F. Spinella¹, E. Greco², A. Victor³, M.G. Minasi², F. Barnes³, C. Zouves³, J. Grifo⁴, E.H. Cheng⁵, S. Munnè⁶, A. Biricik¹, M. Surdo¹, M. Baldi¹, A. Ruberti², F. Fiorentino¹, M. Viotti³.
¹Genoma Group srl, Molecular Genetics Laboratories, Rome, Italy.
²European Hospital, Centre For Reproductive Medicine, Rome, Italy.
³Zouves Fertility Center, Fertility Center, Foster City, U.S.A..
4New York University, Langone Fertility Center, New York, U.S.A..
⁵Lee Women’s Hospital, Lee Women’s Hospital, Taichung, Taiwan R.O.C..
6Cooper Genomics, Cooper Genomics, Livingston, U.S.A..

Study question:
Is the clinical outcome of mosaic embryos influenced by chromosomal constitution?

Summary answer:
Reproductive potential of mosaic embryos is affected by the complexity of and the number of aneuploidy cells present in trophectoderm (TE) biopsy.

What is known already:
Chromosomal mosaic embryos are characterized by the presence of chromosomally different cell lines within the same embryo. While the transfer of these embryos is now offered as an option for women who undergo in vitro fertilization (IVF), several concerns remain. For instance, the limited data on pregnancy outcome and the possibility that intra-biopsy mosaicism in the TE is a poor predictor of the ploidy status of the ICM. Therefore, some argue that mosaicism should be not reported until a clear classification of such embryos in relation with their reproductive potential has been defined.

Study design, size, duration:
We collected the clinical outcomes of 822 mosaic embryos transferred in women underwent IVF between May 2016-May 2019. All embryos were cultured to blastocyst stage; trophectoderm (TE) biopsy was performed on Day-5 of development or Day6/7 for slow growing embryos. The clinical outcome obtained after transfer of mosaic embryos with different chromosomal constitution was compared with each other and with that obtained from a control group of 3781 euploid blastocysts.

Participants/materials, setting, methods:
Preimplantation genetic testing (PGT) was performed using high resolution next generation sequencing (NGS) methodology. TE biopsies were classified as mosaic if they had 20%-80% abnormal cells. For statistical analysis mosaic embryos were divided in groups based on mosaic levels and chromosomal constitution detected in TE: single mosaic aneuploidy (monosomy/trisomy; SM), double mosaic chromosomes (monosomy/trisomy or combination, DM), complex mosaic aneuploidy (>2 different aneuploidies; CM) and mosaic segmental aneuploidy (single and double deletion/insertion >5Mb, MS).

Main results and the role of chance:
The embryos were plotted in 10% increments, representing a progressive increase in the proportion of aneuploid cells in the TE, and linear regression showed a statistically significant decline in rates of implantation and ongoing pregnancy/birth (regression function with respective slopes -0.42 and -0.55, P=0.0381 and 0.0099). Regarding chromosomal constitution, MS had the best outcomes, followed by the group with one affected chromosome, followed by the group with two affected chromosomes, followed by the complex group (implantation P<0.0001, ongoing pregnancy/birth P<0.0001). MS showed a significantly poorer clinical outcomes compared to the euploid control group (implantation 51.3% vs 61.1%, P=0.0004; ongoing pregnancy/birth 42.6% vs 52.7%, P=0.0003).

Limitations, reasons for caution:
Additional clinical data must be obtained to evaluate the contribution of each different chromosome before this approach can be evaluated as an additional tool to choose mosaic embryos for transfer.

Wider implications of the findings:
The study provides the largest dataset of transferred mosaic embryo outcomes reported to date. This compiled analysis conclusively shows that embryos with different pattern of chromosomal mosaicism have a distinct set of clinical outcomes. This findings should be considered for genetic counseling.

Keywords:
mosaic embryos
preimplantation genetic testing for aneuploidy
clinical outcome
Next generation Sequencing