Steady morphokinetic progression is an independent predictor of live birth: a descriptive reference for euploid embryos.

Study question: Can modelling the longitudinal morphokinetic pattern of euploid embryos during time-lapse monitoring (TLM) be helpful for selecting embryos with the highest live birth potential?

Summary answer: Longitudinal reference ranges of morphokinetic development of euploid embryos have been identified, and embryos with steadier progression during TLM are associated with higher chances of live birth.

What is known already: TLM imaging is increasingly adopted by fertility clinics as an attempt to improve the ability of selecting embryos with the highest potential for implantation. Many markers of embryonic morphokinetics have been incorporated into decision algorithms for embryo (de)selection. However, longitudinal changes during this temporal process, and the impact of such changes on embryonic competence remain unknown. Aiming to model the reference ranges of morphokinetic development of euploid embryos and using it as a single longitudinal trajectory might provide an additive value to the blastocyst morphological grade in identifying highly competent embryos.

Study design size duration: This observational, retrospective cohort study was performed in a single IVF clinic between October 2017 and June 2021 and included only autologous single euploid frozen embryo transfers (seFET).

Participants/materials setting methods: Reference ranges were developed from [hours post-insemination (hpi)] of the standard morphokinetic parameters of euploid embryos assessed as tPB2, tPNa, tPNf, t2-t9, tSC, tM, tSB, and tB. Variance in morphokinetic patterns was measured and reported as morphokinetic variance score (MVS). Nuclear errors (micronucleation, binucleation, and multinucleation) were annotated when present in at least one blastomere at the two- or four-cell stages. The blastocyst grade of expansion, trophectoderm (TE), and inner cell mass (ICM) were assessed immediately before biopsy using Gardner's criteria. Pre-implantation genetic diagnosis for aneuploidy (PGT-A) was performed by next-generation sequencing. All euploid embryos were singly transferred in a frozen transferred cycle and outcomes were assessed as live birth, pregnancy loss, or not pregnant. Association of MVS with live birth was investigated with regression analyses.

Main results and the role of chance: TLM data from 340 seFET blastocysts were included in the study, of which 189 (55.6%) resulted in a live birth. The median time for euploid embryos to reach blastulation was 109.9 hpi (95% CI: 98.8-121.0 hpi). The MVS was calculated from the variance in time taken for the embryo to reach all morphokinetic points and reflects the total morphokinetic variability it exhibits during its development. Embryos with more erratic kinetics, i.e. higher morphokinetic variance, had higher rates of pregnancy loss (P = 0.004) and no pregnancy (P < 0.001) compared to embryos with steadier morphokinetic patterns. In the multivariable analysis adjusting for ICM, TE grade, presence of nuclear errors, and time of blastulation, MVS was independently associated with live birth (odds ratio [OR]: 0.62, 95% CI: 0.46-0.84, P = 0.002) along with ICM quality. Live birth rate of embryos with the same ICM grading but different morphokinetic variance patterns differed significantly. Live birth rates of embryos exhibiting low MVS with ICM grades A, B, and C were 85%, 76%, and 67%, respectively. However, ICM grades A, B, and C embryos with high MVS had live birth rates of 65%, 48%, and 21% (P < 0.001). The addition of the MVS to embryo morphology score (ICM and TE grading) significantly improved the model's AUC value (0.67 vs 0.62, P = 0.015) and this finding persisted through repeat cross-validation (0.64 ± 0.08 vs 0.60 ± 0.07, P < 0.001).

Limitations reasons for caution: The exclusion of IVF cases limits, for now, the utility of the model to only ICSI-derived embryos. The utility of these reference ranges and the association of MVS with various clinical outcomes should be further investigated.

Wider implications of the findings: We have developed reference ranges for morphokinetic development of euploid embryos and a marker for measuring total morphokinetic variability exhibited by developed blastocysts. Longitudinal assessment of embryonic morphokinetics rather than static time points may provide more insight about which embryos have higher live birth potential. The developed reference ranges and MVS show an association with live birth that is independent of known morphological factors and could emerge as a valuable tool in prioritizing embryos for transfer.

Study funding/competing interests: This study received no external funding. The authors declare no conflicting interests.

Trial registration number: N/A.