Report of some cases with chromosomal mosaicism in prenatal diagnosis and the corresponding results after birth

Chromosomal mosaicism in prenatal diagnosis is a complex problem that confuses the perception of true mosaicism or pseudomosaicismand often causes difficulties in genetic counseling. In this study, the authors reported 5 cases of chromosomal mosaicism in prenatal karyotype diagnosisand compared them withthe corresponding karyotype results of children after birth. Amniotic fluid and peripheral blood cells were prepared chromosomal metaphase by culture method and chromosomal analysis according to ISCN 2016 standards. Samples were collected and analysed at Hanoi Medical University Hospital from 2017 to 2020. There were 3 cases of abnormal prenatal chromosomal mosaicism, but the postnatal results were normal, two cases of abnormal prenatal chromosome mosaicism, but had abnormal peripheral blood postnatal chromosome results. These results, together with discussion, will provide more valuable information for the prognosis of chromosome mosaicism cases in prenatal diagnosis and give better genetic counseling for the patients.

O-202 Usage of laser during trophectoderm biopsy and its affect on PGT-A results and moscaism

Study question Does laser use during trophectoderm biopsy affect biopsy results on prehatching embryos with regard to mosaicism ? Summary answer According to our findings laser usage during trophectoderm biopsy increases mosaic results on next generation sequencing (NGS) irrespective of embryo quality. What is known already Chromosomal mosaicism, which is a result of mitotic errors after fertilization, is defined as the presence of karyotypically distinct cell lines within an embryo. The introduction of NGS made it possible to detect chromosomal mosaicism at levels as low as 20%. The incidence of mosaicism is highly variable between clinics which reported the incidences between 4-32%. Apart from the biological reasons, there are also various technical factors that may impact the incidence of mosaicism. One of the most emphasized factors is the trophoectoderm biopsy technique. Laser usage and number of laser pulses may cause excessive heat during the procedure Study design, size, duration The mosaicism ratio in embryos in which trophectoderm biopsy was performed with or without laser, between January 2017 December 2020 in Istanbul Memorial Hospital (IMH) were examined retrospectively. A total of 13002 embryos were analyzed. A subgroup analysis was also performed regarding mosaicism ratios in different embryo qualities. Blastocysts were classified according to Gardner’s classification and classified as follows: top quality-TQ (4AA,5AA,6AA), good quality-GQ (3AA, 4,5,6AB,BA) and moderate quality-MQ (3,4,5 BB). Participants/materials, setting, methods The biopsy samples of the cases who had PGT-A in IMH between 2017-2020 were evaluated by NGS method. This method enables the identification of embryos with 20% to 80% mosaicism. The study assessed whether there was an increase in the embryos with mosaic results due to the use of laser during biopsy. The effects of laser use among the TQ (4AA,5AA,6AA), GQ (3AA, 4,5,6AB,BA) and MQ (3,4,5 BB) groups according to Garder classification were analyzed. Main results and the role of chance Trophectoderm biopsy was applied on 13002 embryos within the specified period. During biopsy in 5088 embryos laser was used and in 7843 embryos laser was not used, and biopsy was performed mechanically (flicking method). After observing the biopsy results, 945/5088 (18.5%) of the laser applied embryos; and 1087/7914 (13.7%) of laser not applied embryos were defined as mosaic(p < 0.0001). When mosaicism rates were examined according to embryo qualities, the rate of mosaicism was 19.3%(469/2430), 18.2%(290/1591) and 13.2%( 380/2875), 13.5 (426/3141) respectively in embryos with and without laser in TQ and GQ groups. A statistically high level of significance (p < 0.0001) was observed between the embryos evaluated as top quality and good quality before biopsy. Regarding the evaluation in the moderate group embryos, although the mosaicism rates tended to increase on the laser applied group side 40/248(16.1%), no statistical difference was observed when compared to non-laser group 103/670(15,4%). (P > 0.05) Limitations, reasons for caution The retrospective nature of the data is the main limitation of the study. On the other hand, the large number of NGS based PGT-A tested TQ and GQ embryos from a single center and resuts from single laboratory. However, further studies are required to corroborate our findings. Wider implications of the findings Laser dependent heat effect may increase mosaicism. To reduce the cell damage, teasing of cells should be avoided and a minimum number of laser pulses should be used in order to avoid excessive heat and contact points should be preferably confined to cell junctions Trial registration number None

O-201 Prenatal and postnatal outcome of mosaic embryo transfers: multicentric study of one thousand mosaic embryos diagnosed by preimplantation genetic testing with trophectoderm biopsy

Study question To explore the effect of chromosomal mosaicism detected in preimplantation genetic testing (PGT-A) on prenatal and postnatal outcome of mosaic embryo pregnancies Summary answer No significant difference between euploid and mosaic embryos was observed in terms of weeks of gestation, average weight, and developmental defect of the babies born What is known already Mosaic embryos have the potential to implant and develop into healthy babies. Transfer of these embryos is now offered as an option for women who undergo IVF resulting in no euploid embryos. While, prenatal diagnosis has shown the depletion of chromosomal mosaicism in mosaic embryos, several concerns remain. For instance, the direct effects of different kind of mosaicism on prenatal/postnatal outcome and the possibility that intra-biopsy mosaicism in the TE is a poor predictor of the ploidy status of the ICM. Thus, there is certainly a need for comprehensive analyses of obstetrical and neonatal outcome data of transferred mosaic embryos. Study design, size, duration Compiled analysis from multicenter data on transfers of mosaic embryos (n = 1,000) and their outcome, with comparison to a euploid control group (n = 5,561). To explore the effect of embryonic mosaicism on newborns, we matched mosaic embryos resulting in a birth with a euploid embryo by a series of parameters (maternal age, embryo morphology, and indication for PGT-A). Prenatal tests and birth characteristics of > 200 neonates from mosaic embryo transfers were compared to > 200 euploid embryos. Participants/materials, setting, methods PGT-A was performed on blastocyst-stage embryos with 24-Chromosome whole genome amplification (WGA)-based Next Generation Sequencing (NGS). In accordance with established guidelines, embryos were categorized as mosaic when PGT-A results indicated 20-80% aneuploid content. Prenatal testing where performed in 30% of pregnancies with amniocentesis, 4% did an extra analysis for potential UPD for the suspected mosaic chromosome, and an additional 16% performed chorionic villus sampling (CVS) and 9.5% performed noninvasive prenatal testing (NIPT). Main results and the role of chance Of the 465 mosaic embryos that implanted, about 20% miscarried, and out of those, 75% were early spontaneous abortions. Of the pregnancies, 3 out of 368 were stillborn (2 out of them were twins that were extremely premature at 23 weeks, and the other died during pregnancy from a heart defect). The remaining 99% of those have been born or are late ongoing pregnancies at the time of analysis. Prenatal tests were performed in > 200 pregnancies and the vast majority tested normal. All 5 abnormal cases were amniocentesis tests showing microdeletions or insertions of sizes smaller than the resolution used during PGT-A, so they were unrelated to the mosaicism detected with PGT-A. In fact, in none of the cases did the prenatal test reflect the mosaicism detected at the embryonic stage. Matching each of the 162 mosaic embryos resulting in a birth with a euploid embryo, we found that the length of gestation was similar on average, and so was the average weight of the babies at birth. We also gathered information on the routine physical examination performed on babies at birth, and of those 162 babies from mosaic embryo transfers, none had obvious developmental defects or gross abnormalities. Limitations, reasons for caution Even though newborns resulting from mosaic embryo transfers in this study invariably appeared healthy by routine examination, concerns for long-term health cannot yet be entirely dispelled. The question must therefore be carefully considered by each clinic and patient situation. Wider implications of the findings Prenatal testing of > 200 pregnancies from mosaic embryo transfers showed no incidence of mosaicism that matched the PGT-A findings, indicating the involvement of self-corrective mechanisms. Pregnancy and obstetric data indicates that mosaic embryos prevailing through gestation and birth have similar chromosomal and physiological health compared to euploid embryos. Trial registration number none

P-549 What trophectoderm cells from mosaic embryos tell us about embryonic competence at the transcriptional level

Study question Does transcriptome of remaining trophectoderm (TE) reflect the developmental potential of mosaic blastocysts after preimplantation genetic testing for aneuploidy (PGT-A)? Summary answer TE from low-degree mosaic (Low-mos) and high-degree mosaic (High-mos) blastocysts are transcriptionally equivalent, standing between euploid and aneuploid categories and displaying key deregulated developmental processes. What is known already Blastocysts classified as mosaic by PGT-A are associated with lower implantation and higher miscarriage rates than those classified as euploid, yet they still lead to healthy babies. Unveiling the true developmental identity of these embryos faces a dilemma: understanding to which extent they represent technical artefacts or whether they hold own potential to implant and give rise to normal pregnancies. Current RNA sequencing (RNA-seq) techniques allow for the determination of whole transcriptomic profiles even from single cells, which paves the way for the identification of new molecular keys of embryonic competence. Study design, size, duration Prospective study comparing RNA-seq data of remaining TE from blastocysts classified as euploid (n = 4), Low-mos (n = 5), High-mos (n = 4) and aneuploid (n = 6) by PGT-A. Participants were recruited between October 2018 and November 2019 at IVI-RMA Valencia. Participants/materials, setting, methods Chromosomal mosaicism was defined in the range 30%- < 50% (Low-mos) and 50%- < 70% (High-mos) using a next-generation sequencing (NGS) validated algorithm. Whole TE fractions were separately collected and processed for RNA-seq. Differentially expressed genes (DEGs) were calculated with DESeq2 package [Benjamini-Hochberg (BH)-adjusted p < 0.01 & abs(log2FoldChange)>2 significant]. Fgsea algorithm was used for enrichment analysis on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms (BH-adjusted p < 0.01 significant). Main results and the role of chance For comparisons, TE from euploid blastocysts were used as control. At the gene level, 15 DEGs were found in Low-mos, 20 DEGs in High-mos, and 64 DEGs in aneuploid blastocysts. To address the functional implications of these differences, pathways significantly deregulated according to KEGG and GO categories were identified. TE from aneuploid blastocysts displayed significant downregulation in up to 115 KEGG and GO processes directly involved in processing and integrity maintenance of nuclear and mitochondrial genomes, a reflection of their aberrant chromosomal identity. In addition, TE from High-mos and Low-mos were transcriptionally equivalent (0 DEGs between both groups), with 23 overlapping KEGG and GO processes significantly downregulated compared with control. Importantly, main significantly-affected processes included mitotic sister chromatid segregation, NIK NF-kB activity, regulation of apoptosis, and pathways related to the biosynthesis and metabolism of proteins, fatty acids, carbohydrates and steroid hormones. These findings indicate that mosaic embryos comprise a unique developmental entity, which swims between the euploid and aneuploid waterfronts and may regulate survival by diverse mechanisms, including cell proliferation and apoptosis. Limitations, reasons for caution This is a descriptive, single-center study with limited sample size. TE fractions were obtained by micromanipulation, which may have led to potential cross-contamination with the inner cell mass. Wider implications of the findings Transcriptomic equivalence between Low-mos and High-mos TE fractions questions the biological significance of inferring mosaicism degrees from single biopsies. Deregulated processes in these embryos support their reduced developmental and live birth potential, pointing to mechanisms that may mediate survival in the presence of aneuploid cells, as shown in the mouse. Trial registration number Not applicable

P–549 What trophectoderm cells from mosaic embryos tell us about embryonic competence at the transcriptional level

Study question Does transcriptome of remaining trophectoderm (TE) reflect the developmental potential of mosaic blastocysts after preimplantation genetic testing for aneuploidy (PGT-A)? Summary answer: TE from low-degree mosaic (Low-mos) and high-degree mosaic (High-mos) blastocysts are transcriptionally equivalent, standing between euploid and aneuploid categories and displaying key deregulated developmental processes. What is known already Blastocysts classified as mosaic by PGT-A are associated with lower implantation and higher miscarriage rates than those classified as euploid, yet they still lead to healthy babies. Unveiling the true developmental identity of these embryos faces a dilemma: understanding to which extent they represent technical artefacts or whether they hold own potential to implant and give rise to normal pregnancies. Current RNA sequencing (RNA-seq) techniques allow for the determination of whole transcriptomic profiles even from single cells, which paves the way for the identification of new molecular keys of embryonic competence. Study design, size, duration Prospective study comparing RNA-seq data of remaining TE from blastocysts classified as euploid (n = 4), Low-mos (n = 5), High-mos (n = 4) and aneuploid (n = 6) by PGT-A. Participants were recruited between October 2018 and November 2019 at IVI-RMA Valencia. Participants/materials, setting, methods Chromosomal mosaicism was defined in the range 30%-<50% (Low-mos) and 50%-<70% (High-mos) using a next-generation sequencing (NGS) validated algorithm. Whole TE fractions were separately collected and processed for RNA-seq. Differentially expressed genes (DEGs) were calculated with DESeq2 package [Benjamini-Hochberg (BH)-adjusted p < 0.01 & abs(log2FoldChange)>2 significant]. Fgsea algorithm was used for enrichment analysis on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms (BH-adjusted p < 0.01 significant). Main results and the role of chance For comparisons, TE from euploid blastocysts were used as control. At the gene level, 15 DEGs were found in Low-mos, 20 DEGs in High-mos, and 64 DEGs in aneuploid blastocysts. To address the functional implications of these differences, pathways significantly deregulated according to KEGG and GO categories were identified. TE from aneuploid blastocysts displayed significant downregulation in up to 115 KEGG and GO processes directly involved in processing and integrity maintenance of nuclear and mitochondrial genomes, a reflection of their aberrant chromosomal identity. In addition, TE from High-mos and Low-mos were transcriptionally equivalent (0 DEGs between both groups), with 23 overlapping KEGG and GO processes significantly downregulated compared with control. Importantly, main significantly-affected processes included mitotic sister chromatid segregation, NIK NF-kB activity, regulation of apoptosis, and pathways related to the biosynthesis and metabolism of proteins, fatty acids, carbohydrates and steroid hormones. These findings indicate that mosaic embryos comprise a unique developmental entity, which swims between the euploid and aneuploid waterfronts and may regulate survival by diverse mechanisms, including cell proliferation and apoptosis. Limitations, reasons for caution This is a descriptive, single-center study with limited sample size. TE fractions were obtained by micromanipulation, which may have led to potential cross-contamination with the inner cell mass. Wider implications of the findings: Transcriptomic equivalence between Low-mos and High-mos TE fractions questions the biological significance of inferring mosaicism degrees from single biopsies. Deregulated processes in these embryos support their reduced developmental and live birth potential, pointing to mechanisms that may mediate survival in the presence of aneuploid cells, as shown in the mouse. Trial registration number Not applicable

P-523 Whole-chromosome aneuploidies revealed by transcriptome of trophectoderm biopsied from human pre-implantation blastocyst

Study question Whether mRNA transcriptome of biopsied trophectoderm (TE) in human pre-implantation blastocyst can predict embryo karyotype? Summary answer mRNA transcriptome of biopsied TE can precisely predict whole-chromosome aneuploidies but not mosaicism or segmental aneuploidies. What is known already Karyotype of human pre-implantation blastocyst is well recognized by PGT-A. However, genome can’t demonstrate gene expression level which might infer the development potential of euploidy. Transcriptome of blastocyst by singe-cell RNA-seq has revealed the lineage segregation of human pre-implantation blastocyst. It is not known whether transcriptome of biopsied TE used in PGT-A can infer the karyotype of human pre-implantation blastocyst. Study design, size, duration A total of 74 TE samples were biopsied from 26 blastocysts which were donated from patients who underwent PGT at our Reproductive Medicine Center. All of these embryos have been previously diagnosed as aneuploidies (n = 19) or euploidies (n = 7) with monogenic disorder. Participants/materials, setting, methods The DNA and mRNA of all biopsied TEs were separated independently using a modified oligo-dT bead capture, followed by PGT-A of DNA and smart2-sequencing of mRNA (G&T-seq). Karyotype of biopsied TEs were confirmed with PGT-A performed in MiSeq system (Illumina) in our PGT laboratory with the use of next-generation sequencing. Data of transcriptome was analyzed using Rstudio and R package InferCNV to predict aneuploidies by referring to euploidies which were inferred with corresponding PGT-A results. Main results and the role of chance In human pre-implantation blastocyst, all whole-chromosome aneuploidies could be inferred by transcriptome of biopsied TE, which were consistent with PGT-A result. But chromosomal mosaicism or segmental aneuploidies were hard to be predicted precisely by transcriptome of TE. Limitations, reasons for caution The main limitation of this study lies in the inability to retrieve the exact copy number variations from mRNA transcription. Gene expression is in a great imbalance in such an early development of human pre-implantation blastocyst. Wider implications of the findings Our data suggest that mRNA transcriptome is enough for prediction of whole-chromosome aneuploidies. The method and value for predicting mosaicism and segmental aneuploidies by transcriptome should be further investigated. Trial registration number not applicable

O-204 Is mosaicism affected by an embryologist’s experience in biopsy?

Study question Are there any correlations between blastocyst mosaicism rate and biopsy experience among embryologists? Summary answer Blastocysts biopsied by embryologists with ≥1 year of biopsy experience have significantly lower mosaicism rate compared to those with <1 year of biopsy experience. What is known already It has been reported that the incidence of blastocyst mosaicism is highly variable between centres (PGDIS, 2019). It is also suggested that the technical aptitude of the embryologist performing blastocyst biopsy may give rise to mosaicism. Thus, a retrospective study was conducted to investigate the relationship between blastocyst mosaicism rate and biopsy experience among embryologists in Alpha IVF. Study design, size, duration Thirteen competent embryologists who were trained in blastocyst biopsy were included in this study: 5 have ≥1 year of biopsy experience (Group A; Embryologist A-1, A-2, A-3, A-4, A-5); 8 have <1 year of biopsy experience (Group B; Embryologist B-1, B-2, B-3, B-4, B-5, B-6, B-7, B-8). Embryologists from Group A biopsied a total of 4795 blastocysts while those from Group B biopsied 4869 blastocysts from January 2018 to December 2019. Participants/materials, setting, methods TE biopsy was performed either on Day 5, 6 or 7 using the laser or flicking method. The biopsied cells had Preimplantation Genetic Testing for Aneuploidy (PGT-A) analysed using Next Generation Sequencing (Ion Torrent, USA) and chromosomal mosaicism analysis was done using ReproSeq Mosaic PGS w1.1 workflow. Mosaic blastocysts were reported when 20% - 80% of aneuploid cells are tested in the biopsied samples. Only successfully amplified biopsy samples were included in this study. Main results and the role of chance The mosaicism rate of blastocysts biopsied by embryologists from Group A and B were 17.8% and 19.8% respectively. Blastocysts from Group A showed significantly lower mosaicism rate compared to Group B (p = 0.01). The mosaicism rates of blastocyst biopsied by Embryologist A-1, A-2, A-3, A-4 and A-5 were 17.3%, 19.1%, 16.8%, 15.2%, and 18.9% respectively. The mosaicism rates of blastocyst biopsied by Embryologist B-1, B-2, B-3, B-4, B-5, B-6, B-7, and B-8 were 17.5%, 18.6%, 22.5%, 20.4%, 27.8%, 20.6%, 20.1% and 20.3% respectively. There were no significant differences in blastocyst mosaicism rate between embryologists within Group A (p > 0.05). Contrarily, in Group B, Embryologist B-5 had a significantly higher blastocyst mosaicism rate compared to the other embryologists within the same group (p < 0.05). Limitations, reasons for caution Since this study is retrospective in nature, the biopsy technique (either the laser or flicking method) was not controlled. Hence, further studies to analyse the differences between these 2 biopsy techniques should be carried out to confirm its effect on the occurrence of blastocyst mosaicism. Wider implications of the findings Our study demonstrates that blastocysts biopsied by embryologists with ≥1 year of biopsy experience have significantly lower mosaicism rate compared to those with <1 year of biopsy experience. This indicates that the skill and experience of an embryologist in biopsy may have an impact on the mosaicism rate. Trial registration number Not applicable