Non-invasive preimplantationsexingusingRT-PCRon the spent embryo culture media

Abstract

Introduction Preimplantation sexing is an alternative method for Preimplantation GeneticTesting (PGT) to prevent the birth of children suffering from x-linked genetic disorders.Social sexing for non-medical reasons is also legal for some cases. PGT routinely needsto biopsy of embryonic cells or oocyte polar bodies which are invasive and could lead to implantation failure. SRY gene expression begins in male embryos following genome activation in preimplantation embryos. The aim of this study was sexing of preimplantation embryos without biopsy based on the presence of SRY RNA and DNA in the spent culture medium as a biomarker of sexing of human preimplantation embryos. Materials and Methods In this double-blind study, two groups were evaluated. In the first group, on the third day after the biopsy, the human embryos were cultured individually; we received the spent media of the non-degenerated 5-day embryos those have been underwent sexing by Fluorescent In Situ Hybridization(FISH) method via blastomere biopsy on the third day. In the second group, embryos of ART candidateswere cultivated on the third day individuallyuntil development to blastocyst stage; this group of embryos was not biopsied but the blastocysts were fixed for sexing by FISH. Each group was divided to 2 subgroups; in the first subgroup we extracted the RNA from the spent culture medium of each embryo. Following RNA extraction, the total RNA was immediately reverse transcribed to cDNA. In the second subgroup, PCR was performed directly on the culture medium. PCR was performed for SRY and GAPDH genes. The SRY positive embryos were considered as male embryos and those were GAPDH positive and SRY negative considered as females. The results of sexing based on spent culture media was compared with the results of sexing based on FISH. Results For the first group, 12 samples were evaluated, after comparing the results of PCR and FISH, the results for all the samples were concordant.In the second group, we were able to correctly diagnose 12 of the 14 samples. Two embryos with false diagnosis were females that diagnosed as males by direct PCR. Conclusion Preimplantation sexing without embryo biopsy by RT-PCR on the spent embryo culture media seems to be a reliable tool for non-invasive preimplantation sexing. However, direct PCR-based diagnosis might be lead to false results; this is probably due to DNA contamination.