Time‐lapse systems for embryo incubation in assisted reproduction
Abstract
This is a protocol for a Cochrane Review (Intervention). The objectives are as follows:
To determine the effect of TLS compared to standard embryo incubation on clinical outcomes in women undergoing assisted reproductive technology (ART).
Background
Description of the condition
Embryo incubation is a critical step in all in‐vitro fertilisation (IVF) procedures. Embryo development within the incubator is a dynamic process, moving through the fertilisation stage to cleavage stage and then to the blastocyst stage in some cases. Throughout the incubation period, embryos are usually inspected at specific intervals to provide a brief 'snap‐shot' in order to assess development and morphological features. A consensus on the minimum dataset required for the accurate description of embryo development was recently established by Alpha Scientists in Reproductive Medicine and European Society of Human Reproduction and Embryology (ESHRE) Special Interest Group of Embryology (Alpha 2011). A consensus on timings of observation of fertilized oocytes and embryos was established and deemed critical to the ability to compare results between different laboratories. The checks recommended in hours, give or take one or two hours following insemination are:
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a fertilisation check at 17, a syngamy check at 23
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an early cleavage check at 26 post intracytoplasmic sperm injection (ICSI) or 28 post IVF
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day 2 embryo assessment at 44
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a day 3 embryo assessment at 68
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day 4 embryo assessment at 92
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day 5 embryo assessment at 116
Traditionally this has been achieved by physically removing embryos from the controlled environment of the incubator, to analyse them under a light microscope for assessment of embryo development and quality. This practice exposes embryos to potentially sub‐optimal conditions of the environment outside of the incubator and human handling (Meseguer 2012). Time‐lapse systems (TLS) have evolved over recent years to increase the frequency of morphological observations whilst minimising the impact of the external environment and human handling on embryos.
Description of the intervention
A TLS is a device which takes digital images of embryos at set time intervals. The system can be installed into an existing embryo incubator or can exist as a combined time‐lapse incubation system. The images are compiled using specialist software to create a time‐lapse sequence of embryo development. Images can be digitally displayed on a monitor to allow embryologists to assess the morphology of embryos thus negating the need for the embryologist to remove embryos from the incubator for morphological assessment. Some TLS also utilise computer‐assisted assessment of developmental milestones of embryos, also known as morphokinetic parameters, to offer a semi‐quantative process of embryo evaluation (Conaghan 2013). These cell‐tracking software algorithms have evolved as a non‐invasive, non‐subjective way of attempting to improve the selection of embryos with the highest implantation potential.
There are a number of TLS on the market developed by various manufacturers. TLS are available as devices that can be placed within existing conventional incubators, and some exist with an integrated incubator. The integrated TLS regulates the gas conditions within the incubator by continuously recycling the internal gas volume through filters and monitoring carbon dioxide (CO2) and oxygen concentrations. Oxygen and CO2 concentrations are regulated by mixing CO2 and nitrogen (N2) into the internal airstream. A constant temperature is maintained by direct thermal contact between the culture dish and the dish holder. Images of each embryo can be obtained in a variety of focal planes at various time intervals determined by the embryologist (FertiliTech 2014). Within this integrated TLS, a non‐humid environment is maintained and the imaging of embryos is obtained by internal optics which are light sensitive and designed to work with illumination from a single red LED providing light at 635nm.
How the intervention might work
The potential advantages of TLS can be divided into two distinct categories. First, the ability of TLS to accumulate detailed time‐lapse images of embryo development including timing of cell divisions, intervals between cell cycles and other factors such as the dynamic pronuclei patterns, presence of multinucleation and blastomere symmetry. These detailed images can be utilised either by cell‐tracking software algorithms or by embryologists undertaking morphological assessment, to select the highest morphological quality embryo for transfer. This is important because there is a clear correlation between embryo morphology and viability (Finn 2010; Neuber 2006).
The second advantage is the effect of improved culture conditions, whereby images can be obtained without removing embryos from the incubator environment for conventional bench‐top light microscopy, therefore minimising the exposure of embryos to human handling and changes in air temperature and gas composition.
Therefore the rationale for time‐lapse systems is that they could potentially improve the selection of embryos most likely to implant and develop to term alongside providing embryos with a stable culture environment, both of which may improve IVF live birth rates.
Countering these potential benefits, TLS involve exposing embryos to frequent flashes of light which may be potentially harmful. In addition TLS technology is costly, adding an additional cost to IVF treatments.
Why it is important to do this review
New interventions, such as TLS, should be evaluated by randomised controlled trials to establish their safety, clinical and cost‐effectiveness. Countering the potential benefit outlined in the description of the intervention, TLS involves exposing embryos to light during image aquisition at pre‐determined intervals. This exposure to UV radiation, although likely to be low, does mean that there is potential for harm. Furthermore, authorities responsible for the regulation of fertility clinics and research involving human embryos have a responsibility to provide impartial and authoritative information to prospective and current patients on fertility treatments to allow them to make informed decisions on their care (ACART, HFEA). However, there are currently no systematic reviews comparing time‐lapse systems to standard embryo incubation, despite the novel technology having been adopted by numerous fertility clinics worldwide, many of whom charge their patients to use the technology. Therefore, establishing the technology's success rates in terms of live birth or ongoing pregnancy rate, and safety in terms ofadverse events, will provide vital information for couples seeking fertility treatment and the clinicians and scientists that provide the treatment.
The potential advantages of TLS can be divided into two distinct categories. Firstly the ability of the system to accumulate multiple, detailed, time‐lapse images of embryos which can be utilised either by cell‐tracking software algorithms or by embryologists undertaking morphological assessment, to select the highest quality embryo(s) for transfer. Secondly, the effect of improved culture conditions, whereby human handling is reduced, the air temperature and gas compositions are kept stable, and embryos are not exposed to bench‐top light microscopy. This review aims to establish whether there is evidence of any overall benefit of TLS over current conventional care. Furthermore, this review also aims to assess the evidence from trials that utilise cell‐tracking software algorithms versus morphological assessment of TLS images by an embryologist.
Objectives
To determine the effect of TLS compared to standard embryo incubation on clinical outcomes in women undergoing assisted reproductive technology (ART).
Methods
Criteria for considering studies for this review
Types of studies
Inclusions: Any randomised trial, whether published or not, which in principle can answer questions regarding clinical (post implantation) outcomes. Quasi‐randomised and other concurrently controlled studies will be excluded. Trials that randomise oocytes or embryos will be excluded as it would not be possible to compare clinical outcomes. Cross‐over trials will be excluded as the design is not valid in this context.
Exclusions: trials which have randomised oocytes or embryos over the woman or couple. Cross‐over trials.
Types of participants
Women of any age undergoing assisted reproduction where embryo incubation is required.
Types of interventions
1) TLS versus conventional incubation, with assessment by routine morphological criteria in both arms
2) TLS with cell‐tracking algorithms versus TLS with assessment by routine morphological criteria from TLS images
3) TLS utilising cell‐tracking algorithms versus conventional incubation with assessment by routine morphological criteria
We will be examining time‐lapse embryo imaging systems of any manufacturer, compared with standard incubation of any manufacturer.
Types of outcome measures
Primary outcomes
1. Live birth rate per woman randomly assigned
2. Adverse events such as multiple pregnancy
Secondary outcomes
1. Clinical pregnancy, defined as evidence of a gestational sac, confirmed by ultrasound per woman randomly assigned
Search methods for identification of studies
SA and NA will identify as many relevant RCTs as possible of 'time‐lapse incubation, time‐lapse imaging, EmbryoScope®, time‐lapse monitoring system, time‐lapse cinematography' for 'assisted reproduction', irrespective of their language of publication, publication date and publication status (published, unpublished, in press and in progress). We will use both electronic searches of bibliographic databases and handsearching, as described in the Cochrane Handbook for Systematic Reviews of Interventions.
Electronic searches
We will discuss our search request with the Trials Search Co‐ordinator (TSC) of the Menstrual Disorders and Subfertility Cochrane Review Group in order to implement a comprehensive search strategy to capture as many relevant RCTs as possible in electronic databases. For this purpose we will use a combination of controlled vocabulary (MeSH, Emtree, DeCS, including exploded terms) and free‐text terms (considering spelling variants, synonyms, acronyms and truncation) for 'time‐lapse system' and 'assisted reproduction', with field labels, proximity operators, and boolean operators.
Specifically we will search in the following electronic databases:
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MEDLINEⓇ In‐Process & Other Non‐Indexed Citations, Ovid platform (1946 to present)
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Ovid
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The Cochrane Central Register of Controlled Trials (CENTRAL), Ovid platform, (1991 to present)
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LILACS, IAHx interface (1982 to present)
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PsycINFO, Ovid platform (1946 to present);
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Cumulative Index to Nursing and Allied Health (CINAHL) (inception to present).
We will use for MEDLINE, Cochrane Highly Sensitive Search Strategy for Identifying RCTs: Sensitivity and Precision Maximizing Version (2008 revision), Ovid format (Higgins 2011). The LILACS search strategy will be combined with the RCT filter of the IAHx interface. These searches will be updated within six months before publication of the full review.
Searching other resources
We will attempt to identify additional relevant RCTs by using the following methods:
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searching in the Menstrual Disorders and Subfertility Cochrane Review Group's Specialised Register (using the term 'time‐lapse system' in the title, abstract and keywords), that includes RCTs and controlled clinical trials from 1944 to present, located through electronic searching (MEDLINE, EMBASE and CENTRAL) and handsearching;
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searching in trials registers:
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World Health Organisation (WHO) International Clinical Trials Registry Platform ICTRP portal (apps.who.int/trialsearch/);
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ClinicalTrials.gov (clinicaltrials.gov/)
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searching the Web of Knowledge (inception to present) using key words Topic=(time‐lapse) AND Topic=(system) AND Topic=(assisted reproduction). Refined by: Document Types=(CLINICAL TRIAL). Timespan=All Years;
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searching in Proquest Dissertations and Theses (search.proquest.com) (inception to present);
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searching for grey literature through the System for Information on Grey Literature in Europe 'OpenGrey' (www.opengrey.eu/): 1990, 1992, 1995, 1996 and 1997.
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searching by contact with authors of all RCTs identified by other methods;
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searching by contact with manufacturers of time‐lapse systems;
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handsearching of selected journals in obstetrics, gynaecology and reproductive medicine, as well as conference proceeding (for abstracts) of the European Society for Human Reproduction and Embryology (ESHRE) and the American Society for Reproductive Medicine (ASRM).
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searching through known experts and personal contacts regarding unpublished materials.
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Citation lists of all articles for any relevant reference
Data collection and analysis
Selection of studies
Two authors (SA and NA), will independently scan the titles and abstracts of the articles retrieved by the search. Full texts of potentially eligible studies will be obtained and examined independently by authors for their suitability according to the inclusion criteria. In the case of doubt between the two authors, a third author will be consulted to gain consensus on whether to include the trial or not. The selection process will be documented with a Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) flow chart.
Data extraction and management
The data will be obtained and extracted by two review authors, SA and NA. In the case of disagreement between the two authors, a third author will be consulted to achieve consensus (LC). Data will be extracted using a data extraction form designed and piloted by the authors. If studies are reported in multiple publications, the data will be extracted from the different publications and will be combined into a single data extraction form so no data will be omitted. The following characteristics of included studies will be included in the data extraction form:
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methods
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participants
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interventions
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outcomes, including adverse events
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funding source for studies
Assessment of risk of bias in included studies
For this review, assessment of risk of bias will be conducted by two authors (SA and NA), using the Cochrane risk of bias assessment tool to evaluate all included studies for the following: adequacy of sequence generation and allocation concealment; adequacy of blinding of women, providers and outcome assessors; completeness of outcome data; risk of selective outcome reporting and risk of other potential sources of bias (Higgins 2011).
Disagreements will be resolved by consensus. The results of the assessment of risk of bias will be presented in the characteristics of included studies and in a summary table. These results will be incorporated into the interpretation of review findings by means of sensitivity analyses.
Measures of treatment effect
For dichotomous data (e.g. live birth or not), odds ratios and their standard errors will be calculated and entered into tables.
Unit of analysis issues
If studies have expressed data per cycle or per embryo transfer, the data will be analysed per woman randomised.
Dealing with missing data
If relevant data are missing from an included study, the original investigators of the trial will be contacted to request the missing data. If the original investigator can not be contacted or does not reply, we will determine whether to include or exclude the trial from the meta‐analysis, or to only include the available data.If participants are described as 'lost to follow up' without a specified reason, we will assume the participant did not experience the event or outcome (i.e. did not become pregnant)
Assessment of heterogeneity
We will consider whether the clinical and methodological characteristics of the included studies are sufficiently similar for meta‐analysis to provide a clinically meaningful summary. Statistical heterogeneity will be assessed by measuring the I² statistic. We will assume that there is substantial heterogeneity when I² is calculated to be greater than 50% (Higgins 2011).
Assessment of reporting biases
In view of the difficulty of detecting and correcting for publication bias and other reporting biases, the authors will aim to minimise their potential impact by ensuring a comprehensive search for eligible studies and by being alert to duplication of data. If there are 10 or more studies in an analysis, we will use a funnel plot to explore the possibility of small study effects (a tendency for estimates of the intervention effect to be more beneficial in smaller studies). Within study reporting bias will be assessed, and assessed as low risk if all of the study's pre‐specified primary outcomes have been reported as outlined in the study's protocol.
Data synthesis
Where sufficient data are available, data will be combined for the primary outcomes by using a fixed‐effect model. The following comparisons will be made:
1) Studies that compare the culture conditions but apply consistent embryo selection policies, i.e. Women randomised to either TLS or conventional incubation, but assessment by routine morphological criteria in both arms
2) Studies that use consistent culture conditions but randomise to use the additional information available from TLS to inform selection decisions i.e. Women randomised to either TLS utilising cell‐tracking algorithms, or TLS with assessment by routine morphological criteria from TLS images
3) Studies that compare TLS in its entirety against CI, i.e. Women randomised to either TLS utilising cell‐tracking algorithms, or conventional incubation with assessment by routine morphological criteria
If it was not possible to extract from a trial report dichotomous data suitable for the calculation of ORs then statistical data will be reported in Additional Tables. Where trial results are presented only as graphs, findings will be described in the text. Data will be stratified according to type of TLS (stand alone TLS within a conventional incubator, or combined TLS incubation system) and, for comparisons 1 and 3, by nature of conventional culture conditions.
Subgroup analysis and investigation of heterogeneity
Where sufficient data are available, we will conduct the following subgroup analyses to determine the potential causes of heterogeneity for live birth and clinical pregnancy outcomes:
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Donor oocytes (from donors of any age)
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Fresh cycles (where embryos are replaced either at cleavage stage or blastocyst)
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Frozen cycles (where frozen embryos are replaced in an ART cycle)
If we detect substantial heterogeneity we will explore this by employing the random effects model. We will take any statistical heterogeneity into account when interpreting the results, especially if there is any variation in the direction of effect.
Sensitivity analysis
Sensitivity analyses will be undertaken for the primary review outcomes to determine whether the results are robust to decisions made during the review process. These analyses will include consideration of whether the review conclusions would have differed if:
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the summary effect measure was relative risk rather than odds ratio
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alternative imputation strategies had been implemented
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eligibility was restricted to studies without high risk of bias
Overall quality of the body of evidence: Summary of fndings table
We will prepare a Summary of findings table using GRADEPRO or Guideline Development Tool software. This table will evaluate the overall quality of the body of evidence for the review outcomes (live birth, adverse events and clinical pregnancy), using GRADE criteria (study limitations (i.e. risk of bias), consistency of effect, imprecision, indirectness and publication bias). Judgements about evidence quality (high, moderate or low) will be justified, documented, and incorporated into reporting of results for each outcome.
