Nanopore sequencing with T2T-CHM13 for accurate detection and preventing the transmission of structural rearrangements in highly repetitive heterochromatin regions in human embryos.

BACKGROUND: Structural rearrangements in highly repetitive heterochromatin regions can result in miscarriage or foetal malformations; however, detecting and preventing the transmission of these rearrangements has been challenging. Recently, the completion of sequencing of the complete human genome (T2T-CHM13) has made it possible to accurately characterise structural rearrangements in these regions. We developed a method based on T2T-CHM13 and nanopore sequencing to detect and block structural rearrangements in highly repetitive heterochromatin sequences. METHODS: T2T-CHM13-based "Mapping Allele with Resolved Carrier Status" was performed for couples who carry structural rearrangements in heterochromatin regions. Using nanopore sequencing and the T2T-CHM13 reference genome, the precise breakpoints of inversions and translocations close to the centromere were detected and haplotypes were constructed using flanking single-nucleotide polymorphisms (SNPs). Haplotype linkage analysis was then performed by comparing consistent parental SNPs with embryonic SNPs to determine whether the embryos carried hereditary inversions or balanced translocations. Based on copy number variation and haplotype linkage analysis, we transplanted normal embryos, which were further verified by an amniotic fluid test. RESULTS: To validate this approach, we used nanopore sequencing of families with inversions and reciprocal translocations close to the centromere. Using the T2T-CHM13 reference genome, we accurately detected inversions and translocations in centromeres, constructed haplotypes and prevented the transmission of structural rearrangements in the offspring. CONCLUSIONS: This study represents the first successful application of T2T-CHM13 in human reproduction and provides a feasible protocol for detecting and preventing the transmission of structural rearrangements of heterochromatin in embryos.

Nuclear-cytoplasmic asynchrony in oocyte maturation caused by TUBB8 variants via impairing microtubule function: a novel pathogenic mechanism.

BACKGROUND: TUBB8, a crucial gene encoding microtubule protein, plays a pivotal role in cellular processes. Deleterious TUBB8 variants have been shown to significantly hinder oocyte maturation. In this study, we conducted an in vitro investigation using TUBB8 mutant mouse oocytes to elucidate the pathogenic mechanisms of TUBB8 variants in oocyte nuclear and cytoplasmic maturation. METHODS: A mutant model was successfully established in mouse oocytes via microinjection to further investigate the effects of four novel discovered TUBB8 mutations on the nuclear and cytoplasmic maturation of mouse oocytes. Immunofluorescence and confocal microscopy were performed to observe the cortical polarity and spindle and of mutant oocytes. Active mitochondrial staining was performed to analyze mitochondrial distribution patterns. Endoplasmic reticulum and Ca2+ staining were conducted to assess ER distribution and cytoplasmic calcium ion concentration in oocytes. RESULTS: In mouse oocytes, TUBB8 variants (p.A313V, p.C239W, p.R251Q, and p.G96R) resulted in a reduction of the first polar body extrusion rate, disruption of spindle assembly, and abnormal chromosome distribution. Additionally, these variants induced oocyte organelle abnormalities, including anomalies in mitochondrial redistribution and endoplasmic reticulum stress compared to the wild-type. CONCLUSION: Deleterious TUBB8 variants could disrupt microtubule function, affecting critical processes such as spindle assembly, chromosome distribution, and organelle rearrangement during oocyte meiosis. These disruptions culminate in compromised nuclear-cytoplasmic maturation, consequently giving rise to oocyte maturation defects.

Integrated treatment guided by RNA-seq-based endometrial receptivity assessment for infertility complicated by MEN1.

Background: Preimplantation genetic testing (PGT) serves as a tool to avoid genetic disorders in patients with known genetic conditions. However, once a selected embryo is transferred, implantation success is attained independent of embryo quality. Using PGT alone is unable to tackle implantation failure caused by endometrial receptivity (ER) abnormalities in these patients. Methods: We validated our newly developed RNA-seq-based ER test (rsERT) in a retrospective cohort study including 511 PGT cycles and reported experience in treating an infertile female patient complicated by multiple endocrine neoplasia type 1 (MEN1). Results: Significant improvement in the clinical pregnancy rate was found in the performed personalized embryo transfer (pET) group (CR, 69.7%; P = 0.035). In the rare MEN1 case, pET was done according to the prediction of the optimal time of window of implantation after unaffected blastocysts were obtained by PGT-M, which ultimately led to a healthy live birth. However, none of the mRNA variants identified in the patient showed a strong association with the MEN1 gene. Conclusions: Applying the new rsERT along with PGT improved ART outcomes and brought awareness of the importance of the ER examination in MEN1 infertile female patients. MEN1-induced endocrine disorder rather than MEN1 mutation contributes to the ER abnormality. Trial Registration: Reproductive Medicine Ethics Committee of Xiangya Hospital Registry No.: 2022010.

Nanopore sequencing for detecting reciprocal translocation carrier status in preimplantation genetic testing.

BACKGROUND: Balanced reciprocal translocation (BRT) is one of the most common chromosomal abnormalities that causes infertility, recurrent miscarriage, and birth defects. Preimplantation genetic testing (PGT) is widely used to select euploid embryos for BRT carriers to increase the chance of a healthy live birth. Several strategies can be used to distinguish reciprocal translocation carrier embryos from those with a normal karyotype; however, these techniques are time-consuming and difficult to implement in clinical laboratories. In this study, nanopore sequencing was performed in two reciprocal translocation carriers, and the results were validated using the next-generation sequencing-based method named, "Mapping Allele with Resolved Carrier Status" (MaReCs). RESULTS: The translocation breakpoints in both reciprocal translocation carriers were accurately identified by nanopore sequencing and were in accordance with the results obtained using MaReCs. More than one euploid non-balanced translocation carrier embryo was identified in both patients. Amniocentesis results revealed normal karyotypes, consistent with the findings by MaReCs and nanopore sequencing. CONCLUSION: Our results suggest that nanopore sequencing is a powerful strategy for accurately distinguishing non-translocation embryos from translocation carrier embryos and precisely localizing translocation breakpoints, which is essential for PGT and aids in reducing the propagation of reciprocal translocation in the population.

Chromosomal concordance between babies produced by the preimplantation genetic testing for aneuploidies and trophectoderm biopsies: A prospective observational study.

OBJECTIVES: Contributed to the development of next-generation sequencing (NGS) technology, more and more chromosomally mosaic and aneuploid embryos are discovered during the preimplantation genetic testing for aneuploidy (PGT-A) cycles. Because mosaicism and aneuploidy are routine phenomena throughout human pre- and post-implantation development. The benefit of implanting such mosaicism or aneuploidies detected by precise NGS remains controversial. This study aimed to investigate chromosomal concordance between babies produced by PGT-A and trophectoderm (TE) biopsies, and whether precise NGS resolution would reduce the development of an abnormal embryo in PGT cycles. STUDY DESIGN: Peripheral blood samples from 17 PGT-A babies were collected to compare with TE biopsy results at different NGS resolutions. RESULTS: 16 euploid embryos diagnosed by 10 Mb resolution developed into 16 healthy babies with normal copy number variations (CNVs). One mosaic embryo diagnosed by both 10 Mb and 4 Mb resolution also produced a euploid baby finally. Among them, four euploid embryos diagnosed by 10 Mb NGS, showed segmental aneuploidy at 4 Mb NGS resolution. Four of them developed into euploid babies with normal CNVs finally. CONCLUSIONS: NGS at 10 Mb resolution is accurate enough to diagnose viable embryos. A more precise NGS resolution (e.g., 4 Mb resolution) results in discard of some potentially viable embryos. It is suggested to analyze the TE biopsy at both 10 Mb and 4 Mb resolutions to identify embryos with adverse chromosomal aberrations, but using 10 Mb resolution for guide transfer to increase a development chance of an embryo. TRIAL REGISTRATION: www. CLINICALTRIALS: gov, identifier ChiCTR2100042522.

Arrested Cells/Cellular Debris Expelled from Blastocysts Is Self-Correction Phenomenon During Early Embryonic Development.

Arrested cells/ cellular debris is component left in the zona pellucida after blastocyst hatching. To identify whether expelling arrested cells/cellular debris from blastocysts is a process of human embryo self-correction by eliminating abnormal cells, 21 pairs of trophectoderm (TE) biopsies and the corresponding arrested cells/cellular debris expelled from the blastocysts from July to December 2020 were collected and analyzed using next-generation sequencing (NGS). Then, the NGS results of TE biopsies and the corresponding arrested cells/cellular debris were compared. We identified that 47.6% of blastocysts (10/21) were aneuploidies and mosaicism. A total of 18 groups of arrested cells/cellular debris (85.7%) expelled from blastocysts were abnormal, including nine aneuploid embryos and nine euploid embryos. In the arrested cells/cellular debris, all the chromosomes were affected. In conclusion, mosaicism and aneuploidies are common features of early embryonic development, and the arrested cells/cellular debris expelled from blastocysts provides evidence of early embryonic self-correction.

A novel homozygous mutation in the PADI6 gene causes early embryo arrest.

BACKGROUND: It has been proved that mutations in the PADI6 gene can cause early embryo arrest. This study describes a newly discovered mutation in PADI6 that expands the genetic spectrum of early embryo arrest. METHODS: Peripheral blood of a patient diagnosed with early embryo arrest was collected for whole-exome sequencing. Sanger sequencing was performed to confirm this mutation. The effects of the variant were investigated in human embryonic kidney 293T (HEK293T) cells using western blotting, real-time quantitative polymerase chain reaction, and immunofluorescence. RESULTS: A novel homozygous mutation in PADI6 was identified in the proband. The patient carried a frameshift insertion mutation c.558dupA (p.Thr187Asnfs*48), which was located in the protein arginine deiminase middle domain. The variant destroyed PADI6 protein expression and reduced PADI6 mRNA expression in HEK293T cells. CONCLUSIONS: The newly identified mutation in PADI6 accounts for early embryo arrest. It expands the spectrum of genetic causes and phenotypes of infertility in humans. These findings also provide an additional possible diagnostic marker for patients with recurrent in vitro fertilization/intracytoplasmic sperm injection failure.

Some infertile patients experience multiple in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) failure owing to recurrent early embryo arrest. However, the underlying mechanisms remain largely unknown. Due to the development of whole-exome sequencing, early embryo arrest has been confirmed as a type of Mendelian disease. This study aimed to identify the genetic cause of early embryo arrest in patients and to expand the genetic spectrum. Furthermore, it can help doctors offer better suggestions to such patients and prevent patients from suffering from multiple IVF/ICSI failures.

Mutation analysis of the TUBB8 gene in primary infertile women with oocyte maturation arrest.

BACKGROUND: Oocyte maturation arrest at metaphase I leads to fertilization failure in humans. In early embryos, the tubulin beta 8 class VIII (TUBB8) encodes a ß-tubulin isotype and aids in the assembling of the human oocyte spindle. Mutations in the TUBB8 potentially interfere with human oocyte maturation-a crucial prerequisite for fertilization and subsequent embryonic development. This study aims to investigate the novel mutations in TUBB8 and their prevalence. RESULTS: Hundred fertile women (controls) and eleven infertile women with oocyte maturation arrest were chosen for the study. A total of five TUBB8 heterozygous/homozygous mutations were found in eleven infertile females (p.A313V, p.C239W, p.R251Q, p.P358L, and p.G96R). The Exome Aggregation Consortium (ExAC), SIFT, and PolyPhen-2 analyses revealed that p. A313V has unknown pathogenicity and p.C239W, p.R251Q, p.P358L, and p.G96R have possible pathogenicity. The wild-type (WT) and four mutant gene constructs were transfected to Hela cells. The Western blot analysis indicates that the TUBB8 expression of the p.C239W, p.R251Q, and p.G96R mutations was significantly decreased than that of WT. The immunofluorescence assay showed that the Hela cells transfected with either p.C239W, p.R251Q, or p.G96R mutations exhibited the disrupted microtubule structure, revealing a significant difference in the organization of the microtubule network compared to the WT. CONCLUSIONS: We identified three novel variants and two reported variants out of 11 infertile women with oocyte metaphase I arrest. According to the present data, TUBB8 gene variants account for 31.96% of all participants (109/341) with oocyte maturation arrest.

Identification of factors related to fertilization failure in in vitro fertilization-embryo transfer. / 体外受精-èƒšèƒŽç§»æ¤ä¸­å—ç²¾å¤±è´¥çš„ç›¸å ³å› ç´ .

OBJECTIVES: To investigate the possible factors relevant to fertilization failure in in vitro fertilization-embryo transfer (IVF-ET). METHODS: The medical records of 4 205 infertile patients undergoing IVF-ET treatment at the Reproductive Medicine Center, Xiangya Hospital, Central South University from January 2016 to December 2017 were collected. The patients were divided into a complete fertilization failure group, a low fertilization rate group, and a control group based on fertilization rate. We examined the associations among the 3 groups in terms of female age, duration of infertility, duration of stimulation, gonadotropin (Gn) dosage, follicle-stimulating hormone (FSH) dosage, and total number of retrieved oocytes. According to theincidence factors, the patients were divided into a single female factor group, a single male factor group and a unisex factor group, and the correlation analysis of incidence factor among the 3 groups was performed. The patients were divided into a primary infertility and a secondary infertility in accordance with the type of infertility. We analyzed the correlation of infertility type among the three groups. Risk factors for complete fertilization failure and low fertilization rate in IVF-ET were obtained by stepwise multiple linear regression analysis. RESULTS: Primary infertility, long infertility duration, total number of retrieved oocytes, and unisex factor were associated with completefertilization failure and low fertilization rate in IVF-ET (P<0.05), but female age, duration of stimulation, FSH dosage as well as Gn dosage were not correlated with complete fertilization failure and low fertilization rate in IVF-ET (P>0.05). Stepwise multiple linear regression analysis showed that the incidence factor, type of infertility, and infertility duration were independent influential factors for complete fertilization failure and low fertilization rate. CONCLUSIONS: Complete fertilization failure and low fertilization rate in IVF-ET are related to duration of infertility, total number of retrieved oocytes, cause of onset, and type of infertility, but they are not relevant to female age, duration of stimulation, and Gn and FSH dosage.

Distribution of fragile X mental retardation 1 CGG repeat and flanking haplotypes in a large Chinese population.

Fragile X syndrome is mainly caused by a CGG repeat expansion within the 5' UTR of the fragile X mental retardation 1 (FMR1) gene. Previous analyses of the FMR1 CGG repeat patterns and flanking haplotypes in Caucasians and African Americans have identified several factors that may influence repeat instability. However, the CGG repeat patterns and distribution for FRAXAC2 have not yet been investigated in mainland Chinese. We surveyed the CGG repeat lengths in 1113 Han Chinese (534 males and 579 females), and the CGG repeat patterns of 534 males were determined by sequence analysis. We also explored the flanking haplotypes (DXS548-FRAXAC1-FRAXAC2) in 566 unaffected and 28 unrelated fragile X Chinese males. The most frequent alleles for DXS548 and FRAXAC1 were identical between our Chinese population and other Asian populations. We identified several low-abundance alleles for DXS548 and FRAXAC1 not found in previous studies in mainland Chinese and Taiwanese cohorts. The most frequent allele was (CGG)29 followed by (CGG)30, and the most frequent patterns were 9 + 9 + 9, 10 + 9 + 9, and 9 + 9 + 6 + 9, similar to those in Singaporeans. We identified only one premutation female carrier with 89 CGG repeats in the 1113 Han Chinese. A few associations between the CGG repeat patterns and flanking haplotypes were determined in this study. In general, the Chinese population had a smaller number of alleles and lower expected heterozygosity for all three STR markers and FRAXA locus when compared with Caucasians and African Americans. We identified a novel haplotype 7-3-5 + that is significantly associated with the full mutation.

Mutational analyses of the FMR1 gene in Chinese pediatric population of fragile x suspects: low tolerance for point mutation.

CGG repeat expansion is the most common cause of fragile X syndrome. Numerous efforts have been made to identify novel mutations in patients with intellectual disability, developmental delay, and/or autism. To evaluate the mutational spectrum in the at-risk Chinese population, 60 pediatric patients presenting fragile X traits but normal-sized CGG repeats were sequenced for all 17 exons and regulatory regions in FMR1. A c.879A>C mutation, reported to alter a neighboring splicing, was detected in a severely retarded male and his normal mother. However, the exon junction appears unaffected. A 237-kb deletion covering the entire FMR1 was identified to cause moderate intellectual disability and marked hyperactivity in an 8-year-old boy. The 5' and 3' breakpoints were buried in the surrounding long interspersed and short interspersed elements, respectively. In general, missense mutations do not commonly cause fragile X syndrome, whereas deletions should be considered with caution in patient referrals presenting with developmental delay and/or ordinary retardation.

Cryptic FMR1 mosaic deletion in a phenotypically normal mother of a boy with fragile X syndrome: case report.

BACKGROUND: Increasing number of case reports of mosaic mutations and deletions have better armed clinicians and geneticists with more accurate and focused prenatal diagnoses. Since mosaicism means a significant increase of recurrence risk, detailed parental profiling is essential for risk assessments. CASE PRESENTATION: We here describe a clinically unaffected mother with a son who had fragile X syndrome (FXS) caused by a large deletion that includes the entire FMR1. To assess the recurrence risk regarding her second pregnancy, a series of genetic tests were conducted to establish this mother's status. Routine single nucleotide polymorphism (SNP) array and fluorescence in situ hybridisation (FISH) analyses detected two normal FMR1 copies in her blood. However, in-depth studies across the deleted region revealed varying proportions of mosaic deletion in her somatic tissues: lowest in the blood, moderately higher in the skin, urine sediment and menstrual discharge and highest in her eyebrow. Further FISH analysis of her skin-derived fibroblasts confirmed mosaicism of 13%. CONCLUSION: To our knowledge, this is the first characterized case of a female who was mosaic for an FMR1 deletion and extensive investigation of her mosaic status provided valuable information for her reproduction choices. Our case report may also alert clinicians and geneticists that a cryptic mosaicism with somatic heterogeneity should be carefully considered in families with children having clinically defined 'de novo' mutations, to avoid a second pregnancy with identical genetic abnormalities.

Towards understanding RNA-mediated neurological disorders.

RNA-mediated mechanisms of disease pathogenesis in neurological disorders have been recognized in the context of certain repeat expansion disorders. This RNA-initiated neurodegeneration may play a more pervasive role in disease pathology beyond the classic dynamic mutation disorders. Here, we review the mechanisms of RNA toxicity and aberrant RNA processing that have been implicated in ageing-related neurological disorders. We focus on diseases with aberrant sequestration of RNA-binding proteins, bi-directional transcription, aberrant translation of repeat expansion RNA transcripts (repeat-associated non-ATG (RAN) translation), and the formation of pathological RNA:DNA secondary structure (R-loop). It is likely that repeat expansion disorders arise from common mechanisms caused by the repeat expansion mutations. However, the context of the repeat expansion determines the specific molecular consequences, leading to clinically distinct disorders.

Isolation and characterization of microsatellite loci in the endangered tree Diplopanax stachyanthus (Araliaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed in Diplopanax stachyanthus to investigate the population genetics of this endangered tree. METHODS AND RESULTS: Using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol, 15 microsatellite markers were developed in D. stachyanthus and evaluated for their variability in 25 samples from a natural population. For the 11 polymorphic loci, the number of alleles ranged from two to eight, while the observed and expected heterozygosities ranged from 0.5200 to 0.7600 and 0.4200 to 0.7813, respectively. Their cross-taxa transferability was also examined in Acanthopanax gracilistylus, Tetrapanax papyrifer, Cornus controversa, and Dendrobenthamia japonica var. chinensis, and four to 15 loci proved amplifiable in these species. CONCLUSIONS: These microsatellite markers could be employed to investigate the population genetics of D. stachyanthus, and may potentially be applicable to other related species.