Sofia Makieva¹ , Luca Pagliardini¹ , Laura Corti¹ , Alessandra Alteri¹ , Greta Cermisoni¹ , Enrico Papaleo¹ , Biricik, Anil², Spinella Francesca² , Fiorentino Francesco² , Paola Viganò^1
1.Obstetrics and Gynecology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy , ² GENOMA, Molecular Genetics Laboratories, Rome, Italy

Study question:
Does grouped embryo culture influence ploidy outcomes in PGT-A cycles?

Summary answer:
PGT-A cycles performed using grouped embryo culture had 7.1% (p=0.02) higher euploidy rate compared to single embryo culture.

What is known already:
Animal and human studies have shown that single embryos in culture lack the benefits of embryotrophic factors of group culture. In mice, single culture is associated with suboptimal neonatal outcomes while in humans grouped culture reduces embryo development rates, blastocyst quality and pregnancy rates. Thus, single culture may be detrimental to human embryos compared to grouped culture plausibly due to deprivation of paracrine signaling occurring between neighboring embryos. To date no study has assessed whether grouping embryos can influence their genetic composition and, hence, PGT-A outcomes.

Study design, size, duration:
A retrospective analysis of 342 PGT-A cycles involving 1265 biopsied blastocysts performed between December 2014 and July 2019. The blastocysts were cultured either in groups of 1-6 (grouped culture; n=616) between December 2014 and March 2017 or individually (single culture; n=649) between May 2017 and July 2019, under similar conditions. The biopsy was performed between day 5 and 7 of development. The PGT-A diagnosis and other IVF outcomes were compared between the two groups.

Participants/materials, setting, methods:
PGT-A cycles were performed at the IVF center of San Raffaele Hospital and included patients with an indication for advanced maternal age, recurrent implantation failure or a risk for a genetic disease. Grouped and single embryo culture involved a sequential system using culture media supplemented with SSS. All procedures were performed according to standard clinical practice. The PGT-A diagnosis was based on WGA+aCGH or NGS, which was predominantly performed at the genetics laboratory GENOMA.

Main results and the role of chance:
The analysis of 1265 blastocysts revealed that grouped culture had a higher number of euploid (176/616; 28.5%; p=0.027) and lower number of mosaic (72/616;11.6%; p=0.025) blastocysts when compared to single culture (150/649;23.1% and 104/649;16% respectively). The number of grouped blastocysts biopsied on day 5 (261/616; 42.3%) was higher (p<0.0001) when compared to single blastocysts (166/649;26.9%). Moreover, single cultured blastocysts were more likely to be biopsied on day 6 (414/649;67.2%; p<0.0001) and day 7 (69/649;11.2%; p=0.0015) compared to grouped (366/616;49.6% and 49/616;7.9% respectively). Overall, the rate of euploidy in the 159 cycles in the grouped culture group was significantly higher compared to that in the 183 cycles of single group (28.66 ±29.19% vs 20.95 ±28.45%; p=0.002). No difference was observed in the rate of mosaicism. In the grouped culture cycles, the rate of day 5 biopsy was higher (38.73 ±18.89% vs 24.76 ±19.28%; p<0.0001) and of day 6 lower (52.39±29.07% vs 63.69 ±33.75%; p=0.0012) compared to single culture cycles. A generalised linear estimation corrected for variables known to be associated with ploidy according to the literature. Single embryo culture had 7.1% (13.2-1.1 %CI) lower euploidy rate compared to grouped embryo culture (p=0.02).

Limitations, reasons for caution:
The study is limited by its retrospective design. The grouped embryo culture contained embryos cultured individually when the cycle had 1 embryo in excess. Cycles including at least 1 blastocyst with inconclusive diagnosis were included. The methodology used for assessment of chromosomal constitution may have introduced a bias.

Wider implications of the findings:
Our findings suggest that grouped embryo culture may benefit PGT-A cycles by increasing the number of blastocysts diagnosed as euploid. This phenomenon could be attributed to the paracrine communication that may aid cell correction and free the blastocyst from aneuploid cells, which if persist may result in a mosaic diagnosis.