NON-INVASIVE PGT-A

The measurement of cell-free DNA in the fluid extracted from expanded blastocysts is one of two current approaches in ‘non-invasive PGT-A’, and thus an alternative to the favoured trophectoderm biopsy and analysis by next-generation sequencing. Now, a prospective study following up an earlier investigation testing the validity of blastocoelic fluid as an accurate representative of ploidy status has produced findings which may have implications for embryo selection in both PGT-A and indeed conventional IVF/ICSI cycles.(1)

The group of Gianaroli and Magli in Bologna had ‘surprisingly’ found in an earlier study (to compare results from blastocoel fluid analysis with those from trophectoderm biopsy) that the incidence of positive DNA amplification from blastocoelic fluid analysis was significantly lower with the PGT-A euploid blastocysts (45%) than with the PGT-A aneuploid blastocysts (81%).(2) Moreover, after transfer of euploid blastocysts the ongoing CPR was significantly higher in the group with negative DNA amplification from blastocoel fluid (68%) than from those with positive amplification (31%).

As a follow-up, this latest study, which included 102 consecutive PGT-A patients and 88 conventional IVF/ICSI patients, collected blastocoel fluid from high-grade blastocysts of both groups, the former before trophectoderm biopsy and vitrification and the latter before immediate vitrification. The blastocoel fluid was analysed by whole genome amplification (WGA), and priority for transfer determined by negative or positive results. Only euploid blastocysts were considered for transfer in the PGT-A group, while in both groups priority for transfer was based on WGA results with negative amplification.

Results following transfers in the PGT-A group confirmed suggestions from the initial study, that LBRs at first transfer were higher from those blastocysts with negative amplification in blastocoel fluid than from those with positive amplification (53.3% vs 26.2%), a differential which remained after adjusting for confounders (OR 3.52). In the second group of conventional IVF 30 deliveries after the first transfer resulted from blastocysts with negative amplification in blastocoel fluid (48.4%) and three from the transfer of positive WGA blastocysts (11.5%). Again, the difference remained significant after adjustments (OR 6.89) Indeed, in both groups blastocysts with negative DNA amplification in the blastocoel fluid resulted in a LBR per transfer, per patient and per first transfer which was more than double that of positive WGA blastocysts.

Clearly, the trend that blastocysts with absent genomic amplification in the blastocoel fluid had a greater chance of implantation and live birth than those with positive WGA was evident in both the PGT-A blastocysts and those from conventional IVF - with the suggestion that viability is related to the presence or not of DNA in the blastocoel fluid. ‘The presence of DNA in the [blastocoel fluid] (positive BF-WGA) could be indicative of an abnormal embryo, probably mosaic, that is trying to reach a viable state by marginalizing aneuploid fragments to the periphery of the differentiating embryo,’ suggest the authors by way of explanation. They add that viability in this case would depend on the degree of mosaicism and the amount of energy required to extrude aneuploid cells into the blastocoel fluid - which is why some of the positive amplification blastocysts did indeed implant.

They note that their suggestion may reflect the ‘quiet embryo’ hypothesis of Henry Leese proposed 20 years ago, that more metabolically active embryos (as defined by their uptake of nutrients) had a lower level of viability than those deemed of slow metabolism (and a low uptake of nutrients). Similarly, studies from Elpida Fragouli have suggested that levels of mitochondrial DNA in embryos are indicative of implantation potential, with increased levels linked to aneuploidy, higher levels of metabolism and implantation failure.(3)

While no more than a suggestion, the authors note that DNA amplification in blastocoel fluid might ‘represent a tool to shorten time to delivery’ in all IVF embryos, given that in this study negative amplification was clearly associated with the best clinical outcome. The advantage of an extra method to select embryos for transfer, they add, would even be valuable in patients in a conventional IVF/ICSI cycle. They thus conclude - and until verified in larger trials - that such an easy and cost-effective tool as the analysis of blastocoel fluid by WGA ‘could become a valuable option to offer patients the highest chances of a term pregnancy in the shortest time possible’.


1. Gianaroli L, Perruzza D, Albanese C, et al. Failure to detect DNA in blastocoel fluid is associated with a higher live birth rate in both PGT-A and conventional IVF/ICSI cycles. Hum Reprod 2023;
doi.org/10.1093/humrep/dead088
2. Magli MC, Albanese C, Crippa A, et al. Deoxyribonucleic acid detection in blastocoelic fluid: a new predictor of embryo ploidy and viable pregnancy. Fertil Steril 2019; 111: 77-85.
doi.org/10.1016/j.fertnstert.2018.09.016
3. Fragouli E, Spath K, Alfarawati S, et al. Altered levels of mitochondrial DNA are associated with female age, aneuploidy, and provide an independent measure of embryonic implantation potential. PLoS Genet 2015; 11(6): e1005241. doi.org/10.1371/journal.pgen.1005241