P–160 Sibling oocytes cultured in a time-lapse versus benchtop incubator: limited exposure of embryos outside the incubator improves outcomes

Study question Does the limited exposure of embryos outside the incubator, during evaluation and changeover, have an impact on the blastocyst development, blastocyst quality and euploid outcomes? Summary answer Exposure of embryos outside the incubator, negatively impacts the number, quality and euploidy rate of day 5 blastocysts. What is known already The laboratory environment with its culture conditions is one of the crucial elements of the delicate equation to a successful ART outcome. It has been shown that increased fluctuations in the culture conditions have a considerable impact on the number of blastocysts obtained and cycle outcomes. Compared to conventional benchtop incubators, Time Lapse Technology (TLT) incubators capture images of the embryo and allow morphologic and morphokinetic assessment without disturbance during incubation. Several studies have been published comparing the efficiency, safety and outcome performance between conventional and TLT incubators, however, none of them explored the euploid outcomes. Study design, size, duration An observational sibling oocyte study was performed at ART Fertility Clinics, Abu Dhabi between March 2018 and April 2020 and included data of 796 mature oocytes injected from 42 stimulation cycles. Sibling oocytes were randomly split between 2 different incubators: 12 oocytes were assigned to the twelve wells of the EmbryoscopeTM (ES) and the remaining oocytes were cultured in a conventional benchtop incubator, G185 K-System (KS). Participants/materials, setting, methods Embryos from patients with primary or secondary infertility, who underwent ovarian stimulation for ICSI and PGT-A through NGS on trophectoderm biopsies, were eligible. All patients had at least 16 fresh mature oocytes, randomly allocated to two different incubators after ICSI: 503 (63.2%) oocytes were cultured in ES and 293 (36.8%) in KS. The fertilization, cleavage, useable blastocyst and euploid rates, as well as embryo/blastocyst qualities were assessed to evaluate each incubator’s performance. Main results and the role of chance The fertilization and cleavage rates were similar between incubators. Total useable blastocyst rate (64.8% vs 49.6%, p < 0.001) was significantly higher for embryos cultured in ES, mainly due a higher percentage of blastocysts biopsied on day 5 in ES (67.8% vs 57.0%, p = 0.037), with improved quality (p = 0.008). There was no difference in the total euploid rate between ES and KS (59.9% vs 50.4%, p = 0.314), but a significantly higher euploid rate was seen for blastocysts cultured in ES and biopsied on day 5 (63.5% vs 37.4%, p = 0.001). Day 3 embryo quality and total biopsied blastocyst quality was not different between incubators. No difference was observed in the total useable blastocyst development from good (p = 0.0832) and poor (p = 0.112) quality day 3 cleavage stage embryos. However, when stratifying according to the day of blastocyst development, poor quality embryos on day 3 showed superior blastocyst formation on day 5 when cultured in ES (64.1% vs 39.1% for day 5 and 35.9% vs 60.9% for day 6, p = 0.005). Accordingly, blastocyst formation from poor quality embryos on day 3, was shifted to day 6 for embryos cultured in KS. This difference in the day of blastocyst development was not observed for good quality cleavage stage embryos (p = 0.917). Limitations, reasons for caution The current observational study needs confirmation in a prospective trial and should also include the implantation potential of the euploid blastocysts, which was not followed in the current study. A good prognosis population (≥16 mature oocytes) was studied and may not reflect the outcomes in patients with lower oocyte numbers. Wider implications of the findings: This work builds evidence to the solid introduction of the TLT incubators to the clinical routine, as the reduced exposure of embryos outside the incubator – and hence decreased stress - improves the blastocyst development. Trial registration number NA

P–806 Neogametogenesis via oocyte replication

Study question Can full preimplantation embryo development be achieved from artificial oocytes created through nuclear transfer of a haploid pseudo-blastomere (HpB) into a recipient ooplast? Summary answer It is feasible to replicate the female genome and generate novel sibling oocytes that can yield full preimplantation embryo development, albeit at a reduced rate. What is known already A limitation of assisted reproduction is the number of available oocytes for embryo creation. It is feasible to utilize a somatic cell nucleus to construct novel oocytes through a process known as haploidization, in which a reverse meiosis occurs after SCNT. Similarly, producing haploid parthenogenetic constructs can generate HpBs, useful for genetic testing at the pre-fertilization level or for reproduction. It is feasible to use a HpB as a nuclear donor since it has already completed homologue segregation. Study design, size, duration This is prospective translational animal model study. Over 6 months, 556 oocytes were manipulated for the experimental group, and 158 control oocytes were employed. B6D2F1 HpBs were used to establish the procedure and acquire expertise. FVB HpBs were subsequently introduced for genetic variance. Experimental and control embryos were cultured in a time-lapse incubator (up to 96h). Cleavage parameters were compared to control. Two-sample T-tests and one-way ANOVA with Bonferroni correction were employed for statistical analysis. Participants/materials, setting, methods A cohort of oocytes was harvested from B6D2F1 or FVB superovulated mice and artificially activated by 8% ethanol. At the 8-cell stage, HpBs were exposed to nocodazole. Another cohort of B6D2F1 oocytes was enucleated for recipient ooplasts. HpBs were individually transferred into the perivitelline space of the ooplasts alongside inactivated Sendai virus. After fusion, reconstructed oocytes with spindle development were fertilized by piezo-actuated ICSI using B6D2F1 spermatozoa. Unmanipulated and fertilized B6D2F1 oocytes served as control. Main results and the role of chance A total of 158 control oocytes underwent ICSI with a 67.7% survival rate; of these, 65.4% developed to the blastocyst stage. For artificial oocyte activation (AOA), up to 10 oocytes were activated for each experiment, yielding 8 HpBs per activated oocyte. For the experimental group, 556 oocytes underwent enucleation with a 96.4% survival rate. Nuclear transfer of HpBs resulted in a 93.2% survival rate, consistent for those derived from BDF and FVB. Reconstructed oocytes showed appropriate development of a novel pseudo-meoitic spindle at a rate of 63.7% for B6D2F1 HpBs and 75.5% for FVB HpBs, and ICSI yielded a 67.1% and 57.7% survival rate, respectively. The fertilization rate for the reconstructed oocytes was 64%. Control oocytes underwent ICSI with a 67.7% survival rate. When evaluating time-lapse parameters, reconstructed embryos created via blastomere nuclear transfer showed asynchrony compared to controls beginning as early as the stage of pronuclear fading. While the majority of reconstructed embryos arrested at the 4-cell stage, of those that progressed, 11.3% of those using BDF HpBs and 14.6% of those using FVB HpBs developed to the fully expanded blastocyst stage. This corresponds to a total of 23 reconstructed embryos that developed to the morula or blastocyst stage. Limitations, reasons for caution While we used single-well embryoscope culture for morphokinetic data collection, group culture is superior to single-embryo culture for mice. Thus, developmental rates may be underestimated by this protocol. Implantation and successful pregnancy are also needed to support the clinical utility of this method in generating gametes. Wider implications of the findings: For women with diminished ovarian reserve, oocyte yield and age-related aneuploidy are limitations to achieving genotyped offspring. Nuclear transfer of HpB can generate sibling oocytes while maintaining genetic information. This model represents a promising path for expanding oocyte yield, allowing genetic assessment of sibling oocytes, and enhancing chances of procreation. Trial registration number none