Charles Hanson

Derivation, Characterization, and Differentiation of Human Embryonic Stem Cells

The derivation of human embryonic stem (hES) cells establishes a new avenue to approach many issues in human biology and medicine for the first time. To meet the increased demand for characterized hES cell lines, we present the derivation and characterization of six hES cell lines. In addition to the previously described immunosurgery procedure, we were able to propagate the inner cell mass and establish hES cell lines from pronase‐treated and hatched blastocysts. The cell lines were extensively characterized by expression analysis of markers characteristic for undifferentiated and differentiated hES cells, karyotyping, telomerase activity measurement, and pluripotency assays in vitro and in vivo. Whereas three of the cell lines expressed all the characteristics of undifferentiated pluripotent hES cells, one cell line carried a chromosome 13 trisomy while maintaining an undifferentiated pluripotent state, and two cell lines, one of which carried a triploid karyotype, exhibited limited pluripotency in vivo. Furthermore, we clonally derived one cell line, which could be propagated in an undifferentiated pluripotent state.

Preimplantation genetic screening in women of advanced maternal age caused a decrease in clinical pregnancy rate: a randomized controlled trial

BACKGROUND Advanced maternal age (AMA) is an important parameter that negatively influences the clinical pregnancy rate in IVF, in particular owing to the increased embryo aneuploidy rate. It has thus been suggested that only transferring euploid embryos in this patient group would improve the pregnancy rate. The purpose of this study was to test whether employing preimplantation genetic screening (PGS) in AMA patients would increase the clinical pregnancy rate. METHODS We conducted a two-center, randomized controlled trial (RCT) to analyze the outcome of embryo transfers in AMA patients (>or=38 years of age) after PGS using FISH analysis for chromosomes X, Y, 13, 16, 18, 21 and 22. The PGS group was compared with a control group. The primary outcome measure was clinical pregnancy rate after 6-7 weeks of gestation per randomized patient. RESULTS The study was terminated early as an interim analysis showed a very low conditional power of superiority for the primary outcome. Of the 320 patients calculated to be included in the study, 56 and 53 patients were randomized into the PGS and control groups, respectively. The clinical pregnancy rate in the PGS group was 8.9% (95% CI, 2.9-19.6%) compared with 24.5% (95% CI, 13.8-38.3%) in the control group, giving a difference of 15.6% (95% CI, 1.8-29.4%, P = 0.039). CONCLUSIONS Although the study was terminated early, this RCT study provides evidence against the use of PGS for AMA patients when performing IVF. TRIAL REGISTRATION NUMBER ISRCTN38014610.

The gene map of the Norway rat (Rattus norvegicus) and comparative mapping with mouse and man

The current status of the rat gene map is presented. Mapping information is now available for a total of 214 loci and the number of mapped genes is increasing steadily. The corresponding number of loci quoted at HGM10 was 128. Genes have been assigned to 20 of the 22 chromosomes in the rat. Some aspects of comparative mapping with mouse and man are also discussed. It was found that there is a good correlation between the morphological homologies detectable in rat and mouse chromosomes, on the one hand, and homology at the gene level on the other. For 10 rat synteny groups all the genes so far mapped are syntenic also in the mouse. For the remaining rat synteny groups it appears that the majority of the genes will be syntenic on specific (homologous) mouse chromosomes, with only a few genes dispersed to other members of the mouse karyotype. Furthermore, the data indicate that mouse chromosome 1 genetically corresponds to two rat chromosomes, viz., 9 and 13, equalizing the difference in chromosome number between the two species. Further mappings will show whether the genetic homology will prove to be as extensive as these preliminary results indicate. As might be expected from evolutionary considerations, rat synteny groups are much more dispersed in the human genome. It is clear, however, that many groups of genes have remained syntenic during the period since man and rat shared a common ancestor. One further point was noted. In two cases groups of genes were syntenic in the mouse but dispersed to two chromosomes in rat and man, whereas in a third case a group of genes was syntenic in the rat but dispersed to two chromosomes in mouse and man. This finding argues in favor of the notion that the original gene groups were on separate ancestral chromosomes, which have fused in one rodent species but remained separate in the other and in man.

Ear and hearing in relation to genotype and growth in Turner syndrome

Hearing loss, auricular anomalies and middle ear infections are common findings in many genetic disorders, but the mechanisms have remained unknown. We studied ear and hearing problems in Turners syndrome (TS) in relation to the degree of X chromosome loss (i.e. degree of mosaicism) and growth. One hundred and nineteen girls and women with TS were studied regarding audiometry, fluorescent in situ hybridisation, serum concentration of insulin-like growth factor-1 (IGF-1) and body height. It was found that sensorineural hearing loss and occurrence of auricular anomalies were significantly increased the greater the proportion of 45,X cells in a particular individual (P<0.05 and P<0.001, respectively). Middle ear infections and sensorineural hearing loss were negatively correlated with IGF-1 (P<0.05 and P<0.001, respectively). Hearing correlated positively with height (P<0.01) and IGF-1 independently of age (P<0.05). Height correlated positively with IGF-1 (P<0.001). Auricular malformations, middle ear infections and hearing impairment in TS were interpreted as due to growth disturbances during development. A new hypothesis on the pathophysiology of external, middle and inner ear disorders due to a delayed cell cycle caused by chromosomal aberrations per se and not only to the specific X chromosome deletion is presented.

Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture

There have been recent reports of human embryonic stem cell (hESC) lines developing chromosomal aberrations after long-term culture, indicating an unstable genomic status due to the in vitro milieu. This raises concern, since it would limit their use in therapeutics. In this study the chromosomal status of five well-characterized hESC lines, SA002, SA002.5, AS034.1.1, SA121 and SA461, was monitored during long-term in vitro culture. The criteria of defined hESCs were met by all of the five hESC lines (four diploid and one trisomic for chromosome 13). The genomes were screened for chromosomal aberrations and rearrangements using comparative genomic hybridization (CGH), interphase fluorescence in situ hybridization (FISH) and traditional karyotyping on several occasions while in culture. The genomic integrity was shown to be maintained after repeated freeze-thaw procedures and continuous culture in vitro for up to 22 months (148 passages). We discuss the most common de novo chromosomal aberrations reported in hESCs, as well as their possible origin.

Human embryonic stem cells and chromosome stability

The use of human embryonic stem cells (HESC) in research is increasing exponentially and HESC will certainly be of importance in biological, clinical and toxicological research for many years to come. Once established, HESC lines are expected to be chromosomally stable. However, our own experience of culturing HESC and some published reports indicate that HESC may show chromosomal instability while being cultured continuously in vitro. We conclude that the effects of different culture techniques and long‐term culture on the chromosome stability of HESC still remain to be elucidated and we recommend regular analysis of the chromosome constitution in cell lines using traditional karyotyping, CGH, FISH and PCR. We also recommend freezing of HESC at low passage number and in larger batches after thawing and expansion in order to secure material in case mutations occur in the cell line at a later stage of culture.

Testicular ultrasonography and extended chromosome analysis in men with nonmosaic Klinefelter syndrome: a prospective study of possible predictive factors for successful sperm recovery.

OBJECTIVE To investigate whether extended chromosome analysis or testicular sonography, including flow Doppler imaging, before diagnostic testicular sperm extraction have predictive value for successful sperm retrieval in men with nonmosaic Klinefelter syndrome. DESIGN Prospective clinical study. SETTING IVF clinic and genetics laboratory at a university hospital. PATIENT(S) Nineteen patients with nonmosaic Klinefelter syndrome and azoospermia. INTERVENTION(S) Collection of blood samples; histopathologic examination of testicular tissue; fluorescence in situ hybridization; sonography, including Doppler imaging; and testicular sperm extraction. MAIN OUTCOME MEASURE(S) Testicular volume, serum FSH and serum testosterone levels, percentage of normal XY cells, ultrasound echogenicity, intratesticular blood flow resistance, and sperm recovery. RESULT(S) Testicular volume and levels of serum FSH and serum testosterone levels did not differ significantly. No differences in testicular echogenicity or intratesticular blood flow resistance were found between 47,XXY men in whom sperm recovery was successful and those in whom sperm recovery failed. Significant differences were seen between all patients with the Klinefelter syndrome and controls with normal sperm values. Fluorescence in situ hybridization of peripheral lymphocytes and buccal tissue showed no correlation between frequency of normal 46,XY cells and testicular spermatogenesis. CONCLUSION(S) In azoospermic men with the Klinefelter syndrome, histopathologic findings seem to be predictive for successful sperm recovery. Infertility work-up, including diagnostic testicular sperm recovery, is recommended, and, if possible, viable sperm should be cryopreserved.

Pregnancy rate and outcome in Swedish women with Turner syndrome

Pregnancies occurred in 57 (12%) of 482 Swedish women with Turner syndrome with a liveborn rate of 54% in 124 pregnancies. Spontaneous pregnancies occurred in 40%, mainly in women with 45,X/46,XX mosaicism, and oocyte donation in 53% where miscarriages were less frequent, odds ratio = 0.43 (95% confidence interval 0.17-1.04).

Chromosomal mosaicism mitigates stigmata and cardiovascular risk factors in Turner syndrome.

Objective  To study genotype–phenotype correlations in Turner syndrome (TS) regarding body composition, cardiovascular risk factors, stigmata and age at diagnosis vs. degree of mosaicism estimated as the percentage of 45,X and 46,XX cells.

The influence of karyotype on the auricle, otitis media and hearing in Turner syndrome.

The study has investigated the relationship between the chromosomal aberration and ear and/or hearing disorders in 115 girls/women with Turner syndrome (TS). A dose-response relationship was found between the karyotype and hearing function. Hearing deteriorated more rapidly with increasing age in TS women lacking the whole p-arm of chromosome X (i.e. monosomy 45,X, or isochromosome cases 46,X,i(Xq)) as compared to women having a partial deletion of the p-arm (structural deletions or mosaicism cases), who, in turn, had poorer hearing than a female random population sample (46,XX) (P<0.001). Moreover, TS subjects having total deletion of the p-arm were three times more likely to have auricular anomalies or conductive hearing loss due to otitis media than subjects with partial deletion (P<0. 05). The results support the hypothesis that lack of growth-regulating genes such as the short stature homeobox-containing gene (SHOX), which is located within the pseudo-autosomal region on the p-arm of the X chromosome, may increase the occurrence of auricular malformations and otitis media and also induce an earlier loss of hearing function. Accordingly, the ear and hearing disorders in TS may be a result of growth disturbances of the auricle, the mastoid, the Eustachian tube and the organ of Corti during development. It is suggested that karyotype may be used as a predictor for future ear and hearing problems in TS.