Il Dott. Antonio Capalbo e il team del gruppo G.EN.E.R.A. e del laboratorio di biologia molecolare GENETYX, hanno pubblicato uno studio sulla celebre rivista European Journal of Human Genetics, giornale di settore parte dell’editoriale Nature Publishing Group. Lo studio in questione è incentrato sulla comparazione, in termini di efficienza e accuratezza diagnostica, di due tecniche di screening dei 24cromosomi attualmente in uso per effettuare la diagnosi delle aneuploidie preimpianto (Preimplantetion genetic screening, PGS), l’array comparative genomic hybridization (aCGH) e la quantiative polymerase chain reaction (qPCR). Tale pubblicazione ha richiamato l’attenzione della rivista americana Genome Web, che a seguito di un’intervista rilasciata dal dr Antonio Capalbo e dal dr Nathan Treff, ha sottolineato i punti-chiave toccati dallo studio e le loro implicazioni nel campo della PGS. Nello specifico vengono evidenziati la maggior rapidità di esecuzione della qPCR a fronte di un costo inferiore, l’importanza di una validazione preclinica della tecnica prima della sua introduzione nella pratica clinica, ma soprattutto la possibilità di ridurre la percentuale di falsi positivi dal 7% stimato per l’aCGH allo 0,5% rilevato per la qPCR, tecnica attualmente in uso presso i centri GENETYX e RMA NJ.

Qui di seguito il testo dell’articolo pubblicato sul giornale Genome Web.

In PGD Study, qPCR Largely Concordant with Array CGH, but May Yield Fewer False Positives

By Molika Ashford

NEW YORK (GenomeWeb) — A direct comparison of array comparative genomic hybridization and quantitative PCR methods for detecting aneuploidy in IVF embryos has found the methods to be largely concordant, though qPCR had a lower rate of discordance with well-established SNP arrays.
The study, led by two associated assisted fertility and preimplantation genetics centers in Italy and published last week in the European Journal of Human Genetics, set out to more directly test the validity of array CGH than previous studies have done, and to assess it side-by-side with qPCR, another commonly used technique for preimplantation genetic diagnosis.
While array CGH has been widely adopted in preimplantation genetic screening, the study authors wrote that it had not been evaluated in a preclinical setting in which its false-positive rate or positive predictive value could be directly measured. Meanwhile, hints from clinical studies with unexpectedly high aneuploidy rates from array CGH have raised the question of the potential impact of false-positive results on the PGD IVF process.
According to the authors, recently developed qPCR-based methods offer a potentially more rapid and inexpensive approach compared to both array CGH and SNP-based array technologies for PGS.
“Although many labs use array CGH, there is less published preclinical validation compared to qPCR,” Antonio Capalbo, first author of the study and director of Italy’s Genetyx molecular genetics lab and associated Genera center for reproductive medicine, told PCR Insider in an email. “We wanted to therefore test both methods because our lab was interested in using the most accurate technology for our patients.”
Capalbo said that Genetyx is the first and so far only lab in Europe to adopt qPCR for preimplantation genetics.
In the study, Capalbo and his colleagues compared array CGH — in this case Illumina-owned BlueGnome’s 24sure — with qPCR, using SNP arrays to provide a benchmark upon which to determine the accuracy of any discordant results from the other two methods.
While SNP arrays served as an excellent tiebreaker, Capalbo wrote that they are relatively expensive compared to PCR.
The qPCR method used was originally published by researchers from US-based PGS lab Reproductive Medicine Associates of New Jersey, who are also coauthors of the new study.
To compare the methods, the group tested a total of 122 known aneuploid blastocysts selected from 45 patients that had been tested using array CGH. Among these samples, qPCR was successful in 120.
For 98 of these 120 embryos, about 81 percent, the aneuploidy calls for array CGH and qPCR matched, the authors wrote. But in 22 others, at least one chromosome differed between the two methods.
To resolve these discordant cases, the team then performed blinded reanalysis using a SNP-array technique in a subset of the embryos that could be successfully rewarmed. Out of 18 SNP array-tested samples, there were four in which all three methods were discordant, indicating the presence of mosaicism. In another four cases qPCR seemed to be the incorrect read using the SNP arrays as a benchmark, and in 10 cases, array CGH was discordant.
Of the four miscounts for qPCR, three were cases of mistaken euploidy and one of mistaken aneuploidy. For array CGH, most of the 10 mistaken calls were aneuploidy calls.
To corroborate, the team looked at an additional 39 embryos. In this sample set, the two methods resulted in the same call in 35 of the cases and disagreed with each other in four. SNP array analysis confirmed the qPCR result for all four was concordant, and the array CGH result was discordant.
According to the study authors, considering all the embryos tested, the rate of discordant aneuploidy calls for array CGH was about 7 percent overall, while the rate of discordant aneuploidy calls for qPCR was only 0.5 percent.
Capalbo and his colleagues avoid drawing broad conclusions from their results in terms of the relative utility of array-based and qPCR-based aneuploidy screening. The two methods showed, on a per-chromosome basis, a very high concordance, the authors wrote.
However, the relatively higher discordant aneuploidy call rate for array CGH based on SNP-array reanalysis suggests that the method could contribute to higher rates of false positives in clinical practice.
Avoiding false aneuploidy calls is particularly important, they wrote, for patients of advanced maternal age, or with complicating genetic disorders, for whom fewer competent embryos may be available per IVF cycle.
In lieu of direct evidence of the clinical negative predictive value or false positive rate for array CGH, the results represent the only available evidence in this vein, the authors wrote.
Capalbo said that Genetyx currently sees qPCR as the best-validated, fastest, and most cost-effective method for comprehensive chromosomal screening. However the lab is also involved in an ongoing project with its colleagues at Reproductive Medicine Associates of New Jersey to develop a next-gen sequencing-based method for PGS.


In PGD/PGS – Diagnosi Genetica Pre Impianto   potrete consultare il PDF della rivista dove è stato pubblicato lo studio “Comparison of array comparative genomic hybridization and quantitative real-time PCR-based aneuploidy screening of blastocyst biopsy” in European Journal of Human Genetics di Antonio Capalbo, Nathan R Treff, Danilo Cimadomo, Xin Tao Kathleen Upham,Filippo Maria Ubaldi, Laura Rienzi and Richard T Scott Jr”.

Troverete anche il PDF dell’articolo pubblicato sul giornale americano Genome Web