Quantitative polymerase chain reaction (qPCR)-based patterns consistent with mosaicism/partial aneuploidies(PA) indicate blastocysts with slightly lower reproductive competence: preliminary results from a non-selection study

Study question: Can we set qPCR parameters and thresholds to infer mosaicism and/or large PA in blastocysts on trophectoderm-based analysis’ patterns and predict embryos’ reproductive competence? Summary answer: The developed criteria for qPCR data analysis may indicate mosaicism and/or large PA in trophectoderm biopsies and identify blastocysts with a slightly lower reproductive competence. What is known already: Mosaic blastocysts are made of cells with different karyotypes. Recently, targeted-qPCR has been reported to inherently identify whole chromosome mosaicism in cell-mixture models of trophectoderm biopsies with a considerable sensitivity and specificity. PA are imbalances involving a portion of a chromosome, which targeted-qPCR may also identify when the imbalance involves a large chromosomal region. In this study, we tested whether specific parameters and thresholds compatible with mosaicism and PA applied to clinical blastocyst stage preimplantation genetic diagnosis for aneuploidies (PGD-A) cycles can be valuable to identify embryos with lower reproductive potential. Study design, size, duration: Prospective non-selection study involving 369 vitrified euploid single blastocyst transfers between March 2015 and September 2016. Aneuploidy testing performed through qPCR on trophectoderm biopsies according to a previously published method. Euploid blastocysts to be transferred were chosen independently from putative mosaicism/PA. Participants/materials, setting, methods: Only euploid blastocysts whose qPCR profile plot showed an overall concurrence ≤0.3 and an interchromosome standard deviation ≤0.32 and ≤0.18 for male and female embryos, respectively, were considered as a first inclusion criteria. Putative mosaicism was defined as copy number (CN) ≤1.65 or ≥2.35 and an intrachromosome assays’ concurrence ≤0.25. Putative PA were defined as two consecutive assays on a chromosome arm with a CN ≤1.65 or ≥2.35 and an intrachromosome assays’ concurrence≥0.4. Main results and the role of chance: 3.5%(n = 13/369) and 28.2% (n = 104/369) of the transferred blastocysts were not included due to an overall concurrence and inter-chromosome standard deviation above the defined thresholds, respectively. 78.6% of the blastocysts included in the study showed a qPCR profile not compatible with mosaic and/or PA(n = 198/252;95% CI=73.0%-83.5%), and 21.4% were classified as “putative mosaic” and/or “putative PA” for at least one chromosome(n = 54/252,95%CI=16.5%- 27.0%). Specifically, 15.1% “putative mosaic”(n = 38/252;95%CI=10.9%- 20.1%), 6.3% “putative PA”(n = 16/252; 95%CI=0.4%-10.1%) and 0.4% both (n = 1/252;95%CI=0%-2.2%.). No correlation was found between blastocyst morphology and qPCR profiles compatible with mosaicism/PA (p = 0.4). The positive pregnancy rates per SET were 52.6%(n = 20/38;95%CI=35.8%- 69.0%) and 43.8%(n = 7/16;95%CI=19.8%-70.1%) for “putative mosaic” and “putative PA”, respectively. The biochemical pregnancy loss rates were 10.0%(n = 2/ 20;95%CI=1.2%-31.7%) and 14.3%(n = 1/7;95%CI=0.4%-57.9%). The miscarriage rates were 22.2%(n = 4/18;95%CI=6.4-47.6%) and 16.6%(n = 1/6;95%CI=0.4%- 64.1%). The ongoing pregnancy rates (>12 gestational weeks) per SET were 36.8% (n = 14/38;95%CI=21.8%-54.0%) and 31.3%(n = 5/16;95%CI=11.0%-58.7%). When combining “putative mosaic” with “putative PA” and comparing the clinical outcomes with non-mosaic/PA, the positive pregnancy test rate per SET were 50.0%(n = 27/54;95%CI=36.1%-63.9%) and 60.6%(n = 120/ 198;95%CI=53.4%-67.5%), respectively. The biochemical pregnancy loss rates were 11.1%(n = 3/27,95%CI=0.2%-29.2%) and 8.3%(n = 10/120;95% CI=0.4%-14.8%). The miscarriage rates were 20.8%(n = 5/24;95%CI=7.1%- 42.2%) and 9.1%(n = 10/110; 95%CI=4.5%-16.1%). At last, the ongoing implantation rate per SET was significantly lower for “putative mosaic/PA” blastocysts versus non-mosaic/PA ones (p = 0.05): 35.2%(n = 19/54; 95% CI=22.7%-49.4%) and 50.5%(n = 100/198;95%CI=43.3%-57.7%). Limitations, reasons for caution: Mosaicism on trophectoderm biopsies does not predict mosaicism in the whole blastocyst due to an unavoidable sampling bias. Putative mosaicism/PA were not confirmed on products of conceptions or ongoing pregnancies. Additional biological and technical issues can produce CN profiles resembling mosaicism and have not been tested in this study. Wider implications of the findings: These preliminary outcomes highlighted a slightly lower reproductive potential for embryos showing an intermediate CN consistent with mosaicism/PA. These data need to be corroborated in a higher sample size to define whether the newly developed bioinformatic algorithm can be incorporated in the clinical management of qPCR-based PGD-A cycles. Trial registration number: none.