del, M114T and V115A variants of P2 showed minor differences to the wild-type protein, whereas their thermal stability was reduced. Vesicle aggregation assays revealed no change in membrane stacking characteristics, while the variants showed altered fatty acid binding. Time-lapse imaging of lipid bilayers indicated formation of double-membrane structures induced by P2, which could be related to its function in stacking of two myelin membrane surfaces in vivo. In order to better understand the links between structure, dynamics and function, the crystal structure of perdeuterated P2 was refined from room temperature data using neutrons and X-rays, and the results were compared to simulations and cryocooled crystal structures. Our data indicate similar properties for all known human P2 CMT variants; while crystal structures are nearly identical, thermal stability and function of CMT variants are impaired. Our data provide new insights into the structure-function relationships and dynamics of P2 in health and disease., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Axons form the long-range connections of biological neuronal networks, which are built through the developmental process of axon guidance. Here, we describe a protocol to precisely and non-invasively control axonal growth trajectories in live zebrafish embryos using focal light activation of a photoactivatable Rac1. We outline techniques for photostimulation, time-lapse imaging, and immunohistochemistry. These approaches enable engineering of long-range axonal circuitry or repair of defective circuits in living zebrafish, despite a milieu of competing endogenous signals and repulsive barriers. For complete details on the use and execution of this protocol, please refer to Harris et al. (2020)., Competing Interests: P.A. is a consultant for Herophilus, Foresite Labs, and the New York Stem Cell Foundation. P.A. is a co-founder of Serqet Therapeutics. A patent application is pending based on this work (applicants: President and Fellows of Harvard College; inventors: P.A. and J.M.H). All other authors declare no competing interests., (© 2021.)

Circadian clocks are self-sustained and cell-autonomous oscillators. They respond to various extracellular cues depending on the time-of-day and the signal intensity. Phase Transition Curves (PTCs) are instrumental in uncovering the full repertoire of responses to a given signal. However, the current methodologies for reconstructing PTCs are low-throughput, laborious, and resource- and time-consuming. We report here the development of an efficient and high throughput assay, dubbed Circadian Single-Cell Oscillators PTC Extraction (Circa-SCOPE) for generating high-resolution PTCs. This methodology relies on continuous monitoring of single-cell oscillations to reconstruct a full PTC from a single culture, upon a one-time intervention. Using Circa-SCOPE, we characterize the effects of various pharmacological and blood-borne resetting cues, at high temporal resolution and a wide concentration range. Thus, Circa-SCOPE is a powerful tool for comprehensive analysis and screening for circadian clocks' resetting cues, and can be valuable for basic as well as translational research., (© 2021. The Author(s).)

Experience-dependent formation and removal of inhibitory synapses are essential throughout life. For instance, GABAergic synapses are removed to facilitate learning, and strong excitatory activity is accompanied by the formation of inhibitory synapses to maintain coordination between excitation and inhibition. We recently discovered that active dendrites trigger the growth of inhibitory synapses via CB1 receptor-mediated endocannabinoid signaling, but the underlying mechanism remained unclear. Using two-photon microscopy to monitor the formation of individual inhibitory boutons in hippocampal organotypic slices from mice (both sexes), we found that CB1 receptor activation mediated the formation of inhibitory boutons and promoted their subsequent stabilization. Inhibitory bouton formation did not require neuronal activity and was independent of G i/o -protein signaling, but was directly induced by elevating cAMP levels using forskolin and by activating G s -proteins using DREADDs. Blocking PKA activity prevented CB1 receptor-mediated inhibitory bouton formation. Our findings reveal that axonal CB1 receptors signal via unconventional downstream pathways and that inhibitory bouton formation is triggered by an increase in axonal cAMP levels. Our results demonstrate an unexpected role for axonal CB1 receptors in axon-specific, and context-dependent, inhibitory synapse formation. SIGNIFICANCE STATEMENT Coordination between excitation and inhibition is required for proper brain function throughout life. It was previously shown that new inhibitory synapses can be formed in response to strong excitation to maintain this coordination, and this was mediated by endocannabinoid signaling via CB1 receptors. As activation of CB1 receptors generally results in the suppression of synaptic transmission, it remained unclear how CB1 receptors can mediate the formation of inhibitory synapses. Here we show that CB1 receptors on inhibitory axons signal via unconventional intracellular pathways and that inhibitory bouton formation is triggered by an increase in axonal cAMP levels and requires PKA activity. Our findings point to a central role for axonal cAMP signaling in activity-dependent inhibitory synapse formation., (Copyright © 2021 the authors.)

Sterility and low seed set in bananas is the main challenge to their conventional genetic improvement. The first step to seed set in a banana breeding program depends on pollination at the right time to ensure effective fertilization. This study aimed at determining bract opening time (BOT) to enhance efficient pollination and seed set in bananas. A Nikon D810 digital camera was set-up to take pictures of growing banana inflorescences at five-minute intervals and time-lapse movies were developed at a speed of 30 frames per second to allow real-time monitoring of BOT. Genotypes studied included wild banana (1), Mchare (2), Matooke (4), Matooke hybrid (1), and plantain (1). Events of bract opening initiated by bract lift for female flowers (P < 0.01) started at 16:32 h and at 18:54 h for male flowers. Start of bract rolling was at 18:51 h among female flowers (P < 0.001) and 20:48 h for male flowers. Bracts ended rolling at 02:33 h and 01:16 h for female and flowers respectively (P < 0.05). Total time of bract opening (from lift to end of rolling) for female flowers was significantly longer than that of male flowers (P < 0.001). On average, the number of bracts subtending female flowers opening increased from one on the first day, to between one and four on the fourth day. The number regressed to one bract on day eight before start of opening of bracts subtending male flowers. There was a longer opening interval between bracts subtending female and male flowers constituting spatial and temporal separation. Bract rolling increased from partial to complete rolling from proximal to the distal end of the inflorescence among female flower. On the other hand, bracts subtending male flowers completely rolled. Differences in BOT of genotypes with the same reference time of assessment may be partly responsible for variable fertility. Hand pollination time between 07:00 and 10:00 h is slightly late thus an early feasible time should be tried., (© 2021. The Author(s).)