Chromosomal Mosaicism Research Articles

Feasibility of preimplantation genetic testing for aneuploidy on frozen-thawed embryos following conventional IVF insemination.

Intracytoplasmic sperm injection (ICSI) is commonly employed in preimplantation genetic testing (PGT) to minimize the risk of foreign sperm DNA contamination. Cryopreserved embryos from patients with recurrent miscarriage or repeated implantation failure, who have undergone conventional in vitro fertilization (IVF), can be thawed and biopsied for PGT. Therefore, we aimed to assess the accuracy and effectiveness of preimplantation genetic testing for aneuploidy (PGT-A) on frozen embryos using conventional IVF (c-IVF) insemination methods. From January 2021 to November 2023, our center conducted 107 thawed cryopreserved embryo biopsy cycles to screen for PGT-A. Among them, 58 cycles used c-IVF insemination, and 49 used ICSI insemination. Basic patient clinical information, laboratory data, PGT test results, and clinical outcome data were collected. To minimize the confounding effects of patient characteristics and embryo quality on PGT-A outcomes, clinical outcomes, and contamination assessment, these variables were included in the analysis. We then evaluated the blastocyst euploidy rate, clinical outcomes, and accuracy of PGT-A results between the two groups and analyzed potential contamination in the c-IVF insemination group. A total of 320 blastocysts underwent PGT-A testing, with 179 blastocysts from c-IVF insemination and 141 from ICSI insemination. Considering participants' baseline characteristics and embryological outcomes, no significant differences were found between the two groups regarding infertility type, average age, body mass index, percentage of PGT-A indications, or quality of embryonic development. Regarding PGT-A results, all 320 biopsy samples were successfully analyzed, showing no statistical variance in chromosomal euploidy, abnormality, or mosaicism rates between the two insemination methods. No parental contamination was detected in the c-IVF insemination group. When assessing clinical outcomes, parameters such as biochemical pregnancy, clinical pregnancy, and miscarriage rates did not exhibit significant discrepancies between the two groups, and no misdiagnoses were reported during the study period. Embryo transfer and PGT-A results are not affected by potential parental contamination in frozen-thawed embryos conceived via c-IVF. PGT-A guided embryo transfer in thawed embryos conceived by c-IVF is a viable and clinically effective approach.

Chromosomal abnormalities in oocyte donor candidates: a French survey of over 8200 karyotypes

To study karyotypes of more than 8,200 oocyte donor candidates in nulliparous or multiparous women compared to a reference population. A retrospective observational multicentric study. The study included two cohorts of oocyte donor candidates recruited between January 2005 and October 2021: multiparous women with at least one child at the time of recruitment, and nulliparous women. Both were compared to a reference population composed of female newborns from literature. Not applicable. Blood lymphocyte karyotype. A total of 8229 oocyte donor candidates from 22 fertility centers were included in this study. Nulliparous women (n=1890) and multiparous ones (n=6339) were compared to 8102 female newborns. Overall, 65 candidates were carriers of chromosomal abnormalities and were therefore excluded from the donation process (0.79%, 95% CI: 0.60-0.98). The occurrence of balanced structural chromosomal rearrangements was globally increased in the study population (0.49%, 95% CI: 0.34-0.64) compared to female newborns (0.24%, 95% CI: 0.34-0.64, p=0.0086). The number of reciprocal translocations was increased 5-fold in nulliparous oocyte donor candidates (0.37%, 95% CI: 0.10-0.64, p=0.013). The incidence of sex chromosome mosaicism was notably increased in multiparous oocyte donor candidates, with 17 cases (0.27%, 95% CI: 0.14-0.40, p=0.0052). Among chromosomal aberration carriers only two nulliparous women (one reciprocal translocation and one sex chromosome mosaicism) had fertility issues with a diagnosis of premature ovarian failure. In this comprehensive 16-years French experience of karyotyping in oocyte donor candidates, we confirmed an increased incidence of balanced structural chromosomal rearrangements, especially among those without children at the time of recruitment. Karyotyping could be considered to identify any chromosomal abnormalities that may not be easily detectable through medical questioning. These abnormalities pose an inherent genetic risk for gamete recipients if left undetected.

Low-level chromosomal mosaicism does not explain the spontaneous menarche seen in some women with 45,X turner syndrome

Low-level chromosomal mosaicism does not explain the spontaneous menarche seen in some women with 45,X turner syndrome

Comprehensive diagnosis and genetic analysis of two children with ring chromosome 18

To clarify the genetic diagnosis of two children with ring chromosome 18 and explore their mechanisms and clinical phenotypes. Two patients treated at the Children's Hospital of Henan Province respectively in June 2022 and March 2023 were selected as the study subjects. Genetic testing and diagnosis were carried out through copy number variation sequencing (CNV-seq), G-banded chromosomal karyotyping, and whole exome sequencing (WES). Child 1 had mainly manifested developmental delay, white matter hypoplasia, type 1 diabetes mellitus, and micropenis. He was found to have a chromosomal karyotype of 46,XY,r(18)(p11.21q22.1)[40]/46,XY[7], and CNV-seq results showed that he has a 14.86 Mb deletion at 18p11.21p11.32 and a 14.02 Mb deletion at 18q22.1q23. Child 2 had peculiar facial features, delayed white matter myelination, developmental delay, atrial septal defect, severe sensorineural deafness, and congenital laryngeal stridor. He was found to have a chromosomal karyotype of 46,XY,r(18)(p11.2q23). CNV-seq result proved that he had a 14.86 Mb deletion at 18p11-21p11.32 and a 20.74 Mb deletion at 18q21.32q23. WES has failed to detect single nucleotide variants (SNVs) in either child, but revealed a large segmental deletion at chromosome 18 in both of them. Both children were diagnosed with ring chromosome 18 syndrome. The different size of the deletional fragments in the 18q region and mosaicism of ring chromosome 18 in child 1 may underlay the variation in their clinical phenotypes. The type 1 diabetes mellitus and micropenis noted in both children are novel features for ring chromosome 18 syndrome.

Developmental signals control chromosome segregation fidelity during pluripotency and neurogenesis by modulating replicative stress

Human development relies on the correct replication, maintenance and segregation of our genetic blueprints. How these processes are monitored across embryonic lineages, and why genomic mosaicism varies during development remain unknown. Using pluripotent stem cells, we identify that several patterning signals—including WNT, BMP, and FGF—converge into the modulation of DNA replication stress and damage during S-phase, which in turn controls chromosome segregation fidelity in mitosis. We show that the WNT and BMP signals protect from excessive origin firing, DNA damage and chromosome missegregation derived from stalled forks in pluripotency. Cell signalling control of chromosome segregation declines during lineage specification into the three germ layers, but re-emerges in neural progenitors. In particular, we find that the neurogenic factor FGF2 induces DNA replication stress-mediated chromosome missegregation during the onset of neurogenesis, which could provide a rationale for the elevated chromosomal mosaicism of the developing brain. Our results highlight roles for morphogens and cellular identity in genome maintenance that contribute to somatic mosaicism during mammalian development.

Innovative all-in-one exome sequencing strategy for diagnostic genetic testing in male infertility: Validation and 10-month experience.

Current guidelines indicate that patients with extreme oligozoospermia or azoospermia should be tested for chromosomal imbalances, azoospermia factor (AZF) deletions and/or CFTR variants. For other sperm abnormalities, no genetic diagnostics are recommended. To determine whether exome sequencing (ES) with combined copy number variant (CNV) and single nucleotide variant (SNV) analysis is a reliable first-tier method to replace current methods (validation study), and to evaluate the diagnostic yield after 10 months of implementation (evaluation study). In the validation study, ES was performed on DNA of patients already diagnosed with AZF deletions (n=17), (non-)mosaic sex chromosomal aneuploidies or structural chromosomal anomalies (n=37), CFTR variants (n=26), or variants in known infertility genes (n=4), and 90 controls. The data were analyzed using our standard diagnostic pipeline, with a bioinformatic filter for 130 male infertility genes. In the evaluation study, results of 292 clinical exomes were included. All previously reported variants in the validation cohort, including clinically relevant Y-chromosomal microdeletions, were correctly identified and reliably detected. In the evaluation study, we identified one or more clinically relevant genetic anomalies in 67 of 292 of all cases (22.9%): these included aberrations that could have been detected with current methods in 30 of 67 patients (10.2% of total), (possible) (mono)genetic causes in the male infertility gene panel in 28 of 67 patients (9.6%), and carriership of cystic fibrosis in nine of 67 patients (3.1%). ES is a reliable first-tier method to detect the most common genetic causes of male infertility and, as additional genetic causes can be detected, in our evaluation cohort the diagnostic yield almost doubled (10.2%-19.8%, excluding CF carriers). A genetic diagnosis provides answers on the cause of infertility and helps the professionals in the counseling for treatment, possible co-morbidities and risk for offspring and/or family members. Karyotyping will still remain necessary for detecting balanced translocations or low-grade chromosomal mosaicism.

The Impact of Chromosomal Mosaicisms on Prenatal Diagnosis and Genetic Counseling-A Narrative Review.

Genetic disorders represent a high-impact diagnosis for both patients and their families. Prenatal screening methods and, when recommended, genetic testing allow parents to make informed decisions about the course a pregnancy is going to take. Although offering certainty about the potential evolution and prognosis of the pregnancy, and then the newborn, is usually not possible, genetic counseling can offer valuable insights into genetic disorders. Chromosomal mosaicisms are genetic anomalies that affect only some cell lines in either the fetus or the placenta or both. They can affect autosomal or heterosomal chromosomes, and they can be either numerical or structural. The prognosis seems to be more severe if the genetic alterations are accompanied by malformations visible in ultrasounds. Several genetic techniques can be used to diagnose certain mosaicisms, depending on their nature. A novel approach in prenatal care is non-invasive prenatal screening (NIPS), also known as non-invasive prenatal testing (NIPT), which, although it does not always have diagnostic value, can provide valuable information about potential genetic anomalies, especially numerical, with high sensitivity (Se).

HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos.

Human fertility is suboptimal, partly due to error-prone divisions in early cleavage-stages that result in aneuploidy. Most human pre-implantation are mosaics of euploid and aneuploid cells, however, mosaic embryos with a low proportion of aneuploid cells have a similar likelihood of developing to term as fully euploid embryos. How embryos manage aneuploidy during development is poorly understood. This knowledge is crucial for improving fertility treatments and reducing developmental defects. To explore these mechanisms, we established a new mouse model of chromosome mosaicism to study the fate of aneuploid cells during pre-implantation development. We previously used the Mps1 inhibitor reversine to generate aneuploidy in embryos. Here, we found that treatment with the more specific Mps1 inhibitor AZ3146 induced chromosome segregation defects in pre-implantation embryos, similar to reversine. However, AZ3146-treated embryos showed a higher developmental potential than reversine-treated embryos. Unlike reversine-treated embryos, AZ3146-treated embryos exhibited transient upregulation of Hypoxia Inducible-Factor-1A (HIF1A) and lacked p53 upregulation. Pre-implantation embryos develop in a hypoxic environment in vivo, and hypoxia exposure in vitro reduced DNA damage in response to Mps1 inhibition and increased the proportion of euploid cells in the mosaic epiblast. Inhibiting HIF1A in mosaic embryos also decreased the proportion of aneuploid cells in mosaic embryos. Our work illuminates potential strategies to improve the developmental potential of mosaic embryos.

The density of the inner cell mass is a new indicator of the quality of a human blastocyst: a valid supplement to the Gardner scoring system.

Can the density of the inner cell mass (ICM) be a new indicator of the quality of the human blastocyst? The densification index (DI) developed in this study can quantify ICM density and provide positive guidance for ploidy, pregnancy, and live birth. In evaluating the quality of ICM, reproductive care clinics still use size indicators without further evaluation. The main disadvantage of this current method is that the evaluation of blastocyst ICM is relatively rough and cannot meet the needs of clinical embryologists, especially when multiple blastocysts have the same ICM score, which makes them difficult to evaluate further. This observational study included data from 2272 blastocysts in 1991 frozen-thawed embryo transfer (FET) cycles between January 2018 to November 2021 and 1105 blastocysts in 430 preimplantation genetic testing cycles between January 2019 and February 2023. FET, ICSI, blastocyst culture, trophectoderm biopsy, time-lapse (TL) monitoring, and next-generation sequencing were performed. After preliminary sample size selection, the 11 focal plane images captured by the TL system were normalized and the spatial frequency was used to construct the DI of the ICM. This study successfully constructed a quantitative indicator DI that can reflect the degree of ICM density in terms of fusion and texture features. The higher the DI value, the better the density of the blastocyst ICM, and the higher the chances that the blastocyst was euploid (P < 0.001) and that pregnancy (P < 0.001) and live birth (P = 0.005) were reached. In blastocysts with ICM graded B and blastocysts graded 4BB, DI was also positively associated with ploidy, pregnancy, and live birth (P < 0.05). ROC analysis showed that combining the Gardner scoring system with DI can more effectively predict pregnancy and live births, when compared to using the Gardner scoring system alone. Accurate calculation of the DI value places high demands on image quality, requiring manual selection of the clearest focal plane and exposure control. Images with the ICM not completely within the field of view cannot be used. The association between the density of ICM and chromosomal mosaicism was not evaluated. The associations between the density of ICM and different assisted reproductive technologies and different culture conditions in embryo laboratories were also not evaluated. Prospective studies are needed to further investigate the impact of ICM density on clinical outcomes. ICM density assessment is a new direction in blastocyst assessment. This study explores new ways of assessing blastocyst ICM density and develops quantitative indicators and a corresponding qualitative evaluation scheme for ICM density. The DI of the blastocyst ICM developed in this study is easy to calculate and requires only TL equipment and image processing, providing positive guidance for clinical outcomes. The qualitative evaluation scheme of ICM density can assist embryologists without TL equipment to manually evaluate ICM density. ICM density is a simple indicator that can be used in practice and is a good complement to the blastocyst scoring systems currently used in most centers. This work was supported by the National Key Research & Development Program of China (2021YFC2700603). The authors report no financial or commercial conflicts of interest. N/A.

Clinicopathological and molecular genetic features of confined placental mosaicism

Objective: To investigate the clinicopathological and genetic features of confined placental mosaicism (CPM) and its effect on fetal intrauterine growth. Methods: Fourteen CPM cases of Haidian Maternal and Children Health Hospital were collected from May 2018 to March 2022. Clinicopathological examination on placental specimens and molecular genetic analysis were performed. Results: The age of the parturient women ranged from 27 to 34 years, with an average age of (30.0±3.54) years. The gestational weeks ranged from 35+1 to 41+2 weeks. There were 4 premature births and 10 term births, among which 6 were female and 8 were male fetuses. Nine cases (9/14) had adverse pregnancy outcomes, including 7 cases of fetal growth restriction. The weight of CPM placenta decreased, with 6 cases below the 10th percentile of weight standards and 5 cases between the 10th and 25th percentile. All 14 CPM placental specimens showed morphological changes of perfusion dysfunction to varying degrees, with mainly placental-maternal vascular malperfusion followed by placental-fetal vascular malperfusion. The mosaic chromosomes in different CPM cases varied, with 16-trisomy/monosomy mosaicism being the most common followed by 7-trisomy and 21-trisomy/monosomy mosaicism. The mosaic proportion was unequal in different parts of the same CPM placenta, with the mosaic proportion of umbilical cord, fetal membranes, fetal surface, maternal surface, and edge ranging from 1% to 70%. Conclusions: The mosaic chromosomes in different CPM cases vary, and the mosaic proportion is unequal in different parts of the same CPM placenta. The pathological morphology is mainly manifested as perfusion dysfunction, which can lead to adverse pregnancy outcomes such as fetal growth restriction and preterm birth.

Cell Cycle Kinetics and Sister Chromatid Exchange in Mosaic Turner Syndrome.

Turner syndrome (TS) is caused by a complete or partial absence of an X or Y chromosome, including chromosomal mosaicism, affecting 1 in 2500 female live births. Sister chromatid exchange (SCE) is used as a sensitive indicator of spontaneous chromosome instability. Cells from mosaic patients constitute useful material for SCE evaluations as they grow under the influence of the same genetic background and endogenous and exogenous factors. We evaluated the proliferation dynamics and SCE frequencies of 45,X and 46,XN cells of 17 mosaic TS patients. In two participants, the 45,X cells exhibited a proliferative disadvantage in relation to 46,XN cells after 72 h of cultivation. The analysis of the mean proliferation index (PI) showed a trend for a significant difference between the 45,X and 46,X+der(X)/der(Y) cell lineages; however, there were no intra-individual differences. On the other hand, mean SCE frequencies showed that 46,X+der(X) had the highest mean value and 46,XX the lowest, with 45,X occupying an intermediate position among the lineages found in at least three participants; moreover, there were intra-individual differences in five patients. Although 46,X+der(X)/der(Y) cell lineages, found in more than 70% of participants, were the most unstable, they had a slightly higher mean PI than the 45,X cell lineages in younger (≤17 years) mosaic TS participants. This suggests that cells with a karyotype distinct from 45,X may increase with time in mosaic TS children and adolescents.

Effect of freeze-thawing, cell collection, and laser irradiation cycles on mosaicism occurrence in preimplantation genetic testing for aneuploidy

ObjectiveIn preimplantation genetic testing for aneuploidy, opinions regarding the handling of mosaic embryos vary. In this study, we aimed to investigate the effects of freeze-thawing, the number of cells obtained, and the number of laser irradiation cycles on the degree of embryonic mosaicism. Study designThis study was conducted in three parts. First, we classified specimens into the normal biopsy (control) (119 patients, 304 blastocysts) and thawed-biopsy (TB group) (26 patients, 72 blastocysts)) groups. The control and TB groups were then classified into three categories (euploidy, mosaic and aneuploidy) according to next-generation sequencing (NGS) results, and the number of cells collected and laser irradiation cycles were compared for each category. Subsequently, the effects of differences in the number of cells collected and laser irradiation cycles on NGS results were investigated in the control and TB groups. Finally, data on cell collection and laser irradiation cycles and NGS analysis results for the groups were compared. ResultsThe TB group had a significantly higher incidence of chromosomal mosaicism than the control group. Neither the number of cells collected nor the laser irradiation cycles affected the percentage of chromosomal mosaicism. However, the freeze–thaw process increased the occurrence of mosaicism. ConclusionsThis study showed that repeated freeze–thaw cycles increase the incidence of mosaicism, but the embryos are not aneuploid and are therefore suitable for transfer.

P-502 Mosaic embryos result in equivalent live birth rates when compared to euploid embryos following frozen embryo transfer

Abstract Study question Are mosaic embryos associated with a lower live birth rate (LBR) than euploid embryos? Summary answer In this adequately powered study, mosaic embryos had nearly identical LBR and similar miscarriage rates (MR) when compared to euploid embryos. What is known already While prior data has suggested that mosaic embryos result in almost half the LBR and three-fold higher MR than euploid embryos, all prior data supports mosaic embryo transfers as safe with acceptable LBRs. Recent studies have suggested a revised selection criteria choosing either euploid or mosaic embryos for transfer based on embryo grade, day of freeze, low level (LL) versus high level (HL) mosaicism and segmental versus whole chromosome mosaicism. Given the lower LBR reported with mosaics, we initially offered double embryo transfer (DET). We initiated this study to assess our LBR, MR, aneuploidy and DET outcomes with mosaics. Study design, size, duration This was a retrospective case-control cohort study. All mosaic embryos (51) that underwent frozen embryo transfer (FET) between January 2020 and June 2023 were compared to all euploid embryos (706) among all patients with tested embryos that underwent FET in the same time frame. Our Power analysis confirmed that with the 14:1 ratio of 706/51 euploid/mosaic embryos this study is sufficiently powered to exclude a &amp;gt; 20% lower LBR following mosaic transfer. Participants/materials, setting, methods All patients seen at our IVF center undergoing FET of mosaic or euploid embryos tested with next generation sequencing during the study period were included. Primary outcome was LBR. Further comparison included age, MR, twin rate, freeze day and grade. Embryos were further analyzed for HL versus LL mosaicism, segmental or whole chromosomal mosaicism, freeze date and grade. Statistics employed chi-squared for categorical and student t-test for continuous data with significance set at p &amp;lt; 0.01 Main results and the role of chance Mosaic embryos resulted in nearly identical LBR when compared to euploid embryos, 26/51(50.9%) versus 359/706(50.85%). Implantation rate was also equivalent, 29/51(54.6%) and 376/706(53.3%). Both biochemical pregnancy [mosaics, 8/45(17.8%) versus euploid 127/683(18.6)], and MR [mosaics 3/29(10.3%) versus euploids 24/373(6.4%)] were not significantly different. Among mosaic embryos, the LBR in LL versus HL mosaics were 17/28 (60.7%) and 9/23 (39.1%), respectively. LBR following segmental only versus whole chromosomal defects were 14/27(51.9%) and 11/24(45.8%). LBR following Day 5 versus Day 6/7 freeze were 10/22(45.5%) and 16/29(55.2%) and LBR for favorably graded embryos were 19/34(55.9%) versus fair to poor graded 7/17(41.2%). None of these achieved statistical significance. While Mosaic embryos were transferred in significantly older patients (37.6 vs 36.1 p &amp;lt; 0.01), there was no difference in mean age associated with LBR (36.1 vs 36.3 p =.4). Additionally, there was no statistically significant difference in LBR between age groups, &amp;lt;35yo 143/259(55.2%), 35-37yo 96/202(47.5%), 38-40yo 96/207(46.4%) or &amp;gt; 40yo 50/90(55.6%) among tested embryos. DET was performed more often with mosaic 19/45 (42.2%) than euploid embryos 36/683 (5.3%) p &amp;lt; 0.001, resulting in significantly higher twin births/LB 5/22 (23.8%) versus 6/356 (1.7%) p &amp;lt; 0.001. All DET data sets included the 13 DETs where one euploid and one mosaic embryo were transferred. Limitations, reasons for caution Our small mosaic numbers may have kept the higher LBR with LL, segmental and good quality mosaic embryos from achieving statistical significance, and may be too small to confirm a higher MR with mosaics. Prospective trials are needed to confirm which euploid or mosaic embryo should be selected for transfer. Wider implications of the findings Given the equivalent LBR, mosaic embryos should be used for transfer prior to repeating IVF retrieval for euploids. The high rate of twin delivery following mosaic DET suggests only eSET should be utilized with any tested embryos. Larger prospective trials are needed to further refine euploid versus mosaic embryo selection criteria. Trial registration number not applicable

P-567 Is mosaic embryo transfer a valid alternative for patients lacking euploid embryos? a multicentric study from Argentina

Abstract Study question How does low-level mosaicism embryo transfer affect reproductive outcomes, including pregnancy rates and live birth success, compared to transfers involving euploid embryos? Summary answer Most mosaic embryo transfers do not affect reproductive outcomes, as live birth rates appear to be similar to those observed with euploid embryos. What is known already Preimplantation genetic testing (PGT) is used to identify embryonic genetic abnormalities and, ideally, select euploid embryos for intrauterine transfer, to improve clinical IVF outcomes. Chromosomal mosaicism is defined as the presence of two or more different chromosomal cell lines within an embryo. Numerous sources describe the proportion of mosaic embryos ranging from 4 to 22%. Several publications report that mosaic embryo transfer can lead to successful pregnancies and healthy deliveries, albeit at a lower frequency and with higher rates of pregnancy loss compared to euploid embryos. However, the impact of mosaicism on IVF outcomes remains a matter of controversy. Study design, size, duration Observational and retrospective multi-center study analyzed 6241 embryos from 1218 patients, across 4 IVF centers, who underwent PGT between 2016 and 2023 in a single genetics laboratory. Embryos were categorized as euploid, aneuploid, or mosaic. Patients in group A (n = 894) were transferred an euploid embryo, while in group B (n = 55), a low-grade mosaic embryo, defined as those having less than 40% (2016-2021) or 50% (2021-2023) aneuploid cells, was transferred. Participants/materials, setting, methods The embryos were biopsied and vitrified for later transfer. Blastocyst biopsy DNA was amplified using the SurePlex kit, processed with the VeriSeq kit (Vitrolife), and sequenced on a MiSeq platform. The results were analyzed using BlueFuse-Multi software. Mosaic embryo transfers were conducted only in the absence of a euploid embryo, following genetic counseling. beta-hCG levels &amp;gt;25 IU were considered positive, and clinical pregnancy was confirmed through ultrasound visualization of the gestational sac with heartbeat. Main results and the role of chance Among the analyzed embryos, the proportion of euploid was 46.2% (n = 2883), while mosaic embryos accounted for 19.3% (n = 1207), with 69.5% classified as low-grade and 30.5% as high-grade. Aneuploid embryos comprised 31.3% (n = 1954), and results could not be obtained for 3.2% (n = 197). Fifty-five low-grade mosaic embryos were transferred, including 19 with complete chromosomal mosaicism, 34 with segmental mosaicism, and 2 cases with 2 chromosomes involved in the mosaicism. The positive beta-hCG rate post-transfer was 50.3% for Group A, compared to 49.1% for Group B. Clinical pregnancy rates were 36.9% and 36.4%, respectively. Regarding live births, in Group A 328 (36.7%) children were born, and in Group B, 16 (29.1%), with 2 ongoing pregnancies. Conversely, pregnancy losses in Group B (9.1%) were higher compared to the euploid group (2.9%). Regarding pregnancy losses, in 3 cases no fetal heartbeats were identified, there was one miscarriage &amp;lt;20 weeks, and one twin gestation was lost after a normal non-invasive prenatal testing. In all cases the newborns were apparently healthy after mosaic embryo transfer, with an average gestational week at birth of 39 (37-41), and an average weight of 3197 (2900-4390) grams. Cesarean section was performed for 15/16 births, and one was born by vaginal delivery. Limitations, reasons for caution The study is limited by its retrospective nature and the small number of samples and mosaics transferred. Prenatal and postnatal tests were not conducted. Furthermore, the health of newborns was evaluated only in the initial months of life. Wider implications of the findings Mosaic embryo transfer is a valid alternative for couples lacking euploid embryos. This multicenter study adds evidence that mosaic embryo transfer does not have a clinically relevant impact on pregnancy and live birth rates, similar to what has been reported by other authors. Trial registration number ‘not applicable’

P-563 Improved diagnosis in PGT-A samples with intermediate sex chromosomes copy number changes by adding ploidy and contamination analysis

Abstract Study question Does triple analysis of aneuploidy, ploidy and contamination improve the diagnosis in samples with intermediate copy number changes for the X and the Y chromosomes? Summary answer PGT-A with additional ploidy and contamination analysis improves the diagnosis of samples with intermediate sex chromosomes copy number changes, reducing non-informativity and the re-biopsy need. What is known already Next Generation Sequencing (NGS) in Preimplantation Genetic Testing for Aneuploidies (PGT-A) identifies chromosomal aneuploidies in trophectoderm (TE) biopsies. However, diagnosis limitations exist when the TE biopsy displays intermediate copy number changes for the X and the Y chromosomes, observed in ∼0.3% of biopsies. Few explanations are plausible, including mosaicism for the sex chromosomes, presence of exogenous DNA contamination or triploidy. A second TE biopsy (re-biopsy) could be recommended to obtain a conclusive result. However, additional Single Nucleotide Polymorphism (SNP) analysis to determine the ploidy status of the embryo and the presence of contamination in the biopsy can be an alternative. Study design, size, duration In this retrospective study, the impact on the diagnosis of SNPs analysis with a target panel was evaluated in PGT-A samples showing intermediate copy number changes for the X and the Y chromosomes. A total of 21 TE biopsies from January to December 2023 were included in the study. Participants/materials, setting, methods The TE biopsies were analysed using NGS with a proprietary protocol for library preparation, Ion Chef and Ion S5 System instruments for sequencing and a proprietary bioinformatics pipeline (v2.0) for 24-chromosomes aneuploidy testing. A re-biopsy was analysed in 8 embryos using the same protocol. Eighteen original biopsies were retrospectively evaluated for ploidy and contamination using an Ion AmpliSeq Custom Panel including SNPs (Thermo Fisher Scientific, USA) and a proprietary algorithm (v1.0) for data analysis. Main results and the role of chance The 21 TE biopsies displaying intermediate copy number changes for the X and the Y chromosomes were initially diagnosed as non-informative and candidates for re-biopsy to rescue diagnosis. A total of 8 embryos were re-biopsied (38.1%): 6 re-biopsies (75.0%) showed the same intermediate sex chromosomes copy number changes suggesting a triploid status of the embryo, 1 re-biopsy (12.5%) showed trisomy 22 suggesting DNA contamination in the original biopsy and, 1 re-biopsy (12.5%) displayed a mosaic monosomy X/XY suggesting a diploid-mosaic status of the embryo. In 18 original TE biopsies (85.7%), an additional ploidy and contamination analysis was done: 10 samples were triploid (55.6%), 6 samples showed DNA contamination (33.3%) and 2 samples were diploid (11.1%). In 5 embryos, the re-biopsy with the aneuploidy testing had been done: (i) in 4 triploid embryos the re-biopsies showed the same intermediate copy number changes for the sex chromosomes than the original biopsy, confirming the triploid status of the embryo; (ii) in 1 diploid embryo the re-biopsy showed a mosaic monosomy X/XY, confirming the diploid status and the sex chromosome mosaicism of the embryo. From the 21 embryos initially non-informative, only the 6 contaminated ones remained non-informative and candidates for re-biopsy (33.3%). Limitations, reasons for caution The limited number of samples with both analysis, ploidy/contamination by SNPs in the original sample and aneuploidy by NGS in the re-biopsy precluded to drawn stronger conclusions. This double-check would reinforce the benefit of doing additional ploidy/contamination analysis in these specific cases. Wider implications of the findings A high incidence of samples with intermediate sex chromosomes copy number are triploid, some have exogenous DNA contamination, and few are real mosaics for the sex chromosomes. Additional ploidy and contamination analysis could improve the PGT-A diagnosis and decrease mosaicism and non-informativity rates, reducing the embryos that would need re-biopsy. Trial registration number Not applicable

P-527 Low sperm motility and elevated sperm DNA fragmentation index (DFI) are associated with increased rates of embryo aneuploidy in patients with advanced reproductive age

Abstract Study question Is there a correlation between the paternal age, sperm characteristics, and the prevalence of chromosomal abnormalities in embryos generated through in vitro fertilization (IVF)? Summary answer A correlation exists between advanced paternal age, suboptimal sperm parameters, and elevated rates of embryo aneuploidy in individuals of advanced reproductive age. What is known already The exact mechanisms underlying the relationship between paternal age, sperm quality and embryo ploidy, although a subject of considerable research in assisted reproduction, are not fully elucidated. Some reports, including our group’s, suggest that paternal age may influence sperm quality and segmental embryo aneuploidy, while others suggest a potential association between high DFI(DNA Fragmentation Index) and an increased likelihood of embryo aneuploidy. Studies also show that sperm with optimal motility and fertilization competence are more likely to contribute to the development of euploid embryos, while data on the effect of abnormal sperm parameters on embryo ploidy is scarce and conflicting. Study design, size, duration This was a retrospective cohort study performed in the Reproductive Genetics Department, CReATe Fertility Centre, Toronto, Canada, between January 2019-December 2023. A total of 7628 blastocysts from 1512 cycles (1392 patients) were analyzed using high resolution NGS PGT-A. Sperm motility, concentration and DFI data, as well as, IVF cycle parameter data were collected. In addition, age of gamete providers and any relevant clinical, laboratory and demographic data were obtained for statistical analysis. Participants/materials, setting, methods Embryos were allocated into two groups by oocyte-provider age: OP-≤33 years and OP-≥34 years. Each group was divided into two sub-groups by paternal age: SP-≤39 years (n = 4570); SP-≥40 years (n = 3058). Multiple logistic/ordinal regressions were conducted to evaluate Segmental abnormalities and Segmental mosaicism between these age groups in relation to sperm DFI (GOOD ≤15%; FAIR 15-30%; POOR ≥30%), Motility (HIGH &amp;gt;40%; LOW &amp;lt;40%) and Count (LOW &amp;lt;5 mil/ml; MEDIUM 5-15 mil/ml; HIGH &amp;gt;15 mil/ml). Main results and the role of chance PGT-A analysis of 7628 embryos showed 51.5% euploidy, 36.7% aneuploidy and 11.8% mosaicism. In the OP-≤33 group, there were no significant differences between the two paternal age groups for euploidy, aneuploidy or mosaicism rates. In the OP-≥34 group, decreasing euploidy rates (p &amp;lt; 0.0001 and increasing aneuploidy rates (p &amp;lt; 0.0001) were observed between the two paternal age groups, while mosaicism rates were not different, an expected oocyte age-effect result. No significant differences in aneuploidy rates were observed for each of the sperm parameters (DFI, motility, count) in the groups of the OP-≤33 with SP-≤39, OP-≤33 with SP-≥40 and OP-≥34 with SP-≤39. In the group of OP-≥34 with SP-≥40, in patients with LOW motility vs. HIGH motility the aneuploidy rates were higher (56.8% vs.50.6%, p = 0.005) and euploidy rates were lower (33.8% vs.38.4%, p = 0.03). Each sperm parameter in each age group was further analyzed for chromosomal segmental abnormalities and segmental mosaicism. Higher rates of segmental abnormalities were seen in LOW motility groups with POOR-DFI compared to GOOD DFI in OP-≤33 with SP-≤39 (3.2% vs. 7.4%, p = 0.03). Limitations, reasons for caution The main limitation was the retrospective nature of this study; however, multiple regression analyses were conducted to limit potential bias by adjusting for sperm and oocyte characteristics. In addition, PGT-A analysis cannot provide information on origin of the aneuploidy and all conclusions are deduced from indirect associations to paternal age. Wider implications of the findings The identified associations could stimulate further research into exploring the molecular/cellular processes involved in fertilization, early embryonic development, and the potential impact of sperm-related factors on the chromosomal stability of developing embryos. The broader implications of these findings extend to clinical practice, patient counseling, technological advancements, and ethical considerations. Trial registration number not applicable

P-516 Minimizing mosaicism: impact of trophectoderm (TE) biopsy specimen size on mosaic blastocysts in preimplantation genetic testing for aneuploidy (PGT-A)

Abstract Study question Is the size of TE biopsy specimens related to the chromosomal status of the blastocysts in PGT-A? Summary answer Smaller biopsy specimens are associated with higher proportion and altered composition of mosaic blastocysts in PGT-A. What is known already Concordance between TE biopsy and inner cell mass or whole embryos is influenced by mosaicism type, degree, biopsy location, number of cells biopsied, and cell loading. Particularly, mosaic embryos and those with structural rearrangements have a higher level of inconsistency. Recent studies have demonstrated the potential reclassification of chaotic embryos as euploid, leading to successful live births. This study aimed to investigate the potential influence of TE biopsy specimen size on the chromosomal status of blastocysts undergoing PGT-A. Study design, size, duration This retrospective study, conducted from October, 2023, to January, 2024, included 246 blastocysts from patients undergoing TE biopsy and PGT-A at the blastocyst stage on days 5, 6, or 7. Next-generation sequencing (NGS) was used to obtain PGT-A results, categorizing blastocysts into euploid, aneuploid, and mosaic based on their genetic composition. Participants/materials, setting, methods The biopsy specimen area was measured using the Image J program. Mosaic blastocysts were categorized into either a chaotic mosaic group or another group, which included blastocysts with fewer than three chromosomal aneuploidies and segmental mosaicism. Further analysis was conducted by classifying blastocysts into high-level mosaic (&amp;gt;30% aneuploidy) and low-level mosaic (≤30% aneuploidy) groups. Group differences were analyzed using t-tests and chi-square, with a significance threshold set at p&amp;lt;0.05. Main results and the role of chance In the analysis of expanded mosaic blastocysts, the chaotic mosaic group had smaller biopsy specimens than those in the comparison group (p&amp;lt;0.05). The high-level mosaic group also had significantly smaller biopsy specimens than the low-level mosaic group (p&amp;lt;0.05). When categorizing the biopsied sample size into two groups—below 1350 µM2 (n = 98) and above 1500 µM2 (n = 97)—the rates of mosaic embryos were 37% (36/98) and 30% (29/97), respectively, showing a numerical difference without statistical significance. These findings suggest a correlation between the size of biopsy specimens and the chromosomal status of mosaic blastocysts, particularly indicating that a smaller sample size is associated with an elevated chance of mosaic embryos showing chaotic or high-level mosaic characteristics. Limitations, reasons for caution The biopsy specimens were photographed, and their areas were measured using the Image J program, lacking a comprehensive representation of the three-dimensional sample morphology. This constraint could potentially affect the accuracy and in-depth understanding of the spatial characteristics of the biopsy specimens. Wider implications of the findings The study emphasizes the crucial role of optimizing biopsy specimen size in PGT-A. The identified correlation with the chromosomal status in mosaic embryos, especially the association with chaotic or high-level mosaic embryo outcomes in smaller samples, highlights the need for careful size considerations. Trial registration number not applicable

P-520 Analyses of mosaic embryos reveal critical regions on human chromosome 11 that involve embryo implantation

Abstract Study question Why some mosaic embryos can be implanted, whereas others cannot? What are the secrets hidden in the human genome that control embryo implantation? Summary answer By data-mining genomic information from PGT-A results of 109 mosaic embryos, we have defined the critical regions in chr11 that play essential roles in implantation. What is known already Embryo implantation is usually a bottleneck step for a successful IVF treatment. What factors control the embryo implantation process has been pursued for many years, and the answers remain largely unknown. PGT-A is a widely applied procedure to identify embryos with normal diploid chromosomes to increase the clinical pregnancy rate per embryo transfer. However, some poor responder patients may only have mosaic embryos that can be used for transfer. We thus investigate the critical regions of the human chromosomes that are important for controlling embryo implantation. Study design, size, duration One hundred and nine mosaic blastocysts were analyzed based on PGT-A assay from 94 patients (ages ranging from 30 to 49 years old, mean age = 38.9 years) undergoing IVF between November 2020 and March 2022. The impact of chromosomal regions on implantation was explored by analyzing the sequencing results together with the pregnancy outcome of mosaic embryos. Additional published outcome data of 1000 mosaic embryo transfers was used to confirm our findings. Participants/materials, setting, methods Pregnant outcomes were followed, including hCG examination, sac formation, and live birth. Whole genome data analysis was performed using individual PGT-A results. Different types of chromosome abnormalities were calculated, and heatmap and clustering analysis were used to define the critical chromosome regions for embryo implantation. In addition, gene set enrichment and pathway analyses were conducted to reveal the functions of genes within the critical regions. Main results and the role of chance Among the transferred 109 mosaic embryos, the clinical outcomes of hCG positive, sac formation, and live birth rates were 45.8%, 42.2%, and 32.1%, respectively. There were 74 (67.8%) and 35 (32.1%) embryos carrying single and multiple mosaic chromosomes, respectively, resulting in 182 chromosomal mosaicism events. The trisomy mosaicism (133/182, 73.0%) occurred with higher frequency than monosomy mosaicism (49/182, 26.9%). Using the heatmap and clustering analysis, our data showed clustered monosomy mosaicism in non-pregnant embryos, especially for chr2, chr4, chr11, chr19, and chr22. Further refinement using segmental mosaic embryos has identified the critical regions, ranging from 10M to 40M in size on different chromosomes that are involved in hundreds of protein-coding genes and known dosage-sensitive genes. Importantly, functional analyses of the genes within the critical regions of chromosome 11 revealed enrichments in embryo invasion and angiogenesis, and suggest that loss of the chromosomal copy of these regions may result in implantation failure. Limitations, reasons for caution It’s still preliminary. More data is required to further confirm the conclusion. Wider implications of the findings Our study provides insights into the chromosomal regions involved in human embryo implantation and will improve the embryo prioritizing strategy in future IVF clinics. Importantly it is the first time the genomics information and gene-mapping strategy have been applied in mosaic embryos to uncover mechanisms underlying human embryo implantation. Trial registration number not applicable

O-154 Chromosomal, gestational, and neonatal outcomes of mosaic embryos: analysis of 3074 cases from the international registry of mosaic embryo

Abstract Study question Which are the clinical outcomes and post-natal results of mosaic embryos with low and high-level of mosaicism? Summary answer The level of mosaicism negatively influences implantation and ongoing pregnancy rate. 6/7 cases in which mosaicism persisted in the foetuses were from low-level mosaic. What is known already Chromosomal mosaic embryos are characterized by the presence of chromosomally different cell lines within the same embryo. While the transfer of these embryos is now offered as an option for women who undergo in vitro fertilization (IVF), several concerns remain. For instance, the limited data on pregnancy outcome and the possibility that intra-biopsy mosaicism in the TE is a poor predictor of the ploidy status of the ICM. Therefore, some argue that mosaicism should be not reported until a clear classification of such embryos in relation to their reproductive potential has been defined. Study design, size, duration We collected the clinical outcomes of 3074 mosaic embryos transferred in women who underwent IVF between May 2019-May 2023. All embryos were cultured to blastocyst stage; trophectoderm (TE) biopsy was performed on Day-5 of development or Day6/7 for slow-growing embryos. The clinical outcome obtained after the transfer of mosaic embryos with the different chromosomal constitutions was compared with each other. Prenatal and post-natal outcomes were collected for available cases. Participants/materials, setting, methods Preimplantation genetic testing (PGT) was performed using high-resolution next-generation sequencing (NGS) methodology. TE biopsies were classified as mosaic if they had 20%-80% abnormal cells. For statistical analysis, mosaic embryos were divided into groups based on mosaic levels and chromosomal constitution detected in TE: single mosaic aneuploidy (monosomy/trisomy; SM), double mosaic chromosomes (monosomy/trisomy or combination, DM), complex mosaic aneuploidy (&amp;gt;2 different aneuploidies; CM) and mosaic segmental aneuploidy (single and double deletion/insertion &amp;gt;5Mb, MS). Main results and the role of chance Embryos classified as ‘low-mosaic’ by NGS-based PGT-A have a higher likelihood of achieving implantation compared to ‘high-mosaic’ embryos (48% vs. 39.%; p &amp;lt; 0.05 ), as well as ongoing pregnancy/live birth (40% vs. 29%; p &amp;lt; 0.05). Chromosomal composition of mosaicism abnormalities dictates the success rate of mosaic embryo transfers, with low and high segmental mosaics being preferable over low- and high-mosaics involving whole chromosomes. For 621/670 pregnancies, parental tests and post-natal data were available. The majority (99.75%) of the babies were largely healthy by routine physical inspection by neonatologists (no gross abnormalities in babies from mosaic embryos, n = 495). A combination of NIPT, CVS, and Amniocentesis prenatal testing results were collected for 552 pregnancies of mosaic embryo transfers, with predominantly normal findings. The mosaicism detected at the embryonic stage by PGT-A was reflected in prenatal testing in only 7 out of 552 pregnancies (0.9%), in which the mosaicism identified with PGT-A at the blastocyst stage was reflected in gestation by prenatal chromosomal testing as true fetal mosaicism. Limitations, reasons for caution Additional clinical data must be obtained to evaluate the contribution of each different chromosome before this approach can be evaluated as an additional tool to choose mosaic embryos for transfer. Wider implications of the findings The international registry of mosaic embryo transfers continues to grow in sample size, in turn increasing the power of analysis. The findings of the mosaic embryo transfer registry can help educate the management and selection of embryos in the clinic. Trial registration number no

A Novel Method for Acquired Sex Chromosome Mosaicism.

The phenomenon of sex chromosome loss from hematopoietic cells is an emerging indicator of biological aging. While many methods to detect this loss have been developed, enhancing the field, these existing methods often suffer from being labor-intensive, expensive, and not sufficiently sensitive. To bridge this gap, a novel and more efficient technique is developed, named the SinChro assay. This method employs multiplexed single-cell droplet PCR, designed to detect cells with sex chromosome loss at single-cell resolution. Through the SinChro assay, the age-dependent increase in Y chromosome loss in male blood is successfully mapped. The age-dependent loss of the X chromosome in female blood is also identified, a finding that has been challenging with existing methods. The advent of the SinChro assay marks a significant breakthrough in the study of age-related sex mosaicism. Its utility extends beyond blood analysis, applicable to a variety of tissues, and it holds the potential to deepen the understanding of biological aging and related diseases.