P-757 Trophectoderm biopsy timing standardization via Time Lapse Microscopy and Artificial Intelligence: high consistency across operators and minimal inconclusive results rates
Abstract Study question Can observation through Time Lapse Microscopy (TLM) be leveraged to standardize trophectoderm biopsy outcomes across different operators? Summary answer TLM-based blastocysts’ observation to pinpoint the ideal time-of-biopsy (t-biopsy) indicated comparable inconclusive PGT-A results rates among seven embryologists operating across eight years. What is known already Optimal timing for trophectoderm biopsy in blastocysts lacks clear guidelines, with current literature presenting mixed outcomes regarding its effect on molecular analysis reliability. Inappropriate biopsy timing can potentially yield inconclusive PGT diagnoses due to insufficient blastocyst expansion. Minimizing the need for a second biopsy is essential, as multiple manipulations can affect embryo reproductive competence. Thus, TLM and artificial intelligence (AI) offer valuable tools to objectively define biopsy timing based on blastocyst expansion. Study design, size, duration Observational analysis of 1,943 blastocysts cultured in TLM between 2013-2020 and biopsied with a sequential zona-pellucida opening and trophectoderm biopsy approach. Seven operators were trained to establish the stage of biopsy based on blastocysts’ full-expansion achieved between 110-169 hours-post-insemination (hpi; mean:134±15). AI (CHLOE, Fairtility) was retrospectively applied to annotate all timings and expansion as area of the embryo-proper at the median focal plane in µm2 at the time-of-expanding-blastocyst (tEB) and at the t-biopsy. Participants/materials, setting, methods We assessed all features possibly associated with the inconclusive result rate after trophectoderm biopsy: biopsy-operator, t-biopsy and embryo-proper area, blastocyst quality (based on Gardner and CHLOE embryo-quality score), day of the week (Monday-Sunday), hour of the working day (retrieved through RI electronic-witnessing-system; 8am-8pm), year (2013-2020), comprehensive-chromosome-testing technology (qPCR/NGS). Associations with euploidy and live-birth (LB) per euploid transfer were also tested. Logistic regression and Generalized Estimating Equations analyses were adopted to this end. Main results and the role of chance The overall inconclusive results rate was 3.3% (N = 64/1,943) varying 1%-5% across operators and years. The largest fluctuation occurred when transitioning from qPCR (2013-Oct2017; N = 35/1,222, 2.9%) to NGS (Oct2017-2020; 4%), but decreased to 2.3% (N = 2/88) in 2020. These differences were not-significant, suggesting that fluctuations can be minimized by monitoring operators’ performance systematically. Among all parameters investigated, none associated with inconclusive results. The embryo-proper area at t-biopsy was larger for day6-7 blastocysts (unstandardized coefficient-B day5 to day7:+730µm2, 95%CI:+301 to + 1,158,p<0.001), and smaller for poorer-quality blastocysts (unstandardized coefficient-B AA-grade to CC/BC/CB-grade:-1,115µm2, 95%CI:-1,332 to -897,p<0.001). The year was associated with embryo-proper area at t-biopsy (unstandardized coefficient-B 2013 to 2020:+309µm2, 95%CI:+168 to + 450,p<0.001) becoming significantly larger since 2017 (22,000-23,500µm2 to 24,000-25,000µm2), when NGS was implemented. No difference was reported among biopsy-operators, days of the week, nor hours of the working-day, except for sporadic cases of blastocysts biopsied between 8-9am showing lower expansion. Dispersion plots indicated more pronounced expansion from tEB to t-biopsy among day5 blastocysts, while this appeared less evident among day6-7 blastocysts. When adjusted for maternal-age and blastocyst-quality, embryo-proper area was associated with higher euploidy-rates (each 500µm2-increase OR:1.17,95%CI:1.07-1.28). Only blastocyst-quality and t-biopsy, instead, associated with LB per euploid-transfer. Limitations, reasons for caution Retrospective study to be confirmed prospectively with a larger sample size. Only the protocol entailing simultaneous laser-assisted zona pellucida opening and trophectoderm biopsy plus pulling was adopted. Wider implications of the findings The adoption of standard operating procedures and the continuous monitoring of operators’ performance minimized fluctuations in inconclusive PGT-A results, even when changing CCT technology or introducing new biopsy practitioners. TLM and AI can help monitoring and defining the optimal t-biopsy based on embryo-proper area, enhancing standardization, efficiency and safety. Trial registration number No
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