Background and Aim: Oocyte cryopreservation enables the preservation of fertility for patients, as well as allowing a clinic to store donated oocytes so they are readily available to recipients. Vitrification has significantly increased oocyte survival and increased pregnancy rates. Several vitrification systems are commercially available – some specifying supplementation of post warm culture media with synthetic serum substitute or dextran serum supplement at 20% (v/v), or human serum albumin at 12mg/ml.

Background and Aim: Patients referred for PGT-A are often poor prognosis, and commonly don't have beautiful blastocysts to biopsy. Are we too selective because these blastocysts must undergo trophectoderm biopsy, vitrification and warming to make it to ET?

Background and Aim: The presence of vacuoles has been linked to poor blastocyst formation and embryonic arrest (Ebner et al., 2005). With the use of time-lapse imaging it is possible to track the appearance, time of presentation and impact of vacuoles on outcomes. The spontaneous appearance of vacuoles at the time of compaction has been linked to reduced utilisation and live birth rate (Mayer et al., 2018), this study aims to further investigate the time of appearance of vacuoles and effect on live birth outcomes.

Background and Aim: RFID technology has been used in IVF laboratories for over a decade for sample identification and witnessing at ambient temperatures. RFID-tagging of cryopreserved samples is not routinely used in fertility settings, although its use is established in biobanks providing automation in cryosample identification, reading RFID-tags at temperatures as low as –196°C.

Abstract Study question Can a group-wide quality assurance scheme be developed to effectively determine inter-operator agreement for morphokinetic parameters of interest. Summary answer Very strong agreement was found between all operators except for one, therefore this scheme effectively identified areas of improvement in inter-operator annotations. What is known already Where fertility clinics use embryo morphokinetics to determine viability potential, quality assurance of annotations is essential. Embryo selection algorithms rely on the manual determination of certain morphokinetic parameters. Variations in these parameters can lead to differences in the algorithm score attributed to an embryo thus potentially affecting its fate. It is vital that all embryologists involved in embryo annotation and selection are consistent with their annotation approach through regular quality assurance mechanisms. Study design, size, duration Each participant was required to annotate the same three embryos for morphokinetic parameters of interest, including tPB2, tPNf, t2 to t5, t8, tM, tSB, tB. Participants were also required to grade embryos at 68 hours post insemination (hpi), 112hpi and to assess additional parameters used for embryo selection or future investigations, such as the extent of morula compaction. The aim of this scheme is to release new distribution each quarter to ensure regular participation. Participants/materials, setting, methods All embryologists responsible for embryo annotation in a single, UK fertility group were enrolled onto the scheme. A total of 59 participants from 10 fertility clinics in the UK were included. Inter-operator agreement was assessed using two-way, mixed intraclass correlation coefficient (ICC) for consistency. Five categories of agreement were determined based on ICC score; very weak (0–0.2), weak (0.21–0.4), moderate (0.41–0.6), strong (0.61–0.8) and very strong (0.81–1.0). Main results and the role of chance Very strong agreement (0.81–1.0) was observed between all operators for all parameters assessed except for one operator who showed a weak agreement (0.21–0.4) with all other operators. Descriptive statistics revealed standard deviations (SD) ranging from 0.34 (t3) to 3.43 (t5). For each parameter the SD across the three assessed embryos ranged from 0.34–3.43; tPB2 (0.11–0.98), tPNf (2.06–4.40), t2 (0.22–0.80), t3 (0.16–0.70), t4 (0.39–0.65). t5 (2.40–5.44), t8 (0.33–2.72), tM (1.00–2.72), tSB (1.08–2.67), tB (1.12–1.81). These results indicate a high concordance with less subjective annotations such as the cell stage divisions and more variability with the subjective annotations such as the blastulation parameters. The concordance with less well practiced or understood annotations, such as extent of morula compaction, planar or tetrahedral orientation at the four cell stage as well as angle of extrusion of second polar body in relation to the first polar body, was poorer as indicated using descriptive statistics. This highlighted the need for experience in performing these annotations before drawing conclusions regarding their predictive nature in relation to an embryo’s viability. Limitations, reasons for caution The variability between more subjective parameters would be expected to be higher than others. The participation in these schemes can create false environments which do not reflect how an embryologist would usually score; they may spend longer on some decisions given the nature of the scheme. Wider implications of the findings: Quality assurance of morphokinetic annotations across clinics utilising standardised selection models is crucial. Robust annotation policies and education programmes are essential in achieving consistent results between operators. Quality assurance schemes can identify individuals who lack consistency overall and can identify reliably annotated parameters to inform inclusion in embryo selection algorithms. Trial registration number Not applicable

Abstract Study question Can time-lapse data be used to identify the optimum time to perform the fertilisation check for oocytes cultured in standard incubation? Summary answer The optimum time to perform fertilisation checks for oocytes cultured in standard incubation is 16.5hpi+/–0.5h. What is known already Time-lapse incubation allows the embryologist to retrospectively review collated images of oocytes and embryos to capture important embryological observations that may have otherwise been missed. This is a luxury not available to embryologists when oocytes or embryos are cultured in standard incubation. Traditionally, the optimum time to perform the fertilisation check is 17 hours post insemination (hpi) +/- 1 hour. It was hypothesised that this could be fine-tuned ensuring the maximum number of fertilised oocytes were observed, thereby increasing the number of usable embryos for the patient. Study design, size, duration This was a retrospective, multicentre analysis including data from 27,022 ICSI derived embryos cultured in time-lapse incubation between January 2011 to November 2019. Participants/materials, setting, methods The time of pronuclei appearance and disappearance was recorded using the time-lapse incubation software. The number of oocytes exhibiting normal fertilisation (defined as the presence of two pronuclei) during 30 minute intervals from 15hpi to 20hpi was determined. Main results and the role of chance Between 15–17.5hpi the average number of oocytes exhibiting normal fertilisation was 98.19% with most oocytes having visible pronuclei at 16–16.5hpi (98.32%). At 18–18.5hpi the number of visible pronuclei falls to 95.53% and continues to fall to 87.02% at 19.5–20hpi meaning that over 3000 (11%) normally fertilised oocytes, within this cohort, would not be identified. Limitations, reasons for caution The conclusions of this investigation cannot be effectively extrapolated to IVF embryos as only ICSI embryos were used for the determination of the results. Wider implications of the findings: The optimum time to perform fertilisation checks for oocytes cultured in standard incubation is 16.5hpi+/–0.5h. However, without the use of time-lapse incubation, the fertilisation of at least 2% of embryos that create a fetal heart will be missed, even if the fertilisation check is performed in the optimum window (16.5hpi+/–0.5h). Trial registration number Not applicable

Abstract Study question Can incorporation of novel markers of morphology with known temporal events successfully rank embryos to enable prediction of propensity for live birth? Summary answer Incorporation of variables for trophectoderm and morula grading demonstrably enhanced the model to rank embryos in order of potential for live birth. What is known already Models built using morphokinetic markers of development are widely used to rank embryos within a cohort. Such models include defined temporal parameters which are closely related to morphological grade. However, morphological grading by an embryologist is subjective and is not strongly correlated to outcome. Combining with defined kinetic events has been suggested to improve prediction of outcome. Study design, size, duration Data from 6228 known live birth outcome embryos from 8 UK clinics between 2011 – 2018 were investigated using an exploratory approach to identify novel markers of development. Participants/materials, setting, methods Five significant variables were defined, a derivative of time to start of blastulation; a derivative of trophectoderm grade; a kinetic variable utilising t3, t4, t5 and t8; an interval variable of tB-tSB and a variable based on novel morula classification. To maximise the output, a proxy value was derived for missing datapoints. The model was built using logistical regression and validated using fivefold cross validation with the data split as 80% training and 20% test. Main results and the role of chance An algorithm was developed including the five significant variables identified with an AUC of 0.685 demonstrating reliable prediction of live birth. Without morphological variables, the AUC was 0.674 demonstrating the improvement in the prediction value by including the derivative of the trophectoderm and morula grade. This resulted in ten classes of algorithm scores, 1–10, giving a live birth rate from 2% to 46%, irrespective of patient variables, for chance of live birth. Limitations, reasons for caution Successful application of the algorithm is reliant on stringent quality assurance for maintenance of accurate annotation and grading, and may not be transferable between laboratories with different SOPs. Wider implications of the findings: The addition of a trophectoderm and morula grade in combination with morphokinetic parameters, increases the predictive value of the algorithm in relation to live birth outcome. Using proxy values allows maximization of data for model generation, and allows the model to be applied when missing values are present. Trial registration number Not applicable

Background Uncertainties persist about the magnitude of associations of diabetes mellitus and fasting glucose concentration with risk of coronary heart disease and major stroke subtypes. We aimed to quantify these associations for a wide range of circumstances.Methods We undertook a meta-analysis of individual records of diabetes, fasting blood glucose concentration, and other risk factors in people without initial vascular disease from studies in the Emerging Risk Factors Collaboration. We combined within-study regressions that were adjusted for age, sex, smoking, systolic blood pressure, and body-mass index to calculate hazard ratios (HRs) for vascular disease.Findings Analyses included data for 698 782 people (52765 non-fatal or fatal vascular outcomes; 8.49 million person-years at risk) from 102 prospective studies. Adjusted HRs with diabetes were: 2.00 (95% CI 1.83-2.19) for coronary heart disease; 2.27 (1.95-2.65) for ischaemic stroke; 1.56 (1.19-2.05) for haemorrhagic stroke; 1.84 (1.59-2.13) for unclassified stroke; and 1.73 (1.51-1.98) for the aggregate of other vascular deaths. HRs did not change appreciably after further adjustment for lipid, inflammatory, or renal markers. HRs for coronary heart disease were higher in women than in men, at 40-59 years than at 70 years and older, and with fatal than with non-fatal disease. At an adult population-wide prevalence of 10%, diabetes was estimated to account for 11% (10-12%) of vascular deaths. Fasting blood glucose concentration was non-linearly related to vascular risk, with no significant associations between 3.90 mmol/L and 5.59 mmol/L. Compared with fasting blood glucose concentrations of 3.90-5.59 mmol/L, HRs for coronary heart disease were: 1.07 (0.97-1.18) for lower than 3.90 mmol/L; 1.11 (1.04-1.18) for 5.60-6-09 mmol/L; and 1.17 (1.08-1.26) for 6.10-6.99 mmol/L. In people without a history of diabetes, information about fasting blood glucose concentration or impaired fasting glucose status did not significantly improve metrics of vascular disease prediction when added to information about several conventional risk factors.Interpretation Diabetes confers about a two-fold excess risk for a wide range of vascular diseases, independently from other conventional risk factors. In people without diabetes, fasting blood glucose concentration is modestly and nonlinearly associated with risk of vascular disease.