High-resolution Next-generation Sequencing Research Articles

P-250 Only euploidy does not ensure optimal chances for successful live birth through single blastocyst cryotransfer

Abstract Study question What does a euploid blastocyst require to provide the greatest probability of successful birth? Summary answer Morphokinetic and morphological features of the inner cell mass (ICM) and trophectoderm (TE) are crucial for optimizing live birth chances in cryo-transferred euploid blastocysts. What is known already Embryo selection through preimplantation genetic testing for aneuploidy (PGT-A) alongside frozen embryo transfer strategy enhances outcomes of single-embryo transfer cycles. While euploidy is a crucial indicator, investigations on the identification of additional criteria influencing implantation and live birth rates remain imperative. Studies suggest morphokinetic and morphological quality may supplement predictive factors for euploid embryo success, with varying correlations. The impact of factors like age or gamete origin on euploid blastocyst success remains uncertain, differentiating it from clear effects on non-diagnosed embryos. Optimizing selection criteria involves navigating the intricate interplay of these variables. Study design, size, duration This retrospective study analyses the pregnancy rate (PR) in 596 single embryo cryotransfers of full euploid blastocysts (without detected mosaicism) between February 2019 and September 2023. From these, 437 had a known outcome, and the live birth rate (LBR) was calculated. The study explored the potential impact of gamete origin, oocyte provider age, recipient age, morphokinetics, and morphological quality of ICM or TE (Gardner score) on the reproductive success of the transferred embryo. Participants/materials, setting, methods All embryos were biopsied at the blastocyst stage and successively vitrified. High-resolution next-generation sequencing was used for PGT-A. Only deferred transferences of single post-thawing intact embryos were included. Morphokinetic was assessed using the KIDscoreD5 3.1. Quantitative variables (oocyte provider age, recipient age, and morphokinetic score) were compared using the t-student test, while qualitative variables (gamete origin, morphological quality of ICM and TE) were assessed using the chi-square test. Main results and the role of chance Oocytes leading to pregnancies and live births were derived from women with ages comparable to those without success (Pregnancy: 30.0±7.1 vs. 30.8±6.8, p = 0.174; Live Birth: 30.1±7.1 vs. 30.5±6.9, p = 0.499). Similar age was also observed among recipients achieving pregnancy (41.5±5.4) and live births (41.1±5.1) compared to those who did not (Pregnancy: 41.8±5.1, p = 0.506; Live Birth: 41.5±5.3, p = 0.411). The origin of oocytes and semen (own or donor) did not significantly affect PR (Oocytes: 199/273 vs. 217/323, p = 0.130; Semen: 328/473 vs. 88/123, p = 0.224) or LBR (Oocytes: 113/205 vs. 116/232, p = 0.285; Semen: 183/347 vs. 46/90, p = 0.783). Morphokinetics played a crucial role, with euploid embryos resulting in pregnancy (7.5±1.7) and live births (7.6±1.6) exhibiting significantly higher scores than those without success (6.9±1.7; 7.1±1.7; p < 0.05).199 ICM quality did not significantly impact PR (A:211/296, B:190/275, C:15/25; p = 0.469). However, higher miscarriage rates in C-quality ICM blastocysts significantly reduced the LBR (A:116/211, B:107/205, C:6/21; p < 0.05). TE quality exhibited significant differences in PR (A:221/289, B:184/289, C:11/18; p < 0.05). Since the miscarriage rate was similar among groups (p = 0.787), LBR remained higher in A-quality TE blastocysts (A:124/209, B:98/213, C:7/17; p < 0.05). Limitations, reasons for caution As a retrospective analysis, this study is inherently limited. The influence of expansion grade on success rates remains unexplored, given the use of assisted hatching on day 3 of culture. Performing assisted hatching at the time of biopsy could potentially overcome this limitation. Wider implications of the findings The pursuit of a euploid embryo often becomes the priority in IVF laboratories. However, once the milestone is achieved, morphokinetics and embryonic morphological quality must continue to be considered. This study provides valuable insights for conveying a detailed message to patients with euploid embryos about their prospects for homebirth success. 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 (>2 different aneuploidies; CM) and mosaic segmental aneuploidy (single and double deletion/insertion >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 < 0.05 ), as well as ongoing pregnancy/live birth (40% vs. 29%; p < 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

Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method

PurposeA new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains.MethodsT. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples.ResultsAmong type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing.ConclusionThe new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

High maternal-fetal HLA eplet compatibility is associated with severe manifestation of preeclampsia.

Preeclampsia is responsible for more than 70 000 and 500 000 maternal and fetal deaths, respectively each year. Incomplete remodelling of the spiral arteries in placenta is the most accepted theory of preeclampsia pathogenesis. However, the process is complexed with immunological background, as pregnancy resembles allograft transplantation. Fetus expresses human leukocyte antigens (HLA) inherited from both parents, thus is semiallogeneic to the maternal immune system. Therefore, induction of fetal tolerance is crucial for physiological outcome of pregnancy. Noteworthy, the immunogenicity of discordant HLA antigens is determined by functional epitopes called eplets, which are continuous and discontinuous short sequences of amino acids. This way various HLA molecules may express the same eplet and some HLA incompatibilities can be more immunogenic due to different eplet combination. Therefore, we hypothesized that maternal- fetal HLA incompatibility may be involved in the pathogenesis of gestational hypertension and its progression to preeclampsia. We also aimed to test if particular maternal-fetal eplet mismatches are more prone for induction of anti- fetal HLA antibodies in gestational hypertension and preeclampsia. High resolution next-generation sequencing of HLA-A, -B, -C, -DQB1 and -DRB1 antigens was performed in mothers and children from physiological pregnancies (12 pairs) and from pregnancies complicated with gestational hypertension (22 pairs) and preeclampsia (27 pairs). In the next step HLA eplet identification and analysis of HLA eplet incompatibilities was performed with in silico approach HLAMatchmaker algorithm. Simultaneously maternal sera were screened for anti-fetal HLA class I, class II and anti-MICA antibodies with Luminex, and data were analyzed with HLA-Fusion software. We observed that high HLA-C, -B, and DQB1 maternal-fetal eplet compatibility was associated with severe preeclampsia (PE) manifestation. Both quantity and quality of HLA epletmismatches affected the severity of PE. Mismatches in HLA-B eplets: 65QIA+76ESN, 70IAO, 180E, HLA-C eplets: 193PL3, 267QE, and HLA-DRB1 eplet: 16Y were associated with a mild outcome of preeclampsia if the complication occurred. High HLA-C, HLA-DQB1 and HLA-B eplet compatibility between mother and child is associated with severe manifestation of preeclampsia. Both quantity and quality of maternal-fetal HLA eplet mismatches affects severity of preeclampsia.

Norovirus evolves as one or more distinct clonal populations in immunocompromised hosts.

Noroviruses are a major cause of acute gastroenteritis worldwide and can establish chronic infection in immunocompromised individuals. To investigate the mechanisms of norovirus evolution during chronic infection, we selected seven representative patients from a National Institutes of Health study cohort who sustained norovirus infection for periods ranging from 73 to 1,492 days. Six patients shed viruses belonging to a single genotype (GII.2[PNA], GII.4 New Orleans[P4], GII.4 Den Haag[P4], GII.3[P21], GII.6[P7], or GII.14[P7]) over the period examined, while one patient sequentially shed two genotypes (GII.6[P7] followed by GII.4 Sydney[P31]). Norovirus genomes from consecutive stool samples were sequenced at high resolution (>3,300 reads/nucleotide position) using the Illumina platform and subjected to bioinformatics analysis. Norovirus sequences could be resolved into one or more discrete clonal RNA genomes that persisted within these patients over time. Phylogenetic analyses inferred that clonal populations originated from a single founder virus and not by reinfection with community strains. Estimated evolutionary rates of clonal populations during persistent infection were similar to those of noroviruses from acute infection in the global database, suggesting that inherently higher RNA-dependent polymerase error rates were not associated with the ability to persist. The high-resolution analysis of norovirus diversity and evolution at the population level described here should allow a better understanding of adaptive mutations sustained during chronic infection. IMPORTANCE Noroviruses are an important cause of chronic diarrhea in patients with compromised immune systems. Presently, there are no effective therapies to clear the virus, which can persist for years in the intestinal tract. The goal of our study was to develop a better understanding of the norovirus strains that are associated with these long-term infections. With the remarkable diversity of norovirus strains detected in the immunocompromised patient cohort we studied, it appears that most, if not all, noroviruses circulating in nature may have the capacity to establish a chronic infection when a person is unable to mount an effective immune response. Our work is the most comprehensive genetic data set generated to date in which near full-length genomes from noroviruses associated with chronic infection were analyzed by high-resolution next-generation sequencing. Analysis of this data set led to our discovery that certain patients in our cohort were shedding noroviruses that could be subdivided into distinct haplotypes or populations of viruses that were co-evolving independently. The ability to track haplotypes of noroviruses during chronic infection will allow us to fine-tune our understanding of how the virus adapts and maintains itself in the human host, and how selective pressures such as antiviral drugs can affect these distinct populations.

O-076 Update on the International Registry of Mosaic Embryo Transfers

Abstract Study question Which are the results obtained by the International Registry of Mosaic Embryo Transfers? Summary answer An update of clinical outcomes and post-natal results of mosaic embryos with low and high-level of mosaicism is provided. 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 2045 mosaic embryos transferred in women who undergoing IVF between May 2019-May 2022. 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 outcomes obtained after the transfer of mosaic embryos with the different chromosomal constitutions were compared. Prenatal and post-natal outcome was collected for available cases. Participants/materials, setting, methods Preimplantation genetic testing for aneuploidies (PGT-A) 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 (>2 different aneuploidies; CM) and mosaic segmental aneuploidy (single and double deletion/insertion >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 < 0.05 ), as well as ongoing pregnancy/live birth (40% vs. 29%; p < 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 550/670 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). Prenatal testing performed on pregnancies from mosaic embryo transfers were generally normal. The mosaicism detected at the embryonic stage by PGT-A was reflected in prenatal testing in only 5 out of 550 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 Not applicable

HLA-DQB1*05:02, *05:03, and *03:01 alleles as risk factors for myasthenia gravis in a Spanish cohort.

Myasthenia gravis (MG) is a very heterogenic chronic autoimmune disease caused by the failure of neuromuscular transmission. The HLA gene complex has conventionally been recognized as its main genetic risk and phenotype modifying factor. Our aim was to investigate the prevalence of HLA class I and II alleles and to identify possible risk factors for sporadic MG in a Spanish cohort. We designed a clinical case-control study comparing HLA alleles and haplotype frequencies in a cohort of 234 patients with sporadic autoimmune MG with data from a group of 492 randomly selected healthy subjects. Using a high-resolution next-generation sequencing (NGS)-based HLA genotyping assay, we investigated the contribution of HLA genotypes and haplotypes in the resulting phenotype, especially, the age at onset, sex, onset MGFA class, thymic histopathology, and serological status. We found that the DQB1*05:02 and DQB1*05:03 alleles could be novel risk factors for Spanish MG cases. The HLA alleles A*01:01, B*08:01, DRB1*03:01, DRB1*14:54, and DQB1*02:01 were also risk factors for the disease. DQB1*03:01 acted as a risk factor for EOMG in women with AChR-positive antibodies and thymus hyperplasia. Additionally, several alleles were identified as potential phenotype-modifying factors that could exert a protective effect: HLA-B*35:08, DRB1*13:01, and DQB1*06:03 in MG; HLA-A*24:02 in women and DRB1*07:01 and DQB1*02:02 for early onset. HLA-C*07:01 and haplotype A1-B8-C7-DR3-DQ2 were associated with an early-onset phenotype.

The utility of imputation for molecular mismatch analysis in solid organ transplantation

HLA genotyping has undergone a rapid progression in resolution since the development of DNA-based typing methods. Despite the advent of high-resolution next-generation sequencing, the bulk of solid organ genotyping is performed at intermediate resolution, which provides multiple possible two-field results for each classical HLA loci. As a result, several methodologies have been developed to impute the most likely allele-level (two-field) HLA genotype for the purposes of donor-recipient compatibility analysis. The advent of molecular mismatch analysis, however, has placed a new emphasis on the accuracy of imputation. While seminal molecular mismatch studies have relied on the imputation of intermediate resolution genotyping, several recent studies have performed analysis showing that imputation generates inaccuracies in epitope identification. While the clinical impact of these errors is not clear, it is important that these concerns do not preclude future progress in understanding the utility of molecular mismatch analysis in transplantation. In the future, advances in genotyping methods will result in routine two-field resolution that will abrogate these concerns. In the meantime, however, studies are needed in order to address the role of molecular mismatch in diverse patient populations and to carefully address the potential of molecular mismatch analysis in the context of imputation.

O-086 End-to-end deep learning for recognition of ploidy status using time-lapse videos

Abstract Study question Our Retrospective study is to investigate an end-to-end deep learning model in identifying ploidy status through raw time-lapse video. Summary answer Our deep learning model demonstrates a proof of concept and potential in recognizing the ploidy status. What is known already Since the time-lapse system has been introduced into the IVF lab, the relationship between morphogenetic and ploidy status has been often discussed. However, the result has not yet reached a united conclusion due to some limitations such as human labeling. Besides the statistical approach, deep learning models have been utilized for ploidy prediction. As such approaches are single image-based, the performance remains unpromising as previous statistical-based research. Therefore, in order to move further toward clinical application, better research design and approach are needed. Study design, size, duration A retrospective analysis of the time-lapse videos and chromosomal status from 690 biopsied blastocysts cultured in a time-lapse incubator (EmbryoScope+, Vitrolife) between January 2017 and August 2018 in the Lee Women’s Hospital were assessed. The ploidy status of the blastocyst was derived from the PGT-A using high-resolution next-generation sequencing (hr-NGS). Embryo videos were obtained after normal fertilization through the intracytoplasmic sperm injection or conventional insemination. Participants/materials, setting, methods By randomly dividing the data into 80% and 20%, we developed our deep learning model based on Two-Stream Inflated 3D ConvNets(I3D) network. This model was trained by the 80% time-lapse videos and the PGT-A result. The remaining 20% has been tested by feeding the time-lapse video as input and the PGT-A prediction as output. Ploidy status was classified as Group 1 (aneuploidy) and Group 2 (euploidy and mosaicism). Main results and the role of chance Time-lapse videos were divided into 3-time partitions: day 1, day 1 to 3, and day 1 to 5. Deep learning models have been fed by RGB and optical flow. Combining 3 different time partitions with RGB, optical flow, and fused result from RGB and optical flow, we received nine sets of test results. According to the results, the longest time partition with the fusion method has the highest AUC result as 0.74, which appeared higher than the other eight experimental settings with a maximum increase of 0.17. Limitations, reasons for caution The present study is retrospective and future prospective research would help us to identify more key factors and improve this model. In addition, expanding sample size combined with cross-centered validation will also be considered in our future approach. Wider implications of the findings Group 1 and Group 2 approach provided deselection of aneuploidy embryos, while future deep learning approaches toward high mosaicism, low mosaicism, and euploidy will be needed, in order to provide a better clinical application. Trial registration number CS18082

Behçet disease, new insights in disease associations and manifestations: a next-generation sequencing study

Behçet disease is a multi-system disease associated with human leukocyte antigen (HLA) class I polymorphism. High-resolution next-generation sequencing (NGS) with haplotype analysis has not been performed previously for this disease. Sixty Egyptian patients diagnosed according to the International Study Group (ISG) criteria for Behçet disease and 160 healthy geographic and ethnic-matched controls were genotyped for HLA class I loci (HLA-A, B, C). For HLA class II loci (DRB1, DRB3/4/5, DQA1, DQB1, DPA1, DPB1), 40 control samples were genotyped. High-resolution HLA genotyping was performed using NGS and the results were analyzed. Clinical manifestations were oral ulcers (100%), genital ulcers (100%), eye (55%) and neurological (28%) and vascular involvement (35%). HLA-B*51:08 [odds ratio (OR)=19·75, 95% confidence interval (CI)=6·5-79; P<0·0001], HLA-B*15:03 (OR=12·15, 95% CI=3·7-50·7; P<0·0001), HLA-C*16:02 (OR=6·53, 95% CI=3-14; P<0·0001), HLA-A*68:02 (OR=3·14, 95% CI=1·1-8·9; P<0·01) were found to be associated with Behçet disease, as were HLA-DRB1*13:01 and HLA-DQB1*06:03 (OR=3·39, 95% CI=0·9-18·9; P=0·04 for both). By contrast, HLA-A*03:01 (OR=0·13, 95% CI=0-0·8; P=0·01) and HLA-DPB1*17:01 were found to be protective (OR=0·27, 95% CI=0·06-1·03; P=0·02). We identified strong linkage disequilibrium between HLA-B*51:08 and C*16:02 and A*02:01 in a haplotype associated with Behçet disease. HLA-B*51:08 was significantly associated with legal blindness (OR = 2·98, 95% CI=1·06-8·3; P=0·01). In Egyptian Behçet patients, HLA-B*51:08 is the most common susceptibility allele and holds poor prognosis for eye involvement.

A DNA methylation signature discriminates between excellent and non-response to lithium in patients with bipolar disorder type 1

Lithium (Li) is the cornerstone maintenance treatment for bipolar disorders (BD), but response rates are highly variable. To date, no clinical or biological marker is available to reliably define eligibility criteria for a maintenance treatment with Li. We examined whether the prophylactic response to Li (assessed retrospectively) is associated with distinct blood DNA methylation profiles. Bisulfite-treated total blood DNA samples from individuals with BD type 1 (15 excellent-responders (LiERs) versus 11 non-responders (LiNRs)) were used for targeted enrichment of CpG rich genomic regions followed by high-resolution next-generation sequencing to identify differentially methylated regions (DMRs). After controlling for potential confounders we identified 111 DMRs that significantly differ between LiERs and LiNRs with a significant enrichment in neuronal cell components. Logistic regression and receiver operating curves identified a combination of 7 DMRs with a good discriminatory power for response to Li (Area Under the Curve 0.806). Annotated genes associated with these DMRs include Eukaryotic Translation Initiation Factor 2B Subunit Epsilon (EIF2B5), Von Willebrand Factor A Domain Containing 5B2 (VWA5B2), Ral GTPase Activating Protein Catalytic Alpha Subunit 1 (RALGAPA1). Although preliminary and deserving replication, these results suggest that biomarkers of response to Li may be identified through peripheral epigenetic measures.

T Cell Clonal Dynamics Determined by High-Resolution TCR-β Sequencing in Recipients after Allogeneic Hematopoietic Cell Transplantation.

Delayed reconstitution of the immune system is a long-recognized complication after allogeneic hematopoietic cell transplantation (HCT). Specifically, loss of T cell diversity has been thought to contribute to infectious complications, graft-versus-host disease (GVHD), and disease relapse. We performed serial high-resolution next-generation sequencing of T cell receptor (TCR)-β in 99 related or unrelated donor (57 unrelated, 42 related) allogeneic HCT recipients (55 with reduced-intensity conditioning, 44 with myeloablative conditioning) during the first 3 months after HCT using the immunoSEQ Assay. We measured T cell fraction, clonality (1- Peilou's evenness) and Daley-Smith richness from recipient samples at multiple time points. In agreement with previous studies, we found that although absolute T cell numbers recover relatively quickly after HCT, T cell repertoire diversity remains diminished. Restricted diversity was associated with conditioning intensity, use of antithymocyte globulin, and donor type. Increased number of expanded clones compared to donor T cell clones at day +30 was associated with the incidence of acute GVHD (hazard ratio [HR], 1.11; P=.00005). Even after exclusion of the 12 patients who developed acute GVHD before day +30, the association between acute GVHD and increased clonal expansion at day +30 remained (HR, 1.098; P=.041), indicating that increased clonal T cell expansion preceded the development of acute GVHD. Our results highlight T cell clonal expansion as a potential novel biomarker for acute GVHD that warrants further study.

A Catalogue of 59,732 Human-Specific Regulatory Sequences Reveals Unique-to-Human Regulatory Patterns Associated with Virus-Interacting Proteins, Pluripotency, and Brain Development.

Extensive searches for genomic regions harboring various types of candidate human-specific regulatory sequences (HSRS) identified thousands' HSRS using high-resolution next-generation sequencing technologies and methodologically diverse comparative analyses of human and nonhuman primates' (NHPs) reference genomes. In this study, a comprehensive catalogue of 59,732 genomic loci harboring candidate HSRS has been assembled to facilitate the systematic analyses of genomic sequences that were either inherited from extinct common ancestors (ECAs) or created de novo in human genomes. These analyses identified thousands of candidate HSRS and HSRS-harboring loci that appear inherited from ECAs, yet absent in genomes of our closest evolutionary relatives, chimpanzee and bonobo, presumably due to the incomplete lineage sorting and/or species-specific loss or regulatory DNA. This pattern is particularly prominent for HSRS-harboring loci that have been putatively associated with human-specific gene expression changes in cerebral organoid models. A prominent majority of regions harboring human-specific mutations associated with human-specific expression changes during brain development is highly conserved in chimpanzee, bonobo, and gorilla genomes. Among NHPs, dominant fractions of HSRS-harboring loci associated with human-specific gene expression in both excitatory neurons (347 loci; 67%) and radial glia (683 loci; 72%) are highly conserved in the gorilla genome. Analysis of 4433 genes encoding virus-interacting proteins (VIPs) revealed that 95.9% of human VIPs are components of human-specific regulatory networks that appear to operate in distinct types of human cells from preimplantation embryos to adult dorsolateral prefrontal cortex. These analyses demonstrate that modern humans captured unique genome-wide combinations of regulatory sequences, divergent subsets of which are highly conserved in distinct species of six NHP separated by 30 million years of evolution. Concurrently, this unique-to-human mosaic of genomic regulatory patterns inherited from ECAs was supplemented with 12,486 created de novo HSRS. Genes encoding VIPs appear to represent a principal genomic target during evolution of human-specific regulatory networks, which contribute to fitness of Homo sapiens and affect a functionally diverse spectrum of biological and cellular processes controlled by VIP-containing liquid-liquid phase-separated condensates.

Identification of HLA-A*02:06:01 as the primary disease susceptibility HLA allele in cold medicine-related Stevens-Johnson syndrome with severe ocular complications by high-resolution NGS-based HLA typing

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening acute inflammatory vesiculobullous reactions of the skin and mucous membranes. These severe cutaneous drug reactions are known to be caused by inciting drugs and infectious agents. Previously, we have reported the association of HLA-A*02:06 and HLA-B*44:03 with cold medicine (CM)-related SJS/TEN with severe ocular complications (SOCs) in the Japanese population. However, the conventional HLA typing method (PCR-SSOP) sometimes has ambiguity in the final HLA allele determination. In this study, we performed HLA-disease association studies in CM-SJS/TEN with SOCs at 3- or 4-field level. 120 CM-SJS/TEN patients with SOCs and 817 Japanese healthy controls are HLA genotyped using the high-resolution next-generation sequencing (NGS)-based HLA typing of HLA class I genes, including HLA-A, HLA-B, and HLA-C. Among the alleles of HLA class I genes, HLA-A*02:06:01 was strongly associated with susceptibility to CM-SJS/TEN (p = 1.15 × 10−18, odds ratio = 5.46). Four other alleles (HLA-A*24:02:01, HLA-B*52:01:01, HLA-B*46:01:01, and HLA-C*12:02:02) also demonstrated significant associations. HLA haplotype analyses indicated that HLA-A*02:06:01 is primarily associated with susceptibility to CM-SJS/TEN with SOCs. Notably, there were no specific disease-causing rare variants among the high-risk HLA alleles. This study highlights the importance of higher resolution HLA typing in the study of disease susceptibility, which may help to elucidate the pathogenesis of CM-SJS/TEN with SOCs.

9. ASSESSMENT OF ANEUPLOIDY AND MOSAICISM CONCORDANCE BETWEEN DIFFERENT TROPHECTODERM BIOPSY SITES AND THE INNER CELL MASS EVALUATED WITH NEXT-GENERATION SEQUENCING

Introduction It is recognized that the biopsy of 6-8 TE cells allows for high-resolution next-generation sequencing. However, concerns have been raised as to whether one biopsy sample can be representative of the whole blastocyst regarding aneuploidy and mosaicism. A recent study estimated the TE confined mosaicism rate at 14% and to be due to discordant chromosomal constitution of ICM and TE (Chuang et al., 2018). Another study showed that one clinical TE biopsy was an excellent representative of blastocyst karyotype in cases of whole chromosome aneuploidy, but not for cases with only segmental aneuploidy (Victor et al., 2019). Material and Methods 101 clinical biopsies were taken from a site opposite to the ICM (TE1) and the second sample was taken from thawed blastocysts previously diagnosed as being aneuploid with the informed consent of the patients (TE2). Five aneuploid blastocysts were also biopsied from within the ICM. Both samples were analyzed by the same platform, ReproSeq on Ion Torrent S5 (Thermo Fisher Scientific) next generation sequencing (NGS) to assess ploidy and mosaicism concordance. Blastocysts of good or top-quality belonging to 78 patients were subjected to TE biopsy for PGT for aneuploidy (PGT-A) with the clinical indications of advanced maternal age, repeated implantation failures or a previous history of abnormal fetal karyotype. Blastocysts were then vitrified as to our routine practice. Samples were analyzed by NGS and blastocysts diagnosed as being aneuploid were thawed, re-biopsied and re-analyzed by NGS. Results A total of 101 TE biopsies with whole chromosome aneuploidies were compared with 96 TE biopsy samples taken at another site of the blastocyst and 5 biopsies of the corresponding ICM. 76 samples out of 101 (75.2%) were found to be fully concordant. 25 diagnoses (24.7%) differ between TE1 and TE2: 13 mosaicisms and 10 segmental aneuploidies were either found in TE1 and not in TE2 or vice versa, without altering the overall aneuploid diagnosis. For four blastocysts, whole chromosome aneuploidies differ between TE1 and TE2, the first diagnosis indicating a chaotic aneuploidy and the second one a simple monosomy/ trisomy or vice versa. Regarding the five ICM samples, they were entirely concordant for full chromosome aneuploidies with their corresponding TE samples. However, in one sample, the 30% mosaicism of chromosomes 1, 13 and 16 was not shared by the ICM. Conclusions The high rate of concordance in two separate TE biopsies and ICM observed in this study validates the reliability of PGT-A by NGS. Blastocysts with segmental aneuploidies only may be re-evaluated if no euploids are found, as results suggest that segmental aneuploidies may not be shared among all TE cells. The study involved blastocysts initially diagnosed as being aneuploid, therefore the findings cannot be extrapolated to euploid or euploid-mosaic embryos.

Embryo morphokinetics is potentially associated with clinical outcomes of single-embryo transfers in preimplantation genetic testing for aneuploidy cycles.

Are the morphokinetics of euploid blastocysts evaluated by a generally applicable algorithm associated with the clinical outcomes of single-embryo transfer (SET)? Time-lapse microscopy was used to compare morphokinetic variables between expanded blastocysts derived from preimplantation genetic testing for aneuploidy cycles using high-resolution next-generation sequencing (hr-NGS). The clinical efficacy of the morphokinetic algorithm KIDScore D5 was evaluated after euploid SET. Compared with euploid blastocysts, low-level mosaic blastocysts presented comparable morphokinetic and morphological features. However, high-level mosaic blastocysts exhibited significant delays in t5 (median 51.9 h post insemination (hpi), P = 0.034) (where t is the time for the embryo to reach the specific stage in hours after ICSI or conventional IVF) and t8 (median 58.6 hpi, P = 0.032) accompanied by a prolonged time period for the third cell cycle (median 14.7 h, P = 0.012). A significantly higher incidence (P = 0.011) of multinucleation indicated a susceptibility of high-level mosaic blastocysts to mitotic errors. Only a delay in the time for the embryo to reach the full blastocyst stage (median 106.0 hpi, P = 0.039) was revealed in aneuploid blastocysts, reflecting the reduced formation of good-quality blastocysts (42.6% versus 65.7%, P < 0.001). Euploid blastocysts with specific morphokinetic characteristics were graded using the KIDScore D5 algorithm. Grade C embryos achieved significantly lower rates of clinical pregnancy, implantation and ongoing pregnancy (25%, 25% and 10%, respectively) compared with the grade A (76.2%, 79.4% and 68.3%, respectively) or grade B (62.5%, 66.7% and 62.5%, respectively) embryos (P = 0.0171 to <0.0001). Although morphokinetic features appear dissimilar in embryos with different diploid-aneuploid mosaic levels, predicting chromosomal abnormalities using morphokinetics alone is still insufficient. When combined with hr-NGS, use of the generally applicable KIDScore D5 algorithm has the potential to discriminate euploid blastocysts with different developmental competence.

Clinical outcome: the relationship between mosaicism and advanced maternal age with the use of Next Generation Sequencing (NGS)

Introduction Chromosomal mosaicism refers to the presence of two or more cell lines with a different chromosome content in an individual or tissue sample. The implementation of high resolution NGS for PGS has provided more information that it is not only used for embryonic aneuploidy screening, butdetects the presence of mosaicism in trophectoderm biopsies. Recent studies have shown that transfer of mosaic embryos resulted in lower implantation rates and higher miscarriage rates as compared to euploid transfer. This study aims to evaluate whether there is an association between chromosomal mosaicism and advanced maternal age. It also aims to compare the clinical pregnancy and implantation rate between euploid and whole chromosomal mosaic embryos transfer. Materials and Method Retrospective observational study of 533 blastocyst-stage embryos analysed by NGS from IVF cycles from Sunfert International Fertility Centre over the course of 2015 to 2016. Of those analysed, 197 blastocysts were transferred. The clinical outcome obtained after transfer of whole chromosomal mosaic embryos was compared with those resulting from a control group of 190 euploid blastocysts. Blastocyst-stage embryos underwent biopsy and trophectoderm cells are extracted for aneuploidy screening using IIlumina NGS protocol. Mosaic information for each chromosome was obtained. For this study, only whole chromosomal mosaicism is considered; segmental mosaicism were categorized under euploid embryos. These embryos were further stratified by maternal age group: 40 yo. Result Results are summarized in Figure 1. The result showed that mosaicism rates did not vary with advancing maternal age, staying at an average of 10.14%. Euploid and aneuploidy embryos, however, did show changes in regards to age factor as the former displayed a descending trend whereas the latter displayed an increasing trend. Download : Download high-res image (189KB) Download : Download full-size image Figure 1. Percentages of euploid, aneuploid and whole chromosomal mosaic embryos by age groups. Transfer of mosaic embryos resulted in higher clinical pregnancy rate (60% vs 57.5%) and implantation rate (57% vs 52.9%) than euploid embryos. No incidence of miscarriage was reported. Conclusion In accordance to most of the studies published, our findings showed that mosaicism is not associated with advanced maternal age. Unlike aneuploidy which arises from meiotic error, mosaicism, on the other hand is caused by impaired mitotic chromosome segregation which does not appear to increase with advancing maternal age. Therefore, while there is a direct relationship between advanced maternal age and aneuploidy, age is not a contributor to mosaicism. On the contrary, our findings showed conflicting result in the implantation rate of mosaic embryos. Similar rate of implantation as compared to euploid transfer embryos was found in our clinical study whereas in general, transfer of mosaic embryos have lower implantation rate. This might be the consequence of small sample size of mosaic embryos transfer (

The relationship between chromosomal mosaicism and maternal age in embryos TESTED WITH Next Generation Sequencing (NGS) platform

Introduction Chromosomal mosaicism refers to the presence of two or more cell lines with a differentchromosome content in an individual or tissue sample. Besides aneuploidy, the use of highresolution NGS for Pre-implantation Genetic Diagnosis (PGS) detects the presence of mosaicism introphectoderm biopsies. The transfer of mosaic embryos has been shown to result in lowerimplantation rates and higher miscarriage rates as compared to euploid transfer. This study aims to evaluate whether there is an association between mosaicism and maternal ageand the clinical pregnancy and implantation rate of mosaic embryos transfer. Materials and Method A retrospective observational study of 533 blastocyst-stage embryos analysed by NGS from IVFcycles from Sunfert International Fertility Centre over the course of 2015 to 2016 was performed. Ofthose analysed, 197 blastocysts were transferred. The clinical outcome obtained after the transfer ofwhole chromosomal mosaic embryos was compared with those resulting from a control group 190euploid blastocysts. Blastocyst-stage embryos underwent biopsy and trophectoderm cells were extracted for aneuploidyscreening using IIlumina NGS protocol. Mosaic information for each chromosome was obtained. Forthis study, only whole chromosomal mosaicism was considered; segmental mosaicism wascategorized as euploid embryos. These embryos were further stratified by maternal age group. Result The transfer of mosaic embryos resulted in higher clinical pregnancy rate (60% vs 57.5%) andimplantation rate (57% vs 52.9%) compared to euploid embryos. In our series, no miscarriage wasreported in this group of patients. Conclusion In concordance with most published studies, we found that mosaicism is not associated withadvanced maternal age. Unlike aneuploidy which arises from meiotic error, mosaicism on the otherhand is caused by impaired mitotic chromosome segregation which does not appear to increase withadvancing maternal age. Therefore, while there is a direct relationship between advanced maternalage and aneuploidy, age is not a contributor to mosaicism. Contrary to the experience of other groups, we have observed similar rate of implantation in mosaicembryo transfers as compared to euploid embryo transfers. This may be the consequence of thesmall sample size of mosaic embryos transfer (<10% of total blastocysts transferred) in our series. Similarly, no miscarriage was reported. PGS in its current form does have limitations. Implantation rates can potentially be further improvedby developing a clearer understanding of the impact of mosaic embryos. Chromosomal mosaicism refers to the presence of two or more cell lines with a differentchromosome content in an individual or tissue sample. Besides aneuploidy, the use of highresolution NGS for Pre-implantation Genetic Diagnosis (PGS) detects the presence of mosaicism introphectoderm biopsies. The transfer of mosaic embryos has been shown to result in lowerimplantation rates and higher miscarriage rates as compared to euploid transfer. This study aims to evaluate whether there is an association between mosaicism and maternal ageand the clinical pregnancy and implantation rate of mosaic embryos transfer. A retrospective observational study of 533 blastocyst-stage embryos analysed by NGS from IVFcycles from Sunfert International Fertility Centre over the course of 2015 to 2016 was performed. Ofthose analysed, 197 blastocysts were transferred. The clinical outcome obtained after the transfer ofwhole chromosomal mosaic embryos was compared with those resulting from a control group 190euploid blastocysts. Blastocyst-stage embryos underwent biopsy and trophectoderm cells were extracted for aneuploidyscreening using IIlumina NGS protocol. Mosaic information for each chromosome was obtained. Forthis study, only whole chromosomal mosaicism was considered; segmental mosaicism wascategorized as euploid embryos. These embryos were further stratified by maternal age group. The transfer of mosaic embryos resulted in higher clinical pregnancy rate (60% vs 57.5%) andimplantation rate (57% vs 52.9%) compared to euploid embryos. In our series, no miscarriage wasreported in this group of patients. In concordance with most published studies, we found that mosaicism is not associated withadvanced maternal age. Unlike aneuploidy which arises from meiotic error, mosaicism on the otherhand is caused by impaired mitotic chromosome segregation which does not appear to increase withadvancing maternal age. Therefore, while there is a direct relationship between advanced maternalage and aneuploidy, age is not a contributor to mosaicism.

Concordance between different trophectoderm biopsy sites and the inner cell mass of chromosomal composition measured with a next-generation sequencing platform

STUDY QUESTIONIn PGS, does chromosomal constitution differ among trophectoderm (TE) biopsy sites and between them and the inner cell mass (ICM)?SUMMARY ANSWERThe ploidy concordance between ICM and TE was independent of whether the biopsy site in the TE was near to or far from the ICM.WHAT IS KNOWN ALREADYTE biopsies are considered less harmful to developing embryos than blastomere biopsies. Removal of multi-cellular samples permits high-resolution next-generation sequencing (Veriseq NGS) to detect aneuploidy present in a minority of cells (mosaicism of diploid and aneuploid cells). However, the prevalence of ploidy discrepancies between different TE biopsy sites and the ICM, as well as confined mosaicism (aneuploidy only in a particular area), has not been established.STUDY DESIGN, SIZE, DURATIONBiopsies were taken from a site opposite to the ICM (TE1), near the ICM (TE2) and within the ICM of the same embryo in 33 donated blastocysts obtained from 12 volunteer patients. The samples were analyzed by the Veriseq NGS to assess ploidy concordance.PARTICIPANTS/MATERIALS, SETTING, METHODSThe mean age of the patients was 34.4 years, and samples from all three biopsy sites were achieved in 29 frozen thawed blastocysts. The aneuploid percentage in each sample was interpreted by Veriseq NGS at the finest resolution involving the number of reads after filtering, sample overall noise score, and average quality/alignment scores according to the Veriseq quality control assessment. Ploidy concordance was then assessed between different TE fractions, and between the TE and ICM.MAIN RESULTS AND THE ROLE OF CHANCEThe euploid rates were similar in the TEs and ICM, and no preferential allocation of euploid lineage within a blastocyst was demonstrated. Whether the biopsy site in the TE was near to or far from the ICM, the chromosomal consistency rate was similar [TE1-to-ICM, 86.2% (25/29) versus TE2-to-ICM, 89.7% (26/29); P = 1.0], suggesting that the cells with different chromosomal components may spread randomly throughout the TE. The following two types of inconsistent PGS conclusions between TE and ICM due to confined mosaicism were observed: (i) euploid TE with mosaic ICM (3%) (1/29); and (ii) mosaic TE with euploid ICM (3%) (1/29) or with aneuploid ICM (7%) (2/29). Thus, the overall rate of confined mosaicism was 14% (4/29).LARGE SCALE DATAN/A.LIMITATION, REASONS FOR CAUTIONThe approach used in the present study was affected by biopsy manipulation limitations involving possible cell contamination and the technical challenge of comprehensive chromosomal screening (CCS) procedures.WIDER IMPLICATIONS OF THE FINDINGSThe rate of confined mosaicism in the blastocysts was estimated in this preliminary study, thus, specifying the incidence of biological sampling biases. The results also verified the random distribution of different cell lineages, and the representative value of a single biopsied sample from the TE.STUDY FUNDING AND CONFLICT OF INTEREST(S)No external funding was obtained; all the authors declare no conflicts of interest regarding this study.

Embryos classified as low-grade mosaic (

The Preimplantation Genetic Diagnosis International Society (PGDIS) recommend the use of the high resolution next-generation sequencing (hr-NGS) that can detect mosaicism in excess of 20%. Therefore, embryos with less than 20% aneuploidy in the TE sample are considered as euploid and routinely transferred. Those over 80% as aneuploid and subsequently discarded. The 20-80% range as mosaic and transferred with caution, only in absence of euploid embryos. These cutoffs are currently debated. We recently reported the birth of 17 healthy babies from the transfer of 33 low grade (<50%) mosaic embryos with an overall live birth rate (LBR) and abortion rate (AR) similar to the LBR and AR obtained with euploid embryos (Rubino et al., 2018). The current study reports a grater number of cases. Retrospective analysis of the pregnancy outcome of replaced low rate mosaic embryos (20%-50% abnormal cells in the trophoctoderm biopsy) as diagnosed with the use of hr-NGS. This study included only patients that received a single embryo transfer of low rate mosaic embryos and for which the delivery of a newborn could be confirmed. NGS was performed by Nexgenomics, LLC, Pasadena by means of platform Veryseq NGS (Illumina). Nexgenomics position is to use an artificial cutoff of 50% to classify all the mosaicism events as normal or abnormal. Mosaicism <20% is considered not differentiable from technical noise. Low rate mosaic embryos were transferred when euploid embryos were not available. The patients of the study group were matched 1:2 with the patients of the control group (only single euploid embryo transferred) for age, embryo quality, use of an egg donor and/or gestational carrier and period of IVF treatment. All pregnancies that went to term were confirmed to have a normal karyotype. The results are summarized in Table 1. No birth defects were reported. We reported the birth of 33 babies with confirmed normal karyotype after the transfer of 55 low grade mosaic embryos. LBR and AR were similar to the ones obtained with the transfer of euploid embryos suggesting that low grade mosaic embryos (<50%) can have the same potential to give healthy newborn as euploid embryos and the PGDIS guidelines needs to be reconsidered. As far as we know this is the second study that provides data about the LBR of mosaic embryos (Greco et al., 2015). No deliveries were obtained from the transfer of complex mosaic embryos.Table 1Comparison of the pregnancy outcomes of low rate mosaic and control embryosType of mosaicismSegmental*Single-Double 1-12**Single-Double 13-22**X-YComplexTotalControlsAge38.8±6.440.8±6.936.3±5.049.136.2±4.039.1±6.439.3±6.3N of transfer cycles2215121555110Embryos transferred2215121555110Embryos Implanted16109113773Implantation rate (IR)72.3%66.7%75.0%-067.3%66.4%Abortions11101410Abortion rate (AR)6.3%10.0%11.1%--10.8%13.7%Deliveries1598103363Live birth rate(LBR)68.2%60.0%66.7%--60.0%57.3%*Segmental: regarding a piece of chromosome;**Single-double 1-12: regarding one of two entire chromosomes from number 1 to number 12;***Single-double 13-22: regarding one or two entire chromosomes from number 13 to number 22. Open table in a new tab