Non-invasive PGT-A: many questions remain before clinical application

Published 27 September 2019

More than 170 registrants attended a wide-ranging Campus meeting on the established and emerging techniques of aneuploidy testing and on the latest developments in genomic editing and mitochondrial transfer.

An extremely well attended Campus meeting organised by the SIG Reproductive Genetics in Athens offered a comprehensive technique-by-technique review of embryo selection for IVF, from the very first genetic and epigenetic mechanisms driving early stage development, through chromosomal testing for aneuploidy, and on to the -omics and emerging non-invasive methods. According to Thierry Voet from Leuven, Belgium, PGT-A is now in its third phase of development, which began two decades ago with FISH, day 3 biopsies and limited chromosomal testing, moved on in 2008 (following the clear failure of FISH in a randomised trial) to more comprehensive methods (24 chromosomes with array CGH and SNPs) and most recently with next generation sequencing and trophectoderm biopsy.

The fourth phase, proposed Antonio Capalbo, Laboratory Director of Igenomix in Italy, may well be the evolution of minimally invasive testing. 'Trophectoderm biopsy is a safe and robust procedure,' said Capalbo, 'but non-invasive, easier and more cost-effective strategies are welcome.' The most tested non-invasive method currently, he explained, is genomic DNA sequencing of spent blastocyst medium, which - as in testing of tropectoderm cells - would allow deselection of aneuploid embryos for transfer. Nevertheless, he conceded, the technique poses problems, notably whether the spent culture medium does in fact represent the culture content of the embryo, or even if there is indeed embryonic cell-free DNA detectable there. For example, a study reported from China in 2016 tested (by NGS) the spent culture medium from 42 donated-for-research human blastocysts for ploidy information of all 24 chromosomes found a high correlation for identification of chromosomal abnormalities when the initial results were validated in the corresponding whole blastocyst lysate.(1) However, as Capalbo pointed out, there was no control group in the study to assess efficacy, and there remained a high risk of DNA contamination (mostly of maternal derivation) in the medium. He thus repeated the conclusions of a US study, which identified genetic and mitochondrial DNA in spent culture media, that 'as yet DNA from culture media cannot be used for genetic assessment because embryo-associated structures (such as cumulus cells)release DNA into the culture medium'.(2) Moreover, a number of concordance studies identified by Capalbo showed a wide range in their validation results, with different culture conditions and interpretation criteria. However, a new study just reported by Capalbo and colleagues showed a higher rate of concordance between the spent culture media results and those from trophectoderm biopsies for day 6/7 embryos than day 5.

However, despite glimpses of promise, Capalbo ended his presentation with more questions than answers - on the mechanism whereby cell-free DNA might pass to the culture medium, on the prevention of contamination in the medium, and of course on the validity of the method as tested by a randomised trial. Without any of these answers, he concluded, clinical application could not be supported and remains 'highly discouraged'.

Genome editing and mitochondrial transfer
This state-of-the-art meeting ended with two presentations with more news content than review - and more controversy than even PGT-A can muster. First, Norah Fogarty from the Francis Crick Institute in London, which in 2016 received the first UK licence for application of the genome editing technique CRISPR-Cas9 in human embryos, described the first results from the Institute's research on the earliest stages of human development. The experiments Fogarty reported described how the gene OCT4, transcribed actively from the 8‐cell stage in human embryos and required for pluripotency, was confirmed as necessary for blastocyst formation in human embryos, and that trophectoderm development is compromised in OCT4-silenced human (but not mouse) embryos. Identifying such genes as essential for embryo development may eventually offer clues into the causes of pregnancy failures or birth defects.

There were bold headlines - and a front page of Time magazine - in April this year when Spanish and Greek researchers behind a pilot trial of spindle transfer for the treatment of infertility (and not for the prevention of mitochondrial disease) announced its first live birth from a series aiming for 25 patients. Nuno Costa-Borges from the trial group (and scientific director of a Spanish company called Embryotools), noting that mitochondrial dysfunction is a major cause of decline in oocyte quality, described studies showing first that meiotic spindle transfer in mouse oocytes is feasible without impairing embryo development and later, following preclinical validation, as translational research in human donor oocytes. The latter, Costa-Borges emphasised, was designed with 'authorisation' of the Greek National Authority of Assisted Reproduction and with the informed consent of all patient parties. However, both the pregnancy and delivery were announced by press release - and not in a journal - and there were considerable doubts cast on the ethics of using this mitochondrial replacement technique for purposes other than the prevention of mitochondrial disease. Indeed, in the UK, where the first mitochondrial replacement studies were approved - and ironically in Spain, where the science of this trial was developed - mitochondrial replacement techniques are only allowed to prevent the inheritance of serious mitochondrial disease. It was presumably because of these restrictions that the study is taking place in Greece

In his description of the pilot study Costa-Borges stressed how the trial followed recent protocol advice for the introduction of new technologies, and insisted on the technique's 'experimental' status as defined by the four-point scoring tool of Provoost et al.(4) Following April's live birth announcement ESHRE itself issued a position statement urging caution in the application of spindle transfer in infertility, reiterating ESHRE's endorsement of the HFEA's position paper on new introductions in ART.(5) Moreover, when asked from the floor of this Campus meeting what labs should now do in their own considerations of mitochondrial transfer in infertility, Cost-Borges simple advised 'Do nothing' - at least not until the validation of safety has been assured in clinical trials.

1. Xu J, Fang R, Chen L, et al. Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization. Proc Natl Acad Sci 2016; 113: 11907-11912.

2. Hammond ER, McGillyray BC, Wicker SM, et al. Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified. Fertil Steril 2017; 107: 220-228.

3. Rubio C, Rienzi L, Navarro-Sanchez L, et al. Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications. Fertil Steril 2019; 112: 510-519.

4. Provoost V, Tilleman K, D'Angelo A, et al. Beyond dichotomy: a tool for distinguishing between experimental, innovative and established treatment. Hum Reprod 2014; 29: 413-417.

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