Assessing efficacy of day 3 embryo time-lapse algorithms retrospectively: impacts of dataset type and confounding factors.

This study investigated the efficacy of four published day 3 embryo time-lapse algorithms based on different types of datasets (known implantation data [KID] and single embryo transfer [SET]), and the confounding effect of female age and conventional embryo morphology. Four algorithms were retrospectively applied to three types of datasets generated at Fertility North between February 2013 and December 2014: (a) KID dataset (n = 270), (b) a subset of SET (n = 144, end-point = implantation), and (c) SET (n = 144, end-point = live birth), respectively. All four algorithms showed progressively reduced predictive power (expressed as area under the receiver operating characteristics curve and 95% confidence interval [CI]) after application to the three datasets (a-c): Liu (0.762 [0.701-0.824] vs. 0.724 [0.641-0.807] vs. 0.707 [0.620-0.793]), KIDScore (0.614 [0.539-0.688] vs. 0.548 [0.451-0.645] vs. 0.536 [0.434-0.637]), Meseguer (0.585 [0.508-0.663] vs. 0.56 [0.462-0.658] vs. 0.549 [0.445-0.652]), and Basile (0.582 [0.505-0.659] vs. 0.519 [0.421-0.618] vs. 0.509 [0.406-0.612]). Furthermore, using KID dataset, the association (expressed as odds ratio and 95% CI) between time-lapse algorithms and implantation outcomes lost statistical significance after adjusting for conventional embryo morphology and female age in 3 of the 4 algorithms (KIDScore 1.832 [1.118-3.004] vs. 1.063 [0.659-1.715], Meseguer 1.150 [1.021-1.295] vs. 1.122 [0.981-1.284] and Basile 1.122 [1.008-1.249] vs. 1.038 [0.919-1.172]). In conclusion, SET is a preferred dataset to KID when developing or validating time-lapse algorithms, and day 3 conventional embryo morphology and female age should be considered as confounding factors.

Intracytoplasmic sperm injection using hyaluronic acid or polyvinylpyrrolidone: a time-lapse sibling oocyte study.

This study evaluated the effect of sperm selection and intracytoplasmic sperm injection (ICSI) on subsequent fertilization and embryo development using the hyaluronic acid-based SpermSlow™ (HA-ICSI) compared to injection with polyvinylpyrrolidone (PVP-ICSI). A total of 206 metaphase II oocytes were collected from 21 prospectively enrolled ICSI cycles at Fertility North between July 2014 and March 2015. Sibling oocytes were randomized into HA-ICSI and PVP-ICSI (n = 103 per group). Subsequent fertilization outcomes and embryo development in terms of qualitative and quantitative time-lapse measures following three-day culture in the Embryoscope™ were compared. HA-ICSI resulted in significantly lower abnormal fertilization rates (1.9% vs 9.7%, p = 0.017), and a trend towards increased normal fertilization rates (73.8% vs 62.1%, p = 0.073) with increased injection time (2.5 vs 2.1 min, p = 0.001). No differences between HA-ICSI and PVP-ICSI were observed in (a) the proportion of good conventional morphology embryos (50% vs 53.1%, p = 0.712), (b) time-lapse qualitative measures (p > 0.05) and (c) time-lapse quantitative measures (p > 0.05). In conclusion, HA-ICSI improves fertilization outcomes although sperm injection takes longer to complete. Subsequent embryo development up to day 3 is not affected.

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

OBJECTIVE: To present a time-lapse deselection model involving both qualitative and quantitative parameters for assessing embryos on day 3. DESIGN: Retrospective cohort study and prospective validation. SETTING: Private IVF center. PATIENT(S): 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. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Morphological score on day 3, embryo morphokinetic parameters, abnormal cleavage patterns, and known implantation results. RESULT(S): 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. CONCLUSION(S): 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.

Assessment of human embryos by time-lapse videography: A comparison of quantitative and qualitative measures between two independent laboratories.

A total of 488 Day 3 human embryos with known implantation data from two independent in vitro fertilization laboratories were included for analysis, with 270 from Fertility North (FN) and 218 from Canberra Fertility Centre (CFC). Implanting embryos grew at different rates between FN and CFC as indicated in hours of the time intervals between pronuclear fading and the 4- (13.9 ± 1.1 vs. 14.9 ± 1.8), 5- (25.7 ± 1.9 vs. 28.4 ± 3.7) and 8-cell stages (29.0 ± 3.2 vs. 32.2 ± 4.6), as well as the durations of 2- (10.8 ± 0.8 vs. 11.6 ± 1.1), 3- (0.4 ± 0.5 vs. 0.9 ± 1.2), and 4-cell stages (11.8 ± 1.4 vs. 13.6 ± 2.9), all p<0.05. The application of a previously published time-lapse algorithm on ICSI embryos from the two participating laboratories failed to reproduce a predictive pattern of implantation outcomes (FN: AUC=0.565, p=0.250; CFC: AUC=0.614, p=0.224). However, for the qualitative measures including poor conventional morphology, direct cleavage, reverse cleavage and <6 intercellular contact points at the end of the 4-cell stage, there were similar proportions of embryos showing at least one of these biological events in either implanting (3.1% vs. 3.3%, p>0.05) or non-implanting embryos (30.4% vs. 38.3%, p>0.05) between FN and CFC. Furthermore, implanting embryos favored lower proportions of the above biological events compared to the non-implanting ones in both laboratories (both p<0.01). To conclude, human embryo morphokinetics may vary between laboratories, therefore time-lapse algorithms emphasizing quantitative timing parameters may have reduced inter-laboratory transferability; qualitative measures are independent of cell division timings, with potentially improved inter-laboratory reproducibility.

Time-lapse videography of human embryos: Using pronuclear fading rather than insemination in IVF and ICSI cycles removes inconsistencies in time to reach early cleavage milestones.

Time-lapse videography showed that human early cleavage embryos were quicker following intracytoplasmic sperm injection to reach developmental milestones compared to in vitro fertilization when using insemination as the timing start point (t0), due to differences in the time taken for embryos to reach pronuclear fading (PNF). These differences disappeared when PNF was used as t0. Using a biological rather than procedural t0 will allow a unified assessment strategy to be applied to all cycles irrespective of the insemination method.

Clinical significance of intercellular contact at the four-cell stage of human embryos, and the use of abnormal cleavage patterns to identify embryos with low implantation potential: a time-lapse study.

OBJECTIVE: To investigate the clinical significance of intercellular contact point (ICCP) in four-cell stage human embryos and the effectiveness of morphology and abnormal cleavage patterns in identifying embryos with low implantation potential. DESIGN: Retrospective cohort study. SETTING: Private IVF center. PATIENT(S): A total of 223 consecutive IVF and intracytoplasmic sperm injection treatment cycles, with all resulting embryos cultured in the Embryoscope, and a subset of 207 cycles analyzed for ICCP number where good-quality four-cell embryos were available on day 2 (n = 373 IVF and n = 392 intracytoplasmic sperm injection embryos). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Morphologic score on day 3, embryo morphokinetic parameters, incidence of abnormal biological events, and known implantation results. RESULT(S): Of 765 good-quality four-cell embryos, 89 (11.6%) failed to achieve six ICCPs; 166 of 765 (21.7%) initially had fewer than six ICCPs but were able to establish six ICCPs before subsequent division. Embryos with fewer than six ICCPs at the end of four-cell stage had a lower implantation rate (5.0% vs. 38.5%), with lower embryology performance in both conventional and morphokinetic assessments, compared with embryos achieving six ICCPs by the end of four-cell stage. Deselecting embryos with poor morphology, direct cleavage, reverse cleavage, and fewer than six ICCPs at the four-cell stage led to a significantly improved implantation rate (33.6% vs. 22.4%). CONCLUSION(S): Embryos with fewer than six ICCPs at the end of the four-cell stage show compromised subsequent development and reduced implantation potential. Deselection of embryos with poor morphology and abnormal cleavage revealed via time-lapse imaging could provide the basis of a qualitative algorithm for embryo selection.

Can 'abnormally' fertilized zygotes give rise to viable embryos?

Conventional practice in in vitro fertilization or intracytoplasmic sperm injection is to select the best quality embryos based on their morphology and cleavage status from a cohort of fertilized oocytes in which two pronuclei were observed at the time they were checked for fertilization. However, in a small proportion of cycles, the selection is limited to embryos that appeared to be either unfertilized (displaying zero pronuclei) or abnormally fertilized (displaying one or three pronuclei) at the time they were checked for fertilization. There is a lack of consensus on whether such embryos should be transferred to the uterus. Cytogenetic analysis of embryos from oocytes with one pronucleus has shown a proportion is diploid. Transfer of such embryos has resulted in healthy births. Limited cytogenetic analysis of oocytes that divide despite the absence of pronuclei at fertilization check indicates that a proportion also have a normal cytogenetic constitution. Cytogenetic analysis of embryos from oocytes with three pronuclei has shown high rates of triploidy and chaotic cell divisions. Subsequent foetuses have extremely unfavourable outcomes. Here, we review the published literature on the cytogenetic analysis of 'unfertilized' and 'abnormally fertilized' embryos and discuss possible pathways which lead to their formation. The limited evidence indicates that oocytes with one pronucleus and oocytes that show normal onward division despite the absence of pronuclei may be considered for replacement in certain circumstances.