Molecular cytogenetic investigation of the origin of chromosomal abnormalities arising in human preimplantation embryos and oocytes.

Introduction: Advances in diagnosis and screening of preimplantation embryos or oocytes for chromosomal abnormalities have helped many couples achieve a normal pregnancy. They also pointed to the fact that numerical and structural chromosomal abnormalities are frequent in human preimplantation embryos and can arise at any point during gametogenesis and meiosis through to early embryonic development and mitotic division. However, information coming from studies in this area is far from complete and uniform.;Aim: To investigate aneuploidy and its mechanisms in human preimplantation embryos and oocytes. To develop protocols and improve on existing molecular cytogenetic techniques for the advance of preimplantation genetic diagnosis or screening (PGD/PGS) in routine clinical analysis. To evaluate the impact of PGD and PGS on the treatment of various types of infertility.;Methods: Fluorescent In situ Hybridisation (FISH) and Comparative genomic hybridisation (CGH) were the main methods used. I) Protocols were developed and implemented for the clinical PGD and PGS program. The PGD protocols included 2 couples with rare structural chromosomal abnormalities II) All untransferred embryos were studied and information was obtained for 101 PGS cycles (77 couples-935 embryos) and 18 PGD cycles for structural chromosomal abnormalities. III) Immature and undivided oocytes were studied using CGH from PGS, PGD and routine IVF couples.;Results and discussion: Specific and highly efficient methods and their clinical application to detect a variety of rare and common chromosomal abnormalities in PGD and PGS embryos were achieved. This study adds to the accumulating evidence showing the extent and mechanisms of genetic abnormalities in human oocytes and preimplantation embryos. It is one of the first studies to identify significant differences in the types of chromosomal abnormalities in embryos from couples with different reproductive history suggesting susceptibility to particular types of aneuploidy in these couples. The problems and effectiveness of PGS and PGD are also discussed

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Editoria

Meiotic and mitotic behaviour of a ring/deleted chromosome 22 in human embryos determined by preimplantation genetic diagnosis for a maternal carrier

<p>Abstract</p> <p>Background</p> <p>Ring chromosomes are normally associated with developmental anomalies and are rarely inherited. An exception to this rule is provided by deletion/ring cases. We were provided with a unique opportunity to investigate the meiotic segregation at oogenesis in a woman who is a carrier of a deleted/ring 22 chromosome. The couple requested preimplantation genetic diagnosis (PGD) following the birth of a son with a mosaic karyotype.</p> <p>The couple underwent two cycles of PGD. Studies were performed on lymphocytes, single embryonic cells removed from 3 day-old embryos and un-transferred embryos. Analysis was carried out using fluorescence in situ hybridisation (FISH) with specific probe sets in two rounds of hybridization.</p> <p>Results</p> <p>In total, 12 embryos were biopsied, and follow up information was obtained for 10 embryos. No embryos were completely normal or balanced for chromosome 22 by day 5. There was only one embryo diagnosed as balanced of 12 biopsied but that accumulated postzygotic errors by day 5. Three oocytes apparently had a balanced chromosome 22 complement but all had the deleted and the ring 22 and not the intact chromosome 22. After fertilisation all the embryos accumulated postzygotic errors for chromosome 22.</p> <p>Conclusion</p> <p>The study of the preimplantation embryos in this case provided a rare and significant chance to study and understand the phenomena associated with this unusual type of anomaly during meiosis and in the earliest stages of development. It is the first reported PGD attempt for a ring chromosome abnormality.</p

Prevalence of insertion sequence elements in plasmids relating to mgrB gene disruption causing colistin resistance in Klebsiella pneumoniae

Colistin is a last resort antibiotic for the treatment of carbapenemase producing Klebsiella pneumoniae (CRKP). The disruption of the mgrB gene by insertion sequences (ISs) is a mechanism mediating colistin resistance. Plasmids encode mobilizable IS elements which integrate into the mgrB gene in K. pneumoniae causing gene inactivation and colistin resistance. The species prevalence of mgrB-gene disrupting insertion elements ISL3 (ISKpn25), IS5 (ISKpn26), ISKpn14 and IS903B present on plasmids were assessed. IS containing plasmids were also scanned for antimicrobial resistance genes, including carbapenem resistant genes. Plasmids encoding insertion sequences are abundant in K. pneumoniae. IS903B was found in 28 unique Inc groups, while ISKpn25 was largely carried by IncFIB(pQil) plasmids. ISKpn26 and ISKpn14 were most often found associated with IncFII(pHN7A8) plasmids. Of the 34 unique countries which contained any of the IS elements, ISKpn25 was identified from 26. ISKpn26, ISKpn14, and IS903B insertion sequences were identified from 89.3%, 44.9% and 23.9% plasmid samples from China. Plasmids carrying ISKpn25, ISKpn14, and ISKpn26 IS have a 4.6, 6.0, and 6.6-fold higher carbapenemase gene count respectively, relative to IS903B-carrying plasmids. IS903B bearing plasmids have a 20, 5, and 5-fold higher environmental source isolation count relative to ISKpn25, ISKpn14, and ISKpn26 bearing plasmids. ISKpn25 present on IncFIB(pQil) sourced from clinical settings is established across multiple countries, while ISKpn26, ISKpn14, and IS903B appear most often in China. Carbapenemase presence in tandem with IS elements may help promote an extensively drug resistant profile in K. pneumoniae limiting already narrow therapeutic treatment options

Male and female meiotic behaviour of an intrachromosomal insertion determined by preimplantation genetic diagnosis

<p>Abstract</p> <p>Background</p> <p>Two related family members, a female and a male balanced carrier of an intrachromosomal insertion on chromosome 7 were referred to our centre for preimplantation genetic diagnosis. This presented a rare opportunity to investigate the behaviour of the insertion chromosome during meiosis in two related carriers. The aim of this study was to carry out a detailed genetic analysis of the preimplantation embryos that were generated from the three treatment cycles for the male and two for the female carrier.</p> <p>Patients underwent <it>in vitro </it>fertilization and on day 3, 22 embryos from the female carrier and 19 embryos from the male carrier were biopsied and cells analysed by fluorescent in situ hybridization. Follow up analysis of 29 untransferred embryos was also performed for confirmation of the diagnosis and to obtain information on meiotic and mitotic outcome.</p> <p>Results</p> <p>In this study, the female carrier produced more than twice as many chromosomally balanced embryos as the male (76.5% vs. 36%), and two pregnancies were achieved for her. Follow up analysis showed that the male carrier had produced more highly abnormal embryos than the female (25% and 15% respectively) and no pregnancies occurred for the male carrier and his partner.</p> <p>Conclusion</p> <p>This study compares how an intrachromosomal insertion has behaved in the meiotic and preimplantation stages of development in sibling male and female carriers. It confirms that PGD is an appropriate treatment in such cases. Reasons for the differing outcome for the two carriers are discussed.</p

Genomic analyses of an Escherichia coli and Klebsiella pneumoniae urinary tract co‐infection using long‐read nanopore sequencing.

Escherichia coli (E.coli) and Klebsiella pneumoniae (K. pneumoniae) isolates presenting with the same antimicrobial susceptibility profile were recovered from the same catheter sample of urine (CSU). Both strains were recovered from a patient with a long-standing indwelling urinary catheter. Each isolate had their DNA extracted following culture. Nanopore long-read sequencing was used to build the plasmids and chromosomes from each strain to closure to discern potential horizontal propagation of resistance-encoding plasmids and the relationship between resistance genes and insertion sequences. Plasmids derived from resistance strains in the urinary microbiota remain poorly characterized. The same 11 antimicrobial resistance (AMR) genes were found in plasmids from each strain. The 185,239-bp FIB(K) pKBM1, from the K. pneumoniae strain additionally encoded the 5 AMR genes: sul2, strA, strB, blaTEM-1B, and blaCTX-M-15. A multimeric array of AMR genes and IS26 insertions sequences were found in the plasmids from both isolates. Both plasmids from each isolate were similar. Horizontal transfer of plasmids, followed by subsequent plasmid rearrangement is likely to have occurred during the course of infection. Furthermore, the resistance region in the plasmids shared similarity against the internationally prevalent plasmid, pKPN3-307_typeA, commonly identified in K. pneumoniae ST307. Biofilm formation in catheterized patients may allow close cell-contact between strains. Horizontal propagation of resistance genes may occur, leading to polymicrobial infections

Genomic instability and the link to infertility: A focus on microsatellites and genomic instability syndromes.

Infertility is associated to multiple types of different genomic instabilities and is a genetic feature of genomic instability syndromes. While the mismatch repair machinery contributes to the maintenance of genome integrity, surprisingly its potential role in infertility is overlooked. Defects in mismatch repair mechanisms contribute to microsatellite instability and genomic instability syndromes, due to the inability to repair newly replicated DNA. This article reviews the literature to date to elucidate the contribution of microsatellite instability to genomic instability syndromes and infertility. The key findings presented reveal microsatellite instability is poorly researched in genomic instability syndromes and infertility

Genetic Analyses of Rare ESBL ST628 Klebsiella pneumoniae Detected during a Protracted Nosocomial Outbreak in the United Kingdom

Klebsiella pneumoniae (K. pneumoniae) cultures from a hospital-wide outbreak in the UK, which lasted for over 12 months, were sequenced. We sought to sequence and genetically characterise the outbreak strain. Antibiotic Susceptibility Testing (AST) was performed on 65 K. pneumoniae isolates saved from the outbreak. All isolates were sequenced using the Oxford Nanopore Technologies (ONT) MinION flowcell: 10 isolates, including the isolate with the earliest collection date in 2017, were additionally sequenced on the NovaSeq 6000 platform to build high-accuracy nanopore-illumina assemblies. Among the sequenced strains, 60 were typed as ST628. 96.6% (n = 58/60) ST628 strains harboured a large ~247-kb FIB(K) plasmid carrying up to 11 antimicrobial resistance genes, including the extended-spectrum beta-lactamase (ESBL) gene, blaCTX-M-15. Clonality between the outbreak isolates was confirmed using single nucleotide polymorphism (SNP) typing. The outbreak strains were phylogenetically related to clinical ST628 strains identified in 2012, 6 years prior to the outbreak. A rare ESBL K. pneumoniae K2 ST628 strain harbouring a multi-drug resistant (MDR) plasmid encoding the ESBL gene blaCTX-M-15 was detected across multiple independent wards during the protracted nosocomial outbreak. Surveillance of this strain is recommended to prevent future nosocomial outbreak

A novel isolator-based system promotes viability of human embryos during laboratory processing

In vitro fertilisation (IVF) and related technologies are arguably the most challenging of all cell culture applications. The starting material is a single cell from which one aims to produce an embryo capable of establishing a pregnancy eventually leading to a live birth. Laboratory processing during IVF treatment requires open manipulations of gametes and embryos, which typically involves exposure to ambient conditions. To reduce the risk of cellular stress, we have developed a totally enclosed system of interlinked isolator-based workstations designed to maintain oocytes and embryos in a physiological environment throughout the IVF process. Comparison of clinical and laboratory data before and after the introduction of the new system revealed that significantly more embryos developed to the blastocyst stage in the enclosed isolator-based system compared with conventional open-fronted laminar flow hoods. Moreover, blastocysts produced in the isolator-based system contained significantly more cells and their development was accelerated. Consistent with this, the introduction of the enclosed system was accompanied by a significant increase in the clinical pregnancy rate and in the proportion of embryos implanting following transfer to the uterus. The data indicate that protection from ambient conditions promotes improved development of human embryos. Importantly, we found that it was entirely feasible to conduct all IVF-related procedures in the isolator-based workstations