Chromosomal Copy Number Research Articles

Prenatal diagnosis of two fetuses with Xp22.31 microdeletion syndrome indicated by non-invasive prenatal testing

To assess the value of non-invasive prenatal testing (NIPT) for detecting fetal chromosomal microdeletion/microduplication syndromes by carrying out prenatal diagnoses for two fetuses with Xp22.31 microdeletion indicated by NIPT. Two pregnant women suspected for fetal Xp22.31 microdeletion syndrome who presented at Zaozhuang Maternal and Child Health Care Hospital on December 5, 2017 and October 15, 2020 were selected as the study subjects. Clinical data of the two women were collected, and peripheral venous blood samples were collected for NIPT testing. Amniotic fluid samples were taken for G-banding chromosomal karyotyping analysis and copy number variation sequencing (CNV-seq) for fetus 1, while G-banding chromosomal karyotyping and single nucleotide polymorphism microarray analysis (SNP array) were carried out for fetus 2. Peripheral venous blood samples of couple 1 were collected for CNV-seq to verify the origin of copy number variation . NIPT indicated that fetus 1 had harbored a 1.3 Mb deletion in the Xp22.31 region, while G-banding chromosomal karyotyping had found no abnormality. CNV-seq analysis verified the fetus to be seg[GRCh37]del(X)(p22.31)chrX:g.6800001_7940000del, with a 1.14 Mb deletion at Xp22.31, which was derived from its mother. NIPT indicated that fetus 2 had harbored a 1.54 Mb deletion in the Xp22.31 region, while G-banding chromosomal karyotyping had found no abnormality. SNP array analysis indicated arr[GRCh37]Xp22.31(6458940_8003247)×0, with a 1.54 Mb deletion in Xp22.31 region. NIPT not only has a good performance for detecting fetal trisomies 21, 18 and 13, but also has the potential for detecting chromosomal microdeletion/microduplications. For high risk fetuses indicated by NIPT, prenatal diagnosis needs to be carry out to verify the chromosomal abnormalities.

Clinical phenotype and genetic analysis of a patient with a heterozygous 6p25.3 deletion and partial trisomy 15q

To investigate the clinical phenotype and genetic characteristics of a patient with a heterozygous 6p25.3 deletion and partial trisomy 15q. A patient who had presented at the Genetics and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University on May 14, 2021 was selected as the study subject. Clinical data of the patient was collected, and G-banded chromosomal karyotyping and copy number variation sequencing (CNV-seq) were carried out. The patient's main clinical features have included complete uterine septum, vaginal septum, atrophy of left eyeball, abnormal fingers and toes, and mental retardation. The karyotype of the patient was 46,XX,der(6)t(6;15)(p25.3;q26.1). CNV-seq result has indicated a 1.20 Mb heterozygous deletion in the 6p25.3 region and a 10.20 Mb duplication in the 15q26.1q26.3 region. The deletion segment has included the FOXQ1 gene, which may be related with the abnormal development of the left eye. The duplication segment has a 96.16% overlap with the region associated with 15q26 overgrowth syndrome (including the IGF1R gene), which may be related to the patient' s abnormal development of the Müllerian duct, abnormal fingers and toes, and mental developmental delay. The heterozygous deletion of the 6p25.3 region and duplication of the 15q26.1q26.3 region probably underlay the abnormal clinical phenotype in this patient.

SDPS-27 P16 IMMUNOHISTOCHEMISTRY AS A SCREENING TOOL FOR HOMOZYGOUS CDKN2A DELETIONS IN GLIAL TUMORS

Abstract The 2021 WHO classification of tumors of the central nervous system emphasizes the significance of molecular parameters for an integrated diagnosis. Homozygous deletion of CDKN2A has been associated with an adverse prognosis in IDH-mutant gliomas, supratentorial ependymomas, meningiomas, and MPNST. In this study, we examined the value of p16 protein immunohistochemistry as a screening tool for predicting a homozygous CDKN2A deletion. Genetic analyses for CDKN2A deletion in 30 pleomorphic xanthoastrocytomas, 32 IDH-wildtype high-grade gliomas and 40 supratentorial ependymomas with ZFTA-RELA gene fusion, 10 IDH-mutant astrocytomas, and 10 meningiomas were performed either by a molecular inversion probe assay (MIP), a high-resolution, quantitative technology for the accurate, genome-wide assessment of chromosomal copy number alterations, or by 850k methylation profile-deduced copy number analysis or multiplex ligation-dependent probe amplification analysis. In pleomorphic xanthoastrocytomas and IDH-wildtype high-grade gliomas, p16 immunohistochemistry showed high specificity (100% both), moderate sensitivity (76% and 56%), and a high positive predictive value (PPV; 100% both) for the presence of a homozygous CDKN2A deletion. In supratentorial ependymoma with ZFTA-RELA gene fusion, immunohistochemistry for p16 protein was highly sensitive and specific (90% and 97%) for a homozygous CDKN2A deletion and exhibited a high PPV and NPV (90% and 97%, respectively). For lower-grade meningioma, no correlation between the p16 immunohistochemical status and the CDKN2A deletion status was possible. Therefore, p16 protein expression detected by immunohistochemistry is a useful prescreening tool for detecting a homozygous CDKN2A deletion in glial tumors.

Identification of a novel isolated 4q35.2 microdeletion in a Chinese pediatric patient using chromosomal microarray analysis: a case report and literature review

BackgroundIsolated terminal 4q35.2 microdeletion is an extremely rare copy number variant affecting people all over the world. To date, researchers still have controversial opinions and results on its pathogenicity. Here, we aim to present a Chinese pediatric patient with terminal 4q35.2 microdeletion and use this case to clarify the underlying genotype–phenotype correlation.MethodsA 17-year-old boy from Quanzhou, South China, was recruited as the main subject in this study. Karyotype and single-nucleotide polymorphism (SNP) based microarray analysis were carried out to detect chromosomal abnormalities and copy number variants in this family. Trio whole exome sequencing (Trio-WES) was performed to investigate the potential pathogenic variant in this family.ResultsDuring observation, we identified abnormal clinical phenotypes including upper eyelid ptosis, motor developmental delay, abnormal posturing, abnormality of coordination, attention deficit hyperactivity disorder, and involuntary movements in the patient. SNP array analysis results confirmed a case of 2.0 Mb 4q35.2 microdeletion and parental SNP array verification results indicated that the terminal 4q35.2 microdeletion was inherited from his mother. No copy number variants were detected in his father. In addition, the trio-WES results demonstrated none of pathogenic or likely pathogenic variants in the patient.ConclusionsThis study brings a novel analysis of a case of 2.0 Mb terminal 4q35.2 microdeletion affecting a Chinese individual. In addition, additional clinical symptoms such as upper eyelid ptosis and involuntary movements were first reported to affect a patient with terminal 4q35.2 microdeletion, which may broaden the phenotype spectrum of the condition.

When NIPT meets WES, prenatal diagnosticians face the dilemma: genetic etiological analysis of 2,328 cases of NT thickening and follow-up of pregnancy outcomes.

Objective: To assess the performance of diverse prenatal diagnostic approaches for nuchal translucency (NT) thickening and to investigate the optimal prenatal screening or diagnostic action with a NT thickening of 95th percentile-3.50mm. Methods: A retrospective analysis of 2,328 pregnancies with NT ≥ 95th percentile through ultrasound-guided transabdominal chorionic villus sampling (CVS), amniocentesis, or cordocentesis obtained clinical samples (chorionic villi, amniotic fluid, and cord blood), and real-time quantitative fluorescent PCR (QF-PCR), chromosome karyotyping (CS), chromosome microarray analysis (CMA), or whole exome sequencing (WES) were provided to identify genetic etiologies. Results: In this study, the incidence of chromosomal defects increased with NT thickness. When NT ≥ 6.5mm, 71.43% were attributed to genetic abnormalities. The 994 gravidas with fetal NT thickening underwent short tandem repeat (STR), CS, and CMA. In 804 fetuses with normal karyotypes, CMA detected 16 (1.99%) extra pathogenic or likely pathogenic copy number variations (CNVs). The incremental yield of CMA was only 1.16% (3/229) and 3.37% (10/297) in the group with NT 95th percentile-2.99mm and NT 3.0-3.49mm, separately. Among the 525 gravidas with fetal NT thickening who underwent STR, CMA, and WES, the incremental yield of WES was 4.09% (21/513). In the group of NT 95th percentile-2.99mm, there were no additional single-nucleotide variations (SNVs) detected in WES, while in 143 cases with NT of 3.0-3.49mm, the incremental yield of WES was 5.59% (8/143). Conclusion: In the group of NT 95th percentile-3.0mm, since chromosomal aneuploidy and chromosomal copy number variation were the primary causes and the additional contribution of CMA and WES was not significant, we recommend NIPT-Plus for pregnant women with a NT thickening of 95th percentile-3.0mm first. In addition, comprehensive prenatal genetic testing involving CMA and WES can benefit pregnancies with NT thickening of 3.0-3.49mm.

Genomic analysis of spinal meningiomas: correlation with histopathological grade.

Spinal meningiomas are one of the most common primary intradural tumors of the adult spine. Spinal meningiomas typically have a benign course with low rates of recurrence. Recent advances in genomic profiling have provided valuable information on meningioma biology and natural history, but these studies have focused primarily on cranial meningiomas. Chromosomal copy number analysis of meningiomas has been shown to be a valuable molecular profiling technique for distinguishing benign from aggressive tumors. The Integrated Grade for Meningioma is a novel grading scheme that uses mitotic index and copy-number profile to identify aggressive tumors at high risk for recurrence. The integrated grade has been shown to be a better predictor of tumor behavior than WHO grade alone. The objective of this study was to evaluate the chromosomal copy-number profile of spinal meningiomas, and to correlate these findings with the assigned WHO grades. The authors evaluated 94 spinal meningiomas treated surgically at their institution between 2002 and 2022. The histopathological results including WHO grade, mitotic index, presence of atypical features, and MIB-1 index were recorded. Chromosomal copy number as determined by institutional whole-genome DNA copy-number profiling was available for 57 tumors. The WHO grades of the cohort consisted of 81 (86%) WHO grade 1 tumors and 13 (14%) WHO grade 2 tumors. In tumors for which copy-number profiling was available, 44 (77%) of 57 demonstrated loss of 22q/NF2. Notably absent were frequent high-risk copy number alterations including loss of 1p, 3p, 4p/q, 6p/q, 10p/q, 14q, 18p/q, 19p/q, and focal loss of CDKN2A on 9p. Of the 9 WHO grade 2 tumors for which copy-number profiling was available, 6 tumors were reclassified to a lower risk profile (integrated grade 1). This analysis suggests that spinal meningiomas exhibit overwhelmingly indolent biology, as supported by their benign integrated grade. These findings have implications in the surgical management of these patients in relation to the need for complete Simpson grade I resection, as well as the potential avoidance of adjuvant therapy following surgery in the setting of otherwise frequently benign pathology.

Analysis of potential copy-number variations and genes associated with first-trimester missed abortion

BackgroundCopy number variation sequencing (CNV-seq) was proven to be a highly effective tool in studying of chromosomal copy number variations (CNVs) in prenatal diagnosis and post-natal cases with developmental abnormalities. However, the overall characteristics of missed abortion (MA) CNVs were largely unexplored. MethodsWe retrospectively analyzed the results of CNV-seq in first-trimester MA. The samples included were single pregnancy loss before 13 gestational weeks, and other potential factors affecting embryonic implantation and development had been excluded. Gene ontology and KEGG enrichment analysis was performed on the smallest overlapping regions (SORs) of high-frequency deletion/duplication. ResultOn the basis of strict inclusion and exclusion criteria, only 152 samples were included in our study. 77 (50.7%) samples displayed chromosome number abnormalities, 32 (21%) showed isolated CNVs, and 43 (28.3%) showed no CNVs. A total of 45 CNVs, ranging in size between 300 Kb and 126.56 Mb were identified, comprising 13 segmental aneuploidies CNVs, and 32 submicroscopic CNVs. Among these CNVs, we screened out four SORs (5q31.3, 5p15.33-p15.2, 8p23.3-p23.2, and 8q22.2–24.3), which were potentially associated with first-term MA. 16 genes were identified as potential miscarriage candidate genes through gene-prioritization analysis, including three genes (MYOM2, SDHA and TPPP) critical for embryonic heart or brain development. ConclusionWe identified some potential candidate CNVs and genes associated with first-trimester MA. 5q31.3 duplications, 5p15.33-p15.2 deletions, 8p23.3-p23.2 deletions and 8p22.2-p24.3 duplications are four potential candidate CNVs. Additionally, MYOM2, SDHA and TPPP are potential genes associated with first-trimester MA.

Chromosomal, gestational, and neonatal outcomes of embryos classified as a mosaic by preimplantation genetic testing for aneuploidy

To understand the clinical risks associated with the transfer of embryos classified as a mosaic using preimplantation genetic testing for aneuploidy. Analysis of data collected between 2017 and2023. Multicenter. Patients of infertility treatment. Comparison of pregnancies resulting from embryos classified as euploid or mosaic using the 20%-80% interval in chromosomal intermediate copy numbers to define a mosaic result. Rates of spontaneous abortion, birth weight, length of gestation, incidence of birth defects, and chromosomal status during gestation. Implanted euploid embryos had a significantly lower risk of spontaneous abortion compared with mosaic embryos (8.9% [n = 8,672; 95% confidence interval {CI95} 8.3, 9.5] vs. 22.2% [n = 914; CI95 19.6, 25.0]). Embryos with mosaicism affecting whole chromosomes (not segmental) had the highest risk of spontaneous abortion (27.6% [n = 395; CI95 23.2, 32.3]). Infants born from euploid, mosaic, and whole chromosome mosaic embryos had average birth weights and lengths of gestation that were not statistically different (3,118 g and 267 days [n = 488; CI95 3,067, 3,169, and 266, 268], 3052 g and 265 days [n = 488; CI95 2,993, 3,112, and 264,267], 3,159 g and 268 days [n = 194; CI95 3,070, 3,249, and 266,270], respectively). Out of 488 infants from mosaic embryo transfers (ETs), one had overt gross abnormalities as defined by the Centers for Disease Control and Prevention. Most prenatal tests performed on pregnancies from mosaic ETs had normal results, and only three pregnancies produced prenatal test results reflecting the mosaicism detected at the embryonic stage (3 out of 250, 1.2%; CI95 0.25, 3.5). Although embryos classified as mosaic experience higher rates of miscarriage than euploid embryos (with a particularly high frequency shortly after implantation), infants born of mosaic ETs are similar to infants of euploid ETs. Prenatal testing indicates that mosaicism resolves during most pregnancies, although this process is not perfectly efficient. In a small percentage of cases, the mosaicism persists through gestation. These findings can serve as risk-benefit considerations for mosaic ETs in the fertility clinic.

Primary Cutaneous Epithelioid Mesenchymal Tumor With a Novel ATP2B4::GLI1 Gene Fusion.

GLI1 gene alterations (rearrangement or amplification) have been found in several bone and soft tissue tumors including pericytic tumors, gastric plexiform fibromyxoma, gastroblastoma, and a various group of epithelioid tumors with regional recurrence or distant metastasis. In this article, we describe a case of primary cutaneous epithelioid mesenchymal tumor harboring hitherto not reported ATP2B4::GLI1 gene fusion. A 42-year-old man presented with a growing firm lesion on the left postauricular scalp. Microscopically, the shave biopsy specimen revealed a dermal-based nodular proliferation of relatively monotonous epithelioid cells with round to ovoid nuclei and pale eosinophilic cytoplasm, accompanied by prominent stromal vasculature. Significant cytologic atypia, necrosis, and mitotic activity were absent. The tumor cells were partially positive for CD34 and S-100 protein, but were negative for other markers, including SOX-10, keratins, and myogenic markers. An ATP2B4::GLI1 gene fusion was identified by next-generation sequencing. Array CGH was also performed, but it did not show relevant chromosomal copy number changes. Awareness of this rare cutaneous tumor, and thus, reporting of additional cases is necessary for further delineating its full clinicopathologic spectrum.

Prenatal whole exome sequencing identified two rare compound heterozygous variants in EVC2 causing Ellis-van Creveld syndrome.

Pathogenic mutations in EVC or EVC2 gene can lead to Ellis-van Creveld (EvC) syndrome, which is a rare autosomal recessive skeletal dysplasia disorder. This study aimed to determine pathogenic gene variations associated with EvC syndrome in fetuses showing ultrasound anomalies. A 32-year-old pregnant woman from Quanzhou, China was investigated. In her pregnancy examination, the fetus exhibited multiple fetal malformations, including a narrow thorax, short limbs, postaxial polydactyly, cardiac malformations, and separation of double renal pelvis. Karyotype, chromosomal microarray analysis and whole exome sequencing were performed for prenatal genetic etiology analysis. Chromosome abnormalities and copy number variants were not observed in the fetus using karyotype and chromosomal microarray analysis. Using whole exome sequencing, two compound heterozygous variants NM_147127.5:c.[2484G>A(p.Trp828Ter)];[871-2_894del] in EVC2 gene were identified in the fetus as pathogenic variants inherited from parents. The study is the first to identify two rare compound variants in EVC2 gene in a Chinese family using whole exome sequencing. The application of whole-exome sequencing would be helpful in fetal etiological diagnosis with ultrasound anomalies.

Performance of noninvasive prenatal testing for twin pregnancies in South China

ObjectiveThe purpose of this study was to evaluate the performance of noninvasive prenatal testing (NIPT) for the detection of chromosomal aneuploidies and copy number variations (CNVs) in twin pregnancies.MethodA cohort of 2010 women with twin pregnancies was recruited. 1331 patients opted for NIPT, and 679 patients opted for expanded NIPT (NIPT-plus). All high-risk patients were advised to undergo invasive prenatal diagnosis. All participants were followed up until 6 months after birth.ResultsTwenty-two cases were predicted to have a high risk of chromosome abnormalities by NIPT, of which 14 pregnant women underwent invasive prenatal diagnosis. The 14 cases included 3 cases of trisomy 21, 1 case of trisomy 18, 1 case of trisomy 7, 2 cases of sex chromosome aneuploidies (SCAs), and 7 cases of CNVs, of which the confirmed cases numbered 2, 1, 0, 1, and 0, respectively. Twenty cases were predicted to have a high risk of chromosome abnormalities by NIPT-plus, of which 16 pregnant women underwent invasive prenatal diagnosis. The 16 cases included 1 case of trisomy 21, 1 case of trisomy 7, 7 cases of SCAs, and 7 cases of CNVs, of which were confirmed in 1, 0, 3, and 2, respectively. No false-negative result was reported during the follow-up period.ConclusionThe NIPT/NIPT-plus has excellent performance in the detection of chromosome aneuploidies in twin pregnancies. But for CNVs, the effectiveness of NIPT is poor, and the NIPT-plus have a certain detection efficiency. It is worth noting that pre- and post-genetic counseling is especially important, and the chorionicity, mode of conception, clinical indications, and fetal fraction should be considered as influencing factors.

The adaptive roles of aneuploidy and polyclonality in Leishmania in response to environmental stress.

Aneuploidy is generally considered harmful, but in some microorganisms, it can act as an adaptive mechanism against environmental stress. Here, we use Leishmania-a protozoan parasite with remarkable genome plasticity-to study the early steps of aneuploidy evolution under high drug pressure (using antimony or miltefosine as stressors). By combining single-cell genomics, lineage tracing with cellular barcodes, and longitudinal genome characterization, we reveal that aneuploidy changes under antimony pressure result from polyclonal selection of pre-existing karyotypes, complemented by further and rapid de novo alterations in chromosome copy number along evolution. In the case of miltefosine, early parasite adaptation is associated with independent point mutations in a miltefosine transporter gene, while aneuploidy changes only emerge later, upon exposure to increased drug levels. Therefore, polyclonality and genome plasticity are hallmarks of parasite adaptation, but the scenario of aneuploidy dynamics depends on the nature and strength of the environmental stress as well as on the existence of other pre-adaptive mechanisms.

A clinically applicable molecular classification of oncocytic cell thyroid nodules.

Whole-chromosome instability with near-whole genome haploidization (GH) and subsequent endoreduplication is considered a main genomic driver in the tumorigenesis of oncocytic cell thyroid neoplasms (OCN). These copy number alterations (CNA) occur less frequently in oncocytic thyroid adenoma (OA) than in oncocytic carcinoma (OCA), suggesting a continuous process. The current study described the CNA patterns in a cohort of 30 benign and malignant OCN, observed using a next generation sequencing (NGS) panel that assesses genome-wide loss of heterozygosity (LOH) and chromosomal imbalances using 1,500 single nucleotide polymorphisms (SNP) across all autosomes and the X chromosome in DNA derived from cytological and histological samples. Observed CNA patterns were verified using multiparameter DNA flow cytometry with or without whole-genome SNP array analysis and lesser-allele intensity-ratio (LAIR) analysis. On CNA-LOH analysis using the NGS panel, GH type CNA were observed 4 of 11 (36%) OA and 14 of 16 OCA (88%). Endoreduplication was suspected in 8 of 16 (50%) OCA, all with more extensive GH type CNA (p<0.001). Reciprocal chromosomal imbalance type CNA, characterized by (imbalanced) chromosomal copy number gains and associated with benign disease, were observed in 6 of 11 (55%) OA and one equivocal case of OCA. CNA patterns were different between the histopathological subgroups (p<0.001). By applying the structured interpretation and considerations provided by the current study, CNA-LOH analysis using an NGS panel that is feasible for daily practice may be of great added value to the widespread application of molecular diagnostics in the diagnosis and risk stratification of OCN.

The Clinical Significance of Genetic Variation in Ovarian Cancer.

Genetic variation is a well-known contributor to the onset and progression of cancer. The goal of this study is to provide a comprehensive examination of the nucleotide and chromosomal variation associated with the onset and progression of serous ovarian cancer. Using a variety of computational and statistical methods, we examine the exome sequence profiles of genetic variants present in the primary tumors of 432 ovarian cancer patient samples to compute: (1) the tumor mutational burden for all genes and (2) the chromosomal copy number alterations associated with the onset/progression of ovarian cancer. Tumor mutational burden is reduced in the late vs. early stages, with the highest levels being associated with loss-of-function mutations in DNA-repair genes. Nucleotide variation and copy number alterations associated with known cancer driver genes are selectively favored over ovarian cancer development. The results indicate that genetic variation is a significant contributor to the onset and progression of ovarian cancer. The measurement of the relative levels of genetic variation associated with individual ovarian cancer patient tumors may be a clinically valuable predictor of potential tumor aggressiveness and resistance to chemotherapy. Tumors found to be associated with high levels of genetic variation may help in the clinical identification of high-risk ovarian cancer patients who could benefit from more frequent monitoring.

Probability of malignancy and molecular alterations as determined by ThyroSeq v3 genomic classifier in Bethesda Category IV.

ThyroSeq molecular testing assesses the probability of malignancy (POM) in thyroid fine-needle aspiration cytology (FNAC) with indeterminate cytology. The aim was to investigatewhether Bethesda category IV (BIV) subcategories are associated with specific molecular alterations, molecular-derived risk of malignancy (MDROM), and risk of malignancy (ROM). FNAC slides, associated ThyroSeq, version 3, Genomic Classifier results, and surgical follow-up were retrieved for BIV nodules. Nodules were subcategorized as follicular neoplasm (FN) with or without cytologic atypia or oncocytic follicular neoplasm (OFN). The MDROM, ROM, and frequency of molecular alterations in FN and OFN were analyzed. p < .05 was considered significant. A total of 92 FNAC were identified and subcategorized into 46 FN (15 with and 31 without cytologic atypia) and 46 OFN. The benign call rate and the positive call rate were 49% and 51%, respectively. The MDROM in BIV was 34.3%, trending lower in OFN than in FN. RAS mutations were significantly more frequent in FN when compared to OFN (p = .02). Chromosomal copy number alterationswere more often present in OFN than in FN (p < .01). On histologic follow-up, ROM in OFN was trending lower than in FN (p = .1). The most common diagnosis in OFN was oncocytic adenoma, whereas follicular variant papillary thyroid carcinoma was most common in FN. The MDROM and ROM were trending lower in OFN compared with FN, and the molecular alterations differed between OFN and FN subcategories.

O-307 Aneuploidy in oocytes of women of advanced maternal age: impact on embryo development and evidence for a novel mechanism of meiotic error

Abstract Study question In oocytes of advanced maternal age (AMA) women, what is the association of aneuploidy with embryo development and what are the mechanisms leading to aneuploidy? Summary answer We confirmed precocious separation of sister chromatids as the main cause of aneuploidy. Known chromosome segregation errors explained 90.4% of the observed abnormal chromosome copy numbers in polar bodies. What is known already Meiotic chromosomal aneuploidies in oocytes correlate with AMA (&amp;gt;35 years), and can affect over half of oocytes in this age group. This underlies the rationale for polar body (PB) biopsy as a form of early PGT-A, as performed in the ‘ESHRE STudy into the Evaluation of oocyte Euploidy by Microarray analysis’ (ESTEEM) randomised controlled trial (RCT). So far, chromosome analysis of oocytes and PBs has shown that precocious separation of sister chromatids (PSSC), meiosis II (MII) nondisjunction (ND) and reverse segregation (RS) are the main mechanisms leading to aneuploidy in oocytes. Study design, size, duration Data was sourced from the ESTEEM study, a multicentre RCT from seven European centres to assess the clinical utility of PGT-A on PBs using array comparative genomic hybridisation (aCGH) in patients of AMA (36-40 years). This included data on the chromosome complement in PB pairs, and on embryo morphology in a subset of embryos, up to day 6 post insemination (dpi), from both the intervention (PB biopsy for PGT-A) and from control (no biopsy) arms. Participants/materials, setting, methods ESTEEM recruited 396 AMA patients: 205 in the intervention group and 191 in the control group. Complete genetic data from 693 PB pairs were analysed. Additionally, the morphology from 1034 embryos generated from fertilized oocytes (2 pronuclei) in the PB biopsy group and 1082 in the control group were compared using age-adjusted multilevel mixed-effect ordinal logistic regression for each day of embryo scoring and summarized with adjusted odds ratios (aOR) and 95% confidence intervals (CI). Main results and the role of chance Overall, 461/693 PB pairs showed abnormal segregation in 1162/10810 chromosomes. The main observed abnormal segregations were compatible with PSSC in meiosis I (MI) (n = 568/1162; 48,9%), ND of chromatids in MII or RS (n = 417/1162; 35.9%) and less frequently ND in MI (n = 65/1162; 5.6%). For 112 chromosomes (112/1162; 9,6%), we observed a chromosome copy number in PB1 and PB2 that is not explained by any of the known mechanisms causing aneuploidy in oocytes. We observed that embryos in the PGT-A arm of the RCT did not have a significantly different morphology between 2 and 6 dpi compared to the control group, indicating that polar body biopsy did not affect embryo quality. Following age-adjusted multilevel mixed-effect ordinal logistic regression models performed for each embryo evaluation day (using the before-mentioned embryo scoring subcategories), aneuploidy was associated with a decrease in embryo quality on day 3 (aOR 0.62, 95% CI 0.43-0.90), day 4 (aOR 0.15, 95% CI 0.06-0.39) and day 5 (aOR 0.28, 95% CI 0.14-0.58). Limitations, reason for caution RS cannot be distinguished from normal segregation or MII ND using aCGH. The observed segregations were based on the detected copy number of PB1 and PB2 only and were not confirmed by the analysis of embryos. The embryo morphology assessment was static and single observer. Wider implications of the findings Our finding of frequent unexplained chromosome copy numbers in polar bodies indicates that our knowledge of the mechanisms causing aneuploidy in oocytes is incomplete. It challenges the dogma that aneuploidy in oocytes is exclusively caused by missegregation of chromosomes during meiosis I and II. Study funding/competing interest(s) Data was mined from a study funded by ESHRE. Illumina provided microarrays and other consumables necessary for aCGH testing of polar bodies. None of the authors have competing interests. This work is dedicated to the memory of Professor Joep Geraedts. Trial registration number NCT01532284 Study funding Yes Funding source Funding by national/international organization(s)

O-290 Clinical utility of putative mosaicism detected using concurrent copy-number and genotyping PGT method: outcomes from multisite, prospective, non-selection study including 9828 single embryo transfer cycles

Abstract Study question What is the clinical utility and associated outcomes of mosaic whole chromosome or segmental aneuploidies detected using concurrent copy-number and genotyping analysis in PGT-A cycles? Summary answer Although high-level whole-chromosome mosaicism is linked to reduced sustained implantation, it has limited clinical significance in PGT-A cycles when co-evaluated with other clinical/ embryological factors What is known already NGS-based PGT-A can detect intermediate chromosomal copy number (CN), commonly interpreted as mosaic chromosomal aneuploidies in embryos. A prospective non-selection approach is the most effective way to assess the clinical utility of reporting putative mosaicism findings in PGT-A, wherein the presence of mosaicism is not disclosed and does not influence embryo selection. Conflicting results have been reported previously, possibly due to technological limitations in mosaicism assessment or retrospective analysis methods. This study reports the results of the largest multisite prospective non-selection clinical study examining the predictive value of whole-chromosome and segmental mosaicism, assessed through combined CN and genotyping data analysis. Study design, size, duration A multisite study involving seven IVF clinics was conducted from Feb 2020 to Oct 2022, including 6951 patients and 9828 single embryo transfers. The study involved a prospective non-selection approach, where embryos suspected of having whole chromosomal or segmental mosaicism were reported as negative for non-mosaic aneuploidies. Embryos were chosen for transfer based solely on standard morphological features. The primary outcome was sustained implantation rate (SIR) defined as pregnancy continuing beyond 8 weeks of gestation. Participants/materials, setting, methods In this study, the trophectoderm biopsies were analyzed using a custom, targeted NGS assay that examined approximately 5000 loci across the genome, providing genotyping information to support aneuploidy classification. Mosaicism was identified by any copy number deviation from the expected two copies (LogR plots) and confirmed by corresponding SNP B-allele frequency (BAF) patterns. Confounding factors, such as clinical and embryological variables, were controlled for in the multivariate analysis. Main results and the role of chance The average female age in this cohort was 34.9 years (SD = 4.1), with aneuploidy rate of 30% in embryos and SIR of 61.2%. Of the embryos transferred, 6.5% (636/9828) were whole chromosomal mosaic (WCM) only; 9.6% (947/9828) were segmental mosaic (SM) only and 1% (83/9829) were a combination of WCM and SM. The rate of putative mosaicism ranged from 15%-89%. The SIR of embryos in the control group (non-mosaic), SM and WCM were 62% (5190/8328; 95%CI), 58% (549/947;95%CI) and 50.3% (320/636; 95%CI P &amp;lt; 0.01) respectively. A logistic model found that the level of WCM was associated to SIR, along with other embryological and clinical factors. In particular, WCM with a CN difference &amp;gt;50% as well as poor embryo morphology were associated with lower SIR (OR = 0.5; 95% CI:0.32-0.76), but low-level (&amp;lt;50%) mosaicism was not significant (NS). Notably, female age (OR = 0.98; 95% CI:0.97-0.99 per year), BMI (0.98; 95% CI:0.98-0.99) and previous ET failures (OR = 0.58; 95% CI:0.5-0.68) were strongly associated with SIR. A predictive model, taking into account all relevant variables, yielded a significant stratification of SIR, from 43% to 68%. Given the low incidence of WCM in our clinical setting, the multivariate SIR prediction (AUC) was 0.580 without WCM and 0.585 with mosaicism included. Limitations, reasons for caution This study did not have prenatal and post-natal data available at the time of the abstract's writing, hence conclusions about these outcomes wasn’t possible. Despite the large sample size, chromosome specific analysis was not feasible. Furthermore, this study’s data is platform-specific and cannot be translated to other PGT-A assays. Wider implications of the findings In this non-selection study, WCM of &amp;gt; 50% variation was associated with lower SIR. However, high-level WCM has a minimal overall impact on SIR when co-evaluating with other clinical/ embryological parameters. Decisions on reporting criteria for these findings must weigh the risk of discarding potentially viable embryos with substantial reproductive potential. Trial registration number Not applicable

O-289 Morphology should be prioritised over presumed mosaicism status in PGT-A cycles: data from a non-selection study of 2,621 embryo transfers

Abstract Study question Is morphological grade or presumed low-mosaicism status the most impactful parameter on live birth rate (LBR) when selecting the embryo to transfer in PGT-A cycles? Summary answer Among euploid/presumed low-mosaic embryos, morphology provides a better selection parameter for improving live birth in PGT-A cycles. What is known already NGS-based technologies enable the detection of intermediate chromosomal copy number (CN) values, which are commonly interpreted as embryonic chromosomal mosaicism. So far, studies showed that low morphological grade blastocysts are associated with poorer prognoses. Prospective blinded trials also showed that embryos displaying putative mosaic profiles produce comparable clinical outcomes to uniformly euploid ones. Nonetheless, scientific societies suggest different approaches on embryo prioritisation: PGDIS and COGEN advise a selection based on presence of mosaicism, while ESHRE suggests parallel evaluation of mosaicism and morphology. However, the combined effect of morphology and mosaicism on live birth rate (LBR) hasn’t yet been thoroughly investigated. Study design, size, duration This retrospective multisite cohort analysis is based on a euploid/low-mosaic non-selection study, where the presumed mosaicism was not disclosed to clinics and did not affect the embryo selection procedure. It includes 2,339 consecutive IVF/PGT-A treatments (January2018-December2021) that led to 3,999 transferable blastocysts (euploid or presumed mosaic &amp;lt;50%) and resulted in 2,621 frozen single embryo transfers (SETs). Morphological (i.e., good/poor quality) and chromosomal (i.e., euploid/mosaic) features were assessed. Clinical outcomes were followed up for each subgroup. Participants/materials, setting, methods Morphological grading was assessed using Gardner’s criteria, &amp;lt;BB for poor-quality and &amp;gt;BB for good-quality embryos. PGT-A analysis and CN values calculations were performed on IonTorrentS5 and Ion-Reporter software (ThermoFisher). Based on raw NGS data, CN variations &amp;lt;30% and 30%-50% were internally categorised euploidy and mosaicism, respectively. Nevertheless, embryos belonging to either category were reported to clinics as euploid. The association between morphology, chromosomal status, and LBR was evaluated using Fisher’s exact test and multivariate analysis. Main results and the role of chance In this study population, low-mosaicism had an incidence of 15.8% (N = 630/3,999; 95%CI:14.7-16.9). Overall, LBR and miscarriage rate did not differ between euploid and presumed mosaic SETs: 45.0% vs. 44.8% (P=NS), and 11.2% vs. 13.2% (P=NS), respectively. Also, euploid and presumed mosaic embryos showed similar incidence of poor morphology (7.4% vs. 9.7%; P=NS). In the good-quality category, LBR and miscarriage rate were 46.2% vs. 44.8% (P=NS), and 11.3% vs. 13.1% (P=NS) for euploid and presumed mosaic embryos, respectively. In the poor-quality category, LBR was 21.0% vs. 23.8% (P=NS), respectively; whilst miscarriage rates could not be compared due to insufficient number of cases across the whole morphological category (N = 4/33). Accordingly, the focused scenario that employed poor-quality uniformly euploid embryos instead of good-quality mosaic embryos showed the highest impact on LBR (21.0% vs. 44.8%; P &amp;lt; 10-5). Finally, the proportion of cases where both selection strategies (i.e., mosaicism vs. morphology) could be applied accounted for 6.1% (N = 44/717; 95%CI:4.6-8.1) of all cycles where &amp;gt;1 transferable embryo was available (N = 717/2,339). Limitations, reasons for caution The results were obtained through the analysis of raw NGS data independent from any proprietary diagnostic algorithm or chromosome-specific consideration commonly used by PGT-A laboratories. These findings concern putative whole-chromosome mosaic aneuploidies only and cannot be extended to segmental mosaic configuration. Follow-up studies in prenatal diagnosis are needed. Wider implications of the findings This study suggests that, when selecting embryos for transfer, morphological grading should be considered as a higher criterion than low-mosaic conformation (i.e., mosaicism &amp;lt;50% of copy-number). Reporting mosaicism based on intermediate copy-number categorization ranges &amp;lt;50% appears to provide no clinical utility in the current technological landscape. Trial registration number not applicable

O-181 Utilisation of monopronucleated (1PN) embryos and potential clinical benefits for patients

Abstract Study question Is it beneficial to culture 1PN embryos for clinical use? Summary answer Culturing 1PN embryos to blastocyst for potential clinical use is beneficial to all patients, especially those with low prognosis. What is known already 1PN embryos are those with only a single pronucleus visible at the time of fertilisation check. It is generally assumed that such embryos are haploid, having only 23 chromosomes in each of their cells. Given that haploid embryos are not viable, current guidelines recommend only transfering embryos where two pronuclei are visualised. Embryo biopsy at the blastocyst stage, followed by testing with next-generation sequencing (NGS), combined with analysis of DNA polymorphisms, can reveal whether 1PN embryos are truly haploid, or whether normal fertilsation has occurred. Previous studies have shown that in some cases a diploid chromosome number may be present. Study design, size, duration A new clinical policy was put into practice to culture all 1PN embryos to day 5/6. This is an ongoing study initiated in April 2022. The data reported are from an eight month period, up until December 2022. During this time, we cultured 288 1PN embryos from 203 patients. Trophectoderm biopsy specimens were shipped to a specialist genetics laboratory where they underwent PGT-A using a method that assesses chromosome copy number and polymorphisms in tandem. Participants/materials, setting, methods If embryos were assessed at fertilisation check and found to be 1PN they were not discarded, but rather they were maintained in culture. If any such embryos successfully produced a blastocyst the patients received counselling from their fertility consultant and an embryologist, giving them the options to either go ahead with transfer (only IVF derived), have the embryos genetic status clarified using PGT-A, freeze without testing, or to discard the embryo. Main results and the role of chance Of the 288 1PN embryos cultured (IVF and ICSI derived) 85 displayed signs of blastulation (29.5%). 134 of these were derived from ICSI with 20 forming blastocysts (14.9%). 154 were derived from IVF with 65 forming blastocysts (42.2%). Three IVF derived 1PN embryos have been transferred. Currently one is an ongoing singleton pregnancy, one is an ongoing twin pregnancy and the third has resulted in a live birth. 35 embryos were biopsied and underwent genetic testing to confirm their ploidy status. Six of these were ICSI derived, four of which were found to be haploid and two diploid. The other 29 tested embryos were derived from IVF. Of these, 27 were found to be diploid (93.1%) and two were shown to be triploid (6.9%). None were confirmed as haploid. Traditionally, PGT-A methods using NGS examine relative chromosome copy number, but in cases of haploidy/triploidy all of the chromosomes are decreased/increased in number, meaning there is no change in the relative number of individual chromosomes. We established embryo ploidy status using an advanced PGT method, genotyping thousands of polymorphisms scattered across the genome, which are essential for accurate diagnosis of haploidy/triploidy. Limitations, reasons for caution The sample size for genetically tested ICSI derived 1PN’s is currently too small to clearly determine whether culture and testing of these embryos is beneficial to the patient. Patients who decided to transfer an embryo categorised as 1PN were made fully aware of the potential risks in doing so. Wider implications of the findings These findings show that the culture of 1PN embryos is clinically beneficial to a large number of patients, especially those with poor prognosis who would otherwise have had a failed cycle. Genetic testing demonstrates that IVF derived 1PN embryos that reach the blastocyst stage are likely to have fertilised normally. Trial registration number *

P-240 High mitochondrial DNA content in Day6 blastocyst may impair ongoing pregnancy rate

Abstract Study question Is there an association between mitochondrial DNA (mtDNA) content and Ongoing Pregnancy Rate (OPR) in D5 or D6 euploid blastocyst? Summary answer Day6 OPR is decreased in high mtDNA content blastocyst. Day5 and Day6 mtDNA levels are independent of maternal age or standard morphology. What is known already Preimplantation development is an energy-demanding process and mitochondrial ATP production is crucial for cellular activity in fast replicating cells. Mitochondrial content in preimplantation embryo has been proposed as a marker of embryo potential in term of viability and implantation success though the dynamics and distribution of mitochondria in human embryo is still debated. In recent years, it has been suggested that low mtDNA levels were associated with euploid chromosomal complement and higher implantation rate while other studies failed to find a correlation between mtDNA content and reproductive outcome. Study design, size, duration This study is a retrospective cohort analysis from 2020 to 2022 including 343 Day5 and 187 D6 single euploid blastocyst transfer. Primary endpoint was OPR beyond pregnancy week 16. Day5 and Day6 frozen-thawed transfer were divided based on blastocyst mtDNA content in four groups: Day5-high, Day5-low, D6-high, D6-low. Secondary endpoints were relationship between mtDNA content, maternal age and standard morphology in all obtained blastocysts. Statistical differences were compared by Chi-Square test. Participants/materials, setting, methods 771 couples performed IVF cycles with next-generation sequencing (NGS)-preimplantation genetic testing of aneuploidy (PGT-A) (age 19-47yrs; Mean age 37.8yrs). Following culture in sequential media 700 euploid blastocyst were obtained and mitochondrial and chromosomal DNA copy number variation were examined simultaneously with next generation sequencing (NGS) methodology. mtDNA copy numbers was based on the observed ratios of sequence coverages between mtDNA and nuclear DNA. Main results and the role of chance In our clinical setting OPR is similar in Day5 vs. Day6 transfers (55% vs. 49% n.s.). However when mtDNA content is considered D6-low OPR is comparable to D5-high or low (57.3%; 53.3%; 57,3% respectively n.s.) while D6-high transfers yield significantly decreased OPR (31,2% p &amp;lt; 0.05). When stratified by age only Advanced Maternal Age (AMA) patients ( &amp;gt; = 38yrs) showed this decrease at a significant level (Day6 high OPR 20% p &amp;lt; 0,05).This result in transferred blastocyst is obtained despite high:low ratio of all biopsied blastocyst is constant in Day5 (1:1) (47%-53%) and markedly decreased in Day6 (1:4) (25%-75%) either overall or in younger (n.s.) and AMA patients (n.s.). Moreover when standard morphology distribution is evaluated according to Gardner classification criteria top, fair and poor quality blastocysts display the same 1:1 high:low blastocysts mtDNA ratio in Day5 and 1:4 in Day 6 overall and in younger and AMA patients. Hence, the mtDNA blastocyst content is independent of patients’ age and blastocyst quality. The origin of Day6 OPR decrease could therefore depend on the number of Day6-high blastocyst transferred in AMA patients and ultimately in the number of blastocyst available to transfer for each patient. Limitations, reasons for caution Due to the small numbers of Day6-high mtDNA blastocyst, a larger sample size is required to confirm these preliminary findings. Moreover, the retrospective nature of the study may introduce some bias mainly in patients’ characteristics and the strategy of embryo selected for transfer. Wider implications of the findings While Day5-high blastocyst yield higher OPR the persistence of higher mtDNA levels in Day6 blastocyst may hamper their implantation potential. Our findings may help to select the embryo to transfer to maximize transfer success. D6 blastocysts with low mtDNA content should be preferred whenever a choice is possible. Trial registration number Not Applicable