Morphokinetic analysis and embryonic prediction for blastocyst formation through an integrated time-lapse system.

To describe the events associated with the blastocyst formation and implantation that occur in embryos during preimplantation development based on the largest sample size ever described with time-lapse monitoring. Observational, retrospective, single-center clinical study. University-affiliated private IVF center. A total of 7,483 zygotes from 990 first treatments of intracytoplasmic sperm injection (ICSI; 627 of oocyte donor vs. 363 autologous oocyte cycles), of which 832 blastocysts were transferred. No patient intervention. Embryos were cultured in a time-lapse monitoring system, and the embryos were transferred on day 5 after ICSI. Embryo selection was based on the multivariable model previously developed and on blastocyst morphology. Using a time-lapse system, embryo images were acquired every 15 minutes for 120 hours. Embryos cleavage time points up to the 9-cell stage (t2-t9) as well as to the morula stage (tM) and blastocyst formation (tB) were registered in hours after ICSI. Additionally, duration of the cell cycle and synchrony of the second and third cell cycles were defined. As a result, we have monitored the embryonic development of a total of 3,215 blastocysts, of which 832 were transferred. Finally, we analyzed the characteristics of embryonic development of blastocyst (phase 1) and of implanted and not implanted (phase 2) embryos as finally validated in an independent data set (phase 3). A detailed retrospective analysis of cleavage times was made for 7,483 zygotes. We analyzed 17 parameters and found several significantly correlated with subsequent blastocyst formation and implantation. The most predictive parameters for blastocyst formation were time of morula formation, tM (81.28-96.0 hours after ICSI), and t8-t5 (≤8.78 hours) or time of transition of 5-blastomere embryos to 8-blastomere embryos with a receiver operating characteristic curve (ROC) value = 0.849 (95% confidence interval [CI], 0.835-0.854; phase 1). These parameters were less predictive of implantation, with a ROC value = 0.546 (95% CI, 0.507-0.585). We also observed that time for expansion blastocyst, tEB (107.9-112.9 hours after ICSI), and t8-t5 (≤5.67 hours after ICSI) predict blastocyst implantation, with a ROC value = 0.591 (95% CI, 0.552-0.630; phase 2). The model was validated on an independent data set and gave a ROC of 0.596 (0.526-0.666; phase 3). The inclusion of kinetic parameters into score evaluation may improve blastocyst selection criteria and can predict blastocyst formation with high accuracy. We propose two multivariable models based on our findings to classify embryos according to their probabilities of blastocyst stage and implantation in the largest data set ever reported of human blastocysts.

Motato Y ，de los Santos MJ ，Escriba MJ ，Ruiz BA ，Remohí J ，Meseguer M ... -  《-》

A time to look back: analysis of morphokinetic characteristics of human embryo development.

To describe the times associated with the morphological changes that occur in the embryo during preimplantation development based on the largest sample size described with time lapse. Cohort study. University-affiliated private center. A total of 9,530 embryos from 1,806 intracytoplasmic sperm injection (ICSI) cycles. None. Using a time-lapse system, embryo images were acquired for at least 68 hours, in some cases reaching 120-130 hours. Embryo cleavage time points up to 8-cell-stage (t2-t8) as well as morulae (tM) and blastocyst formation (tB) were registered in hours after ICSI. Additionally, duration of the cell cycle (cc) and synchrony (s) of the second and third cell cycles were defined. Finally, four subgroups of embryos were considered: the "regular divisions" group excluded embryos with a direct cleavage from 1 to 3 or 2 to 5 cells, and the "viable 8-cell," the "viable blastocyst," and "implanted embryos" groups included only embryos viable to the 8-cell stage, blastocyst stage, or transferred and successfully implanted, respectively. Averages of times in the general population were: t2 = 27.9 hours, t3 = 38.2 hours, t4 = 40.7 hours, t5 = 51.0 hours, t6 = 54.1 hours, t7 = 56.7 hours, t8 = 59.1 hours, tM = 86.6 hours, tB = 104.1 hours, cc2 = 10.3 hours, cc3 = 12.8 hours, s2 = 2.7 hours, and s3 = 9.9 hours. Comparison between groups showed significant differences between regular divisions and viable 8 cells for t2, t3, t5, cc2, cc3, s2, and s3; between 8 cells and blastocyst for t5, t8, tM, cc3, and s2; and between blastocyst and implanted embryos for t8, tM, tB, and s2. Differences in timing related to morphology of cleavage- and blastocyst-stage embryos were detected. A time-lapse monitoring system applied to embryology allows accuracy and objectivity when defining the basis of embryo development within a clinic. The sample size is the largest ever described that provides consistent information about the normal distribution of embryo developmental timings.

Herrero J ，Tejera A ，Albert C ，Vidal C ，de los Santos MJ ，Meseguer M ... -  《-》

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

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. Observational, retrospective, multicenter cohort study. University-affiliated private IVF center. 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. None. 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. 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. 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.

Aparicio-Ruiz B ，Basile N ，Pérez Albalá S ，Bronet F ，Remohí J ，Meseguer M ... -  《-》

Influence of different oocyte insemination techniques on early and late morphokinetic parameters: retrospective analysis of 500 time-lapse monitored blastocysts.

To determine how standard IVF vs. intracytoplasmic sperm injection (ICSI) fertilization influences early and late morphokinetic parameters during prolonged embryo culture. Five-hundred expanded blastocysts that were monitored in a time-lapse monitoring incubator were analysed retrospectively. Early (pronuclear fading [PNf], t2-t9) and late (start of blastulation, expanded blastocyst) morphokinetic variables were scored according to published consensus criteria. Private infertility clinic. A total of 209 consecutive infertile patients (mean ± SD age, 38.4 ± 4 years; range, 28-47 years) undergoing 238 natural IVF/minimal ovarian stimulation cycles during 2012-2014. Minimal ovarian stimulation, oocyte retrieval, fertilization with standard IVF or ICSI, prolonged embryo culture in a time-lapse monitoring incubator. Differences in morphokinetic parameters according to insemination techniques. In total, 29% and 71% of the whole cohort was fertilized with standard IVF and ICSI, respectively. During early cleavage stages (PNf to t4) there was a statistically significant delay (+1.5 to +1.1 hours) among IVF-fertilized embryos. By contrast, at the expanded blastocyst stage IVF-fertilized embryos showed faster development (-3.3 to -4.1 hours). After normalizing to the time point of PNf, differences in cleavage-stage parameters disappeared, but those at all blastocyst stages increased even further in favor of IVF-fertilized embryos (-3.2 to -5.7 hours). The observed 1.5-hour time difference between standard IVF- and ICSI-fertilized embryos is an artificial phenomenon. At the blastocyst stages, however, genuine timing differences arise between IVF- and ICSI-fertilized embryos, possibly related to their different quality. Normalization to a common time point permits the joint analysis of IVF- and ICSI-fertilized embryos, thus increasing the size of studied cohorts.

Bodri D ，Sugimoto T ，Serna JY ，Kondo M ，Kato R ，Kawachiya S ，Matsumoto T ... -  《-》

Time-lapse deselection model for human day 3 in vitro fertilization embryos: the combination of qualitative and quantitative measures of embryo growth.

To present a time-lapse deselection model involving both qualitative and quantitative parameters for assessing embryos on day 3. Retrospective cohort study and prospective validation. Private IVF center. A total of 270 embryos with known implantation data (KID) after day 3 transfer from 212 IVF/intracytoplasmic sperm injection (ICSI) cycles were retrospectively analyzed for building the proposed deselection model, followed by prospective validation using an additional 66 KID embryos. None. Morphological score on day 3, embryo morphokinetic parameters, abnormal cleavage patterns, and known implantation results. All included embryos were categorized either retrospectively or prospectively into 7 grades (A+, A, B, C, D, E, F). Qualitative deselection parameters included poor conventional day 3 morphology, abnormal cleavage patterns identified via time-lapse monitoring, and <8 cells at 68 hours postinsemination. Quantitative parameters included time from pronuclear fading (PNF) to 5-cell stage and duration of 3-cell stage. KID implantation rates of embryos graded from A+ to F were 52.9%, 36.1%, 25.0%, 13.8%, 15.6%, 3.1%, and 0 respectively (area under the curve [AUC] = 0.762; 95% confidence interval [CI], 0.701-0.824), and a similar pattern was seen in either IVF (AUC = 0.721; 95% CI, 0.622-0.821) or ICSI embryos (AUC = 0.790; 95% CI, 0.711-0.868). Preliminary prospective validation using 66 KID embryos also showed statistically significant prediction in Medicult (AUC = 0.750; 95% CI, 0.588-0.912) and Vitrolife G-Series (AUC = 0.820; 95% CI, 0.671-0.969) suites of culture media. The proposed model involving both qualitative and quantitative deselection effectively predicts day 3 embryo implantation potential and is applicable to all IVF embryos regardless of insemination method by using PNF as the reference starting time point.

Liu Y ，Chapple V ，Feenan K ，Roberts P ，Matson P ... -  《-》