Natalia Basile

Increasing the probability of selecting chromosomally normal embryos by time-lapse morphokinetics analysis

OBJECTIVE To study the differences in the cleavage time between chromosomally normal and abnormal embryos and to elaborate an algorithm to increase the probability of noninvasively selecting chromosomally normal embryos. DESIGN Retrospective cohort study. SETTING University-affiliated infertility center. PATIENT(S) Preimplantation genetic screening patients (n = 125; n = 77 with ET), including cases of repeated implantation failure or recurrent miscarriage. A total of 504 embryos were analyzed. INTERVENTION(S) Embryo culture within a time-lapse system. MAIN OUTCOME MEASURE(S) Kinetic variables included the time to 2 (t2), 3 (t3), 4 (t4), and 5 (t5) cells as well as the length of the second (cc2 = t3 - t2) and third (cc3 = t5 - t3) cell cycle, the synchrony in the division from 2 to 4 cells (s2 = t4 - t3), and the interval t5 - t2. Implantation and clinical pregnancy rates were also analyzed. RESULT(S) A logistic regression analysis identified t5 - t2 (odds ratio [OR] = 2.853; 95% confidence interval [CI], 1.763-4.616), followed by cc3 (OR = 2.095; 95% CI, 1.356-3.238) as the most relevant variables related to normal chromosomal content. On the basis of these results, an algorithm for embryo selection is proposed to classify embryos from A to D. Each category exhibited significant differences in the percentage of normal embryos (A, 35.9%; B, 26.4%; C, 12.1%; D, 9.8%). CONCLUSION(S) Chromosomally normal and abnormal embryos have different kinetic behavior. On the basis of these differences, the proposed algorithm serves as a tool to classify embryos and to increase the probability of noninvasively selecting normal embryos.

Type of culture media does not affect embryo kinetics: a time-lapse analysis of sibling oocytes

STUDY QUESTION Are the morphokinetics of growing embryos affected by the type of culture media utilized? SUMMARY ANSWER Morphokinetic parameters used for embryo selection are not affected between the two different concept culture media analyzed. WHAT IS KNOWN ALREADY Studies on the effect of culture media on human embryos have focused on evaluating different in-house and commercially available media as well as comparing outcomes among different commercial media. Nonetheless, the evaluation of embryo development in these studies was based on static observations and very little is known from a dynamic point of view. STUDY DESIGN, SIZE, DURATION Prospective cohort study, October 2010 and April 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS University-affiliated infertility center. Patients undergoing egg donation (n = 75) in which embryos were cultured with two different types of media in a time-lapse system. Embryo development was analyzed with time-lapse imaging for single step media (Global®) and sequential media (Sage® Cleavage). Variables studied included the timing to two cells (t2), three cells (t3), four cells (t4) and five cells (t5) as well as the length of the second cell cycle (cc2 = t3 - t2) and the synchrony in the division from two to four cells (s2 = t4 - t3). Implantation and clinical pregnancy rates were also analyzed. MAIN RESULTS AND THE ROLE OF CHANCE No statistically significant differences were observed between the two media for all the variables analyzed. When analyzing the percentage of embryos falling within the optimal ranges proposed for s2, cc2 and t5, we did not find significant differences between the two media. Pregnancy and implantation rates were similar for the three types of transfers: 48.0% (CI 95% 28.4-67.6) and 42.0% (CI 95% 22.5-61.4) with Global media; 58.8% (CI 95% 35.4-82.2) and 38.2% (CI 95% 15.0-61.4) with Cleavage media; and 58.1% (CI 95% 40.7-75.4) and 37.1% (CI 95% 22.1-52.1) with mixed transferred, respectively. Multiple implantations (twins) were also similar among the three groups, with 24.0% (CI 95% 9.3-45.1) for transfers with embryos cultured in Global media, 17.6% (CI 95% 3.7-43.3) for transfers with embryos cultured in Cleavage media and 22.5% (CI 95% 9.5-41.0) with mixed transfers. LIMITATIONS, REASONS FOR CAUTION The study was not powered to test differences in pregnancy rates between the two culture media, as this was not the hypothesis tested. Results are based on observations with embryos from oocyte donors and need to be repeated with embryos from infertile patients of different ages. WIDER IMPLICATIONS OF THE FINDINGS The absence of differences in morphokinetics between two different media concepts validates the algorithm for embryo selection in diverse culture conditions. STUDY FUNDING/COMPETING INTEREST(S) No specific funding was obtained for this study; it was solely funded by IVI. None of the authors have any economic affiliation with Unisense Fertilitech A/S but IVI is a minor shareholder in Unisense Fertilitech A/S.

Type of chromosome abnormality affects embryo morphology dynamics

OBJECTIVE To study the differences in the cleavage time between types of embryo chromosomal abnormalities and elaborate algorithm to exclude aneuploid embryos according to the likelihood to be euploid. DESIGN Retrospective cohort study. SETTING University affiliated private center. PATIENT(S) Preimplantational genetic screening patients (n = 112) including cases of advanced maternal age, repeated implantation failure, and recurrent miscarriage. A total of 485 embryos were analyzed. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) All biopsied embryos were cultured in an incubator with time-lapse technology, cleavage timing from insemination to day 3 and all kinetic parameters that have been described in previous studies by our group. RESULT(S) Logistic regression analysis were used to identify morphokinetic parameters and some were strongly associated with complex aneuploid embryos; t3 (odds ratio = 0.590, 95% confidence interval 0.359-0.971) and t5-t2 (odds ratio = 0.151, 95% confidence interval 0.082-0.278). CONCLUSION(S) Embryo morphokinetics are affected by chromosome aneuploidy and further analysis of the chromosome content reveals higher differences when the complexity in the chromosome disorders is increased. The use of time-lapse monitoring, although not able to detect an abnormal embryo, may be potentially useful to discard those embryos with high risk of complex chromosomal abnormalities.

Automatic time-lapse instrument is superior to single-point morphology observation for selecting viable embryos: retrospective study in oocyte donation

OBJECTIVE To correlate the different categories provided by a commercial diagnostic test with blastocyst formation, quality, implantation potential, and ongoing pregnancy (OPR) for the purpose of validating the automatic annotations and the classification algorithm. DESIGN Observational, retrospective, multicenter cohort study. SETTING University-affiliated private IVF center. PATIENT(S) A total of 3,002 embryos, including 521 transferred embryos with known implantation, from 626 IVF cycles that were incubated in a conventional incubator and monitored with an automatic time-lapse test. INTERVENTIONS(S) None. MAIN OUTCOME MEASURE(S) Embryo selection was based on morphology and the classification provided by a commercial diagnostic test. Implantation was the primary end point, and OPR, blastocyst formation (BR), and embryo morphology were secondary end points. RESULT(S) BR and number of optimal blastocysts were related to the classification test. This correlation was also observed when analyzing implantation rates (day 3 transfer: high 38.2%, medium 31.7% and low 26.1%; day 5 transfer: high 66.7%, medium 50%, low 31%). Patients where no high embryos were transferred (n = 75) had an OPR of 46.70%, and those patients where at least one high embryo was transferred (n = 109) significantly increased OPR to 67%. A logistic regression analysis studying other confounding factors (day of transfer, number of oocytes obtained, and embryo morphology classification) was included. In that model, if at least one of the embryos was labeled as high, OPR was 2.567 times higher than a cycle where no high embryos were transferred. CONCLUSION(S) Our study presents, to our knowledge, the largest set of transferred embryos after time-lapse analysis with the use of an automatic time-lapse test. The provided classification was related to reproductive outcome. Our results suggest that the automated embryo diagnostic test provided extra information to the embryologist to select the best embryos, independently from clinical features of the patient or day of transfer.

What does morphokinetics add to embryo selection and in-vitro fertilization outcomes?

Purpose of review Time lapse technology represents a new tool in the in-vitro fertilization (IVF) laboratory. It can aid the embryologist in the detection of objective and quantifiable markers associated with embryo viability and implantation. The purpose of this review is to explain how embryo morphokinetics can be used as an adjunct to standard morphological assessment and to evaluate its potential value to improve IVF outcomes. Recent findings Several algorithms have been developed. Some utilize early kinetic markers, whereas others rely more on later stages of embryo development. Even though over a handful of randomized control trials are in progress, at this point, only one has been published demonstrating a significant increase in implantation rates and ongoing pregnancy rates when selecting embryos on the basis of a combination of morphological assessment and morphokinetics. Summary We believe that standard morphological assessment should remain the gold standard to initiate embryo evaluation; however, if possible, it can be complemented with the use of morphokinetics. This new approach will allow the embryologist to perform a more accurate and objective embryo selection and therefore reduce the number of embryos transferred while maintaining or even improving clinical results.

Systematic review on clinical outcomes following selection of human preimplantation embryos with time-lapse monitoring.

Sir, We have read with great interest the recently published systematic review authored by Kaser and Racowsky (2014). This review enlightens the reader on the problem of utilizing time-lapse technology as a clinical tool based on the absence of ‘high quality’ data and advises the users to keep it as an experimental tool. Although we agree with the authors’ general comment that standardization in embryo annotation is necessary, and that the existing literature does not yet provide any certainty on the improvement in live birth rates permitted by timelapse monitoring (TLM), we would like to address some issues raised in this paper. First, the literature described as not having ‘high quality’ data represents, in our opinion, the irreplaceable starting point of future prospective studies, essential for elaborating relevant randomized controlled trials (RCTs) to demonstrate the clinical usefulness of embryo morphokinetics. First, the morphokinetic differences between implanted and nonimplanted embryos have been described and used to build algorithms (Mesegueret al., 2011). The benefit of this strategyover standard morphology has then been confirmed retrospectively (Meseguer et al., 2012), allowing the design of a ‘high quality’ RCT with the appropriate sample size and power (Rubio et al., 2014). Second, the authors state that current studies available on TLM demonstrate that faster cleaving embryos have a higher implantation potential, consistently with all previously published studies using conventional morphology, implying that TLM would have limited superiority over morphology. This statement should be nuanced, as there is some evidence that TLM can also provide some relevant exclusion criteria for embryo de-selection, regardless of embryo morphology (Rubio et al., 2012). In this view, one can postulate that future TLM would not only predict which embryo has the highest implantation potential, but also help the embryologist to discard the ones that have very low implantation potential. To go further, preliminary work recently published on the increased possibility of selecting chromosomally normal embryos by TLM paves the way for future studies aiming at identifying morphokinetic markers relevant for both embryo selection and de-selection for transfer (Basile et al., 2014). Third, we agree that single embryo transfer (SET) is the gold standard for studies aiming at revealing a link between embryological aspects and implantation. However, most studieson TLM used the concept of known implantation data (KID) embryos, embryos with known implantation. Whether excluding cycles with partial implantation negatively impacts validity should be debated, as external validation can be conducted in KID embryos too. One can also argue that any study conducted with a SET policy should not be extrapolated to double embryo transfer cycles, which still represent the large majority of IVF activity throughout the world. It would thus be interesting to check the SET proportion in published studies using the KID concept. Fourth, the authors mention concerns regarding light exposure in TLM. Embryo exposure to light during incubation in a time-lapse system has already been compared favorably with light exposure on standard microscope (Ottosen et al., 2007). Fifth, we fully agree that standardization in time-lapse nomenclature is necessary. However, whether tcf1 (identification of first cytokinesis furrow) is unequivocally identifiable and should be the standard reference can be debated, as pronucleus fading has been shown to offer an accurate alternative (Cruz et al., 2013). Sixth, we would like to insist on one aspect of clinical embryology that the authors shortly recall in their introduction. Indeed, conventional morphology assessment only allows moderate prediction of the embryos’ implantation capability, and suffers from relatively limited specificity and sensitivity, with significant inter/intra observer variability. We obviously fully agree with this statement, especially as, to our knowledge, no RCT has evaluated the clinical interest of morphology evaluation. Moreover, few studies conducted in humans without any embryo selection, for example for legal reasons, showed that high cumulative pregnancy rates could also be obtained (Ubaldi et al., 2010). It should also be noted that variability is largely present in studies based on conventional morphology (media, atmosphere. . .); however, this has not invalidated its usefulness as the gold standard in embryology. Therefore, if one considers that any embryo assessment method not supported by ‘high quality’ evidence of its efficiency should be considered an experimental strategy subject to Internal Review Board approval, then all IVF labs across the world should reconsider most of the procedures that are routinely used including the way they choose embryos for transfer. It should also be recalled that morphology represents the first step of TLM-based embryo selection. In summary, we are confident that some conclusions drawn by the authors of this review will very soon be partially dismissed by ‘high quality’ clinical prospective studies, ruling out the statement that ‘TLM should remain an experimental strategy subject to institutional review and approval’.

The state of “freeze-for-all” in human ARTs

The recent development of vitrification technologies and the good outcomes obtained in assisted reproduction technologies have supported new indications for freezing and segmentation of treatment. Beyond OHSS prevention and avoidance of embryo transfers in the setting of an adverse endocrinological profile or endometrial cavity, we have witnessed a trend to shift fresh embryo transfers to frozen embryo transfers in many programs. We critically review the available evidence and suggest that freeze-all is not “for all,” but should be individualized.

Time-lapse in the IVF lab: how should we assess potential benefit?

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Multinucleation on day 2 affects embryos kinetics but it is not correlated with their chromosomal content

Conclusions: Ongoing implantation rates after day-3 and day5/6 vitrification are comparable between SET and DET, therefore SET should be recommended. Vitrification of blastocysts derived from non 8-cell embryos (assessed within fixed time frames) significantly increases ongoing implantation rates, resulting much quicker in a live birth.