Minimally invasive preimplantation genetic testing using blastocyst culture medium

STUDY QUESTION: Is minimally invasive chromosome screening (MICS) using blastocyst culture medium (BCM) sufficiently fast and accurate for preimplantation genetic testing (PGT) SUMMARY ANSWER: A new assay for MICS, named MICS-Inst achieved high-resolution, comprehensive chromosome ploidy detection using BCM. WHAT IS KNOWN ALREADY: BCM is a viable source of genomic DNA for use in PGT. STUDY DESIGN, SIZE, DURATION: Forty-one vitrified blastocysts donated by 22 couples known to carry a chromosome rearrangement and 21 vitrified blastocysts donated from 8 couples with normal karyotypes were used in this study. Good-quality blastocysts, defined as Day 5 and Day 6 embryos >= BB (AA, AB, BA, BB) based on the Gardner system were used for analysis. Recruitment took place from May 2018 to August 2018. We performed PGT for structural rearrangements (PGT-SR) on 41 BCM, trophectoderm (TE) biopsy and blastocyst-stage embryo (BE) samples as well as PGT for aneuploidies (PGT-A) on 21 BCM, TE biopsy and BE samples. PARTICIPANTS/MATERIALS, SETTING, METHODS: We made several significant modifications to the BCM composition (mixing blastocoel fluid and spent blastocyst medium) as well as the pre-existing multiple annealing and looping-based amplification cycles (MALBAC) techniques and library generation procedures. The design of a quasilinear preamplification (Pre-AMP) primer and AMP primers 1 and 2 enables the preparation of a next-generation sequencing library after the exponential amplification stage by introducing the Illumina P5 and P7 primers into the final products, which are then ready for sequencing. Sequencing was performed on the Illumina Hiseq 2500 platform with 2.0 Mb raw reads generated for each sample. MAIN RESULTS AND THE ROLE OF CHANCE: For PGT-A, BCM and TE biopsy samples showed 90% and 86% clinical concordance with the corresponding BE samples, respectively. In addition, both BCM and TE biopsy samples showed 76% karyotype concordance with the corresponding BE samples. For PGT-SR, we successfully obtained ploidy information for all 23 chromosomes with the exception of any rearrangements involving the Y chromosome. Both BCM and TE biopsy samples showed 100% clinical concordance with the corresponding BE samples in detecting chromosomal rearrangements. BCM and TE biopsy samples showed 90% and 100% karyotype concordance with the corresponding BE samples, respectively. Additionally, no statistically significant differences were detected in the aforementioned values of the BCM and TE biopsy samples in either PGT-A or PGT-SR (P > 0.05). Moreover, we achieved accurate quantification of segmental abnormalities using BCM samples. In addition, MICS-Inst reduced the number of steps required for library preparation through the use of new primer designs, resulting in an overall time reduction of 7.5 h. This time reduction allows for the performance of fresh blastocyst transfers. LIMITATIONS, REASONS FOR CAUTION: The main limitation is that BE, rather the inner cell mass, was used as the standard to evaluate the chromosome screening results. WIDER IMPLICATIONS OF THE FINDINGS: These results show that MICS-Inst is effective in procedure and precision for PGT, and that it is possible to achieve fresh blastocyst transfer following PGT. The implications are significant, as these findings may lead to minimally invasive PGT methods in the future.