Time-lapse systems for embryo incubation and assessment in assisted reproduction(review)

Transcription

1 Cochrane Database of Systematic Reviews Time-lapse systems for embryo incubation and assessment in assisted reproduction(review) ArmstrongS,ArrollN,CreeLM,JordanV,FarquharC ArmstrongS,ArrollN,CreeLM,JordanV,FarquharC. Time-lapse systems for embryo incubation and assessment in assisted reproduction. Cochrane Database of Systematic Reviews 2015, Issue 2. Art. No.: CD DOI: / CD pub2. Time-lapse systems for embryo incubation and assessment in assisted reproduction(review) Copyright 2015 The Cochrane Collaboration. Published by John Wiley& Sons, Ltd.

2 T A B L E O F C O N T E N T S HEADER ABSTRACT PLAIN LANGUAGE SUMMARY SUMMARY OF FINDINGS FOR THE MAIN COMPARISON BACKGROUND OBJECTIVES METHODS RESULTS Figure Figure Figure Figure Figure Figure ADDITIONAL SUMMARY OF FINDINGS DISCUSSION AUTHORS CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES CHARACTERISTICS OF STUDIES DATA AND ANALYSES FEEDBACK WHAT S NEW HISTORY CONTRIBUTIONS OF AUTHORS DECLARATIONS OF INTEREST SOURCES OF SUPPORT DIFFERENCES BETWEEN PROTOCOL AND REVIEW INDEX TERMS i

3 [Intervention Review] Time-lapse systems for embryo incubation and assessment in assisted reproduction Sarah Armstrong 1, Nicola Arroll 1, Lynsey M Cree 1, Vanessa Jordan 1, Cindy Farquhar 1 1 Department of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand Contact address: Sarah Armstrong, Department of Obstetrics and Gynaecology, University of Auckland, Park Rd, Grafton, Auckland, New Zealand. s.armstrong@auckland.ac.nz. Editorial group: Cochrane Gynaecology and Fertility Group. Publication status and date: Edited (no change to conclusions), comment added to review, published in Issue 10, Citation: Armstrong S, Arroll N, Cree LM, Jordan V, Farquhar C. Time-lapse systems for embryo incubation and assessment in assisted reproduction. Cochrane Database of Systematic Reviews 2015, Issue 2. Art. No.: CD DOI: / CD pub2. Background A B S T R A C T Embryo incubation and assessment is a vital step in assisted reproductive technology (ART). Traditionally, embryo assessment has been achieved by removing embryos from a conventional incubator daily for assessment of quality by an embryologist, under a light microscope. Over recent years time-lapse systems (TLSs) have been developed which can take digital images of embryos at frequent time intervals. This allows embryologists, with or without the assistance of computer algorithms, to assess the quality of the embryos without physically removing them from the incubator. The potential advantages of a TLS include the ability to maintain a stable culture environment, therefore limiting the exposure of embryos to changes in gas composition, temperature and movement. Additionally a TLS has the potential advantage of improving embryo selection for ART treatment by utilising additional information gained through monitoring embryo development. Objectives To determine the effect of a TLS compared to conventional embryo incubation and assessment on clinical outcomes in couples undergoing ART. Search methods A comprehensive search of all the major electronic databases, including grey literature, was undertaken in co-ordination with the Trials Search Co-ordinator of the Cochrane Menstrual Disorders and Subfertility Group in July 2014 and repeated in November 2014 to confirm that the review is up to date. Selection criteria Two authors (SA and NA) independently scanned the titles and abstracts of the articles retrieved by the search. Full texts of potentially eligible randomised controlled trials (RCTs) were obtained and examined independently by the authors for their suitability according to the review inclusion criteria. In the case of doubt between the two authors, a third author (LC) was consulted to gain consensus. The selection process is documented with a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow chart. Data collection and analysis Data were obtained and extracted by two authors. Disagreement was resolved by consensus. Trial authors were contacted by to obtain further study information and data. All extracted data were dichotomous outcomes and odds ratios (OR) were calculated on an intention-to-treat basis. Where enough data were available, meta-analysis was undertaken. 1

4 Main results Three studies involving 994 women were found for inclusion. Data from all three studies were used to address comparison one, TLS with or without cell-tracking algorithms versus conventional incubation. No studies were found to address comparison two, TLS utilising cell-tracking algorithms versus TLS not utilising cell-tracking algorithms. There was only one study which reported live birth (n = 76). The results demonstrated no conclusive evidence of a difference in live birth rate per couple randomly assigned to the TLS and conventional incubation arms of the study (OR 1.1, 95% CI 0.45 to 2.73, 1 RCT, n = 76, moderate quality evidence). All three studies reported miscarriage (n = 994). There was no conclusive evidence of a difference in miscarriage rates per couple randomly assigned to the TLS and conventional incubation arms (OR 0.70, 95% CI 0.47 to 1.04, 3 RCTs, n = 994, I 2 = 0%, low quality evidence). Only one study reported stillbirth rates (n = 76). There were equal numbers of stillbirths in both the TLS and conventional incubation arms of the study. Therefore, there was no evidence of a difference in the stillbirth rate per couple randomly assigned to TLS and conventional incubation (OR 1.0, 95% CI 0.13 to 7.49, 1 RCT, moderate quality evidence). All three studies reported clinical pregnancy rates (n = 994). There was no conclusive evidence of a difference in clinical pregnancy rate per couple randomly assigned to the TLS and conventional incubation arms (OR 1.23, 95% CI 0.96 to 1.59, 3 RCTs, n = 994, I 2 = 0%, low quality evidence). None of the included studies reported cumulative clinical pregnancy rates. Authors conclusions There is insufficient evidence of differences in live birth, miscarriage, stillbirth or clinical pregnancy to choose between TLS and conventional incubation. Further data explicitly comparing the incubation environment, the algorithm for embryo selection, or both, are required before recommendations for a change of routine practice can be justified. P L A I N L A N G U A G E S U M M A R Y Time-lapse systems for embryo incubation and embryo assessment for couples undergoing IVF Review question We reviewed the evidence for time-lapse systems (TLSs) for embryo incubation and embryo assessment for couples undergoing in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). Background Embryo incubation and assessment are vital steps in IVF and ICSI. Traditionally, embryos are removed from a conventional incubator for assessment of quality and stage of development under a light microscope. In this review, we have called that conventional incubation. TLSs can take digital images of embryos at frequent time intervals. This allows assessment of embryos without removing them from the incubator. The use of a TLS often adds a significant extra cost onto an IVF or ICSI cycle. We wanted to determine whether a TLS would improve the chances of a pregnancy and live born baby, and whether a TLS reduces the risk of miscarriage and stillbirth. Study characteristics The evidence was current to November The three included studies involved 994 couples; 62 couples were undergoing IVF and the remainder ICSI. These studies took place in Spain, Hungary and Turkey. One study is ongoing but the other studies were 6 and 17 months in duration. Some couples received donor eggs from younger women. Two studies (138 couples) replaced one embryo at a time, at the blastocyst stage; 856 couples had multiple embryos replaced at a time (1.86 per couple on average), at the cleavage stage or blastocyst stage, in the other study. The age of women ranged between 20 and 38 years. One study disclosed that the instrumentation and utensils used were fully paid for by IVI. IVI is a shareholder in UnisenseFertiliTech A/S, a manufacturer of a TLS, but none of the authors had any economic affiliation with UnisenseFertiliTech A/S. Key results The results did not demonstrate evidence of a difference in the live birth, miscarriage, stillbirth or clinical pregnancy rate per couple randomly assigned to either a TLS or conventional incubation. 2

5 Quality of the evidence The quality of the evidence was moderate or low. This was due to the scarcity of studies, two of which were very small in terms of numbers of participants, with high risk of bias in the largest study. 3

6 S U M M A R Y O F F I N D I N G S F O R T H E M A I N C O M P A R I S O N [Explanation] TLS with or without cell- tracking algorithms versus conventional incubation for embryo incubation in assisted reproduction Patient or population: embryo incubation in assisted reproduction Settings: Intervention: TLS with or without cell-tracking algorithms Comparison: conventional incubation Outcomes Illustrative comparative risks* (95% CI) Relative effect (95% CI) Assumed risk incuba- conventional tion Corresponding risk TLS with or without cell- tracking algorithms Live birth 500 per per 1000 (310 to 732) Miscarriage 143 per per 1000 (73 to 148) Stillbirth 53 per per 1000 (7 to 294) OR 1.11 (0.45 to 2.73) OR 0.7 (0.47 to 1.04) OR 1 (0.13 to 7.49) No of participants (studies) 76 (1 RCT) 994 (3 RCTs) 76 (1 RCT) Quality of the evidence (GRADE) MODERATE 1 LOW 2,3,4 MODERATE 1 Comments Clinical pregnancy 558 per per 1000 (548 to 668) OR 1.23 (0.96 to 1.59) 994 (3 RCTs) LOW 2,3,4 * The basis for the assumed risk is the median control group risk across studies. The corresponding risk (and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI). CI: Confidence interval 4

7 GRADE Working Group grades of evidence High quality: Further research is very unlikely to change our confidence in the estimate of effect. M oderate quality: Further research is likely to have an important impact on our confidence in the estimate of effect and may change the estimate. Low quality: Further research is very likely to have an important impact on our confidence in the estimate of effect and is likely to change the estimate. Very low quality: We are very uncertain about the estimate. 1 Serious risk of imprecision secondary to small sample size and wide confidence intervals 2 Overall high risk of selection, performance, attrition and reporting bias. 3 The largest study used donor and autologous oocytes, whereas the remaining two studies used autologous oocytes only. Donor oocytes were generally from young women, which may behave differently to the usual population of oocytes and embryos of couples undergoing ART 4 Following further clarification by the study author, Kovacs 2013 did utilise a cell-tracking algorithm. xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx 5

8 B A C K G R O U N D Description of the condition Embryo incubation is a critical step in all in vitro fertilisation (IVF) procedures. Embryo development within media in culture dishes in an 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 time intervals to provide a brief snap shot in order to assess morphological features. A consensus on the minimum data set 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 & ESHRE SIG 2011). A consensus on timings of observation of fertilised oocytes and embryos was established and deemed critical to the ability to compare results between different laboratories. The recommended checks, in hours, following insemination are: a fertilisation check at 17 hours, a syngamy check at 23 hours; an early cleavage check at 26 hours post-intracytoplasmic sperm injection (ICSI) or 28 hours post-ivf; day 2 embryo assessment at 44 hours; day 3 embryo assessment at 68 hours; day 4 embryo assessment at 92 hours; day 5 embryo assessment at 116 hours. Traditionally the checks have 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 the embryos to the potentially suboptimal conditions of the environment outside of the incubator and human handling (Meseguer 2012a). Timelapse systems (TLSs) have evolved over recent years to increase the frequency of morphological observations whilst minimising the impact of the external environment and human handling on embryo development. Description of the intervention A TLS is a device which takes digital images of embryos at set time intervals, for example every 5 to 15 minutes. 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 TLSs also utilise computer-assisted assessment of developmental milestones of embryos, also known as morphokinetic parameters, to offer a semi-quantitative 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 commercially available TLSs developed by various manufacturers. TLSs are available as devices that can be placed within existing conventional incubators, and some exist with an integrated incubator. The integrated TLS combines both the time-lapse cameras and the incubator in one device. How the intervention might work There are two potential benefits of a TLS. Firstly, an advantage may lie with the undisturbed nature of the culture conditions, whereby images can be obtained without removing embryos from the incubator environment for conventional bench-top light microscopy (which may or may not include heated microscope stages). This minimises the exposure of embryos to both human handling and changes in air temperature and gas composition. This may lead to improved culture conditions. A second potential advantage may be owing to the ability of a TLS to accumulate detailed time-lapse images of embryo development at regular time intervals. This includes the timing of cell divisions, intervals between cell cycles, and other factors such as dynamic pronuclei patterns, presence of multinucleation and fragmentation, and blastomere symmetry. Many of these features may be missed by using standard morphological assessment. 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 ability to select the highest quality embryo at an optimal stage of development for replacement first in an ART cycle may lead to a reduction in time to pregnancy for a couple, and reduced need for subsequent embryo transfers. Why it is important to do this review New interventions, such as TLSs, should be evaluated by randomised controlled trials to establish their safety, clinical effectiveness and cost-effectiveness. Countering the potential benefit outlined in the description of the intervention, a TLS involves exposing embryos to light during image acquisition, at pre-determined intervals. Exposure to ultraviolet (UV) radiation, albeit in low doses, means that there is potential for harm. Furthermore, the 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 aid them in making informed 6

9 decisions on their care (ACART; HFEA). However, there are currently no systematic reviews comparing TLSs 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 of adverse events will provide vital information both to couples seeking fertility treatment and to clinicians and scientists providing the treatment. This review aimed to establish whether there is evidence of any overall benefit of a TLS over current conventional care. Furthermore, this review aimed to assess the evidence from trials that utilised cell-tracking software algorithms versus morphological assessment of TLS images by an embryologist. 1b) TLSs not utilising cell-tracking algorithms versus conventional incubation. 2) TLSs utilising cell-tracking algorithms versus TLS not utilising cell-tracking algorithms. We examined the time-lapse embryo imaging systems of any manufacturer, compared with standard incubation using the incubators of any manufacturer. Types of outcome measures Primary outcomes 1. Live birth rate per couple randomly assigned 2. Miscarriage and stillbirth O B J E C T I V E S To determine the effect of a TLS compared to conventional embryo incubation and assessment on clinical outcomes in couples undergoing assisted reproductive technology (ART). Secondary outcomes 3. Clinical pregnancy, defined as evidence of a gestational sac, confirmed by ultrasound per couple randomly assigned 4. Cumulative clinical pregnancy rate, per couple randomly assigned M E T H O D S Criteria for considering studies for this review Types of studies Inclusions: any randomised controlled trial (RCT), whether published or not, which in principle could answer questions regarding clinical (post-implantation) outcomes. Exclusions: quasi-randomised and other concurrently controlled studies will be excluded. Trials that randomised oocytes or embryos were excluded as it would not be possible to compare clinical outcomes. Cross-over trials were excluded as the design is not valid in this context. Types of participants Couples of any age undergoing assisted reproduction where embryo incubation was required. Types of interventions 1) TLSs with or without cell-tracking algorithms versus conventional incubation: 1a) TLSs utilising cell-tracking algorithms versus conventional incubation, Search methods for identification of studies SA and NA identified as many relevant RCTs as possible of timelapse 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 used both electronic searches of bibliographic databases and handsearching as described in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011). Electronic searches We discussed 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 in electronic databases as possible. For this purpose we used 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 searched in the following electronic databases: MEDLINE In-Process & Other Non-Indexed Citations, Ovid platform (1946 to present); Ovid; Cochrane Central Register of Controlled Trials (CENTRAL), Ovid platform (1991 to present); LILACS, IAHx interface (1982 to present); 7

10 PsycINFO, Ovid platform (1946 to present); Cumulative Index to Nursing and Allied Health (CINAHL) (inception to present). For MEDLINE, we used the Cochrane highly sensitive search strategy for identifying RCTs: sensitivity and precision maximizing version (2008 revision), Ovid format (Higgins 2011). The LILACS search strategy was combined with the RCT filter of the IAHx interface. These searches were updated within the six months before publication of the full review. Please see Appendix 1; Appendix 2; Appendix 3; Appendix 4; Appendix 5 for details of the search strategies. The following search terms were used: searching in the Cochrane Menstrual Disorders and Subfertility Group Specialised Register (using the term timelapse system in the title, abstract and keywords), which includes RCTs and controlled clinical trials from 1944 to present, located through electronic searching (MEDLINE, EMBASE and CENTRAL) and handsearching; searching in trials registers, the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) portal ( and ClinicalTrials.gov (clinicaltrials.gov/); searching the Web of Knowledge (inception to present) using the key words Topic=(time-lapse) AND Topic=(system) AND Topic=(assisted reproduction), refined by Document Types=(CLINICAL TRIAL), Timespan=All Years; searching in Proquest Dissertations and Theses (search.proquest.com) (inception to present); searching for grey literature through the System for Information on Grey Literature in Europe OpenGrey ( , 1992, 1995, 1996 and Searching other resources We attempted to identify additional relevant RCTs by using the following methods: contact with authors of all RCTs identified by other methods; contact with manufacturers of TLSs; handsearching of selected journals in obstetrics, gynaecology and reproductive medicine, as well as conference proceedings (for abstracts) of the European Society for Human Reproduction and Embryology (ESHRE) and the American Society for Reproductive Medicine (ASRM); contacting known experts and personal contacts regarding unpublished materials; searching the citation lists of all identified articles for any relevant reference. Data collection and analysis Selection of studies Two authors (SA and NA) independently scanned the titles and abstracts of the articles retrieved by the search. Full texts of potentially eligible studies were obtained and examined independently by the authors for their suitability according to the inclusion criteria. In the case of doubt between the two authors, a third author (LC) was consulted to gain consensus on whether to include the trial or not. The selection process was documented with a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow chart. Data extraction and management The data were obtained and extracted by two review authors (SA and NA). In the case of disagreement between the two authors, they consulted a third author to achieve consensus (LC). They extracted data using a data extraction form designed and piloted by the authors. If studies were reported in multiple publications, the data were extracted from the different publications and were combined into a single data extraction form so no data were omitted. The following characteristics of included studies were included in the data extraction form: methods; participants; interventions; outcomes, including adverse events; funding source for studies. Assessment of risk of bias in included studies For this review, assessment of risk of bias was conducted by two authors (SA and NA) using the Cochrane risk of bias assessment tool. All included studies were evaluated for the following: adequacy of sequence generation and allocation concealment; adequacy of blinding of couples, providers and outcome assessors; completeness of outcome data; risk of selective outcome reporting; and risk of other potential sources of bias (Higgins 2011). Disagreements were resolved by consensus. The results of the assessment of risk of bias are presented in the Characteristics of included studies table and a Summary of findings table. Measures of treatment effect For dichotomous data (for example live birth or not), odds ratios (ORs) and their confidence intervals (CIs) were calculated. Unit of analysis issues The data were analysed per couple randomised. Studies randomising oocytes or embryos were excluded. 8

11 Dealing with missing data If relevant data were missing from an included study, the original investigators of the trial were contacted to request the missing data. All original investigators were contacted. If participants were described as lost to follow up without a specified reason, we assumed the participant did not experience the event or outcome (that is did not become pregnant). Assessment of heterogeneity We considered whether the clinical and methodological characteristics of the included studies were sufficiently similar for metaanalysis to provide a clinically meaningful summary. Statistical heterogeneity was assessed by measuring the I² statistic. We assumed that there was substantial heterogeneity when I² was 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 aimed to minimise their potential impact by ensuring a comprehensive search for eligible studies and by being alert to duplication of data. Within study reporting bias was assessed, and assessed as low risk if all of the study s pre-specified primary outcomes were reported as outlined in the study s protocol. Data synthesis Where sufficient data were available, data were combined for the primary outcomes by using a fixed-effect model in the following comparisons. 1) TLS with or without cell-tracking algorithms versus conventional incubation: 1a) TLS utilising cell-tracking algorithms versus conventional incubation, 1b) TLS not utilising cell-tracking algorithms versus conventional incubation. 2) TLS utilising cell-tracking algorithms versus TLS not utilising cell-tracking algorithms. We aimed to stratify data according to type of TLS (stand alone TLS within a conventional incubator or combined TLS incubation system) and by the nature of the conventional culture conditions, however the small number of included studies precluded us from achieving this. Subgroup analysis and investigation of heterogeneity Where sufficient data were available, we aimed to conduct the following subgroup analyses to determine the potential causes of heterogeneity for the live birth and clinical pregnancy outcomes: donor oocytes (from donors of any age) versus autologous oocytes (from women of any age); fresh cycles (where embryos were replaced either at cleavage stage or blastocyst) versus frozen cycles (where frozen embryos were replaced in an ART cycle). If we detected substantial heterogeneity we hoped to explore this by employing the random-effects model. We aimed to take any statistical heterogeneity into account when interpreting the results, especially if there was any variation in the direction of effect. Sensitivity analysis We undertook sensitivity analyses for the review outcomes to determine whether the results were robust to decisions made during the review process. These analyses included consideration of whether the review conclusions would have differed if: the summary effect measure was relative risk rather than odds ratio; alternative imputation strategies had been implemented; eligibility was restricted to studies without high risk of bias. Overall quality of the body of evidence: summary of findings table We prepared a summary of findings table using Guideline Development Tool software. This table evaluates the overall quality of the body of evidence for the review outcomes (live birth, miscarriage and stillbirth, and clinical pregnancy) using GRADE criteria (study limitations (that is risk of bias), consistency of effect, imprecision, indirectness and publication bias). Judgements about evidence quality (high, moderate or low) were justified, documented and incorporated into reporting of the results for each outcome. R E S U L T S Description of studies Results of the search The search retrieved 33 articles in total, 29 from database searches and four from handsearching. Ten studies (12 articles) were potentially eligible and were retrieved in full text. Three studies met our inclusion criteria, one of which is an ongoing study but has published interim results. We also identified one ongoing study. We excluded the remaining eight studies as they were not RCTs or did not randomise couples (Figure 1 Excluded studies). 9

12 Figure 1. PRISMA flow diagram of studies. 10

13 Included studies Study design and setting Three parallel-design RCTs were included. One study was a multicentre RCT conducted in Spain (Rubio 2014). Another was a single-centre RCT conducted in Turkey (Kahraman 2013). The final included study is an interim analysis of data from an ongoing single-centre RCT in Hungary (Kovacs 2013). Participants The studies included 994 infertile couples undergoing ART; the studies by Rubio 2014 and Kahraman 2013 included couples undergoing their first or second cycle of intracytoplasmic sperm injection (ICSI) and the study by Kovacs 2013 included couples with fewer than two previous failed IVF cycles undergoing IVF. The study by Rubio 2014 contributed the majority of participants (856), with the studies by Kahraman 2013 and Kovacs 2013 contributing much smaller numbers (76 and 62 participants respectively). The study by Rubio 2014 included couples undergoing ART with autologous or donor oocytes whereas the studies by Kahraman 2013 and Kovacs 2013 included couples utilising autologous oocytes only. The studies by Kahraman 2013 and Kovacs 2013 included couples undergoing single embryo transfer; the study by Rubio 2014 included couples receiving multiple embryos per transfer. The age range of women included in these studies was 20 to 38 years old and all women had a body mass index (BMI) of < 30 kg/m 2. The main cause of infertility was described as tubo-peritoneal factor in the study by Kahraman 2013, but was not described by the remaining studies. Interventions All three studies compared time-lapse imaging versus conventional incubation. The studies by Rubio 2014 and Kahraman 2013 both utilised an integrated TLS. The study by Kovacs 2013 utilised a TLS which fits inside a conventional incubator. Images were obtained by the TLS every 15 to 20 minutes in all three studies. Embryo transfer took place at the blastocyst stage in the studies by Kahraman 2013 and Kovacs The study by Rubio 2014 undertook embryo transfer on days 3 and 5. The studies by Kahraman 2013 and Kovacs 2013 sought to assess the TLS versus conventional incubation, with assessment by routine morphological criteria in both arms. This meant that in the TLS arm of both trials the TLS images were assessed by routine morphological criteria by an embryologist to determine the embryo quality. The study by Rubio 2014 sought to assess whether a TLS utilising cell-tracking algorithms held any advantage over conventional incubation with routine morphological embryo assessment. All three studies aimed to undertake fresh embryo transfers. All autologous oocytes were fertilised fresh and most donor oocytes in the Rubio 2014 study were used in fresh cycles, however some donor oocytes were obtained from an oocyte bank and were therefore vitrified. Outcomes All three studies reported clinical pregnancy rates per couple and miscarriage. Kahraman 2013 also reported live birth, stillbirth and ectopic pregnancy data. Excluded studies Eight studies were excluded from the review for the following reasons: six studies randomised oocytes as opposed to couples; two studies were not RCTs. Risk of bias in included studies Allocation Sequence generation Two studies were at low risk of selection bias related to sequence generation; Kahraman 2013 used a computer generated randomisation list from random.org and Kovacs 2013 undertook paired randomisation of the intervention. Trial authors clarified paired randomisation as a system whereby two envelopes containing time-lapse or control group assignments were prepared and the first patient was randomly assigned to one of the groups and the next patient received the other assignment. This was repeated with patient numbers three and four, and so on. Expert advice was sought from the Cochrane Fellow of the Menstrual Disorders and Subfertility Group to determine the risk of bias associated with this method of sequence generation. Rubio 2014 was deemed high risk in this domain because although it undertook adequate random sequence generation some women were able to request the intervention, and in some cases this request was granted. The authors of this study assured us that this preferential allocation occurred on a minority of occasions and the vast majority of participants were truly randomised, therefore we have maintained that this is an RCT. 11

14 Allocation concealment Kahraman 2013 described allocation concealment which was at low risk of selection bias; the randomisation list was held by one of the investigators who was not involved clinically with the patients or routinely working in the embryology laboratory. The randomisation list from random.org was printed out into sequentially numbered lists where the groups were masked and not revealed until the recruitment of each patient. The remaining two studies were at high risk in this domain; Kovacs 2013 described that randomisation was carried out by the principal investigator who was involved in the study, and Rubio 2014 described that in some cases the allocation was non-random. Blinding Blinding of participants and personnel (performance bias) The study by Kahraman 2013 was deemed at low risk of performance bias as couples were blinded and this blinding was not broken unless participants withdrew from the study. Clinicians involved in the study were blinded until after embryo transfer. The studies by Kovacs 2013 and Rubio 2014 were deemed high risk because the couples were not blinded. The gynaecologist and the statistician were blinded in the Rubio 2014 study, however the lack of blinding of the couples still meant that this study was at high risk of performance bias. In all studies the embryologists were not blinded, but this would have been impossible. Blinding of outcome assessors (detection bias) All three studies were deemed at low risk of detection bias because the outcomes (live birth, clinical pregnancy, miscarriage and stillbirth) cannot be influenced by the person detecting the outcome. Incomplete outcome data The following studies were deemed to be at low risk for attrition bias: Kahraman 2013 because the 12 couples who dropped out after randomisation were accounted for, and the reasons were clearly stated; Rubio 2014 because the 13 couples who were excluded following randomisation were accounted for and were a very small proportion of the total number of couples randomised. The study by Kovacs 2013 was deemed at high risk of attrition bias because a large proportion of the couples recruited were excluded from the trial (15 so far out of 62 couples randomised). On communication with the author it was made clear that these excluded couples were dropouts and not merely an artefact of interim analysis. Reasons for dropouts were provided, however with such a high attrition rate this study is at high risk of attrition bias. All couples excluded from these three studies were included in an intention-to-treat analysis of the dichotomous outcomes in this review. Selective reporting The three studies were at low risk of reporting bias because they reported and published all outcomes they set out to investigate. Other potential sources of bias Kovacs 2013 has undertaken interim reporting and analysis of results, which was not disclosed as being planned in ClinicalTrials.gov. We found no potential sources of within-study bias in Rubio 2014 or Kahraman For details of the risk of bias assessments, see Figure 2, Figure 3. 12

15 Figure 2. Risk of bias summary: review authors judgements about each risk of bias item for each included study. 13

16 Figure 3. Risk of bias graph: review authors judgements about each risk of bias item presented as percentages across all included studies. Effects of interventions See: Summary of findings for the main comparison TLS with or without cell-tracking algorithms versus conventional incubation; Summary of findings 2 TLS utilising cell-tracking algorithms versus conventional incubation; Summary of findings 3 TLS not utilising cell-tracking algorithms versus conventional incubation 1. TLS with or without cell-tracking algorithms versus conventional incubation All three studies were included in this comparison (n = 994) ( Kahraman 2013; Kovacs 2013; Rubio 2014). Primary outcomes 1.1 Live birth There was only one study which reported live birth (Kahraman 2013). Of the 76 couples randomised in this study, 39 had a live birth: 20 out of 38 couples randomised to TLS and 19 out of 38 couples randomised to conventional incubation. There was no conclusive evidence of a difference in live birth rate per couple randomly assigned to the TLS and conventional incubation arms (OR 1.1, 95% CI 0.45 to 2.73, 1 RCT, n = 76, moderate quality evidence). We were not able to perform any planned subgroup or sensitivity analyses as only one study reported this outcome. 1.2 Miscarriage and stillbirth All three studies reported on miscarriage (n = 994). Of the 512 couples randomised to TLS, there were 50 miscarriages; of the 482 couples randomised to conventional incubation, there were 64 miscarriages. There was no conclusive evidence of a difference in miscarriage rates per couple randomly assigned to the TLS and conventional incubation arms (OR 0.70, 95% CI 0.47 to 1.04, 3 RCTs, n = 994, I 2 = 0%, low quality evidence) (Analysis 1.2; Figure 4). 14

17 Figure 4. Forest plot of comparison: 1 TLS with or without cell-tracking algorithms versus conventional incubation, intervention: 1.2 Miscarriage. Only one study reported stillbirth rates (Kahraman 2013). There were equal numbers of stillbirths in both the TLS and conventional incubation arms of the study, 2 out of the 38 couples in both arms. Therefore, there was no evidence of a difference in stillbirth rate per couple randomly assigned to the TLS and conventional incubation arms (OR 1.0, 95% CI 0.13 to 7.49, 1 RCT, moderate quality evidence). We did not perform any planned subgroup analyses for miscarriage as there was no detected heterogeneity between studies; nor for stillbirth as there was only one study that reported this outcome. Secondary outcomes 1.3 Clinical pregnancy All three studies reported clinical pregnancy rates (n = 994). Of the 512 couples randomised to TLS there were 313 clinical pregnancies; of the 482 couples randomised to conventional incubation there were 270 pregnancies. There was no conclusive evidence of a difference in clinical pregnancy rate per couple randomly assigned to the TLS and conventional incubation arms (OR 1.23, 95% CI 0.96 to 1.59, 3 RCTs, n = 994, I 2 = 0%, low quality evidence) (Analysis 1.4; Figure 5). No studies reported cumulative pregnancy rates. Figure 5. Forest plot of comparison: 1TLS with or without cell-tracking algorithms versus conventional incubation, intervention : 1.3 Clinical pregnancy. We did not perform any planned subgroup analyses for clinical pregnancy as there was no detected heterogeneity between studies. 1a) TLS utilising cell-tracking algorithms versus conventional incubation One study undertook this comparison (n = 856) (Rubio 2014). Data were not available for those 13 couples excluded following randomisation for any of the following outcomes. Primary outcomes 1a.1 Live birth There were no studies found that undertook this comparison and examined this outcome. 1a.2 Miscarriage and stillbirth The included study reported miscarriage. Of the 444 couples randomised to TLS, 45 experienced a miscarriage; of the 412 couples randomised to conventional incubation, 59 experienced a miscarriage. There was no conclusive evidence of a difference in miscarriage rates per couple randomly assigned to TLS utilising cell- 15

18 tracking algorithms and to conventional incubation (OR 0.67, 95% CI 0.45 to 1.02, 1 RCT, n = 856, very low quality of evidence). We were not able to perform any planned subgroup or sensitivity analyses as only one study reported this outcome. Stillbirth was not reported in this study. following outcomes. Data on clinical pregnancy rate for the 15 couples excluded from the Kovacs 2013 study were obtained from the authors of the systematic review by Polanski 2014, who sought the information from the authors of the study. 1a.3 Clinical pregnancy The included study reported clinical pregnancy. Of the 444 couples randomised to TLS, there were 272 pregnancies; of the 412 couples randomised to conventional incubation, there were 230 pregnancies. There was no conclusive evidence of a difference in clinical pregnancy rates per couple randomly assigned to TLS utilising cell-tracking algorithms and to conventional incubation (OR 1.25, 95% CI 0.95 to 1.64, 1 RCT, n = 865, very low quality evidence); however, this result was very close to being significant and there is the suggestion that with further RCT evidence we may see an increase in clinical pregnancy rates with this technology. We were not able to perform any planned subgroup or sensitivity analyses as only one study reported this outcome. 1b) TLS not utilising cell-tracking algorithms versus conventional incubation Two studies were included in this comparison (n = 138) ( Kahraman 2013; Kovacs 2013). Data from the 12 couples from the Kahraman 2013 study that were excluded following randomisation were obtained from the authors and included for all of the Primary outcomes 1b.1 Live birth As described above, only one study reported live birth (Kahraman 2013), the results of which are described under TLS with or without cell-tracking algorithms versus conventional incubation. 1b.2 Miscarriage and stillbirth Both studies reported miscarriage rates (n = 138). Of the 68 couples randomised to TLS, there were 5 miscarriages; of the 70 couples randomised to conventional incubation, there were 5 miscarriages. There was no evidence of a difference in miscarriage rates per couple randomly assigned to TLS utilising routine morphological assessment of TLS images and to conventional incubation (OR 1.01, 95% CI 0.28 to 3.72, 2 RCTs, n = 138, I 2 = 0%, low quality evidence) (Analysis 3.2; Figure 6). As described above, only one study reported stillbirth (Kahraman 2013), the results of which are described under TLS with or without cell-tracking algorithms versus conventional incubation. Figure 6. Forest plot of comparison:1b TLS not utilising cell-tracking algorithms versus conventional incubation, intervention: 1b.1 Miscarriage. Secondary outcomes 1b.3 Clinical pregnancy Both studies reported clinical pregnancy. Of the 68 couples randomised to TLS, there were 41 clinical pregnancies; of the 70 couples randomised to conventional incubation, there were 40 pregnancies. There was no evidence of a difference in clinical pregnancy rate per couple randomly assigned to TLS utilising routine morphological assessment of TLS images and to conventional incubation (OR 1.13, 95% CI 0.65 to 2.26, 2 RCTs, n = 138, I 2 = 0%, low quality evidence). We did not perform any planned subgroup analyses for clinical pregnancy as there was no detected heterogeneity between studies. 16

19 2. TLS utilising cell-tracking algorithms versus TLS not utilising cell-tracking algorithms There were no studies found that undertook this comparison or interventions. Sensitivity analysis The conclusions of this review did not differ when relative risk rather than OR was used as the summary effect measure. Restricting analysis to studies without high risk of bias was not a feasible option in this review owing to there only being three included studies, two of which were considered to be at high risk of bias. The one remaining study without high risk of bias had low numbers of participants. 17

20 A D D I T I O N A L S U M M A R Y O F F I N D I N G S [Explanation] TLS utilising cell- tracking algorithms versus conventional incubation for embryo incubation in assisted reproduction Patient or population: embryo incubation in assisted reproduction Settings: Intervention: TLS utilising cell-tracking algorithms Comparison: conventional incubation Outcomes Illustrative comparative risks* (95% CI) Relative effect (95% CI) Assumed risk incuba- conventional tion Corresponding risk TLS utilising celltracking algorithms Miscarriage 143 per per 1000 (70 to 146) Clinical pregnancy 558 per per 1000 (546 to 675) OR 0.67 (0.45 to 1.02) OR 1.25 (0.95 to 1.64) No of participants (studies) 856 (1 RCT) 856 (1 RCT) Quality of the evidence (GRADE) VERY LOW 1,2,3 VERY LOW 1,2,3 Comments * The basis for the assumed risk is the median control group risk across studies. The corresponding risk (and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI). CI: Confidence interval GRADE Working Group grades of evidence High quality: Further research is very unlikely to change our confidence in the estimate of effect. M oderate quality: Further research is likely to have an important impact on our confidence in the estimate of effect and may change the estimate. Low quality: Further research is very likely to have an important impact on our confidence in the estimate of effect and is likely to change the estimate. Very low quality: We are very uncertain about the estimate. 1 High risk of selection and performance bias 2 Serious risk of imprecision secondary to small sample size and wide confidence intervals 3 Serious risk of indirectness as study included donor oocytes from young women, which may behave differently to the usual population of oocytes and embryos of couples undergoing ART 18

21 TLS not utilising cell- tracking algorithms versus conventional incubation for embryo incubation in assisted reproduction Patient or population: embryo incubation in assisted reproduction Settings: Intervention: TLS utilising routine morphological assessment Comparison: conventional incubation Outcomes Illustrative comparative risks* (95% CI) Relative effect (95% CI) Assumed risk incuba- conventional tion Corresponding risk TLS utilising routine morphological assessment Live birth 500 per per 1000 (310 to 732) Miscarriage 68 per per 1000 (20 to 214) Stillbirth 53 per per 1000 (7 to 294) OR 1.11 (0.45 to 2.73) OR 1.01 (0.28 to 3.72) OR 1 (0.13 to 7.49) No of participants (studies) 76 (1 RCT) 138 (2 RCTs) 76 (1 RCT) Quality of the evidence (GRADE) MODERATE 3 LOW 1,2,4 MODERATE 3 Comments Clinical pregnancy 561 per per 1000 (417 to 743) OR 1.13 (0.56 to 2.26) 138 (2 RCTs) LOW 1,2,4 1 Overall high risk of selection, performance, attrition and other bias 2 The two included studies were small with very wide confidence intervals 3 Serious risk of imprecision secondary to small sample size and wide confidence intervals 4 Following further clarification by the study author, Kovacs 2013 did utilise a cell-tracking algorithm. In the next update of this review, Kovacs 2013 will be reclassified as a TLS utilising a cell-tracking algorithm 19

22 D I S C U S S I O N Summary of main results For all types of TLS, with or without cell-tracking algorithms, versus conventional embryo incubation there was no conclusive evidence of a difference in live birth, miscarriage, stillbirth and clinical pregnancy rates per couple randomised (Analysis 1.1; Analysis 1.2; Analysis 1.3; Analysis 1.4). This review only found three RCTs from which to extract data, one of which makes up 86% of the participants (Rubio 2014). Live birth and stillbirth data were only available from one small study (Kahraman 2013) and revealed no evidence of a difference between a TLS and conventional incubation. The third study is an interim analysis of data from an ongoing study and is, again, a small study (Kovacs 2013). None of the three studies presented cumulative pregnancy rate data. The additional comparison we set out to assess (TLS utilising celltracking algorithms versus TLS not utilising cell-tracking algorithms) yielded no studies for inclusion. Overall completeness and applicability of evidence The three studies in this review all provided data on miscarriage and clinical pregnancy, however only one study (Kahraman 2013) addressed live birth and stillbirth outcomes. On communication with the author it was established that live birth data will eventually be published from the large Rubio 2014 study, which will add significantly to the data presented in this review. The majority of participants were couples undergoing ICSI, with only one study utilising couples undergoing IVF. Further studies that recruit couples undergoing IVF are needed to ensure the results of this review are relevant in the future for couples embarking on all types of ART, both IVF and ICSI. The large study by Rubio 2014 recruited couples receiving both autologous and donor oocytes whereas the other studies recruited couples receiving only autologous oocytes. All three studies undertook frozen cycles on some couples who were unable to undergo fresh embryo transfer. Therefore, in order to subgroup autologous, donor and frozen oocytes, future studies will need to present their results under these subgroups and state explicitly how many couples underwent these interventions. Elective single embryo transfer was undertaken in two studies (Kahraman 2013; Kovacs 2013) however the large Rubio 2014 study undertook multiple embryo transfers. We were unable to obtain figures from the authors of this study on exactly what proportion of couples received multiple embryo transfer in each arm of the study. Given that the Rubio 2014 study contributed a large proportion of the data on miscarriage and clinical pregnancy, it is important to recognise that the results presented here may reflect rates of clinical outcomes in keeping with multiple embryo transfer as opposed to single embryo transfer. The included studies were able to address TLS with or without celltracking algorithms versus conventional incubation. There were no studies found that addressed a TLS utilising cell-tracking algorithms versus a TLS not utilising cell-tracking algorithms. A search of online clinical trials registers ( revealed one ongoing RCT which addresses this comparison (Desai 2014). Quality of the evidence The quality of the evidence overall for all outcomes presented in this review is moderate or low, given the scarcity of studies. Two of these studies had low numbers of participants, and we assessed in the largest study as at high risk of bias. For the comparison TLS with or without cell-tracking algorithms versus conventional incubation, the quality of the evidence for live birth and stillbirth is moderate with the remaining outcomes being of low quality (Summary of findings for the main comparison). The study that assessed live birth and stillbirth was downgraded for imprecision. For results related to miscarriage and clinical pregnancy, quality was downgraded because of an overall high risk of bias and indirectness (Figure 2). The serious risk of indirectness came about due to one large study (Rubio 2014) utilising donor oocytes from young women in a proportion of participants. Donor oocytes of young women may result in improved clinical outcomes when compared to oocytes of infertile couples undergoing ART. For the comparison TLS utilising cell-tracking algorithms versus conventional incubation, the quality of the evidence for the two reported outcomes, miscarriage and clinical pregnancy, is very low (Summary of findings 2). The study quality was downgraded due to a high risk of bias, indirectness and imprecision. The study was at high risk of selection bias and performance bias and some participants used donor oocytes which may behave differently to autologous oocytes of couples undergoing ART. For the comparison TLS not utilising cell-tracking algorithms versus conventional incubation, the quality of the evidence for miscarriage and clinical pregnancy is low (Summary of findings 3). The quality of the results was downgraded due to the presence of a high risk of bias within the two included studies, imprecision secondary to the small number of participants within the two studies, and the very wide CIs around the results. Potential biases in the review process We aimed to identify all eligible studies for inclusion in this review, and the included study authors have been contacted on many occasions to seek as much information for inclusion as possible. Authors of all three studies have been forthcoming with further study information which has helped us to accrue a full picture of 20