Inconclusive chromosomal assessment after blastocyst biopsy: prevalence, causative factors and outcomes after re-biopsy and re-vitrification. A multicenter experience

Danilo Cimadomo, Laura Rienzi, Valeria Romanelli, Erminia Alviggi, Paolo Emanuele Levi-Setti, Elena Albani, Ludovica Dusi, Letizia Papini, Claudia Livi, Francesca Benini, Antonella Smeraldi, Cristina Patassini, Filippo Maria Ubaldi and Antonio Capalbo

Hum Reprod. 2018 Sep 18; Submitted on June 27, 2018; resubmitted on August 6, 2018; accepted on August 22, 2018

STUDY QUESTION: Can a second round of biopsy, vitrification and chromosomal testing provide a valid diagnosis where the first attempt fails? SUMMARY ANSWER: The risk of inconclusive chromosomal-assessment after trophectoderm biopsy was 2.5% but a further biopsy and vitrification-warming appeared not to impair the competence of euploid blastocysts.

WHAT IS KNOWN ALREADY: The increasing implementation of multicell trophectoderm biopsy has significantly reduced the risk of inconclusive diagnosis after preimplantation-genetic-testing (PGT). Yet, few reports have defined the variables that influence the risk of failure or described the technical and clinical outcomes after re-biopsy.

STUDY DESIGN, SIZE, DURATION: Retrospective multicenter study involving 8990 blastocyst biopsies conducted between April 2013 and September 2017 at six IVF centers but analyzed at a single genetic laboratory. A total of 206 blastocysts were successfully re-biopsied after warming and re-expansion, then re-vitrified. And 49 of these blastocysts were diagnosed euploid and used in single-embryo-transfers (SETs). Logistic regression analyses were conducted.

PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 3244 PGT-for-aneuploidies (PGT-A) cycles with a freeze-all approach, vitrification and qPCR-based analysis were performed by 2687 consenting couples. DNA amplification failure (AF) or non- concurrent data resulted in inconclusive diagnoses. In case of DNA amplification, the cellularity of the biopsy was estimated according to a previously validated method. Euploid SETs were performed. Clinical pregnancy, miscarriage, live birth rates (LBR) and perinatal outcomes were monitored.

MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 2.5% of trophectoderm biopsies resulted in an inconclusive diagnosis (N = 228/8990). Specifically, 2% (N = 176/8990) resulted in AF and 0.5% (N = 52/8990) in non-concurrent results. The only parameters signifi- cantly associated with inconclusive diagnoses were the IVF center and the embryo age (days) at biopsy. Among samples with successful ampli- fication, the number of cells in the biopsy and the day of biopsy were critical to limit non-concurrent results. In total, 213 blastocysts with an inconclusive diagnosis were warmed for re-analysis and the survival rate was 96.7% (N = 206/213). The euploidy rate in blastocysts biopsied twice was 51.9% (N = 107/206) and the euploid embryos were re-vitrified. Overall, 49 euploid embryos were warmed for replacement and all survived. The LBR after SET was 38.8% (N = 19/49). No minor/major obstetrical/perinatal complication was reported.

LIMITATIONS, REASONS FOR CAUTION: A single aneuploidy-testing method was adopted in this retrospective analysis. A more pow- ered report of the clinical and obstetrical/perinatal outcomes after re-biopsied and re-vitrified blastocysts euploid SET requires a larger sample size.

WIDER IMPLICATIONS OF THE FINDINGS: It is important to re-biopsy and re-vitrify undiagnosed blastocysts since healthy live births can result from them.

STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: None.

Key words: blastocyst / trophectoderm biopsy / preimplantation-genetic-testing / undiagnosed embryo / inconclusive results

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