Nondisjunction Research Articles

Drosophila ring chromosomes interact with sisters and homologs to produce anaphase bridges in mitosis.

Ring chromosomes are known in many eukaryotic organisms, including humans. They are typically associated with a variety of maladies, including abnormal development and lethality. Underlying these phenotypes are anaphase chromatin bridges that can lead to chromosome loss, nondisjunction and breakage. By cytological examination of ring chromosomes in Drosophila melanogaster we identified five causes for anaphase bridges produced by ring chromosomes. Catenation of sister chromatids appears to be the most common cause and these bridges frequently resolve during anaphase, presumably by the action of topoisomerase II. Sister chromatid exchange and chromosome breakage followed by sister chromatid union also produce anaphase bridges. Mitotic recombination with the homolog was rare, but was another route to generation of anaphase bridges. Most surprising, was the discovery of homolog capture, where the ring chromosome was connected to its linear homolog in anaphase. We hypothesize that this is a remnant of mitotic pairing and that the linear chromosome is connected to the ring by multiple wraps produced through the action of topoisomerase II during establishment of homolog pairing. In support, we showed that in a ring/ring homozygote the two rings are frequently catenated in mitotic metaphase, a configuration that requires breaking and rejoining of at least one chromosome.

Unambiguous chromosome identification reveals the factors impacting irregular chromosome behaviors in allotriploid AAC Brassica.

The major irregular chromosome pairing and mis-segregation were detected during meiosis through unambiguous chromosome identification and found that allotriploid Brassica can undergo meiosis successfully and produce mostly viable aneuploid gametes. Triploids have played a crucial role in the evolution of species by forming polyploids and facilitating interploidy gene transfer. It is widely accepted that triploids cannot undergo meiosis normally and predominantly produce nonfunctional aneuploid gametes, which restricts their role in species evolution. In this study, we demonstrated that natural and synthetic allotriploid Brassica (AAC), produced by crossing natural and synthetic Brassica napus (AACC) with Brassica rapa (AA), exhibits basically normal chromosome pairing and segregation during meiosis. Homologous A chromosomes paired faithfully and generally segregated equally. Monosomic C chromosomes were largely retained as univalents and randomly entered daughter cells. The primary irregular meiotic behaviors included associations of homoeologs and 45S rDNA loci at diakinesis, as well as homoeologous chromosome replacement and premature sister chromatid separation at anaphase I. Preexisting homoeologous arrangements altered meiotic behaviors in both chromosome irregular pairing and mis-segregation by increasing the formation of A-genomic univalents and A-C bivalents, as well as premature sister chromatid separation and homologous chromosome nondisjunction. Meiotic behaviors depended significantly on the genetic background and heterozygous homoeologous rearrangement. AAC triploids mainly generated aneuploid gametes, most of which were viable. These results demonstrate that allotriploid Brassica containing an intact karyotype can proceed through meiosis successfully, broadening our current understanding of the inheritance and role in species evolution of allotriploid.

Drosophila ring chromosomes interact with sisters and homologs to produce anaphase bridges in mitosis.

Ring chromosomes are known in many eukaryotic organisms, including humans. They are typically associated with a variety of maladies, including abnormal development and lethality. Underlying these phenotypes are anaphase chromatin bridges that can lead to chromosome loss, nondisjunction and breakage. By cytological examination of ring chromosomes in Drosophila melanogaster we identified five causes for anaphase bridges produced by ring chromosomes. Catenation of sister chromatids is the most common cause and these bridges frequently resolve during anaphase, presumably by the action of topoisomerase II. Sister chromatid exchange and chromosome breakage followed by sister chromatid union also produce anaphase bridges. Mitotic recombination with the homolog was rare, but was another route to generation of anaphase bridges. Most surprising, was the discovery of homolog capture, where the ring chromosome was connected to its linear homolog in anaphase. We hypothesize that this is a remnant of mitotic pairing and that the linear chromosome is connected to the ring by multiple wraps produced through the action of topoisomerase II during establishment of homolog pairing. In support, we showed that in a ring/ring homozygote the two rings are frequently catenated in mitotic metaphase, a configuration that requires breaking and rejoining of at least one chromosome.

Double Autosomal Aneuploidy: A Case of Trisomy 18 and 21 Mosaicism in A Neonate with Clinical Down Syndrome

Background: Double aneuploidy is the phenomenon where two aneuploidies co-exist in the same individual, usually involving one autosomal chromosome and one sex chromosome. Double autosomal aneuploidy is rare and usually results in spontaneous abortions. There are only six published case reports of liveborn with trisomy 18 and trisomy 21 and all of which involve mosaicism. Case Presentation: This case report documents an infant born at 35 weeks with phenotypic features of Down Syndrome. However, cytogenetic analysis showed a mosaic of both trisomy 18 and 21. The patient initially had patent ductus arteriosus requiring operative closure and has congenital hypothyroidism. Conclusion: We describe five possible pathways leading to this phenomenon, including error during meiosis, meiotic nondisjunction, mitotic nondisjunction, sequential mitotic segregation and chimerism. This case report reiterates the significance of traditional cytogenetic analysis in children with features of Down Syndrome to detect any further abnormalities.

Folate gene polymorphisms CBS 844ins68 and RFC1 A80G and risk of Down syndrome offspring in young Iranian women: A cross-sectional study

Background: Cytogenetics and association studies showed that folate gene polymorphisms can increase the risk of chromosomal nondisjunction and aneuploidies. The folate-metabolizing gene polymorphisms in Down syndrome mothers (DSM) have been assessed in a variety of populations. Reduced folate carrier 1 (RFC1) and cystathionine beta-synthase (CBS) are key enzymes in folate metabolism.
 Objective: 2 common polymorphisms, CBS 844ins68 and RFC1 A80G, were analyzed to determine their probable risk for having Down syndrome (DS) babies in young mothers of Khuzestan province, Iran.
 Materials and Methods: This study was conducted on 100 mothers who had trisomy 21 DS children. 100 age- and ethnic-matched mothers with at least 2 healthy children and no history of abnormal pregnancies were considered as control. The samples were collected from all the mothers from June 2019 to April 2021. Genomic DNA was extracted from peripheral blood. The CBS-844ins68 and RFC1-A80G were genotyped using polymerase chain reaction-electrophoresis and restriction fragment length polymorphism, respectively.
 Results: The frequency of RFC1 AG and GG genotypes in DSM was significantly higher than the control mothers (odds ratio [OR] of 2.38 and 3.07, respectively). The heterozygote genotype of CBS 844ins68 was significantly more prevalent among DSM than the control (OR: 2.419). The OR was significantly increased to 6.667 when the homozygote of both variants was found together.
 Conclusion: Studying polymorphisms possibly increases the susceptibility of having a DS child. However, ethnicity, nutrition, and epistatic interactions are considerable factors to be evaluated in future studies.
 Key words: Down syndrome, Folic acid, Polymorphism, CBS, RFC1.

The severity of meiotic aneuploidy is associated with altered morphokinetic variables of mouse oocyte maturation.

Is there an association between morphokinetic variables of meiotic maturation and the severity of aneuploidy following in vitro maturation (IVM) in the mouse? The severity of meiotic aneuploidy correlates with an extended time to first polar body extrusion (tPB1) and duration of meiosis I (dMI). Morphokinetic variables measured using time-lapse technology allow for the non-invasive evaluation of preimplantation embryo development within clinical assisted reproductive technology (ART). We recently applied this technology to monitor meiotic progression during IVM of mouse gametes. Whether there is a relationship between morphokinetic variables of meiotic progression and aneuploidy in the resulting egg has not been systematically examined at the resolution of specific chromosomes. Next-generation sequencing (NGS) is a robust clinical tool for determining aneuploidy status and has been reverse-translated in mouse blastocysts and oocytes. Therefore, we harnessed the technologies of time-lapse imaging and NGS to determine the relationship between the morphokinetics of meiotic progression and egg aneuploidy. Cumulus-oocyte complexes were collected from large antral follicles from hyperstimulated CD-1 mice. Cumulus cells were removed, and spontaneous IVM was performed in the absence or presence of two doses of Nocodazole (25 or 50 nM) to induce a spectrum of spindle abnormalities and chromosome segregation errors during oocyte meiosis. Comprehensive chromosome screening was then performed in the resulting eggs, and morphokinetic variables and ploidy status were compared across experimental groups (control, n = 11; 25 nM Nocodazole, n = 13; 50 nM Nocodazole, n = 23). We monitored IVM in mouse oocytes using time-lapse microscopy for 16 h, and time to germinal vesicle breakdown (tGVBD), tPB1, and dMI were analyzed. Following IVM, comprehensive chromosome screening was performed on the eggs and their matched first polar bodies via adaptation of an NGS-based preimplantation genetic testing for aneuploidy (PGT-A) assay. Bioinformatics analysis was performed to align reads to the mouse genome and determine copy number-based predictions of aneuploidy. The concordance of each polar body-egg pair (reciprocal errors) was used to validate the results. Ploidy status was categorized as euploid, 1-3 chromosomal segregation errors, or ≥4 chromosomal segregation errors. Additionally, aneuploidy due to premature separation of sister chromatids (PSSC) versus non-disjunction (NDJ) was distinguished. We applied and validated state-of-the-art NGS technology to screen aneuploidy in individual mouse eggs and matched polar bodies at the chromosome-specific level. By performing IVM in the presence of different doses of Nocodazole, we induced a range of aneuploidy. No aneuploidy was observed in the absence of Nocodazole (0/11), whereas IVM in the presence of 25 and 50 nM Nocodazole resulted in an aneuploidy incidence of 7.69% (1/13) and 82.61% (19/23), respectively. Of the aneuploid eggs, 5% (1/20) was due to PSSC, 65% (13/20) to NDJ, and the remainder to a combination of both. There was no relationship between ploidy status and tGVBD, but tPB1 and the dMI were both significantly prolonged in eggs with reciprocal aneuploidy events compared to the euploid eggs, and this scaled with the severity of aneuploidy. Eggs with ≥4 aneuploid chromosomes had the longest tPB1 and dMI (P < 0.0001), whereas eggs with one to three aneuploid chromosomes exhibited intermediate lengths of time (P < 0.0001). N/A. We used Nocodazole in this study to disrupt the meiotic spindle and induce aneuploidy in mouse oocytes. Whether the association between morphokinetic variables of meiotic progression and the severity of aneuploidy occurs with other compounds that induce chromosome segregation errors remain to be investigated. In addition, unlike mouse oocytes, human IVM requires the presence of cumulus cells, which precludes visualization of morphokinetic variables of meiotic progression. Thus, our study may have limited direct clinical translatability. We validated NGS in mouse eggs to detect aneuploidy at a chromosome-specific resolution which greatly improves the utility of the mouse model. With a tractable and validated model system for characterizing meiotic aneuploidy, investigations into the molecular mechanisms and factors which may influence aneuploidy can be further elaborated. Time-lapse analyses of morphokinetic variables of meiotic progression may be a useful non-invasive predictor of aneuploidy severity. This work was supported by the Bill & Melinda Gates Foundation (INV-003385). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. The authors have no conflict of interest to disclose.

Whole transcriptome screening for novel genes involved in meiosis and fertility in Drosophila melanogaster

Reproductive success requires the development of viable oocytes and the accurate segregation of chromosomes during meiosis. Failure to segregate chromosomes properly can lead to infertility, miscarriages, or developmental disorders. A variety of factors contribute to accurate chromosome segregation and oocyte development, such as spindle assembly and sister chromatid cohesion. However, many proteins required for meiosis remain unknown. In this study, we aimed to develop a screening pipeline for identifying novel meiotic and fertility genes using the genome of Drosophila melanogaster. To accomplish this goal, genes upregulated within meiotically active tissues were identified. More than 240 genes with no known function were silenced using RNA interference (RNAi) and the effects on meiosis and fertility were assessed. We identified 94 genes that when silenced caused infertility and/or high levels of chromosomal nondisjunction. The vast majority of these genes have human and mouse homologs that are also poorly studied. Through this screening process, we identified novel genes that are crucial for meiosis and oocyte development but have not been extensively studied in human or model organisms. Understanding the function of these genes will be an important step towards the understanding of their biological significance during reproduction.

MTHFR (C677T) polymorphism and its association with cytogenetic and clinical profile in individuals with primary amenorrhea

BackgroundAbnormal folate metabolism is a risk factor for DNA hypomethylation and chromosomal nondisjunction. MTHFR is a candidate gene for folliculogenesis and ovarian development. In the present study, we aimed to investigate the distribution of the MTHFR C677T polymorphism in individuals with primary amenorrhea and it’s association with the cytogenetic and clinical profile. The MTHFR polymorphism (C677T) was checked in 45 females with PA and 45 control females (age-matched) with regular menstrual cycles using polymerase chain reaction-restriction fragment length polymorphism.ResultsWe observed the CC genotype in 84.4% (n = 38) of the control group females and 48.9% (n = 22) of the case group females, CT genotype in 13.3% (n = 6) in the control group females and 24.4% (n = 11) in the case group females (p = 0.039; χ2 value 4.253; odd ratio 0.316, 95%CI 0.103–0.973) and TT genotype in 2.2% (n = 1) in the control group females and 26.7% (n = 12) in the case group females (p = 0.000266; χ2 value 13.294; odd ratio 0.048, 95%CI 0.006–0.397). Out of 45 case group females, 26 females showed 46,XX karyotype, 4 females showed male karyotype, 3 females showed pure Turner karyotype, 2 females were mosaic Turner and the rest of the females showed structural abnormalities like deletion, isochromosome and normal variants. The serum values revealed significantly lower levels (p = 0.032) of progesterone in the individuals with the TT genotype as compared to the CC genotype and the radiology profile showed a significant role of the MTHFR gene in ovarian development (p = 0.024).ConclusionWe suggest that the MTHFR polymorphism (C677T) might be responsible for the chromosomal nondisjunction in monosomy X females. It also influences the progesterone level and ovarian development, thus affecting folliculogenesis and the ovarian reserve responsible for primary amenorrhea.

An update on Telomeres Dynamics in Reproduction; Specifically in Females &amp; Ageing: A Narrative Review

The oocyte portrays a postmitotic cell having a longer life working as locus of reproductive ageing in women. Female germ cells replication occurs in fetal life which age right through reproductive period. Modes behind oocyte ageing are oxidative injury, mitochondrial impairment and disturbed protein inclusive of cohesion. BobEdwards invented the “generation line” at the time of oogonial proliferation in mouse oogenesis, where the initial oocytes to leave the oogonial proliferation while fetal life are the ones which undergo ovulation first at puberty and the last oocytes to leave the oogonial proliferation are the last to undergo ovulation in adult. Hence Kalmbach K group posited ‘’second hit’’ hypothesis of reproductive ageing for incorporating to plethora of characteristics of the oocyte ageing. The 1st hit being oocyte continuing in the older women pass via more cell cycles while fetal oogenesis. The 2nd hit was oocytes which continued had&gt;accrual of environmental &amp; endogenous oxidative injury right through woman’s life. Telomeres (T’s) might modulate both parts of oocyte ageing. T’s offer a mitotic clock, with T debilitation becoming a granted inimical sequelae of cell division due to end replication cell botheration. The guanine enrichment of the telomeres makes them more vulnerable to oxidative injury, in post mitotic cell also. Since telomerase,/ the reverse transcriptase is more adept in sustenance rather than T lengthening. Furthermore telomerase continues to be inactive in maximum oogenesis &amp; early generation. Thus oocytes possess small T’s on the border of viability. To corroborate this posit mice having induced T debilitation and women having natural telomeropathies have diminished ovarian reserve (DOR), aberrant embryonic generation &amp; infertility. Conversely sperms form all through life of male by ALT, have telomerase active progenitor, spermatogonia, longest body T’s. Numerous queries arise i) does normal T debilitation of human oocytes aid in meiotic nondisjunction (MN)? ii) does recombination dependent T lengthening make embryos more prone to MN / mosaicism? iii) can certain features of T’s work as oocyte quality markers.

Saxagliptin, a selective dipeptidyl peptidase-4 inhibitor, alleviates somatic cell aneugenicity and clastogenicity in diabetic mice.

Diabetes-related complications are becoming increasingly common as the global prevalence of diabetes increases. Diabetes is also linked to a high risk of developing cancer. This raises the question of whether cancer vulnerability is caused by diabetes itself or the use of antidiabetic drugs. Chromosomal instability, a source of genetic modification involving either an altered chromosomal number or structure, is a hallmark of cancer. Saxagliptin has been approved by the FDA for diabetes treatment. However, the detailed in vivo effects of prolonged saxagliptin treatment on chromosomal instability have not yet been reported. In this study, streptozotocin was used to induce diabetes in mice, and both diabetic and non-diabetic mice received saxagliptin for five weeks. Fluorescence in situ hybridization was conducted in combination with a bone marrow micronucleus test for measuring chromosomal instability. Our results indicated that saxagliptin is neither mutagenic nor cytotoxic, under the given treatment regimen. Diabetic mice had a much higher incidence of micronuclei formation, and a centromeric DNA probe was present inside the majority of the induced micronuclei, indicating that most of these were caused by chromosome nondisjunction. Conversely, diabetic mice treated with saxagliptin exhibited a significant decrease in micronuclei induction, which were centromeric-positive and centromeric-negative. Diabetes also causes significant biochemical changes indicative of oxidative stress, such as increased lipid peroxidation and decreased reduced/oxidized glutathione ratio, which was reversed by saxagliptin administration. Overall, saxagliptin, the non-mutagenic antidiabetic drug, maintains chromosomal integrity in diabetes and reduces micronuclei formation by restoring redox imbalance, further indicating its usefulness in diabetic patients.

A Method for Physical Analysis of Recombination Intermediates in Saccharomyces cerevisiae.

Meiosis is a process through which diploid cells divide into haploid cells, thus promoting genetic diversity. This diversity arises from the formation of genetic crossovers (COs) that repair DNA double-strand breaks (DSBs), through homologous recombination (HR). Deficiencies in HR can lead to chromosomal abnormality resulting from chromosomal nondisjunction, and genetic disorders. Therefore, investigating the mechanisms underlying effective HR is crucial for reducing genome instability. Budding yeast serves as an ideal model for studying HR mechanisms due to its amenability to gene modifications and the ease of inducing synchronized meiosis to yield four spores. During meiosis, at the DNA level, programmed DSBs are repaired as COs or non-crossovers (NCOs) through structural alterations in the nascent D-loop, involving single-end invasions(SEIs) and double-Holliday junctions(dHJs). This repair occurs using homologous templates rather than sister templates. This protocol, using Southern blotting, allows for the analysis and monitoring of changes in DNA structures in the recombination process. One-dimensional (1D) gel electrophoresis is employed to detect DSBs, COs, and NCOs, while two-dimensional (2D) gel electrophoresis is utilized to identify joint molecules (JMs). Therefore, physical analysis is considered the most effective method for investigating the HR mechanism. Our protocol provides more comprehensive information than previous reports by introducing conditions for obtaining a greater number of cells from synchronized yeast and a method that can analyze not only meiotic/mitotic recombination but also mitotic replication.

Aneuploidy in oocytes from women of advanced maternal age: analysis of the causal meiotic errors and impact on embryo development.

In oocytes of advanced maternal age (AMA) women, what are the mechanisms leading to aneuploidy and what is the association of aneuploidy with embryo development? Known chromosome segregation errors such as precocious separation of sister chromatids explained 90.4% of abnormal chromosome copy numbers in polar bodies (PBs), underlying impaired embryo development. Meiotic chromosomal aneuploidies in oocytes correlate with AMA (>35 years) and can affect over half of oocytes in this age group. This underlies the rationale for PB biopsy as a form of early preimplantation genetic testing for aneuploidy (PGT-A), as performed in the 'ESHRE STudy into the Evaluation of oocyte Euploidy by Microarray analysis' (ESTEEM) randomized controlled trial (RCT). So far, chromosome analysis of oocytes and PBs has shown that precocious separation of sister chromatids (PSSC), Meiosis II (MII) non-disjunction (ND), and reverse segregation (RS) are the main mechanisms leading to aneuploidy in oocytes. Data were sourced from the ESTEEM study, a multicentre RCT from seven European centres to assess the clinical utility of PGT-A on PBs using array comparative genomic hybridization (aCGH) in patients of AMA (36-40 years). This included data on the chromosome complement in PB pairs (PGT-A group), and on embryo morphology in a subset of embryos, up to Day 6 post-insemination, from both the intervention (PB biopsy and PGT-A) and control groups. ESTEEM recruited 396 AMA patients: 205 in the intervention group and 191 in the control group. Complete genetic data from 693 PB pairs were analysed. Additionally, the morphology from 1034 embryos generated from fertilized oocytes (two pronuclei) in the PB biopsy group and 1082 in the control group were used for statistical analysis. Overall, 461/693 PB pairs showed abnormal segregation in 1162/10 810 chromosomes. The main observed abnormal segregations were compatible with PSSC in Meiosis I (MI) (n = 568/1162; 48.9%), ND of chromatids in MII or RS (n = 417/1162; 35.9%), and less frequently ND in MI (n = 65/1162; 5.6%). For 112 chromosomes (112/1162; 9.6%), we observed a chromosome copy number in the first PB (PB1) and second PB (PB2) that is not explained by any of the known mechanisms causing aneuploidy in oocytes. We observed that embryos in the PGT-A arm of the RCT did not have a significantly different morphology between 2 and 6 days post-insemination compared to the control group, indicating that PB biopsy did not affect embryo quality. Following age-adjusted multilevel mixed-effect ordinal logistic regression models performed for each embryo evaluation day, aneuploidy was associated with a decrease in embryo quality on Day 3 (adjusted odds ratio (aOR) 0.62, 95% CI 0.43-0.90), Day 4 (aOR 0.15, 95% CI 0.06-0.39), and Day 5 (aOR 0.28, 95% CI 0.14-0.58). RS cannot be distinguished from normal segregation or MII ND using aCGH. The observed segregations were based on the detected copy number of PB1 and PB2 only and were not confirmed by the analysis of embryos. The embryo morphology assessment was static and single observer. Our finding of frequent unexplained chromosome copy numbers in PBs indicates that our knowledge of the mechanisms causing aneuploidy in oocytes is incomplete. It challenges the dogma that aneuploidy in oocytes is exclusively caused by mis-segregation of chromosomes during MI and MII. Data were mined from a study funded by ESHRE. Illumina provided microarrays and other consumables necessary for aCGH testing of PBs. None of the authors have competing interests. Data were mined from the ESTEEM study (ClinicalTrials.gov Identifier NCT01532284).

FRI452 A Rare Case of Klinefelter Syndrome, 48XXYY

Abstract Disclosure: P. Kachhadia: None. S. Khan: None. S. Aldasouqi: None. A. Abu Limon: None. Introduction: Klinefelter Syndrome (KS), 47XXY, is the most commonly seen chromosomal abnormality causing infertility, or primary hypogonadism. Other chromosomal variants are rare. We present a very rare case of nondisjunction of the sex chromosome resulting in a 48XXYY KS variant. Case Presentation: The patient is a 45-year-old male was referred to our clinic for evaluation of early onset osteoporosis. The patient was accompanied by his sister, given his cognitive delay. On initial physical exam, there was a concern about acromegaly with signs of frontal bossing, increased hand size, increased ring size and increased shoe size. More recent pictures of the patient showed significant changes in his facial appearance from few years prior. Other symptoms included night sweats. Physical exam revealed decreased testicular volume (less than 3mL bilaterally). Pituitary labs showed elevated prolactin, elevated LH, FSH and low free and total testosterone. GH, IGF-1 levels were within normal range. Chromosomal analysis showed an unexpected pattern of 48XXYY. In addition, MRI of the pituitary was done which showed increased infundibular stalk and increase in size of the pituitary gland which was thought to be secondary to pituitary microadenoma. DEXA scan confirmed osteoporosis. Patient was treated with testosterone injections and alendronate. The rationale for alendronate therapy was considered as a bridge to prevent fractures while the bone density would expectedly improve with androgen therapy, over time. Discussion: Although KS is commonly seen at a frequency of 1:500-1:1000 males, less common and variable phenotypical presentations can occur and can lead to a delayed diagnosis. The reason for the delay of diagnosis in these rare KS variants is the cognitive impairment which leads to delayed evaluation of fertility or hypogonadism, as contrasted to patients with the common variant, KS 47XXY. Additionally, unlike the classic KS variant, these rare variants are often associated with learning disabilities and gross bony features. Conclusion: Clinicians should be familiar with different clinical presentations of the less common KS genetic variants, in contrast to classical KS variants. Patients with classical KS typically present early in view of recognizable complaints of infertility, while patients with the less common KS variants may not present early on due to cognitive delay. Thus, familiarity with the additional manifestations of the less common KS variants (e.g., gross bony features) can help clinicians to suspect the possibility of these variants, prompting chromosomal analysis for further evaluation in such patients. Presentation Date: Friday, June 16, 2023

Risk factors in the origin of Down syndrome

Down syndrome (DS) is the most common autosomal aneuploidy and the leading cause of intellectual disability of genetic origin worldwide. It is identified as a syndrome in which the variability of its clinical manifestations and the severity of its phenotype have a multifactorial origin. Worldwide prevalence ranges between 1 per 700 live births and several factors that may be involved in the origin of DS have been proposed. Our objective was to describe updates regarding risk factors in the cytogenetic origin or cause of DS. We conducted a narrative review study in which a literature search was carried out from January to June 2022 in databases such as PubMed, EBSCO, Medigraphic, ClinicalKey, and meta-search engines such as Elsevier and Evidence Alerts. Only articles published in the last 10 years in English and Spanish were included. The search terms used were: Down syndrome, risk factors, prevention. Although DS is a very common chromosomal pathology worldwide, there is no single risk factor at the origin of meiotic or mitotic nondisjunction of chromosome 21, but rather each of the associated risk factors contributes to a greater or lesser degree to a cytogenetic predisposition in the etiology of trisomy 21. During the review it was identified that the main established risk factor associated with DS is still advanced maternal age (≥ 35 years).

Nondisjunction in Trisomy 21: Origin and Mechanisms

Down’s Syndrome is a chromosomal abnormality with a gain of a third copy of chromosome 21, characterized by craniofacial abnormalities, intellectual developmental delay and growth retardation. In the present study, one-day-old neonate and a patient with an aborted fetus were presented to rule out genetic aberrations via CMA screening. DNA was extracted using from the case samples and evaluated by CMA Affymetrix platform. Chromosomal abnormalities in the fetus such as trisomy emerges due to several factors including maternal age, genetic mutational events, non-disjunction of chromosomes and epigenetic changes. Genetic Cause of trisomy 21 is chromosomal aneuploidy and gain of three copies of chromosome 21. The Trisomy 21 can occur due to Robertsonian translocation, Mosaicisms or duplication of critical region of chromosome 21. The trisomy 21 is the result of nondisjunction of homologous chromosomes 21 during gametogenesis at the time of embryo development. CMA is an effective molecular diagnostic tool for predicting and diagnosing chromosomal abnormalities which needs to be promoted in India for healthy pregnancy outcomes. We report two cases of confirmed post-natal Trisomy 21. The main aim of this study is to focus on genetic diagnosis and its awareness which is still lacking with 100% coverage, in developing countries to reduce the burden of genetic diseases and associated emotional and economic consequences.

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

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

Down-Syndrome-Related Maternal Dysbiosis Might Be Triggered by Certain Classes of Antibiotics: A New Insight into the Possible Pathomechanisms

Down syndrome (DS) is a leading human genomic abnormality resulting from the trisomy of chromosome 21. The genomic base of the aneuploidy behind this disease is complex, and this complexity poses formidable challenges to understanding the underlying molecular basis. In the spectrum of the classic DS risk factor associations, the role of nutrients, vitamins, and, in general, the foodborne-associated background, as part of the events ultimately leading to chromosome nondisjunction, has long been recognized as a well-established clinical association. The integrity of the microbiome is a basic condition in these events, and the dysbiosis may be associated with secondary health outcomes. The possible association of DS development with maternal gut microbiota should therefore require more attention. We have hypothesized that different classes of antibiotics might promote or inhibit the proliferation of different microbial taxa; and hence, we might find associations between the use of the different classes of antibiotics and the prevalence of DS through the modification of the microbiome. As antibiotics are considered major disruptors of the microbiome, it could be hypothesized that the consumption/exposure of certain classes of antibiotics might be associated with the prevalence of DS in European countries (N = 30). By utilizing three different statistical methods, comparisons have been made between the average yearly antibiotic consumption (1997–2020) and the estimated prevalence of people living with DS for the year 2019 as a percentage of the population in European countries. We have found strong statistical correlations between the consumption of tetracycline (J01A) and the narrow-spectrum, beta-lactamase-resistant penicillin (J01CF) and the prevalence of DS.

Deletions of DNA in cancer and their possible uses for therapy.

Despite advances in treatments over the last decades, a uniformly reliable and free of side effects therapy of human cancers remains to be achieved. During chromosome replication, a premature halt of two converging DNA replication forks would cause incomplete replication and a cytotoxic chromosome nondisjunction during mitosis. In contrast to normal cells, most cancer cells bear numerous DNA deletions. A homozygous deletion permanently marks a cell and its descendants. Here, we propose an approach to cancer therapy in which a pair of sequence-specific roadblocks is placed solely at two cancer-confined deletion sites that are located ahead of two converging replication forks. We describe this method, termed "replication blocks specific for deletions" (RBSD), and another deletions-based approach as well. RBSD can be expanded by placing pairs of replication roadblocks on several different chromosomes. The resulting simultaneous nondisjunctions of these chromosomes in cancer cells would further increase the cancer-specific toxicity of RBSD.

Identifying parental and cell-division origins of aneuploidy in the human blastocyst

Preimplantation genetic testing commonly employs simplistic copy-number analyses to screen for aneuploidy in blastocyst trophectoderm biopsies. Interpreting intermediate copy number alone as evidence of mosaicism has led to suboptimal estimation of its prevalence. Because mosaicism originates from mitotic nondisjunction, utilizing SNP microarray technology to identify the cell-division origins of aneuploidy might provide a more accurate estimation of its prevalence. The present study develops and validates a method of determining the cell-division origin of aneuploidy in the human blastocyst by using both genotyping and copy-number data in parallel. The concordance of predicted origins with expected results was demonstrated in a series of truth models (99%-100%). This included determination of X chromosome origins from a subset of normal male embryos, determination of the origins of translocation chromosome-related imbalances via embryos from couples with structural rearrangements, and prediction of either mitotic or meiotic origins via multiple rebiopsies of embryos with aneuploidy. In a cohort of blastocysts with parental DNA (n= 2,277), 71% were euploid, 27% were meiotic aneuploid, and 2% were mitotic aneuploid, indicating a low frequency of bona fide mosaicism in the human blastocyst (mean maternal age: 34.4). Chromosome-specific trisomies in the blastocyst were also consistent with observations previously established in products of conception. The ability to accurately identify mitotic-origin aneuploidy in the blastocyst could benefit and better inform individuals whose IVF cycle results in all aneuploid embryos. Clinical trials with this methodology might also help provide a definitive answer regarding the reproductive potential of bona fide mosaic embryos.

Postnatal Edward’s syndrome

Edward's syndrome or trisomy 18 syndrome is due to meiotic nondisjunction with multisystemic involvement. It is characterized by craniofacial, skeletal, cardiovascular, central nervous system and genitourinary malformations. Developmental delay is common in all children. Most cases are diagnosed before birth based on the mother's age and the detection of ultrasound abnormalities in the second and third trimesters. A careful examination is therefore very important to confirm the diagnosis and to help identify unexpected abnormalities. There is very little data on Edward's syndrome. This case report can contribute to the literature and compare it to other known genetic disorders. Keywords : Trisomy 18 syndrome, Edwards syndrome, diagnosis, genetic disorders.