Genome-wide haplotyping embryos developing from 0PN and 1PN zygotes increases transferrable embryos in PGT-M

Abstract

STUDY QUESTIONCan genome-wide haplotyping increase success following preimplantation genetic testing for a monogenic disorder (PGT-M) by including zygotes with absence of pronuclei (0PN) or the presence of only one pronucleus (1PN)?SUMMARY ANSWERGenome-wide haplotyping 0PNs and 1PNs increases the number of PGT-M cycles reaching embryo transfer (ET) by 81% and the live-birth rate by 75%.WHAT IS KNOWN ALREADYAlthough a significant subset of 0PN and 1PN zygotes can develop into balanced, diploid and developmentally competent embryos, they are usually discarded because parental diploidy detection is not part of the routine work-up of PGT-M.STUDY DESIGN, SIZE, DURATIONThis prospective cohort study evaluated the pronuclear number in 2229 zygotes from 2337 injected metaphase II (MII) oocytes in 268 cycles. PGT-M for 0PN and 1PN embryos developing into Day 5/6 blastocysts with adequate quality for vitrification was performed in 42 of the 268 cycles (15.7%). In these 42 cycles, we genome-wide haplotyped 216 good quality embryos corresponding to 49 0PNs, 15 1PNs and 152 2PNs. The reported outcomes include parental contribution to embryonic ploidy, embryonic aneuploidy, genetic diagnosis for the monogenic disorder, cycles reaching ETs, pregnancy and live birth rates (LBR) for unaffected offspring.PARTICIPANTS/MATERIALS, SETTING, METHODSBlastomere DNA was whole-genome amplified and hybridized on the Illumina Human CytoSNP12V2.1.1 BeadChip arrays. Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the embryonic genome architecture. Bi-parental, unaffected embryos were transferred regardless of their initial zygotic PN score.MAIN RESULTS AND THE ROLE OF CHANCEA staggering 75.51% of 0PN and 42.86% of 1PN blastocysts are diploid bi-parental allowing accurate genetic diagnosis for the monogenic disorder. In total, 31% (13/42) of the PGT-M cycles reached ET or could repeat ET with an unaffected 0PN or 1PN embryo. The LBR per initiated cycle increased from 9.52 to 16.67%.LIMITATIONS, REASONS FOR CAUTIONThe clinical efficacy of the routine inclusion of 0PN and 1PN zygotes in PGT-M cycles should be confirmed in larger cohorts from multicenter studies.WIDER IMPLICATIONS OF THE FINDINGSGenome-wide haplotyping allows the inclusion of 0PN and 1PN embryos and subsequently increases the cycles reaching ET following PGT-M and potentially PGT for aneuploidy (PGT-A) and chromosomal structural rearrangements (PGT-SR). Establishing measures of clinical efficacy could lead to an update of the ESHRE guidelines which advise against the use of these zygotes.STUDY FUNDING/COMPETING INTEREST(S)SymBioSys (PFV/10/016 and C1/018 to J.R.V. and T.V.), the Horizon 2020 WIDENLIFE: 692065 to J.R.V., T.V., E.D., A.D. and M.Z.E. M.Z.E., T.V. and J.R.V. co-invented haplarithmisis (‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’), which has been licensed to Agilent Technologies. H.M. is fully supported by the (FWO) (ZKD1543-ASP/16). The authors have no competing interests to declare.

In vitro fertilization does not increase the incidence of de novo copy number alterations in fetal and placental lineages

“…The foregoing, and the number of cells per embryo analyzed with those technologies, can affect the reported embryo mosaicism rates in studies, with those studies analyzing all cells from a single embryo showing higher rates of mosaicism 10 . In the current study, we applied the same wet-and dry-lab technologies as in our preimplantation embryo studies 3,34,35 and further validated our approach using sensitive ddPCR and standard copy number analysis methods 36,37 , allowing us to rule out technology-driven bias. Third, the mosaicism rate is reported to be influenced by the fertility clinic where the IVF procedure is carried out 38 ; one key component that can explain differences in the reported mosaicism rate is embryo culture conditions 39 .…”

HiVA: an integrative wet- and dry-lab platform for haplotype and copy number analysis of single-cell genomes

“…1 and 2 ). As such the applications of the tool range from genome-wide haplotype reconstruction and genetic variant imputation, to deciphering the nature of allelic imbalances in single cells, including their parental origin as well as their mitotic or meiotic segregational origin 19–21 (Fig. 2).…”

“…2). Moreover, the methodology has been clinically implemented for genetic diagnosis of preimplantation embryos by imputation of Mendelian disease variants 6, 21, 22 (Fig. 2).…”

Should we transfer monopronuclear embryos? More data and even more questions

“…Monopronuclear (1PN) embryos may result from asynchronous PN formation, fusion of maternal and paternal PN, parthenogenetic activation, or premature breakdown of PN (1). These embryos are generally considered to have undergone abnormal fertilization and to not be capable of progressing to a normal embryo (2). Most studies suggest they are more likely to be aneuploid compared with 2PN embryos (3,4).…”

“…Most studies suggest they are more likely to be aneuploid compared with 2PN embryos (3,4). Conversely, fewer data suggest that 1PN and 2PN embryos may have similar aneuploid rates (2). When timelapse imaging diagnosed 1PN development, only 17% of the embryos assessed were euploid and only a single blastocyst embryo resulted in a live birth (1).…”