Meiotic outcome in two carriers of Y autosome reciprocal translocations: selective elimination of certain segregants

BACKGROUND: Reciprocal Y autosome translocations are rare but frequently associated with male infertility. We report on the meiotic outcome in embryos fathered by two males with the karyotypes 46,X,t(Y;4)(q12;p15.32) and 46,X,t(Y;16)(q12;q13). The two couples underwent preimplantation genetic diagnosis (PGD) enabling determination of the segregation types that were compatible with fertilization and preimplantation embryo development. Both PGD and follow up analysis were carried out via fluorescence in situ hybridization (FISH) or array comparative genomic hybridization (aCGH) allowing the meiotic segregation types to be determined in a total of 27 embryos. RESULTS: Interestingly, it was seen that the number of female embryos resulting from alternate segregation with the chromosome combination of X and the autosome from the carrier gamete differed from the corresponding balanced males with derivative Y and the derivative autosome by a ratio of 7:1 in each case (P = 0.003) while from the adjacent-1 mode of segregation, the unbalanced male embryos with the combination of der Y and the autosome were seen in all embryos from couple A and in couple B with the exception of one embryo only that had the other chromosome combination of X and derivative autosome (P = 0.011). In both cases the deficit groups have in common the der autosome chromosome that includes the segment Yq12 to qter. CONCLUSION: The most likely explanation may be that this chromosome is associated with the X chromosome at PAR2 (pseudoautosomal region 2) in the sex-body leading to inactivation of genes on the autosomal segment that are required for the meiotic process and that this has led to degeneration of this class of spermatocytes during meiosis

The improvement of the best practice guidelines for preimplantation genetic diagnosis of cystic fibrosis : toward an international consensus

Cystic fibrosis (CF) is one of the most common indications for preimplantation genetic diagnosis (PGD) for single gene disorders, giving couples the opportunity to conceive unaffected children without having to consider termination of pregnancy. However, there are no available standardized protocols, so that each center has to develop its own diagnostic strategies and procedures. Furthermore, reproductive decisions are complicated by the diversity of disease-causing variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and the complexity of correlations between genotypes and associated phenotypes, so that attitudes and practices toward the risks for future offspring can vary greatly between countries. On behalf of the EuroGentest Network, eighteen experts in PGD and/or molecular diagnosis of CF from seven countries attended a workshop held in Montpellier, France, on 14 December 2011. Building on the best practice guidelines for amplification-based PGD established by ESHRE (European Society of Human Reproduction and Embryology), the goal of this meeting was to formulate specific guidelines for CF-PGD in order to contribute to a better harmonization of practices across Europe. Different topics were covered including variant nomenclature, inclusion criteria, genetic counseling, PGD strategy and reporting of results. The recommendations are summarized here, and updated information on the clinical significance of CFTR variants and associated phenotypes is presented

Investigation of chromosomal abnormalities during colorectal tumour development by somatic cell hybrid and fluorescence in-situ hybridisation analysis

Familial adenomatous polyposis (FAP) is a dominantly inherited disorder where affected individuals usually develop hundreds of polyps in the colon. Cancer inevitably follows as a result of an accumulation of mutations within the adenoma, (the adenoma to carcinoma sequence.) The events leading to colorectal cancer in individuals with FAP, and individuals with 'sporadic' colorectal polyps or cancers were investigated for this thesis. Two approaches were undertaken: Somatic cell hybrid studies in individuals with FAP, and Fluorescence In Situ Hybridisation (FISH), with alpha-satellite probes. Somatic cell hybrids were generated from 6 separate fusions of human tissue from four different FAP patients with the Chinese hamster cell line A23. The human tissues used were one secondary cancer of the liver, two adenomas, two lymphocyte preparations, and one fibroblast cell line. The lymphocyte preparations and the fibroblast cell line were from two FAP patients one known to carry a constitutional abnormality and the other with a somatic clonal abnormality involving chromosome 5. Somatic cell analysis was carried out by PCR using human specific primers, and by FISH painting experiments. The aim was to try to rescue possible chromosomes of interest from the fresh human tissue samples, since the setting up of primary cultures proved unsuccessful. However somatic cell hybrids made from fresh human tissue resulted in a high degree of fragmentation of human chromosomes and therefore these hybrids did not appear to be of any use, at least in the isolation of chromosome 5 marker chromosomes derived from the tumour tissue. For the lymphocyte preparations and for the fibroblast cell line fusions, the aim was to try to separate the normal chromosome 5 from the rearranged one. From these fusions only one somatic cell hybrid clone, P2c, was generated with possible future use in mapping experiments in the region of the gene causing FAP (the APC gene). A second approach was to use FISH with alpha-satellite centromeric probes in order to determine the number of chromosomes 7, 17, and 18 within colorectal polyp and cancer tissue samples from FAP and non FAP individuals. These chromosomes have been implicated in the development of colorectal cancer. Normal mucosa was used as a control to determine the significance of the results. Three colon adenoma cell lines, LIM1215, HCA7, and JW2 were also investigated and compared with their karyotypes. These results were also compared to DNA analysis that had been carried out on the same samples. The results show that although loss of heterozygosity can be detected by DNA probes on these chromosomes, this is not due to whole chromosome loss in many cases, since the centromeric probes did not pick up this loss. In some cases however, chromosome loss was detected by FISH in a significant percentage of cells, but not by DNA analysis, indicating that more than one clone of cells was present in the tissue. The presence of different clones of cells within a single tumour may be important in the development of the tumour as a whole. The FISH data seemed to reflect the basic principle that tumours develop independently even when they are derived from a single individual. The results did not show any strong relationship between patient age, sex, tumour type, size, or site and the presence of cells with numerical chromosomal abnormalities. The main numerical chromosomal change detected by FISH in this study were an increase in chromosome 7 and a loss of chromosome 17 in colorectal tumours from sporadic and FAP patients. Normal mucosa from FAP patients also showed numerical chromosomal changes compared to mucosa from sporadic colorectal patients, indicating that FAP patients may suffer an inherent chromosome instability due to germline mutations in the APC gene. Numerical changes in chromosome 18 were rarely detected but, monosomy of this chromosome appeared to be associated with feeder independence in cell line JW2

Differentially expressed microRNAs in aneuploid preimplantation blastocysts: a systematic review

IntroductionMicroRNAs are small noncoding genes with gene expression regulatory function. Their emergence as potential diagnostic biomarker for many diseases has gained a specific interest among researchers. Observations of changes in miRNA levels correlating with aneuploidy in early embryos raise the prospective of employing miRNA as biomarkers to assess the embryo quality.MethodTo identify and gather the miRNAs with potential link to chromosomal abnormalities in embryos from previous research, we conducted a systematic search using four databases, including Embase, Medline, Web of Science, and Cochrane databases in accordance with PRISMA guidelines.ResultsOut of 200 identified records, only seven met the inclusion criteria. Seven miRNAs: miR-19b, miR-517c, miR-518e, miR-522, miR-92a, and miR-106a exhibited persistent downregulation in aneuploid blastocysts in the included studies. These miRNAs are members of important miRNA clusters, associated with abnormal expression in studies on reproductive failure. Pathway analysis revealed their involvement in regulating gene transcription, as well as cell cycle progression and apoptosis.DiscussionThe changes detected in the miRNA expression in aneuploid embryos across different studies support the aneuploidy and miRNA relationship and prospect miRNA as a valuable tool for the assessment of embryo quality. Collectively, these observations highlight the role of miRNAs in embryonic development, and their involvement in genetic abnormalities that occur in embryos, such as aneuploidy, indicating their potential implementation to improve the embryo selection and reproductive outcomes

Functional assessment for elimination of mismatches in nuclear and whole cell extracts obtained from mouse and human blastocysts

<p>Preimplantation embryos may have an increased risk of having mismatches due to the rates of cell proliferation and DNA replication. Elimination of mismatches in human gametes and embryos has not been investigated. In this study we developed a sensitive functional assay to examine the repair or elimination of mismatches in both commercially available cell extracts and extracts obtained from preimplantation embryos. Heteroduplex molecules were constructed using synthetic oligonucleotides. Efficiency of the repair of mismatches was semi-quantitatively analysed by exposure to nuclear/whole cell extracts (as little as 2.5 µg) and extracts obtained from pooled mouse and human blastocysts to investigate the repair capacity in human embryos. A cell free <i>in vitro</i> assay was successfully developed to analyze the repair of mismatches using heteroduplex complexes. The assay was further optimized to analyze repair of mismatches in cell extracts obtained from oocytes and blastocysts using minute amounts of protein. The efficiency of mismatch repair was examined in both mouse and human blastocysts (2.5 µg). The blastocysts were observed to have a lower repair efficiency compared to commercially available nuclear and whole cell extracts. In conclusion, a sensitive, easy, and fast <i>in vitro</i> technique was developed to detect the repair of mismatch efficiency in embryos.</p

Next Generation Sequencing Detects Premeiotic Errors in Human Oocytes

Autosomal aneuploidy is the leading cause of embryonic and foetal death in humans. This arises mainly from errors in meiosis I or II of oogenesis. A largely ignored source of error stems from germinal mosaicism, which leads to premeiotic aneuploidy. Molecular cytogenetic studies employing metaphase fluorescence in situ hybridization and comparative genomic hybridisation suggest that premeiotic aneuploidy may affect 10&ndash;20% of oocytes overall. Such studies have been criticised on technical grounds. We report here an independent study carried out on unmanipulated oocytes that have been analysed using next generation sequencing (NGS). This study confirms that the incidence of premeiotic aneuploidy in an unselected series of oocytes exceeds 10%. A total of 140 oocytes donated by 42 women gave conclusive results; of these, 124 (88.5%) were euploid. Sixteen out of 140 (11.4%) provided evidence of premeiotic aneuploidy. Of the 140, 112 oocytes were immature (germinal vesicle or metaphase I), of which 10 were aneuploid (8.93%); the remaining 28 were intact metaphase II - first polar body complexes, and six of these were aneuploid (21.4%). Of the 16 aneuploid cells, half contained simple errors (one or two abnormal chromosomes) and half contained complex errors. We conclude that germinal mosaicism leading to premeiotic aneuploidy is a consistent finding affecting at least 10% of unselected oocytes from women undergoing egg collection for a variety of reasons. The importance of premeiotic aneuploidy lies in the fact that, for individual oocytes, it greatly increases the risk of an aneuploid mature oocyte irrespective of maternal age. As such, this may account for some cases of aneuploid conceptions in very young women