Embryos not showing pronuclei by single static observation arise from atypical pronuclear dynamics or rare unfertilized oocytes with abortive cleavage behaviour

Study question: Is the non-pronuclear (0PN) fertilization pattern, as assessed by single static observation during the 16- to 18-h post-insemination (hpi) interval, compatible with embryo developmental competence? Summary answer: A few zygotes show pronuclei (PN) solely outside the static fertilization assessment time interval and can develop to blastocyst stage, while rare cases of cleavage without pronuclear formation always result in early developmental arrest or degeneration. What is known already: Recently, the use of atypically pronucleated zygotes-showing no, one, or three PN (0PN, 1PN, and 3PN), or other less frequent patterns-has been increasingly proposed as a measure to maximize the number of embryos available for treatment and the cumulative clinical outcome per cycle. The use of such zygotes poses important clinical and ethical concerns; it also raises scientific questions, such as the hypothesis that the 0PN pattern can be compatible with pre- and post-implantation development in the absence of actual formation of PN. Allowing detailed observation of embryo morphokinetics, time-lapse technology (TLT) can be decisive in resolving such questions. Study design, size, duration: Retrospective observational study (2013-2020) including 6035 oocytes inseminated by ICSI and cultured in a time-lapse imaging incubator system. Zygotes (N = 4479), classified by PN-type, were divided into sub-groups based on timings of PN appearance (tPNa: <16hpi, >16hpi) and PN fading (tPNf: <16hpi, 16-18hpi, 18-20hpi, >20hpi). Their development was monitored until blastocyst stage. The remaining 1556 injected oocytes were either degenerated (N = 801) or did not show PN (true-0PN; N = 755). Among the latter, 186 showed ≥1 cleavage(s) and then degenerated. Their videos were analysed to assess events preceding developmental arrest. Participants/materials, setting, methods: Ovarian stimulation with GnRH-antagonist and hCG/agonist trigger was performed, with all patients undergoing ICSI and blastocyst culture. The study endpoints were the blastulation rates across different PN-appearance and -fading thresholds, as well as a thorough description of the events during cell division(s) in 0PN embryos that experienced early arrest. Regression analyses were conducted, adjusting for confounders such as maternal age, male factor, and PN-type, with associations confirmed using generalized estimating equations. Main results and role of chance: Zygotes showing two PN (2PN, N = 4479) monitored by TLT were distributed in sub-groups based on tPNf (<16 hpi, N = 11; 16-18 hpi, N = 44; 18-20 hpi, N = 309; >20 hpi, N = 4115). In a few cases (N = 85), PN formation occurred after 16 hpi, up until 31 hpi. Single static observation during the 16- to 18-hpi conventional interval would have missed the zygotes undergoing very early PNf and at least part of those undergoing late PNa. Comparison between the above sub-groups showed that pronuclear fading occurring at 18-20 hpi was associated with the highest blastocyst formation rate (36.4%, 47.7%, 65.4%, and 53.3%, for the diverse tPNf, respectively; P < 0.001). Late tPNa (>16 hpi) was accompanied by reduced blastocyst formation rate (54.5% vs 29.4%; P < 0.001). tPNf occurring at 18-20 hpi was also associated with a higher blastocyst euploidy rate. Of 755 true-0PN oocytes, 186 (24.6%) underwent ≥1 cleavage(s). However, all of them arrested before compaction, often displaying irregular cleavage(s) (including direct, reverse, and chaotic), vacuolization, and/or extensive fragmentation. Limitations, reasons for caution: The study data are derived from a single, although large, dataset. Therefore, they require independent confirmation. In addition, assessment of fertilization morphokinetics can only be applied to ICSI insemination, leaving fertilization morphokinetics achieved by standard IVF unexplored. Wider implications of the findings: TLT confirms the limitations of single static observation, which fails to detect a significant proportion of zygotes, especially if executed relatively late. This has implications for fertilization assessment, prediction of blastulation, and utilization of atypically pronucleated zygotes. Moreover, TLT demystifies the belief that blastocyst development may occur in the absence of pronuclear formation. Study funding/competing interest(s): None. Trial registration number: None.