STUDY QUESTION Can chromosomal abnormalities beyond copy-number aneuploidies (i.e. ploidy level and microdeletions (MDs)) be detected using a preimplantation genetic testing (PGT) platform? SUMMARY ANSWER The proposed integrated approach accurately assesses ploidy level and the most common pathogenic microdeletions causative of genomic disorders, expanding the clinical utility of PGT. WHAT IS KNOWN ALREADY Standard methodologies employed in preimplantation genetic testing for aneuploidy (PGT-A) identify chromosomal aneuploidies but cannot determine ploidy level nor the presence of recurrent pathogenic MDs responsible for genomic disorders. Transferring embryos carrying these abnormalities can result in miscarriage, molar pregnancy, and intellectual disabilities and developmental delay in offspring. The development of a testing strategy that integrates their assessment can resolve current limitations and add valuable information regarding the genetic constitution of embryos, which is not evaluated in PGT providing new level of clinical utility and valuable knowledge for further understanding of the genomic causes of implantation failure and early pregnancy loss. To the best of our knowledge, MDs have never been studied in preimplantation human embryos up to date. STUDY DESIGN, SIZE, DURATION This is a retrospective cohort analysis including blastocyst biopsies collected between February 2018 and November 2021 at multiple collaborating IVF clinics from prospective parents of European ancestry below the age of 45, using autologous gametes and undergoing ICSI for all oocytes. Ploidy level determination was validated using 164 embryonic samples of known ploidy status (147 diploids, 9 triploids, and 8 haploids). Detection of nine common MD syndromes (-4p=Wolf-Hirschhorn, -8q=Langer-Giedion, -1p=1p36 deletion, -22q=DiGeorge, -5p=Cri-du-Chat, -15q=Prader-Willi/Angelman, -11q=Jacobsen, -17p=Smith-Magenis) was developed and tested using 28 positive controls and 97 negative controls. Later, the methodology was blindly applied in the analysis of: (i) 100 two pronuclei (2PN)-derived blastocysts that were previously defined as uniformly euploid by standard PGT-A; (ii) 99 euploid embryos whose transfer resulted in pregnancy loss. PARTICIPANTS/MATERIALS, SETTING, METHODS The methodology is based on targeted next-generation sequencing of selected polymorphisms across the genome and enriched within critical regions of included MD syndromes. Sequencing data (i.e. allelic frequencies) were analyzed by a probabilistic model which estimated the likelihood of ploidy level and MD presence, accounting for both sequencing noise and population genetics patterns (i.e. linkage disequilibrium, LD, correlations) observed in 2504 whole-genome sequencing data from the 1000 Genome Project database. Analysis of phased parental haplotypes obtained by single-nucleotide polymorphism (SNP)-array genotyping was performed to confirm the presence of MD. MAIN RESULTS AND THE ROLE OF CHANCE In the analytical validation phase, this strategy showed extremely high accuracy both in ploidy classification (100%, CI: 98.1–100%) and in the identification of six out of eight MDs (99.2%, CI: 98.5–99.8%). To improve MD detection based on loss of heterozygosity (LOH), common haploblocks were analyzed based on haplotype frequency and LOH occurrence in a reference population, thus developing two further mathematical models. As a result, chr1p36 and chr4p16.3 regions were excluded from MD identification due to their poor reliability, whilst a clinical workflow which incorporated parental DNA information was developed to enhance the identification of MDs. During the clinical application phase, one case of triploidy was detected among 2PN-derived blastocysts (i) and one pathogenic MD (-22q11.21) was retrospectively identified among the biopsy specimens of transferred embryos that resulted in miscarriage (ii). For the latter case, family-based analysis revealed the same MD in different sibling embryos (n = 2/5) from non-carrier parents, suggesting the presence of germline mosaicism in the female partner. When embryos are selected for transfer based on their genetic constitution, this strategy can identify embryos with ploidy abnormalities and/or MDs beyond aneuploidies, with an estimated incidence of 1.5% (n = 3/202, 95% CI: 0.5–4.5%) among euploid embryos. LIMITATIONS, REASONS FOR CAUTION Epidemiological studies will be required to accurately assess the incidence of ploidy alterations and MDs in preimplantation embryos and particularly in euploid miscarriages. Despite the high accuracy of the assay developed, the use of parental DNA to support diagnostic calling can further increase the precision of the assay. WIDER IMPLICATIONS OF THE FINDINGS This novel assay significantly expands the clinical utility of PGT-A by integrating the most common pathogenic MDs (both de novo and inherited ones) responsible for genomic disorders, which are usually evaluated at a later stage through invasive prenatal testing. From a basic research standpoint, this approach will help to elucidate fundamental biological and clinical questions related to the genetics of implantation failure and pregnancy loss of otherwise euploid embryos. STUDY FUNDING/COMPETING INTEREST(S) No external funding was used for this study. S.C., M.F., F.C., P.Z., I.P., L.G., C.P., M.P., D.B., J.J.-A., D.B.-J., J.M.-V., and C.R. are employees of Igenomix and C.S. is the head of the scientific board of Igenomix. A.C. and L.P. are employees of JUNO GENETICS. Igenomix and JUNO GENETICS are companies providing reproductive genetic services. TRIAL REGISTRATION NUMBER N/A.

Study question What is the prevalence of egg donors resulting in oocyte maturation/fertilization/blastulation rates significantly poorer than expected? Summary answer 6.7% of the egg donors (N = 34/504) elicited outcomes significantly poorer than expected mostly due to low oocyte maturation rates (N = 27/34, 79.4%). What is known already 5-10% of donor oocyte cycles produce no blastocyst involving challenging follow-up counseling. Recent research outlined a statistical model identifying women subject to IVF outcomes poorer than expected. These infertile women underwent whole-exome-sequencing (WES) to identify genes involved in oocyte-maturation-defects (OOMD) and/or preimplantation-embryo-lethality (PREMBL). Oocyte donors are the ideal population to apply this model, because they are young, theoretically-fertile, and undergo multiple retrievals of numerous oocytes. This model can find clinical application in improving egg banks’ management, social application in pinpointing young women potentially-infertile because of oocyte defects, and improving knowledge on the molecular pathways involved in human oocyte/embryo competence. Study design, size, duration Retrospective study involving oocyte donors recruited at three private IVF centers in 2020-2021. 504 donors (25.6 ± 4.7 years; range: 18-35) undergoing 1684 retrievals (N = 32056 cumulus-oocyte-complexes, 24288 metaphase-II) and whose metaphase-II were used (N = 6996 to date) in 487 fresh and 445 frozen ICSI cycles, respectively, were included. Recipient couples affected from severe-male-factor infertility were excluded. Blastocyst culture was systematically conducted. We leveraged clinical/embryological key-performance-indicators to pinpoint outliers subject to outcomes significantly poorer than expected. Participants/materials, setting, methods We quantitatively described donors’ performance as oocyte maturation, fertilization and blastulation rates. The outlier identification was based on the probabilistic modeling of the rates as independent binomial processes, whose baseline success probabilities were estimated from the whole dataset and accounted both for donor age and oocyte type (fresh/vitrified-warmed). The model assigns low probability to individuals with unusually large numbers of immature/unfertilized oocytes or arrested embryos. Donors with low probability (p < 0.001) were designated as outliers. Main results and the role of chance Oocyte donors underwent 3.3 ± 1.9 retrievals (1-8) collecting 63.6 ± 44.4 cumulus-oocyte-complexes (5-241), of which 48.2 ± 34.6 (3-162) metaphase-II (maturation-rate: 75 ± 13%,19-100). 13.9 ± 10.5 (3-75) metaphase-II were used during 1.8 ± 1.3 (1-9) cycles per donor, 1.0 ± 1.0 (0-6) cycles with 7.1 ± 7.6 (0-45) fresh metaphase-II oocytes and 0.9 ± 1.0 (0-7) cycles with 6.8 ± 8.1 (0-59) vitrified-warmed metaphase-II oocytes. Overall, 10.1 ± 8.0 (0-58) 2PN-zygotes were obtained per donor (fertilization-rate: 72 ± 16%,0-100), 5.1 ± 5.8 (0-34) in fresh (fertilization-rate: 72 ± 17%,0-100) and 5.0 ± 6.1 (0-41) in frozen cycles (fertilization-rate: 74 ± 16%,20-100). Overall, 5.5 ± 5.0 (0-31) blastocysts were obtained per donor (blastulation-rate: 55 ± 25%,0-100), 3.0 ± 3.8 (0-25) in fresh (blastulation-rate: 59 ± 25%,0-100) and 2.5 ± 3.6 (0-31) in frozen cycles (50 ± 24%,0-100). The model applied to this dataset designated 34 outliers (N = 34/504,6.7%, p < 0.001, FDR<0.1%). Most of them (N = 27) showed oocyte maturation-rates significantly lower than expected (51 ± 10%,19-66). These women were 26.9 ± 5.1 years old (19-35) and underwent 4.3 ± 1.7 retrievals, collecting 91.2 ± 45.0 cumulus-oocyte-complexes (25-210), of which 49.1 ± 31 (8-127) were metaphase-II oocytes. Three outliers showed fertilization-rates significantly poorer than expected (19 ± 17%,0-31). They were 21 ± 4.4 years old (18-26) and their metaphase-II oocytes (12.7 ± 4.9,7-16) were inseminated in 1.3 ± 0.6 donation cycles (1-2) resulting in 3 ± 2.6 (0-5) 2PN-zygotes. Four outliers showed blastulation-rates significantly poorer than expected (13 ± 7%,7-22SAME). They were 26.3 ± 6.2 years old (19-34) and their oocytes resulted in 28.5 ± 20.5 (14-58) 2PN-zygotes across 4.5 ± 3.1 (2-9) donation cycles that developed into 4.5 ± 5.7 (1-13) blastocysts. Limitations, reasons for caution Euploidy, another pivotal measure of oocyte competence, was not assessed. Larger studies are warranted, also accounting for putative additional confounders on all embryological outcomes under investigation. Nonetheless, gathering well-structured data on the cumulative performance of donor oocytes collected across multiple retrievals and through multiple IVF cycles is challenging and remarkable. Wider implications of the findings These outlier oocyte donors could benefit from follow-up investigation aimed at underpinning OOMD/PREMBL-related genetic factors. The development of genetic prediction tools to this end will improve egg donation cycles’ management and equity and might pave the way to preventive screening strategies and precision reproductive interventions in theoretically-fertile young women. Trial registration number not applicable

Study question What are the incidence and origin of ploidy abnormalities in embryos derived from 2 pronuclei (2PN) zygotes, and is their risk linked to maternal age? Summary answer While embryo haploidy is usually due to male meiosis errors, triploidy is mainly caused by female meiosis II errors, whose incidence increases with maternal age. What is known already Normal fertilization is denoted by the appearance of 2PN 16-18 hours after insemination. Deviation from 2PN is considered evidence of abnormal fertilization and ploidy anomalies. Nonetheless, even 2PN embryos can be diagnosed with ploidy abnormalities during preimplantation genetic testing (PGT). The incidence of 3PN has already been linked to advanced maternal age. However, no conclusive and well-powered studies have yet investigated the incidence, origin, and maternal age correlation of genetically-diagnosed ploidy abnormalities. A targeted NGS-based approach that simultaneously analyzes copy number and genotyping data provides a comprehensive ploidy status assessment and helps address numerous basic and clinical research questions. Study design, size, duration Retrospective study including 96,660 trophectoderm biopsies analyzed between 2020 and 2022 using a targetedNGS-based PGT platform. A total of 1,063 embryos carrying haploidy or triploidy were used to investigate maternal age correlation, and parental/meiotic origin of the anomaly. Of these, 57 embryos were from concomitant PGT-M (PGT for monogenic disorders) cycles where also parental DNA was available. These trios (embryo-parents) were used to independently estimate parental/meiotic contribution to the ploidy abnormality and genome-wide recombination events. Participants/materials, setting, methods Targeted-NGS’s accuracy in genotyping and copy number (CN) detection were previously validated on triploid cell lines and multifocal blastocyst biopsies with known ploidy status derived from abnormally fertilized oocytes. Parental and meiotic origins were estimated using two independent approaches. First, they were inferred using sex chromosomes CN from 1,063 embryos with altered ploidy status. Secondly, they were directly calculated using genotyping data from 57 trios (embryo and parents) from PGT-M cycles. Main results and the role of chance The prevalence of ploidy abnormalities in 2PN-derived embryos was 1.1% (n = 1,063/96,660), with triploids accounting for 83.0% (n = 882/1,063) and haploids for 17.0% (n = 181/1,063). Remarkably, the incidence of ploidy anomalies is positively correlated with maternal age (OR = 1.046 per year; p < 0.001). Triploidy showed strong correlation with age (OR = 1.059 per year; p < 0.001), while haploidy did not (OR = 0.96 per year; p = 0.1). Based on sex chromosomes CN analysis, the extra haploid set of triploid embryos was almost completely of maternal origin (94.6%; 95%CI:93.0-96.1), with male errors accounting for only 5.4% (95%CI:4.0-7.1). On the other hand, haploid embryos were the result of paternal errors in 97.8% of cases (95%CI:94.4-99.4), with the missing haploid set being of maternal origin in the remaining 2.2% (95%CI:0.6-5.6). In terms of triploidy’s meiotic origin, two-thirds of the errors occurred during MII (95%CI:63.4-69.8), while one-third occurred during MI (95%CI:30.2-36.5). An independent method using genotyping data of 57 trios confirmed the predominance of paternal error in haploidy (n = 12/14) and the exclusively maternal origin of all embryonic triploidies (n = 43/43). The extra haploid set resulted from an error during MI in 27.9% (n = 12/43) and during MII in 72.1% (n = 31/43) of cases. Interestingly, 16.3% of triploids (n = 7/43) showed no genome-wide recombination events. Limitations, reasons for caution Despite the uniquely large sample size, the inference based on sex chromosomes CN suffers the limitations of the modeling assumptions (independence between parental and meiotic errors), which require further validation. Due to the low prevalence of paternal errors, clinical correlation with male factor could not be investigated with sufficient power. Wider implications of the findings Thanks to the exceedingly high sample size, this is the first study to reveal an increased risk of triploid conception with advancing female age (76% higher at age 40 than at age 30), providing meaningful insights for patients counseling. Importantly, the meiotic origin of ploidy anomalies in embryos were unveiled. Trial registration number not applicable