66. THE DETECTION OF MITOCHONDRIAL DNA IN THE BASTOCOELIC FLUID OF EXPANDED BLASTOCYSTS

• Published in: Reproductive biomedicine online 2019-08, Vol.39, p.e67-e68
• Main Authors: Carano, F., Magli, M.C., De Fanti, S., Terzuoli, G., Azzena, S., Albanese, C., Gianaroli, L.
• Format: Article
• Language: English
• Subjects: blastocoelic fluid; mitochondrial DNA; PGT
• ISSN: 1472-6483; 1472-6491
• DOI: 10.1016/j.rbmo.2019.04.119

The pivotal role of mitochondria in ATP production, as well as the high mutation rate of their genome (mtDNA) are well known. Although variants fixation within the population depends to the phenotype functionality, some recurrent variants with deleterious effect survive to natural selection.We already described a relation between mitochondrial haplogroups and aneuploidy susceptibility. Since the segregation process is strictly dependent on adequate ATP provision, the mutation load analysis of mtDNA in blastocysts may be informative of embryo viability. The aim of this study was to verify whether mtDNA analysis could be conducted on the fluid extracted from the blastocoelic cavity through a procedure that is moderately invasive compared with the conventional forms of biopsy. In the first part of the study, the blastocoelic fluid (BF) retrieved from 10 expanded blastocysts previously inferred as aneuploid by a-CGH was amplified for the mitochondrial D-loop region and sequenced by Sanger. Maternal age of the 10 patients ranged between 37 and 41 years. In the second part of the study, D-loop sequencing was carried out in five additional sets of sequential biopsies including both polar bodies (PB I; PB II), BF, trophectoderm (TE) and the corresponding whole embryo (EM), accounting for a total of 25 samples. Also in this case, the 5 sets had been diagnosed as aneuploid by chromosome analysis on PBs. All sequences were aligned to mtDNA reference sequence prior to haplogroup inference. In the first part of the study, the complete D-loop amplification and sequencing was successful for all the BF samples. Haplogroups were inferred with 60% of lineages belonging to Macro-haplogroup H, while the remaining 40% was represented by the lineages I, W and K. These haplogroups are characteristic of the European population.In the second part of the study, D-loop amplification and sequencing was possible in all samples except for the PBs from one sample set. The intra-group sequence comparison highlighted full correspondence in 4 sets, while the remaining set showed a heteroplasmic position in one PB I. The feasibility to amplify and sequence mtDNA from the BF may offer a reliable alternative to other forms of conventional biopsy. In this context, it would be interesting to validate the method also to the complete mitochondrial genome (16Kb), testing in this way also its integrity.