Modelling a risk classification of aneuploidy in human embryos using non-invasive morphokinetics
TL;DR: No significant differences were observed in first or second cell-cycle length, synchrony of the second or third cell cycles, duration of blastulation, multinucleation at the 2-cell stage and irregular division patterns between euploid and aneuploid embryos.
Abstract: This study determined whether morphokinetic variables between aneuploid and euploid embryos differ as a potential aid to select euploid embryos for transfer. Following insemination, EmbryoScope time-lapse images from 98 blastocysts were collected and analysed blinded to ploidy. The morphokinetic variables were retrospectively compared with ploidy, which was determined fol- lowing trophectoderm biopsy and analysis by array comparative genomic hybridization or single-nucleotide polymorphic array. Multiple aneuploid embryos were delayed at the initiation of compaction (tSC; median 85.1 hours post insemination (hpi); P = 0.02) and the time to reach full blastocyst stage (tB; median 110.9 hpi, P = 0.01) compared with euploid embryos (tSC median 79.7 hpi, tB median 105.9 hpi). Embryos having single or multiple aneuploidy (median 103.4 hpi, P = 0.004 and 101.9 hpi, P = 0.006, respec- tively) had delayed initiation of blastulation compared with euploid embryos (median 95.1 hpi). No significant differences were observed in first or second cell-cycle length, synchrony of the second or third cell cycles, duration of blastulation, multinucleation at the 2-cell stage and irregular division patterns between euploid and aneuploid embryos. This non-invasive model for ploidy clas- sification may be used to avoid selecting embryos with high risk of aneuploidy while selecting those with reduced risk. RBMOnline
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TL;DR: The strategy of culturing and selecting embryos in the integrated EmbryoScope time-lapse monitoring system improves reproductive outcomes.
261 citations
TL;DR: Correlations were observed, in that euploid human blastocysts showed a higher percentage with top quality inner cell mass (ICM) and trophectoderm (TE), higher expansion grades and shorter time to start of blastulation, expansion and hatching, compared to aneuploid ones.
Abstract: Study question Are there correlations among human blastocyst ploidy status, standard morphology evaluation and time-lapse kinetics? Summary answer Correlations were observed, in that euploid human blastocysts showed a higher percentage with top quality inner cell mass (ICM) and trophectoderm (TE), higher expansion grades and shorter time to start of blastulation, expansion and hatching, compared to aneuploid ones. What is known already Embryo quality has always been considered an important predictor of successful implantation and pregnancy. Nevertheless, knowledge of the relative impact of each morphological parameter at the blastocyst stage needs to be increased. Recently, with the introduction of time-lapse technology, morphokinetic parameters can also be evaluated. However, a large number of studies has reported conflicting outcomes. Study design, size, duration This was a consecutive case series study. The morphology of 1730 blastocysts obtained in 530 PGS cycles performed from September 2012 to April 2014 that underwent TE biopsy and array comparative genomic hybridization was analyzed retrospectively. A total of 928 blastocysts were cultured in a time-lapse incubator allowing morphokinetic parameters to be analyzed. Partcipants/materials, setting, method Mean female age was 36.8 ± 4.24 years. Four hunderd fifty-four couples were enrolled in the study: 384, 64 and 6 of them performed single, double or triple PGS cycles, respectively. In standard morphology evaluation, the expansion grade, and quality of the ICM and TE were analyzed. The morphokinetic parameters observed were second polar body extrusion, appearance of two pronuclei, pronuclear fading, onset of two- to eight-cell divisions, time between the two- and three-cell (cc2) and three- and four-cell (s2) stages, morulae formation time, starting blastulation, full blastocyst stage, expansion and hatching timing. Main results and the role of chance Of the 1730 biopsied blastocysts, 603 were euploid and 1127 aneuploid. We observed that 47.2% of euploid and 32.8% of aneuploid blastocysts showed top quality ICM (P Limitations, reasons for caution The main limitation of morphology assessment is that it is a static system and can be operator-dependent. In this study, eight embryologists performed morphology assessments. The main limitation of the time-lapse technology is that it is impossible to rotate the embryos making it very difficult to observe them in case of blastomere overlapping or increased cytoplasmic fragmentation. Wider implications of the findings Although there seems to be a relationship between the ploidy status and blastocyst morphology/development dynamics, the evaluation of morphological and morphokinetic parameters cannot currently be improved upon, and therefore replace, PGS. Our results on ongoing pregnancy and miscarriage rates suggest that embryo evaluation by PGS or time-lapse imaging may not improve IVF outcome. However, time-lapse monitoring could be used in conjunction with PGS to choose, within a cohort, the blastocysts to analyze or, when more than one euploid blastocyst is available, to select which one should be transferred. Study funding/competing interests No specific funding was obtained for this study. None of the authors have any competing interests to declare.
228 citations
TL;DR: The clinical relevance of the aneuploidy risk classification model is demonstrated and a novel, non-invasive method of embryo selection to yield higher implantation and live birth rates without PGS is introduced.
Abstract: Time-lapse imaging of human preimplantation IVF embryos has enabled objective algorithms based on novel observations of development (morphokinetics) to be used for clinical selection of embryos. Embryo aneuploidy, a major cause of IVF failure, has been correlated with specific morphokinetic variables used previously to develop an aneuploidy risk classification model. The purpose of this study was to evaluate the effectiveness and potential impact of this model for unselected IVF patients without biopsy and preimplantation genetic screening (PGS). Embryo outcomes - no implantation, fetal heart beat (FHB) and live birth (LB) - of 88 transferred blastocysts were compared according to calculated aneuploidy risk classes (low, medium, high). A significant difference was seen for FHB (P<0.0001) and LB (P<0.01) rates between embryos classified as low and medium risk. Within the low-risk class, relative increases of 74% and 56%, compared with rates for all blastocysts, were observed for FHB and LB respectively. The area under the receiver operating characteristic curve was 0.75 for FHB and 0.74 for LB. This study demonstrates the clinical relevance of the aneuploidy risk classification model and introduces a novel, non-invasive method of embryo selection to yield higher implantation and live birth rates without PGS.
208 citations
TL;DR: The results indicate that a universal algorithm for optimal timing of development might not be feasible and the apparent negative significance of division patterns that differ from the expected may imply that time-lapse will facilitate de-selection of embryos.
Abstract: STUDY QUESTION Do early time-lapse parameters predict which embryos will develop to high-quality blastocysts and does timing of development differ between embryos that implant and those that do not. SUMMARY ANSWER Development to high-quality blastocysts could be predicted within the first 48 h of culture, whereas time-lapse parameters could not predict pregnancy. WHAT IS KNOWN ALREADY Historical cohort studies on embryos from unselected groups of patients have suggested several putative kinetic markers of viability. Before well-designed randomized studies can be conducted, relevant selection models based on solid data must be developed. So far conclusions from the previous studies are ambiguous. STUDY DESIGN, SIZE, DURATION A prospective cohort study conducted from February 2011 to June 2012. A total of 571 ICSI embryos from 92 patients were included in the blastocyst development analysis and 84 single embryo transfers were included in the pregnancy outcome analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS Embryos from women aged <38 years, with no endometriosis and ≥ 8 oocytes retrieved. University affiliated clinic. Embryos were cultured in a time-lapse incubator till Day 6. Logistic regression analysis was performed with variables selected based on indication. MAIN RESULTS AND THE ROLE OF CHANCE Duration of the first cytokinesis, duration of the 3-cell stage and direct cleavage to 3-cells predicted development to high-quality blastocyst. We found no difference in timing between implanted and non-implanted embryos. LIMITATIONS, REASONS FOR CAUTION A larger study might detect differences in timing between implanted and non-implanted embryos. The cohort consisted of good prognosis patients only and may not be representative of the entire IVF population. WIDER IMPLICATIONS OF THE FINDINGS Our results in context with the lack of consistency in previous studies and the presumed influences of different external factors indicate that a universal algorithm for optimal timing of development might not be feasible. The apparent negative significance of division patterns that differ from the expected may imply that time-lapse will facilitate de-selection of embryos. STUDY FUNDING/COMPETING INTEREST(S) Funding for the present study was provided by Aarhus University, the Lippert Foundation, the Toyota Foundation, the Aase og Einar Danielsen foundation and by an unrestricted grant from MSD and Ferring. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER The study was registered at ClinicalTrial.gov with accession number NCT01139268.
200 citations
TL;DR: The adoption of the proposed guidelines for defining annotation practice and universal nomenclature would help unify time-lapse monitoring practice, allow validation of published embryo selection algorithms and facilitate progress in this field.
Abstract: Study question Can the approach to, and terminology for, time-lapse monitoring of preimplantation embryo development be uniformly defined in order to improve the utilization and impact of this novel technology? Summary answer The adoption of the proposed guidelines for defining annotation practice and universal nomenclature would help unify time-lapse monitoring practice, allow validation of published embryo selection algorithms and facilitate progress in this field. What is known already An increasing quantity of publications and communications relating to time-lapse imaging of in vitro embryo development have demonstrated the added clinical value of morphokinetic data for embryo selection. Several articles have identified similar embryo selection or de-selection variables but have termed them differently. An evidence-based consensus document exists for static embryo grading and selection but, to date, no such reference document is available for time-lapse methodology or dynamic embryo grading and selection. Study design, size and duration A series of meetings were held between September 2011 and May 2014 involving time-lapse users from seven different European centres. The group reached consensus on commonly identified and novel time-lapse variables. Participants/materials, setting, methods Definitions, calculated variables and additional annotations for the dynamic monitoring of human preimplantation development were all documented. Main results and the role of chance Guidelines are proposed for a standard methodology and terminology for the of use time-lapse monitoring of preimplantation embryo development. Limitations, reasons for caution The time-lapse variables considered by this group may not be exhaustive. This is a relatively new clinical technology and it is likely that new variables will be introduced in time, requiring revised guidelines. A different group of users from those participating in this process may have yielded subtly different terms or definitions for some of the morphokinetic variables discussed. Due to the technical processes involved in time-lapse monitoring, and acquisition of images at varied intervals through limited focal planes, this technology does not currently allow continuous monitoring such that the entire process of preimplantation embryo development may be visualized. Wider implications This is the first time that a group of experienced time-lapse users has systematically evaluated current evidence and theoretical aspects of morphokinetic monitoring to propose guidelines for a standard methodology and terminology of its use and study, and its clinical application in IVF. The adoption of a more uniform approach to the terminology and definitions of morphokinetic variables within this developing field of clinical embryology would allow practitioners to benefit from improved interpretation of data and the sharing of best practice and experience, which could impact positively and more swiftly on patient treatment outcome. Study funding/competing interests There was no specific funding for the preparation of these proposed guidelines. Meetings were held opportunistically during scientific conferences and using online communication tools. H.N.C. is a scientific consultant for ESCO, supplier of Miri TL. I.E.A. is a minor shareholder in Unisense Fertilitech, supplier of the EmbryoScope. Full disclosures of all participants are presented herein. The remaining authors have no conflict of interest.
195 citations
Cites background or result from "Modelling a risk classification of ..."
...…several confounding factors on morphokinetic variables, such as age (Leibenthron et al., 2012; Hampl and Stěpán, 2013), ploidy (Chavez et al., 2012; Campbell et al., 2013a,b), ovarian reserve (Fréour et al., 2012), infertility indication (Wissing et al., 2012), ovarian response to stimulation…...
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...For ICSI/IMSI, where the time-lapse monitoring system and practice allows, the time of the sperm injection may be recorded, per oocyte but otherwise, it is the mid-time point from when injection begins and ends for that patient’s cohort of oocytes....
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...For example ‘irregular divisions’ have been collectively referred to as those from one to three and/or two to five cells in ,5 h (Campbell et al., 2013a,b); the former has also been defined as abrupt division (Meseguer et al., 2011), direct (Rubio et al., 2012), tri-polar cleavage (Wong et al.,…...
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...…of the zona pellucida, at the early cleavage stage, to facilitate herniation of trophectoderm for biopsy, has been reported not to impact downstream development to the full blastocyst stage, compared with unbreached controls (Campbell et al., 2013a). tSB ¼ initiation/start of blastulation....
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...…between embryo kinetics and embryo viability have been demonstrated in various studies (Pribenszky et al., 2010; Wong et al., 2010; Cruz et al., 2011; Meseguer et al., 2011; Azzarello et al., 2012; Campbell et al., 2013b; Chamayou et al., 2013; Herrero et al., 2013; Aguilar et al., 2014)....
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References
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TL;DR: It is expected that the definition of common terminology and standardization of laboratory practice related to embryo morphology assessment will result in more effective comparisons of treatment outcomes.
Abstract: Background: Many variations in oocyte and embryo grading make inter-laboratory comparisons extremely difficult. This paper reports the proceedings of an international consensus meeting on oocyte and embryo morphology assessment.
Methods: Background presentations about current practice were given.
Results: The expert panel developed a set of consensus points to define the minimumcriteria for oocyte and embryomorphology assessment.
Conclusions: It is expected that the definition of common terminology and standardization of laboratory practice related to embryo morphology assessment will result in more effective comparisons of treatment outcomes. This document is intended to be referenced as a global consensus to allow standardized reporting of the minimum data set required for the accurate description of embryo development.
1,189 citations
TL;DR: Evidence suggests that cohesin acts as a novel topological device that traps chromosomal DNA within a large tripartite ring formed by its core subunits.
Abstract: The cohesin complex is a major constituent of interphase and mitotic chromosomes. Apart from its role in mediating sister chromatid cohesion, it is also important for DNA double-strand-break repair and transcriptional control. The functions of cohesin are regulated by phosphorylation, acetylation, ATP hydrolysis, and site-specific proteolysis. Recent evidence suggests that cohesin acts as a novel topological device that traps chromosomal DNA within a large tripartite ring formed by its core subunits.
917 citations
"Modelling a risk classification of ..." refers background in this paper
...Although the cause of a temporal delay in aneuploid embryos compared with their euploid counterparts is not yet fully explained, there exist error detection and repair systems within the cell to prevent aneuploidy (Nasmyth and Haering, 2009)....
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TL;DR: A multivariable model is proposed based on the findings to classify embryos according to their probability of implantation and it is proposed that the image acquisition and time-lapse analysis system makes it possible to determine exact timing of embryo cleavages in a clinical setting.
Abstract: background: Time-lapse observation presents an opportunity for optimizing embryo selection based on morphological grading as well as providing novel kinetic parameters, which may further improve accurate selection of viable embryos. The objective of this retrospective study was to identify the morphokinetic parameters specific to embryos that were capable of implanting. In order to compare a large number of embryos, with minimal variation in culture conditions, we have used an automatic embryo monitoring system. methods: Using a tri-gas IVF incubator with a built-in camera designed to automatically acquire images at defined time points, we have simultaneously monitored up to 72 individual embryos without removing the embryos from the controlled environment. Images were acquired every 15 min in five different focal planes for at least 64 h for each embryo. We have monitored the development of transferred embryos from 285 couples undergoing their first ICSI cycle. The total number of transferred embryos was 522, of which 247 either failed to implant or fully implanted, with full implantation meaning that all transferred embryos in a treatment implanted. results: A detailed retrospective analysis of cleavage times, blastomere size and multinucleation was made for the 247 transferred embryos with either failed or full implantation. We found that several parameters were significantly correlated with subsequent implantation (e.g. time of first and subsequent cleavages as well as the time between cleavages). The most predictive parameters were: (i) time of division to 5 cells, t5 (48.8–56.6 h after ICSI); (ii) time between division to 3 cells and subsequent division to 4 cells, s2 (≤0.76 h) and (iii) duration of cell cycle two, i.e. time between division to 2 cells and division to 3 cells, cc2 (≤11.9 h). We also observed aberrant behavior such as multinucleation at the 4 cell stage, uneven blastomere size at the 2 cell stage and abrupt cell division to three or more cells, which appeared to largely preclude implantation. conclusions: The image acquisition and time-lapse analysis system makes it possible to determine exact timing of embryo cleavages in a clinical setting. We propose a multivariable model based on our findings to classify embryos according to their probability of implantation. The efficacy of this classification will be evaluated in a prospective randomized study that ultimately will determine if implantation rates can be improved by time-lapse analysis.
738 citations
TL;DR: It is found that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA).
Abstract: We report studies of preimplantation human embryo development that correlate time-lapse image analysis and gene expression profiling. By examining a large set of zygotes from in vitro fertilization (IVF), we find that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA). These parameters can be reliably monitored by automated image analysis, confirming that successful development follows a set of carefully orchestrated and predictable events. Moreover, we show that imaging phenotypes reflect molecular programs of the embryo and of individual blastomeres. Single-cell gene expression analysis reveals that blastomeres develop cell autonomously, with some cells advancing to EGA and others arresting. These studies indicate that success and failure in human embryo development is largely determined before EGA. Our methods and algorithms may provide an approach for early diagnosis of embryo potential in assisted reproduction.
714 citations
TL;DR: This is the first description of aCGH fully integrated with a clinical IVF program to select single blastocysts for fresh SET in good prognosis patients and highlights the inherent imprecision of SET when conventional morphology is used alone.
Abstract: Single embryo transfer (SET) remains underutilized as a strategy to reduce multiple gestation risk in IVF, and its overall lower pregnancy rate underscores the need for improved techniques to select one embryo for fresh transfer. This study explored use of comprehensive chromosomal screening by array CGH (aCGH) to provide this advantage and improve pregnancy rate from SET. First-time IVF patients with a good prognosis (age <35, no prior miscarriage) and normal karyotype seeking elective SET were prospectively randomized into two groups: In Group A, embryos were selected on the basis of morphology and comprehensive chromosomal screening via aCGH (from d5 trophectoderm biopsy) while Group B embryos were assessed by morphology only. All patients had a single fresh blastocyst transferred on d6. Laboratory parameters and clinical pregnancy rates were compared between the two groups. For patients in Group A (n = 55), 425 blastocysts were biopsied and analyzed via aCGH (7.7 blastocysts/patient). Aneuploidy was detected in 191/425 (44.9%) of blastocysts in this group. For patients in Group B (n = 48), 389 blastocysts were microscopically examined (8.1 blastocysts/patient). Clinical pregnancy rate was significantly higher in the morphology + aCGH group compared to the morphology-only group (70.9 and 45.8%, respectively; p = 0.017); ongoing pregnancy rate for Groups A and B were 69.1 vs. 41.7%, respectively (p = 0.009). There were no twin pregnancies. Although aCGH followed by frozen embryo transfer has been used to screen at risk embryos (e.g., known parental chromosomal translocation or history of recurrent pregnancy loss), this is the first description of aCGH fully integrated with a clinical IVF program to select single blastocysts for fresh SET in good prognosis patients. The observed aneuploidy rate (44.9%) among biopsied blastocysts highlights the inherent imprecision of SET when conventional morphology is used alone. Embryos randomized to the aCGH group implanted with greater efficiency, resulted in clinical pregnancy more often, and yielded a lower miscarriage rate than those selected without aCGH. Additional studies are needed to verify our pilot data and confirm a role for on-site, rapid aCGH for IVF patients contemplating fresh SET.
506 citations
"Modelling a risk classification of ..." refers background in this paper
...Recently, it has been shown that the incidence of live birth in young, good-prognosis women, with a theoretically low risk of embryo aneuploidy is enhanced by PGS (Yang et al., 2012)....
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