Non-Invasive Chromosome Screening for Embryo Preimplantation Using Cell-Free DNA

Preimplantation genetic testing (PGT) is widely adopted to select embryos with normal ploidy but requires invasive embryo biopsy procedures. Therefore, non-invasive PGT (niPGT) detection of cell-free DNA (cfDNA) in blastocyst culture medium has gradually become a hot area in the field of assisted reproduction. This chapter will systematically summarize how researchers use embryonic cfDNA to conduct niPGT detection worldwide. It will also thoroughly review the factors that affect the accuracy of the test and its underlying issues, as well as prospective applications. We hope to provide a useful reference for the standardized operation of non-invasive PGT that can be widely applied in clinical practice.

Can expelled cells/debris from a developing embryo be used for PGT?

Background Preimplantation genetic testing (PGT) is offered to a wide range of structural and numerical chromosomal imbalances, with PGT- polymerase chain reaction (PCR), as the method of choice for amplifying the small DNA content achieved from the blastomere biopsy or trophectoderm (TE) biopsy, that might have a detrimental impact on embryonic implantation potential. Since human embryos cultured until Day-5–6 were noticed to expel cell debris/ fragments within the zona pellucida, we aimed to examine whether these cell debris/ fragments might be used for PGT, as an alternative to embryo biopsy. Methods Blastocysts, which their Day-3 blastomere biopsy revealed an affected embryo with single-gene defect, and following hatching leaved cell debris/fragments within the zona pellucida were analyzed. Each blastocyst and its corresponding cell debris/fragments were separated and underwent the same molecular analysis, based on multiplex PCR programs designed for haplotyping using informative microsatellites markers. The main outcome measure was the intra-embryo congruity of Day-3 blastomere biopsy and its corresponding blastocyst and cell debris/fragments. Results Fourteen affected embryos from 9 women were included. Only 8/14 (57.2%) of embryos demonstrated congruent molecular genetic results between Day-3 embryo and its corresponding blastocyst and cell debris/fragments. In additional 6/14 (42.8%) embryos, molecular results of the Day-3 embryos and their corresponding blastocysts were congruent, while the cell debris/fragments yielded no molecular diagnoses (incomplete diagnoses). Conclusions It might be therefore concluded, that in PGT cycles, examining the cell debris/fragments on Day-4, instead of Day-3 blastomere or Day-5 TE biopsies, is feasible and might avoid embryo biopsy with its consequent detrimental effect on embryos’ implantation potential. Whenever the latter results in incomplete diagnosis, TE biopsy should be carried out on Day-5 for final genetic results. Further large well-designed studies are required to validate the aforementioned PGT platform.

P–582 High level of concordance between invasive and non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) at day5 and day6–7

Study question To explore ploidy concordance between invasive and non-invasive PGTA (niPGT-A) at different embryo culture time. Summary answer High level (>84%) of concordance rate for ploidy and sex, sensitivity (>88%), and specificity (76%) were obtained for both day6/7 samples and day5 samples. What is known already The analysis of embryo cell free DNA (cfDNA) that are released into culture media during in vitro embryo development has the potential to evaluate embryo ploidy status. However, obtaining sufficient quality and quantity of cfDNA is essential to achieve interpretable results for niPGT-A. More culture time is expected to be directly proportional to the release of more cfDNA. But embryo culture time is limited due to in-vitro embryo survival potential. Therefore, it is important to estimate the duration of the culture that will provide the maximum cfDNA that can be obtained without adversely affecting the development of the embryo. Study design, size, duration A total of 105 spent culture media (SCM) from day5-day7 blastocyst stage embryos have been included in this cohort study. The cfDNA of SCM samples were amplified and analyzed for niPGT-A by NGS analysis. The SCM samples were divided into 2 subgroups according the embryo culture hours (Day5 and Day6/7 group). The DNA concentration, informativity and euploidy results have then been compared with their corresponding embryos after trophectoderm biopsy (TE) and PGT-A analysis by NGS Participants/materials, setting, methods Embryos cultured until Day3 washed and cultured again in 20µl fresh culture media until embryo biopsy on Day5, 6, or 7. After biopsy SCM samples were immediately collected in PCR tubes and conserved at –20 °C until whole genome amplification by MALBAC® (Yicon Genomics). The TE and SCM samples were analyzed by next-generation sequencing (NGS) using Illumina MiSeq® System. NGS data analysis has been done by Bluefuse Multi Software 4.5 (Illumina) for SCM and TE samples Main results and the role of chance Only the SCM samples which have an embryo with a conclusive result were included in this cohort (n = 105). Overall 97.1% (102/105) of SCM samples gave a successful DNA amplification with a concentration ranging 32.4–128.5ng/µl. Non-informative (NI) results including a chaotic profile (>5 chromosome aneuploidies) were observed in 17 samples, so 83.3%(85/102) of SCM samples were informative for NGS data analysis. Ploidy concordance rate with the corresponding TE biopsies (euploid vs euploid, aneuploid vs aneuploid) was 84.7% (72/85). Sensitivity and specificity were 92,8% and 76,7%, respectively with no significant difference for all parameters for day 6/7 samples compared with day 5 samples. The false-negative rate was 3.5% (3/85), and false-positive rate was 11.7% (10/85). Limitations, reasons for caution The sample size is relatively small. Larger prospective studies are needed. As this is a single-center study, the impact of the variations in embryo culture conditions can be underestimated. Maternal DNA contamination risk cannot be revealed in SCM, therefore the use of molecular markers would increase the reliability. Wider implications of the findings: Non-invasive analysis of embryo cfDNA analyzed in spent culture media demonstrates high concordance with TE biopsy results in both early and late culture time. A non-invasive approach for aneuploidy screening offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population. Trial registration number Not applicable

P–546 Exome sequencing and preimplantation genetic testing for unexplained recurrent fetal malformations

Study question Could patient suffering unexplained recurrent fetal malformations be benefit of PGT-M by exome sequencing mutations identification? Summary answer Patients suffering unexplained recurrent fetal malformations could be benefit of the use of exome sequencing in combination to PGT-M to have a healthy live birth. What is known already Fetal malformations account for approximately 3% of live births and causes include: chromosomal abnormalities, exposure to toxic substances or teratogens and infections. Recently, studies have shown that several monogenic diseases are linked to fetal abnormalities. However, because of the large number of potential genes, genetic testing is challenging. Exome sequencing is widely used to detect genetic mutations and has emerged as a useful tool for finding the genetic cause of fetal abnormalities. The aim of this study was to show how exome sequencing in patients suffering unexplained recurrent fetal malformations in combination to PGT-M could lead to successful healthy newborn. Study design, size, duration Case report of a non-consanguineous couple with unexplained, recurrent fetal malformations. Couple were recruited during clinical consultation for unexplained recurrent fetal malformations at a private reproductive medicine clinic. The couple had two malformed fetus with the same congenital abnormalities: hydrocephalus, cerebellar vermis agenesis, cerebellar hypoplasia and enlarged cisterna magna. Patients signed written informed consent regarding to exome testing. For fetal sample, informed consent was obtained from parents. Participants/materials, setting, methods Sample of the affected fetus were provided. Parental genomic DNA was extracted from peripheral blood. Exome sequencing was performed using TrusightOne (Illumina®). FASTAQ data were processed through BWA and GATK algorithm. VCF files were analysed using Variant Interpreter software. After genetic counselling, PGT-M was performed using linkage polymorphic markers analysis and mutation sequencing. Embryo biopsy was carried at blastocyst stage. Embryos were vitrified and one healthy embryo was thaw and transfer in a subsequent cycle. Main results and the role of chance An homozygous novel pathogenic mutation c.641 C>T (p.Ala214Val) in FVLCR2 gene was found. The parents were heterozygous carriers revealing that the detected variant follow an autosomal recessive pattern. The FLVCR2 (14q24.3) gene encodes a transmembrane protein that belongs to the major facilitator superfamily of secondary carriers that transport small solutes in response to chemiosmosis ion gradients, such as calcium. Mutations in this gene are related to fetal central nervous system defects. This disorder is diagnosed prenatally and is lethal. PGT-M was recommended during genetic counselling. After control ovarian stimulation 14 oocytes were retrieved and finally 4 embryos were suitable for embryo biopsy at blastocyst stage. One embryo was diagnosed as healthy, two affected and one heterozygous carrier. The healthy embryo was thaw and transferred and a healthy male baby was born. Limitations, reasons for caution Exome sequencing has technical limitations: only covers mutations in coding regions and does not cover noncoding regions of the genome. It also cannot reliably detect copy-number variants at single gene level. Wider implications of the findings: This study offers strong evidence of exome-sequencing as a new diagnostic strategy and powerful tool discovering the underlying etiology of recurrent fetal malformations and identifying new genes important for human development. Using this strategy in combination with PGT-M, clinicians can help couples with recurrent fetal malformations to have healthy newborns. Trial registration number Not applicable