Blastocysts from abnormally-fertilized zygotes can be euploid/diploid and are reproductively competent: live-births from preimplantation genetic diagnosis of aneuploidy/polyploidy in embryos with abnormal pronuclear morphology

Study question: Is it possible to rescue abnormally fertilized zygote (AFZs) if the risk of polyploidy is evaluated by a novel combined protocol for simultaneous aneuploidy/polyploidy assessment? Summary answer: Polyploidy assessment in AFZ-derived blastocysts is an effective approach to rescue diploid embryos from monopronuclear/tripronuclear- derived blastocysts and may improve the live-birth rate in IVF cycles. What is known already: Approximately 10% of inseminated oocytes fertilized abnormally and the embryos derived from them are discarded all over the world in the absence of a reliable approach for the monitoring of the genetic risk for polyploidy. Here, we report the development and application of an improved comprehensive chromosomal diagnosis protocol for the detection of 24-chromosome aneuploidy and parallel polyploidy assessment. The combined test was implemented clinically, with the aim of rescuing AFZs in poor prognosis patients to maximize live-birth rate per IVF cycle. Study design, size, duration: Longitudinal cohort study performed between January2015-September2016 involving 553 patients (39.2 ± 3.2 yr) and 719 consecutive PGD-A cycles. Blastocyst PGD-A was offered to patients of advanced female age ( > 35 years), a history of unsuccessful IVF ( > two failed IVF cycles) or previous miscarriages ( > two). The combined genetic analysis was performed when a blastocyst from monopronuclear(1PN) or tripronuclear(3.1PN) zygote was obtained. Euploid-diploid embryos obtained from AFZs were considered suitable for replacement if others from normally-fertilized zygotes (NFZs) were not available. Participants/materials, setting, methods: Injected oocytes were individually cultured and fertilization was assessed between 16–18 hours. 1PN zygotes were defined when a single pronucleus was present. Zygotes showing one extra smaller pronucleus on the top of two evenly sized pronuclei were defined as 3.1PN. Blinded ploidy assessment using single nucleotide polymorphism allele ratios was conducted on trophectoderm biopsies from AFZ-derived blastocysts by targeted-NGS (1:1 = diploid; 2:1 = triploid; loss of heterozygosity = haploid). Samples of known ploidy were included as controls Main results and the role of chance: For validation, a blinded analysis of previously-established triploid and diploid cells was performed. The allele ratio was 2.12 ± 0.12(1.97–2.31) and 1.29 ± 0.15(1.18–1.50) for triploidy and diploidy, respectively(p < 0.01). Furthermore, the DNA products from one triploid miscarried blastocyst and two sibling unknown-ploidy vitrified euploid blastocysts were blindly submitted to ploidy assessment. The results correctly identified the triploid blastocyst. In the clinical phase, 3785 zygotes (200 1PN-AFZs;27 3.1PN-AFZs;3558 2PN-NFZs) from 719 PGD-A cycles were assessed. In 25.2% (n = 181/719) and 3.6% (n = 26/719) of the cycles AFZs and AFZ-derived blastocysts were observed, respectively. Significantly less blastocysts were obtained from 1PNAFZs (n = 13/200, 6.5%) with respect to both 3.1PN-AFZs and 2PN-NFZs (n = 14/27, 51.9%; n = 1667/3558, 46.9%; p < 0.05), mainly due to a lower cleavage rate at the first cell division after fertilization. Ploidy analysis identified 23.1% (n = 3/13), 69.2%(n = 9/13) and 7.7% (n = 1/13) of the 1PN-AFZ-derived blastocysts as haploid, diploid and triploid, respectively. A normal diploid constitution was observed in 85.7% (n = 12/14) of the 3.1PN-AFZ-derived blastocysts, while 2 (3%) were triploid. In 0.8% of the cycles (n = 6/719) AFZ-derived euploid/diploid blastocysts were identified. Four of them were transferred and resulted in 2 healthy pregnancies (one from a 1PN-AFZ and one from a 3.1PN-AFZ). Limitations, reasons for caution: Gestational and neonatal outcomes of AFZ-derived pregnancies still need to be investigated in a larger sample size. Only 1/3 of the pronuclei check on day1 was performed in a time lapse system. However,AFZs were re-evaluated within 4 hours from the first check by an independent embryologist to confirm the pronuclear abnormality Wider implications of the findings: Overall, AFZs account for ~10% of the zygotes in IVF and are generally discarded. Here, we report the development and validation of a reliable protocol for simultaneous aneuploidy and ploidy assessment from a single trophectoderm biopsy and the first clinical experience that resulted in two healthy pregnancies from AFZs. Trial registration number: none.