A comparison of morphokinetic models and morphological selection for prioritizing euploid embryos: a multicentre cohort study.

STUDY QUESTION: Are morphokinetic models better at prioritizing a euploid embryo for transfer over morphological selection by an embryologist? SUMMARY ANSWER: Morphokinetic algorithms lead to an improved prioritization of euploid embryos when compared to embryologist selection. WHAT IS KNOWN ALREADY: PREFER (predicting euploidy for embryos in reproductive medicine) is a previously published morphokinetic model associated with live birth and miscarriage. The second model uses live birth as the target outcome (LB model). STUDY DESIGN, SIZE, DURATION: Data for this cohort study were obtained from 1958 biopsied blastocysts at nine IVF clinics across the UK from January 2021 to December 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ability of the PREFER and LB models to prioritize a euploid embryo was compared against arbitrary selection and the prediction of four embryologists using the timelapse video, blinded to the morphokinetic time stamp. The comparisons were made using calculated percentages and normalized discounted cumulative gain (NDCG), whereby an NDCG score of 1 would equate to all euploid embryos being ranked first. In arbitrary selection, the ploidy status was randomly assigned within each cycle and the NDGC calculated, and this was then repeated 100 times and the mean obtained. MAIN RESULTS AND THE ROLE OF CHANCE: Arbitrary embryo selection would rank a euploid embryo first 37% of the time, embryologist selection 39%, and the LB and PREFER ploidy morphokinetic models 46% and 47% of the time, respectively. The AUC for LB and PREFER model was 0.62 and 0.63, respectively. Morphological selection did not significantly improve the performance of both morphokinetic models when used in combination. There was a significant difference between the NDGC metric of the PREFER model versus embryologist selection at 0.96 and 0.87, respectively (t = 14.1, P < 0.001). Similarly, there was a significant difference between the LB model and embryologist selection with an NDGC metric of 0.95 and 0.87, respectively (t = 12.0, P < 0.001). All four embryologists ranked embryos similarly, with an intraclass coefficient of 0.91 (95% CI 0.82-0.95, P < 0.001). LIMITATIONS, REASONS FOR CAUTION: Aside from the retrospective study design, limitations include allowing the embryologist to watch the time lapse video, potentially providing more information than a truly static morphological assessment. Furthermore, the embryologists at the participating centres were familiar with the significant variables in time lapse, which could bias the results. WIDER IMPLICATIONS OF THE FINDINGS: The present study shows that the use of morphokinetic models, namely PREFER and LB, translates into improved euploid embryo selection. STUDY FUNDING/COMPETING INTEREST(S): This study received no specific grant funding from any funding agency in the public, commercial or not-for-profit sectors. Dr Alison Campbell is minor share holder of Care Fertility. All other authors have no conflicts of interest to declare. Time lapse is a technology for which patients are charged extra at participating centres. TRIAL REGISTRATION NUMBER: N/A.

Association between a morphokinetic ploidy prediction model risk score and miscarriage and live birth: a multicentre cohort study.

OBJECTIVE: To determine whether the aneuploidy risk score from a morphokinetic ploidy prediction model, Predicting Euploidy for Embryos in Reproductive Medicine (PREFER), is associated with miscarriage and live birth outcomes. DESIGN: Multicentre cohort study. SETTING: Nine in vitro fertilization clinics in the United Kingdom. PATIENTS: Data were obtained from the treatment of patients from 2016-2019. A total of 3587 fresh single embryo transfers were included; preimplantation genetic testing for aneuploidy) cycles were excluded. INTERVENTION: PREFER is a model developed using 8,147 biopsied blastocyst specimens to predict ploidy status using morphokinetic and clinical biodata. A second model using only morphokinetic (MK) predictors was developed, P PREFER-MK. The models will categorize embryos into the following three risk score categories for aneuploidy: "high risk," "medium risk," and "low risk." MAIN OUTCOME MEASURES: The primary outcomes are miscarriage and live birth. Secondary outcomes include biochemical clinical pregnancy per single embryo transfer. RESULTS: When applying PREFER, the miscarriage rates were 12%, 14%, and 22% in the "low risk," "moderate risk," and "high risk" categories, respectively. Those embryos deemed "high risk" had a significantly higher egg provider age compared with "low risk," and there was little variation in risk categories in patients of the same age. The trend in miscarriage rate was not seen when using PREFER-MK; however, there was an association with live birth, increasing from 38%-49% and 50% in the "high risk," "moderate risk," and "low risk" groups, respectively. An adjusted logistic regression analysis demonstrated that PREFER-MK was not associated with miscarriage when comparing "high risk" to "moderate risk" embryos (odds ratio [OR], 0.87; 95% confidence interval [CI], 0.63-1.63) or "high risk" to "low risk" embryos (OR, 1.07; 95% CI, 0.79-1.46). An embryo deemed "low risk" by PREFER-MK was significantly more likely to result in a live birth than those embryos graded "high risk" (OR, 1.95; 95% CI, 1.65-2.25). CONCLUSION: The PREFER model's risk scores were significantly associated with live births and miscarriages. Importantly, this study also found that this model applied too much weight to clinical factors, such that it could no longer rank a patient's embryos effectively. Therefore, a model including only MKs would be preferred; this was similarly associated with live birth but not miscarriage.

A comparison of 12 machine learning models developed to predict ploidy, using a morphokinetic meta-dataset of 8147 embryos.

STUDY QUESTION: Are machine learning methods superior to traditional statistics in predicting blastocyst ploidy status using morphokinetic and clinical biodata? SUMMARY ANSWER: Mixed effects logistic regression performed better than all machine learning methods for ploidy prediction using our dataset of 8147 embryos. WHAT IS KNOWN ALREADY: Morphokinetic timings have been demonstrated to be delayed in aneuploid embryos. Machine learning and statistical models are increasingly being built, however, until now they have been limited by data insufficiency. STUDY DESIGN, SIZE, DURATION: This is a multicentre cohort study. Data were obtained from 8147 biopsied blastocysts from 1725 patients, treated from 2012 to 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: All embryos were cultured in a time-lapse system at nine IVF clinics in the UK. A total of 3004 euploid embryos and 5023 aneuploid embryos were included in the final verified dataset. We developed a total of 12 models using four different approaches: mixed effects multivariable logistic regression, random forest classifiers, extreme gradient boosting, and deep learning. For each of the four algorithms, two models were created, the first consisting of 22 covariates using 8027 embryos (Dataset 1) and the second, a dataset of 2373 embryos and 26 covariates (Dataset 2). Four final models were created by switching the target outcome from euploid to aneuploid for each algorithm (Dataset 1). Models were validated using internal-external cross-validation and external validation. MAIN RESULTS AND THE ROLE OF CHANCE: All morphokinetic variables were significantly delayed in aneuploid embryos. The likelihood of euploidy was significantly increased the more expanded the blastocyst (P < 0.001) and the better the trophectoderm grade (P < 0.01). Univariable analysis showed no association with ploidy status for morula or cleavage stage fragmentation, morula grade, fertilization method, sperm concentration, or progressive motility. Male age did not correlate with the percentage of euploid embryos when stratified for female age. Multinucleation at the two-cell or four-cell stage was not associated with ploidy status. The best-performing model was logistic regression built using the larger dataset with 22 predictors (F1 score 0.59 for predicting euploidy; F1 score 0.77 for predicting aneuploidy; AUC 0.71; 95% CI 0.67-0.73). The best-performing models using the algorithms from random forest, extreme gradient boosting, and deep learning achieved an AUC of 0.68, 0.63, and 0.63, respectively. When using only morphokinetic predictors the AUC was 0.61 for predicting ploidy status, whereas a model incorporating only embryo grading was unable to discriminate aneuploid embryos (AUC = 0.52). The ploidy prediction model's performance improved with increasing age of the egg provider. LIMITATIONS, REASONS FOR CAUTION: The models have not been validated in a prospective study design or yet been used to determine whether they improve clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS: This model may aid decision-making, particularly where pre-implantation genetic testing for aneuploidy is not permitted or for prioritizing embryos for biopsy. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was sought for this study; university funds supported the first author. A.Ca. is a minor shareholder of participating centres. TRIAL REGISTRATION NUMBER: N/A.

Morphological and morphokinetic associations with aneuploidy: a systematic review and meta-analysis.

BACKGROUND: A time lapse system (TLS) is utilized in some fertility clinics with the aim of predicting embryo viability and chance of live birth during IVF. It has been hypothesized that aneuploid embryos display altered morphokinetics as a consequence of their abnormal chromosome complement. Since aneuploidy is one of the fundamental reasons for IVF failure and miscarriage, attention has focused on utilizing morphokinetics to develop models to non-invasively risk stratify embryos for ploidy status. This could avoid or reduce the costs associated with pre-implantation genetic testing for aneuploidy (PGT-A). Furthermore, TLS have provided an understanding of the true prevalence of other dysmorphisms. Hypothetically, the incorporation of morphological features into a model could act synergistically, improving a model's discriminative ability to predict ploidy status. OBJECTIVE AND RATIONALE: The aim of this systematic review and meta-analysis was to investigate associations between ploidy status and morphokinetic or morphological features commonly denoted on a TLS. This will determine the feasibility of a prediction model for euploidy and summarize the most useful prognostic markers to be included in model development. SEARCH METHODS: Five separate searches were conducted in Medline, Embase, PubMed and Cinahl from inception to 1 July 2021. Search terms and word variants included, among others, PGT-A, ploidy, morphokinetics and time lapse, and the latter were successively substituted for the following morphological parameters: fragmentation, multinucleation, abnormal cleavage and contraction. Studies were limited to human studies. OUTCOMES: Overall, 58 studies were included incorporating over 40 000 embryos. All except one study had a moderate risk of bias in at least one domain when assessed by the quality in prognostic studies tool. Ten morphokinetic variables were significantly delayed in aneuploid embryos. When excluding studies using less reliable genetic technologies, the most notable variables were: time to eight cells (t8, 1.13 h, 95% CI: 0.21-2.05; three studies; n = 742; I2 = 0%), t9 (2.27 h, 95% CI: 0.5-4.03; two studies; n = 671; I2 = 33%), time to formation of a full blastocyst (tB, 1.99 h, 95% CI 0.15-3.81; four studies; n = 1640; I2 = 76%) and time to expanded blastocyst (tEB, 2.35 h, 95% CI: 0.06-4.63; four studies; n = 1640; I2 = 83%). There is potentially some prognostic potential in the degree of fragmentation, multinucleation persisting to the four-cell stage and frequency of embryo contractions. Reverse cleavage was associated with euploidy in this meta-analysis; however, this article argues that these are likely spurious results requiring further investigation. There was no association with direct unequal cleavage in an embryo that progressed to a blastocyst, or with multinucleation assessed on Day 2 or at the two-cell stage. However, owing to heterogeneous results and poor-quality evidence, associations between these morphological components needs to be investigated further before conclusions can be reliably drawn. WIDER IMPLICATIONS: This first systematic review and meta-analysis of morphological and morphokinetic associations with ploidy status demonstrates the most useful morphokinetic variables, namely t8, t9 and tEB to be included in future model development. There is considerable variability within aneuploid and euploid embryos making definitively classifying them impossible; however, it is feasible that embryos could be prioritized for biopsy. Furthermore, these results support the mechanism by which algorithms for live birth may have predictive ability, suggesting aneuploidy causes delayed cytokinesis. We highlight significant heterogeneity in our results secondary to local conditions and diverse patient populations, therefore calling for future models to be robustly developed and tested in-house. If successful, such a model would constitute a meaningful breakthrough when accessing PGT-A is unsuitable for couples.

Evolution of embryo selection for IVF from subjective morphology assessment to objective time-lapse algorithms improves chance of live birth.

RESEARCH QUESTION: Does using an objective time-lapse imaging algorithm (TLIA) after IVF relate to conventional morphological assessment of human blastocysts as a prognosticator for live birth? DESIGN: Prospective use of a TLIA to select embryos in multicentre IVF clinics all using the same strictly controlled laboratory protocols. Each blastocyst was given a ranking from A to D, with the highest rank preferred for fresh transfer. This ranking was retrospectively compared with a given morphological score, which was blinded to the TLIA rank; all embryos were cultured under the same conditions. RESULTS: Using multiple variable logistic regression models, TLIA embryo rank enabled greater discrimination between cycles with and without live births than the conventional morphology grade, even when considered in isolation, and when adjusting for covariates related to treatment and patient criteria. Of the 1810 cycles of single blastocyst transfer, 894 (49.4%) resulted in a live birth. A Vuong non-nested test including covariates showed strong evidence of the superiority of the embryo rank model compared with the transfer grade model (P = 0.0008 [raw], P = 0.0003 [Akaike information criterion - corrected]). From the receiver operating characteristic (ROC) curves across all possible thresholds the TLIA rank showed better true positive and true negative rates and had a higher area under the curve [AUC] of 67.43% compared with 61.74% for the blastocyst morphology grade. The same analysis but excluding covariates demonstrated an AUC of 62.86% versus 54.02%, respectively. CONCLUSION: Objective TLIA is superior for selecting embryos for their propensity to generate a live birth over a conventional, subjective blastocyst morphology scoring system.

Time-lapse imaging algorithms rank human preimplantation embryos according to the probability of live birth.

RESEARCH QUESTION: Can blastocysts leading to live births be ranked according to morphokinetic-based algorithms? DESIGN: Retrospective analysis of 781 single blastocyst embryo transfers, including all patient clinical factors that might be potential confounders for the primary outcome measure of live birth, was weighed using separate multi-variable logistic regression models. RESULTS: There was strong evidence of effect of embryo rank on odds of live birth. Embryos were classified A, B, C or D according to calculated variables; time to start (tSB) and duration (dB{tB - tSB}) of blastulation. Embryos of rank D were less likely to result in live birth than embryos of rank A (odds ratio [OR] 0.3046; 95% confidence interval [CI] 0.129, 0.660; P < 0.005). Embryos ranked B were less likely to result in live birth than those ranked A (OR 0.7114; 95% Cl 0.505, 1.001; P < 0.01), and embryos ranked C were less likely to result in live birth than those ranked A (OR 0.6501, 95% Cl 0.373, 1.118; P < 0.01). Overall, the LRT (Likelihood Ratio Test) p-value for embryo rank shows that there is strong evidence that embryo rank is informative as a whole in discriminating between live birth and no live birth outcomes (p = 0.0101). The incidence of live birth was 52.5% from rank A, 39.2% from rank B, 31.4% from rank C and 13.2% from rank D. CONCLUSIONS: Time-lapse imaging morphokinetic-based algorithms for blastocysts can provide objective hierarchical ranking of embryos for predicting live birth and may have greater discriminating power than conventional blastocyst morphology assessment.

Perivitelline threads in cleavage-stage human embryos: observations using time-lapse imaging.

Time-lapse imaging of the human preimplantation embryo in vitro has revealed a transient phenomenon involving the appearance of perivitelline threads, commonly observed at the two-cell stage. These threads span the perivitelline space, arising at the specific area where the cytoplasmic membrane contacts the zona pellucida, before any perivitelline space is formed. The threads persist as the cytoplasmic membrane retracts from the zona pellucida to form the first cleavage furrow. In this observational report, these structures and their incidence are described. A total of 834 time-lapse videos from IVF treatment cycles, one per patient, were retrospectively analysed for perivitelline threads, from pronuclear formation until completion of the first cell cycle. Threads were observed in 56.4% (470/834) of embryos and varied from a single to an array spanning an area of the zona pellcida. A total of 91.9% (432/470) were seen to form after cytoplasmic membrane-zona-pellucida contact. A total of 76.4% (359/470) were visible at the first cleavage furrow; 77% (362/470) were associated with cytoplasmic fragments at the two-cell-stage. Presence or absence of threads did not affect embryo development. This descriptive study is limited; further characterization of these structures is needed to elucidate their potential role in early human embryo development.

Live births after embryo selection using morphokinetics versus conventional morphology: a retrospective analysis.

The increasing corpus of clinical studies using time-lapse imaging for embryo selection demonstrates considerable variation in study protocols and only limited-sized study cohorts. Outcome measures are based on implantation or clinical pregnancy; some predict blastulation from early cleavage-stage data, and few have evaluated live birth. Erroneously, most studies treat the embryos as independent variables and do not include patient or treatment variables in the statistical analyses. In this study, cohort size was 14,793 patients and 23,762 cycles. The incidence of live birth (n = 973 deliveries) after embryo selection by objective morphokinetic algorithms was compared with conventional embryology selection parameters (n = 6948 deliveries). A 19% increase in the incidence of live birth was observed when morphokinetic data were used to select embryos for the patient cohort aged younger than 38 years (OR 1.19 with 95% CI 1.06 to 1.34) using their own eggs, and an increase of 37% for oocyte recipients aged over 37 years (OR 1.370; 95% Cl 0.763 to 2.450). This is the largest study of the prospective use of time-lapse imaging algorithms in IVF reporting on live birth outcome, although the nature of purely a closed system versus standard incubation could not be assessed.

Outbreak of escherichia coli O157: H7 infections after Petting Zoo visits, North Carolina State Fair, October-November 2004.

OBJECTIVES: To identify cases, describe the outbreak, implement control measures, and identify factors associated with infection or protection from infection, including contact with animals and hand hygiene practices. DESIGN: Case finding, a case-control study of 45 cases and 188 controls, environmental investigation, and molecular subtyping of clinical and environmental Escherichia coli O157:H7 isolates. SETTING: The 2004 North Carolina State Fair. PARTICIPANTS: Case patients were fair visitors who had laboratory-confirmed E coli O157 infections, hemolytic uremic syndrome (HUS) diagnoses, or bloody diarrheal illnesses. Control subjects were recruited from a randomized list of persons who had purchased fair tickets online. Environmental samples from the fairgrounds were obtained from locations that had held animals during the fair. Main Exposure Visiting a petting zoo. MAIN OUTCOME MEASURES: Case finding: Summary descriptive statistics of suspected, probable, or confirmed E coli O157:H7 infections, signs, symptoms, and HUS. Environmental investigation: E coli O157:H7 isolates, pulsed-field gel electrophoresis patterns, and spatial distribution of source locations. Case-control study: Odds ratios (ORs) comparing reported fair-related activities, hygiene practices, and zoonotic disease knowledge with outcome. RESULTS: A total of 108 case patients were ascertained, including 41 with laboratory-confirmed illness and 15 who experienced HUS. Forty-five case patients and 188 controls were enrolled in the case-control study. Visits to a petting zoo having substantial environmental E coli O157:H7 contamination were associated with illness (age-adjusted OR, 8.2; 95% confidence interval [CI], 3.3-20.3). Among children 5 years or younger who had visited the implicated petting zoo, contact with animal manure (OR, 6.9; 95% CI, 2.2-21.9) and hand-to-mouth behaviors (OR, 10.6; 95% CI, 2.0-55.0) were associated with illness. Reported hand hygiene practices did not differ significantly (OR, 1.8; 95% CI, 0.3-9.5). Reported awareness of the risk for zoonotic disease was protective (OR, 0.1; 95% CI, 0.03-0.5). Environmental samples from the petting zoo implicated in the case-control study yielded E coli O157:H7, with indistinguishable pulsed-field gel electrophoresis patterns from the predominant strain. CONCLUSIONS: We describe one of the largest petting zoo outbreaks of E coli O157:H7 to date. Persons became infected after contact with manure and engaging in hand-to-mouth behaviors in a petting zoo having substantial E coli O157:H7 contamination. Use of alcohol-based hand-sanitizing gels was not protective, although knowledge of the risk for zoonotic infection was protective. Future investigations in similar outbreaks should assess risks for infection and protective measures (eg, physical barriers separating visitors from animal manure, education, and appropriate hand hygiene practices).

Spindle position assessment prior to ICSI does not benefit fertilization or early embryo quality.

Intracytoplasmic sperm injection (ICSI) is traditionally performed with the first polar body at 6 or 12 o'clock, and the injection pipette inserted at 3 or 9 o'clock. This positioning aims to direct the path of the injection pipette at a distance from the presumed metaphase II spindle position. Since spindles can now be imaged directly in living oocytes using computer-assisted polarized light microscopy, the effectiveness of this positioning precaution was studied. Patients undergoing oocyte collection and ICSI had their oocytes non-invasively imaged for spindles prior to ICSI. The spindle position relative to the first polar body at 6 o'clock was assessed using an analogue clock face as an approximation. Fertilization and embryo quality were recorded blind to spindle position. Polar body displacement and spindle position at ICSI did not significantly affect fertilization or embryonic quality. The highest frequency of normally fertilized oocytes and good quality embryos developed from oocytes with spindles located in or near the plane of injection at ICSI (the 3, 4, 8 and 9 o'clock positions). This study questions the usefulness of spindle imaging and the relevance of positioning the first polar body at 6 o'clock during ICSI.