Evaluation of the impact of the pulling and flicking trophectoderm biopsy procedures on the integrity of the biopsied cells and their correlation to PGT-A results

Abstract

Blastocyst biopsy is currently the gold standard in PGT-A. However, because trophectoderm (TE) cell excision is technically challenging, results can vary depending on how the procedure is performed. To ensure successful results, it is important not only to avoid harming the blastocyst during the biopsy procedure, but also to ensure a minimal damage on the biopsied cells. In this study, we aimed to evaluate the impact of two different TE biopsy techniques (pulling and flicking) on the integrity of the biopsied cells and to correlate their status with the chromosome screening results of the two methods. This is a retrospective observational study that includes the data analysis of 268 TE biopsies performed on blastocysts from 83 patients (mean age – 39.1 y/o) that underwent a PGT-A cycle between October 2018 and April 2019. Chromosome screening analysis were carried out by an associated genetics laboratory. Indications for PGT-A cycles included advanced maternal age, recurrent implantation failure, recurrent miscarriage and/or severe male factor. Trophectoderm biopsies were performed with the assistance of a dynamic laser. Assisted hatching was performed on Day 3 and blastocysts with herniating cells or completely hatched were biopsied with the “pulling” or “flicking” techniques. In the pulling method, blastocysts were held firmly with the holding pipette and the biopsy needle used to pull TE cells away from the blastocyst, while laser pulses were applied. In the flicking method, laser pulses were used to allow TE cells to be drawn inside the biopsy pipette and subsequently the TE cells were excised with a quick movement of the biopsy pipette against the holding pipette. Biopsied cells were then photographed and classified according to their integrity status as follows: intact (A); partially lysed (B); completely lysed (C). After biopsy, the cells were washed and transferred into PCR tubes to be processed for chromosome screening by NGS. Statistical significance was assessed by Students t-test or Fisher’s exact test. A total of 118 blastocysts were biopsied with the pulling method and 150 with the flicking technique and no differences were found in terms of mean age of the patients (39.5±1.1 and 38.5±3.2, respectively) or average number of laser pulses used (4.2±1.1 vs 3.9±0.9, respectively). Overall, the pulling technique resulted in higher (p= 0.0009) percentage of pieces graded as A (74.6%, n=88) than the flicking method (54.7%, n=82), but no differences were found among the two groups in terms of euploidy rates (28% and 36%, respectively). Regardless of the technique used, all cells graded as C were majorly (80-100%) diagnosed as chromosomally abnormal compared to those that were classified as morphologically intact (43.9-62.5%) or partially lysed (52.4-62.5%). These results indicate that the pulling and flicking methods do not differ in terms of rates of chromosomally normal blastocysts as long as both procedures are applied correctly. The integrity of the cells seems to affect the results of aneuploidy rates, which might depend on blastocyst morphology.