Comprehensive Notes on Time-Lapse Imaging in IVF

Time-Lapse Imaging and Morphokinetic Analysis in IVF

Introduction to Time-Lapse Imaging (TLI)

• Time-lapse imaging (TLI) is a non-invasive method that allows continuous monitoring of embryo development without disturbing culture conditions.
• TLI involves recording a large amount of morphokinetic data, describing the dynamics of the embryo during its in vitro development.
• Annotation of the exact time at which developmental events occur is a unique feature of TLI that cannot be performed using conventional incubation.
• Accurate timing of events like early cleavage, cell cycle intervals, synchronicity of cell divisions, and start of blastulation are associated with better pregnancy outcomes.
• Predictive parameters on developmental kinetics have been identified and used to create predictive models for implantation, live birth, and aneuploidy.

Traditional Embryo Selection Methods and Their Limitations

• Traditionally, embryos are selected based on morphological parameters at single time points, a subjective methodology.
• On day 3, embryos are classified by cell number, fragmentation, cell symmetry, multinucleation, and other alterations.
• Day 5 blastocysts are assessed by expansion degree, number, cohesion and homogeneity of trophectoderm cells, and compaction and area of inner cell mass.
• There is no strong correlation between morphology and embryonic competence due to high interobserver and intraobserver variability.
• Embryo status can change within a few hours, and significant events may be missed between observational time points.
• Increasing observations may improve information but expose the embryo to undesirable environmental changes.

Materials and Methods

• A systematic search of PubMed, Embase, the Web of Science, and the Cochrane Library was performed up to April 2023.
• Medical subject headings and free text terms used: ‘‘time-lapse technology,’’ ‘‘timelapse monitoring,’’ ‘‘time-lapse imaging,’’ ‘‘morphokinetics,’’ ‘‘human embryo kinetics,’’ ‘‘blastocyst grade,’’ ‘‘IVF outcome,’’ ‘‘embryo transfer,’’ ‘‘implantation,’’ ‘‘pregnancy,’’ ‘‘live birth,’’ and ‘‘embryo ploidy outcome.’’
• Three researchers (C.G., L.C., and L.M.) performed literature searches. After exporting the search results to a citation manager (EndNote) and removing duplicates, the articles were screened by two researchers (C.G. and L.C.).
• Eligibility criteria included studies reporting IVF outcomes for embryos cultured in TLI incubators. Analyzed outcomes: blastocyst development grade, implantation outcome, pregnancy outcome, live birth outcome, and embryo ploidy outcome.
• Exclusion criteria were case reports and letters.
• Data extraction was performed independently by two reviewers (C.G. and L.C.).
• Extracted information included study description, participant characteristics, types of TLI used, and outcomes.

Results of the Study Selection

• A total of 1,395 citations were identified, with 105 duplicates removed.
• 112 citations were selected for full-text evaluation.
• 47 articles were included in the review.

Characteristics of Included Studies

• The included studies were published between 1997 and 2023 and were clinical-based.
• Studies included 1 guideline, 11 prospective studies, and 35 retrospective studies.
• The number of embryos analyzed per publication participant varied from 75 to 41,000.

Embryo Culture in Time-Lapse (TL) Systems

• Time-lapse imaging systems have emerged as a technology that allows integration of the most stable embryo culture conditions.
• Since the first application of TLI in 1997, different options for TLI systems have developed.
• TLI systems include: Embryoscope, Embryoscopeþ, Geri, Geriþ, Miri TL, CCM-iBIS, Primo Vision ,and the Eeva Test.
• Culture in TLI systems does not compromise embryonic development and newborn outcomes compared with conventional systems.
• Whether TL incubators offer improvements in clinical outcomes over conventional incubators is still debated.
• Some studies have reported no significant improvement in terms of fertilization rate, embryo quality, and pregnancy rate.

Potential Benefits of TLI Incubators

• TLI incubators provide a more stable environment.
• The potential benefits of reducing disruption and providing a more stable environment with TLI incubators are more likely to be realized when embryos are incubated until the blastocyst stage.
• TLI systems can improve IVF outcomes compared with conventional incubation according to numerous strong studies.
• A meta-analysis showed that ongoing pregnancy and live birth rates were significantly increased using TLI.
• Early pregnancy loss was significantly reduced, and no differences were found in stillbirth rates.

Transition of TLI to a Selection and Prediction Tool

• Time-lapse imaging systems provide an opportunity for optimizing embryo selection on the basis of morphological grading and novel kinetic parameters.
• Morphokinetics may further improve the accurate selection of viable embryos and, therefore, IVF outcomes.

Morphokinetic Parameters and Abnormal Phenotypes

• The vast amount of morphokinetic data collected as a result of TLI has been proposed as a possible predictor of IVF treatment outcomes.
• Guidelines were proposed by Ciray et al. (38) to standardize the nomenclature and annotation of morphokinetic parameters.
• TL videos have been shown to be useful in the detection of atypical features of embryonic development that can compromise competence.
• Abnormal phenotypes include those affected by direct cleavage, reverse cleavage, fragmentation, internalization of cellular fragments, multinucleation, cell exclusion, or blastocyst collapse.
• Direct cleavage, or trichotomous mitosis, can consist of either the division of the zygote into three cells or the division of a blastomere into three cells in the first or second cell division cycle.
• Reverse cleavage is defined as the fusion of two cells into one blastomere or the failure of cytokinesis.

Blastocyst Development Studies

• Studies have been conducted to associate morphokinetic markers with good-quality embryos and predict blastocyst formation.
• The first study analyzing the relationship between morphokinetics and blastocyst formation was performed by Wong et al. (39) in 2010.
• High-scoring blastocysts exhibited significantly shorter completion times for these divisions.
• Faster fertilization kinetics were associated with better embryonic development.

Implantation, Pregnancy, and Live Birth Studies

• The scientific community has attempted to correlate kinetic markers with embryo implantation and live birth.
• Meseguer et al. (12) performed the first study of TLI in a clinical environment, associating morphokinetics with implantation.
• This study introduced the notion of optimal ranges for kinetic parameters.
• The quantitative parameters predicting implantation potential were cc2 and t5.
• Time to pronuclei fading (tPNf) has been described as a potential predictor of implantation in different studies.
• The morphokinetic prediction model (KIDScore Day 5) relates t2, t3, t4, t5, and tB with the chances of pregnancy and live birth after blastocyst transfer.
• Higher embryo scores were significantly associated with better clinical outcomes after IVF treatment.

Aneuploidy Studies

• Transferring only euploid embryos should increase live birth rates by increasing implantation rates and/or decreasing miscarriage rates.
• The correlation between embryo ploidy and embryo kinetics has been explored.
• Euploid embryos exhibited precise timing during the initial cell divisions up to the four-cell stage.
• The most notable variables associated with embryo ploidy were t8, t9, tB, and tHN.
• A study showed that the percentage of embryos affected by direct cleavage decreased throughout development either because they had reduced viability and/or because aneuploid cell lineages were purged or failed to propagate.

Discussion

• The main challenge of in vitro culturing human embryos is to keep them as close to their natural environment as possible.
• Specialized plates and closed TL units with image capture have been the subject of many studies in the context of embryo culture and manipulation.
• TLI does not have any negative impact on embryonic development compared with conventional methods using sibling oocytes.
• An essential benefit of TLI is its capability to document and evaluate embryo morphology and the timing of developmental events through continuous live image tracking.
• TLI simplifies the logistics of the IVF laboratory, enabling the evaluation and grading of embryos at any desired time point during the daily workflow.
• Information on developmental progression obtained through TLI can aid in training embryologists to assess embryo quality.
• By utilizing a single culture medium approach, embryos can remain within the TL system continuously, starting from intracytoplasmic sperm injection or fertilization and denudation until the day of transfer or cryopreservation.
• Many studies have attempted to assess the embryo on the basis of different potential morphokinetic parameters.
• Moreover, TLI opens avenues for conducting comparative studies.
• In the last decade, other studies have attempted to correlate different kinetic markers and events with embryo ploidy detection.
• Continuous recording of embryo development provides high-quality images that allow embryologists to identify precise markers to determine embryo quality and to choose the best embryo for transfer.

Artificial Intelligence (AI) in Embryo Selection

• Several algorithms have been developed using morphokinetic parameters to improve embryo evaluation and selection.
• Innovative AI techniques have the potential to shift the focus of study from a restricted set of independent variables to a large collection of big data.
• The utilization of artificial neural networks has witnessed a significant sevenfold increase in precision, yielding promising outcomes.
• The application of AI has demonstrated potential for automating embryo evaluation and enhancing the efficacy of manual sorting.
• TLI in conjunction with AI could be useful in the selection of the embryo for transfer that is most likely to be euploid and viable.

Combination of Morphokinetics with Other Technologies

• Combination of morphokinetics with proteomics to identify relevant embryonic features.
• Combination of morphokinetics and the oxidative status of the spent embryo culture medium to develop a noninvasive selection algorithm.
• The levels of oxidative parameters on day 5 of incubation were significantly higher in successfully implanted embryos than in those that did not result in pregnancy.
• The combination of technologies may be key to improving the results of IVF treatments in the future.

Conclusion

• Morphology has been the primary criterion for assessing embryonic development since the beginning of IVF practice.
• Research on noninvasive screening of the embryo is an ongoing process, and the focus is on identifying new biomarkers to support morphological methods.
• Time-lapse imaging provides valuable information and has enormous potential to enhance our understanding of embryonic development.
• Considerable knowledge has been accumulated to describe the morphokinetic dynamics during different stages in human embryos.
• The combination of morphokinetics obtained using TLI with other technologies and their study using AI has been a turning point in embryo evaluation, allowing the emergence of new studies that allow an objective assessment of embryos.