P–581 The day of blastocyst biopsy and the chromosomal constitution. Evaluation of 5125 embryos by Next Generation Sequency (NGS)

Study question Is there a relationship between the day of blastocyst biopsy and the results NGS analysis? Summary answer Embryos biopsied on day 6 or 7 are associated with the increased probability of being an aneuploidy embryo and less likely to be mosaic embryo. What is known already There is controversy about whether an embryo that reaches the blastocyst stage on day 5 has a higher chance of being euploid than embryos which are biopsied later. In our study, chromosome constitution was evaluated by next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) and confounding factors were eliminated. Study design, size, duration Data was collected retrospectively from June 2016 to January 2020 Participants/materials, setting, methods In total, 5125 blastocyst (day 5=2914, day 6 N = 2154 and day7 N = 57), generated from 1318 cycles were analysed with PGT-A. The chromosome constitution for each embryo was classified as euploid, aneuploid and mosaic. A multilevel model was made and associations betwwen variables by logistic regression were adjusted according to maternal age, SART blastocyst grade, fertilization method, biopsy operator and blastocyst stage. Main results and the role of chance The mean maternal age was 36.2 ± 4.2. Euploid rate was 62.1% and 37.9% (day 5 and day 6–7 respectively), aneuploidy rate was 47.0% and 53.0% (day 5 and day 6–7, respectively), mosaicism rate was 59.6% and 40.4% (day 5 and day 6–7, respectively) (p < 0.001). Embryos biopsied on day 6–7 have a significantly lower probability to be euploid and mosaicism than embryos biopsied on day 5 ((OR = 0.76 [0.68–0.86]); (OR = 0.84 (0.73 – 0.96) respectively) (p < 0.001). On the contrary, embryos biopsy on day 5 were significantly more likely to be euploid than day 6–7 (OR = 1.63[1.42–1.86]) (p < 0.001). Limitations, reasons for caution The results observed in this study should be confirmed using a larger number of samples. For the NGS analysis, a chromosome with a variation between 20 to 80% was considered mosaic. Wider implications of the findings: The present study revealed that embryos that reach blastocyst classified as full to hatched on day 5 are more like to be euploid compared to slow growing embryos. Trial registration number non-clinical trials

P–532 Embryo quality needs to be considered as a main criterion when selecting mosaic embryos for transfer

Study question Should we consider embryo quality as one of the most important criteria to follow when transferring a mosaic embryo? Summary answer Embryo quality is an implantation biomarker both for euploid and mosaic embryos, and also a determinant for selecting the most eligible mosaic for transfer. What is known already Several studies show the benefit of transferring mosaic embryos when there are no euploid embryos to transfer, and they still result in ongoing pregnancies and what is more important is that they result in healthy babies. Studies and guidelines suggest prioritizing mosaic embryos based on maternal age, chromosomes impacted, percentage of aneuploidy, number of chromosomes involved, type of mosaic (simple vs complex, segmental vs complete, monosomy vs trisomy) but embryo quality is never part of these criteria. Studies claim that mosaic implantation rate is lower than euploid embryos, but they never show if both populations are comparable in terms of quality. Study design, size, duration This is a retrospective observational study performed in a private centre between February 2018 and January 2020. The study includes the data analysis of 96 euploid blastocysts and 14 low risk mosaic blastocysts (defining low risk regarding chromosome syndromes and less than 50% level mosaicism). All transferred in single embryo transfer (SET) to 105 patients after PGT-A (mean maternal age 38,9 years). The SET factor enables us to track the implantation outcome of all embryos. Participants/materials, setting, methods PGT-A with NGS technology was offered to patients of advanced maternal age and/or with repeated IVF failures. Trophectoderm biopsies were performed on day 5 and/or day 6 embryos, with laser assistance. Blastocyst morphology was scored in 3 groups: A: excellent (AA, AB, BA), B: good (BB), C: average and poor-quality embryos (BC, CB, CC). (Gardner-Schoolcraft classification) Low risk mosaic embryo transfer was offered to patients with no euploid embryos to transfer. Main results and the role of chance We found no significant differences between both populations (euploid and mosaic embryos) in terms of embryo quality (Chi^2 p-value =0,0975) so we were able to compare the overall implantation of similar quality populations. Despite euploid implantation being higher as described in most studies, no statistical differences (Chi^2 p-value = 0,4344) were found in terms of implantation rates between mosaic (57,0%) and euploid (67,6%) blastocysts during the same period. There are no differences between the mean age of both groups (39,7 vs 38,8 years, respectively). The implantation rates for euploid blastocysts were 79,5% (n = 39), 62,7% (n = 51) and 33,3% (n = 6) in the A, B and C blastocyst quality groups, respectively, showing significative differences among the three groups. The implantation rates of low-risk mosaic blastocysts were 100% (n = 3), 62,5% (n = 8) and 0,0% (n = 3) in the A, B and C blastocyst morphology groups, respectively, showing also still significant differences among the three groups despite the small population. (Chi^2 p-values according to implantation: Euploid =0,0434; Mosaic=0,0419) We have also compared the three quality categories between both populations showing no significative differences (Chi^2 p-values according to quality: A = 0,4344; B = 0,9894; C = 0,2568), concluding that same quality embryos behave the same way despite being euploid or mosaic. Limitations, reasons for caution The study is limited by its retrospective nature and the low number of mosaic embryos transferred as they are the last option for transfer. Additionally, it is common to transfer more than one mosaic embryo to increase the chances of pregnancy, therefore losing implantation track. Wider implications of the findings: Embryo quality has always been a strong biomarker predictable for implantation and this is also true for mosaic embryos as well. It is a simple concept, but we cannot compare implantation potential of euploid embryos with mosaic embryos without describing both populations in terms of quality. Trial registration number Not applicable

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–564 Analysis of efficiency and outcomes of assisted reproductive technology (ART) programs after embryo transfer with a low-level of mosaicism

Study question How does embryo transfer with a low-level of mosaicism affect the success of ART programs, pregnancy, and live birth in comparison with euploid embryo transfer? Summary answer The transfer of mosaic embryos results in the delivery of a healthy baby however significantly decreases the outcome of ART programs and live birth rate. What is known already Present methods of preimplantation genetic testing of aneuploidy (PGT-A) allow detecting a mixture of euploid and aneuploid cells at the blastocyst stage with high accuracy. Such embryos are classified as mosaics with varying levels according to the guidelines of the International Society for Preimplantation Genetic Diagnosis (PGDIS). Numerous sources describe that number of mosaic embryos can vary from 4 to 22%. Several publications report that mosaic embryos can lead to successful pregnancies and healthy childbirth, but with a lower frequency and higher rates of pregnancy loss compared to euploid embryos. Nevertheless, the effect of mosaicism on ART outcomes remains controversial. Study design, size, duration It has been analyzed 2506 embryos from 648 patients undergoing the ART program with PGT-A at the Institute of Reproductive Medicine for 2018 - 2019. Embryos after PGT-A were classified as euploid, aneuploid, and having mosaicism of less than 40% as low level and more than 40% as high level following PGDIS guidelines. Patients of (group A) were transferred 467 single euploid embryos, and 43 patients (group B) underwent single low-level mosaic embryo transfer. Participants/materials, setting, methods The embryos on day 5 or 6 were graded by Gardner Scoring System. Approximately 5–10 TE cells were biopsied from good quality blastocysts and subsequently vitrified. PGT-A was performed utilizing an array comparative genomic hybridization (aCGH) (Agilent). The transfer of mosaic embryos was performed in the absence of an alternative, only after medical genetic counseling with a risk explanation and the subsequent signing of an informed agreement. Statistical tests processed by Pearson’s chi-squared test. Main results and the role of chance Of all analyzed embryos, the proportion of euploid embryos was 48.6% (n = 1002), the total number of mosaics was 18.6% (n = 384) and aneuploid ones were 32.8% (n = 676). Depending on the level of mosaicism, the ratio between embryos with low-level mosaicism (≤40%) / high-level (≥40%) was 38.3% / 61.7%, respectively. According to the study, there was a significant decrease in the indicator of clinical pregnancy rate after embryo transfer with a low-level of mosaicism of 44.1% versus 63.2% transferred euploid embryo (р<0,01), however, despite an increase losses pregnancy in the group B (26.3%) there was no significant difference (p = 0.16) in comparison with the control group (15.4%). The live birth rate (LBR) significantly decreased (p < 0.001) after the transfer of the mosaic embryo by 32.5%, while in the control group the indicator was 53.9%. In all cases, after the transfer of the mosaic embryo, healthy babies were born. There were 2 cases of high-level mosaic embryo transfer as a result of which pregnancy did not occur. According to the survey, about 70% of patients agree to replant mosaic embryos, 20% are ready to go to the new program, and 10% cannot make a decision. Limitations, reasons for caution The number of patients in group B was significantly lower than in group A. Not enough cases of embryo transfer with a high-level of mosaicism. Wider implications of the findings: The current study might help to develop and to select a more appropriate strategy for transfer mosaic embryos. The next series of studies should focus on obstetric and neonatal outcome data from mosaic embryo transfer to gain a better understanding of the chromosomal and physiological health of children. Trial registration number Not applicable