Re-biopsy combined with double vitrification during preimplantation-genetic-testing cycles: comprehensive description of blastocysts rescued after failing the first round of diagnosis

Study question: Which are the technical and clinical results of re-biopsied blastocysts that failed the first round of qPCR-based aneuploidy testing on trophectoderm biopsies? Summary answer: The risk of inconclusive diagnoses is <3%.Re-biopsied blastocysts after double vitrification show similar survival, euploidy and implantation rates as blastocysts diagnosed at the first analysis. What is known already: The implementation of multicellular trophectoderm biopsy approach has significantly reduced the risk for amplification failure and inconsistent results during preimplantation-genetic-testing cycles. Yet, to date few reports defined the rate of re-biopsy procedures required, as well as their related technical and clinical results. On average, 2-6% of the blastocysts were reported to result in an inconclusive diagnosis after comprehensivechromosome- testing (CCT), of which 1-2% due to DNA amplification failure. The technical and clinical outcomes seem comparable to blastocysts with a conclusive result after the first biopsy. Study design, size, duration: 8991 blastocyst biopsy procedures were conducted between April 2013 and September 2017 at 7 IVF centres in Italy and analysed by qPCR at a single genetic lab. 206 blastocysts were successfully rebiopsied after warming and re-expansion, and then re-vitrified. 41 frozen single-embryo-transfers (SETs) of blastocysts diagnosed euploid after trophectoderm re-biopsy were performed to date. Logistic regression analyses were performed to investigate the parameters associated with an inconclusive diagnosis. Participants/materials, setting, methods: Only preimplantation-genetictesting- for-aneuploidies (PGT-A) cycles with a freeze-all approach were included. Vitrification was performed within 30 min from trophectoderm biopsy on collapsed blastocysts. qPCR-based CCT method was adopted. Amplification failure or nonconcurrent molecular data resulted in an inconclusive diagnosis. Only frozen euploid SETs were performed ≈2 hr after warming. Clinical pregnancy, miscarriage (<20weeks) and ongoing pregnancy rates were monitored. Main results and the role of chance: 8991 blastocysts were included (3244 PGT-A cycles conducted from 2687 couples, mean maternal age: 38.5 ± 3.9, 25-45). 2.6% of the trophectoderm biopsies resulted in an inconclusive diagnosis (n = 228/8991, 95%CI:2.2%-2.9%), of which 2% (n = 176/8991) due to amplification failure and 0.6% (n = 52/8991) due to nonconcurrent results. The only parameters significantly associated with the risk of obtaining an inconclusive diagnosis were the IVF centre (OR = 1.13, 95%CI:1.1-1.2 from the one performing the highest to the one performing the lowest number of procedures; p<0.01) and the day-of-biopsy (OR = 0.55, 95%CI:0.43-0.69 from day 5 to day7; p<0.01). 213 blastocysts were warmed to be re-analysed. The survival rate after warming was 98.1% (n = 209/213) and the survival rate after re-biopsy was 98.6% (n = 206/209). All the re-biopsied blastocysts resulted in a conclusive diagnosis, among them the euploidy rate was 51.9% (n = 107/206) (The euploidy rate of blastocysts biopsied only once was 45.7% (n = 4000/4778)). 41 blastocysts diagnosed euploid after re-biopsy have been warmed to date. All of them survived (100%, n = 41/41) and were transferred. 51.2% clinical pregnancy (n = 21/41), 9.5% miscarriage (n = 2/21) and 46.3% ongoing pregnancy (n = 19/41) rates were reported. Limitations, reasons for caution: Only targeted-qPCR-based CCT method was adopted in this retrospective analysis. A more powered report of the clinical (and possibly obstetrical and perinatal) outcomes after frozen euploid SET of re-biopsied blastocysts requires a larger sample size. Ideally, these clinical data should be clustered also per blastocysts’ quality and day of full-blastulation. Wider implications of the findings: Trophectoderm biopsy approach is confirmed technically-consistent and clinically-secure. The residual risk for inconclusive diagnoses (2.6%) seems related to day-of-biopsy (possibly the number/volume of retrieved cells) and IVF centres’ expertise. Yet, it is worth re-biopsying undiagnosed blastocysts since the survival, euploidy and ongoing implantation rates seem comparable to the control. Trial registration number: None.