Increased susceptibility to maternal aneuploidy demonstrated by comparative genomic hybridization analysis of human MII oocytes and first polar bodies.

Single cell comparative genomic hybridization (CGH) was employed to extensively investigate 24 unfertilized or in vitromatured meiosis II oocytes and their corresponding first polar bodies (PBs), to determine how and whether all 23 chromosomes participate in female meiosis I errors and to accurately estimate the aneuploidy rate in the examined cells. Results were obtained for 15 oocytes and 16 PBs, representing 23 eggs (MII oocyte-PB complexes) donated from 15 patients (average age 32.2 years). Abnormalities were detected in ten eggs, giving an overall aneuploidy rate of 43.5%. In all, fourteen anomalies were scored, with the fertilized oocyte being at risk of monosomy in eight cases and at risk of trisomy in six; chromosomes of various sizes participated. CGH was able to give a comprehensive aneuploidy rate, as both absence of chromosomal material and the presence of extra copies were accurately scored. The aneuploidy mechanisms determined were: classical whole univalent non-disjunction; chromatid predivision prior to anaphase I, leading to metaphase II imbalance. There was also evidence of germinal mosaicism for a trisomic cell line. Three patients appeared to be predisposed to meiosis I errors, based on the presence of either multiple abnormalities in one or more of their examined cells, or of the same type of abnormality in all of their cells. Exclusion of these susceptible patients reduces the aneuploidy rate to 20%. Various hypotheses are put forward to explain these observations in order to stimulate research into the complex nature of female meiotic regulation.

Sister chromatid exchange frequency in human epidermal cells in culture treated with 8-methoxypsoralen and long-wave UV radiation.

The effects of 8-methoxypsoralen with long-wave ultraviolet radiation on the sister chromatid exchange frequency in human epidermal cells in culture was investigated. With a constant amount of radiation the number of exchanges increased in an approximately linear manner with increasing concentrations of 8-methoxypsoralen up to 0.3 micrograms/ml. Above this concentration there were fewer dividing cells and an apparent departure from linearity in the dose-response curve. These results show that 8-methoxypsoralen concentrations equivalent to those found in the serum of patients undergoing photochemotherapy, in conjunction with UVA radiation, cause striking increases in sister chromatid exchange frequency in human epidermal cells in vitro.

Comparative studies of spermatogenesis in fertile and subfertile men.

Testicular biopsy specimens from 16 fertile and 10 subfertile men with normal male karyotype were studied quantitatively to provide histological and cytogenetic data for a basis of reference in assessing abnormalities of spermatogenesis. Histological studies included estimation of the proportion and activity of germinal epithelium and an assessment of tubular morphology. In cytogenetic preparations, counts were made of cells at different stages of meiosis. Studies of cells at diakinesis included chiasma counts and percentage of cells with dissociated sex chromosomes. One fertile and six subfertile men showed decreased germinal activity; the six subfertile men also had decreased MII/MI ratios. Other findings were similar in the two groups.

Trisomy 21 in transient myeloproliferative disorder.

Transient leukemia in phenotypically normal children is rare. A newborn child in whom fever and tachypnea developed at age 2 days had a white blood cell count of 20.1 x 10(9)/L and many abnormal blast cells. Chromosome analysis of spontaneously dividing cells from the blood showed these to have trisomy 21, and 80% of cells in the marrow were also trisomic. No trisomic cells were present in skin fibroblast cultures. At age 6 months, at which time the blood film appeared normal, trisomic cells were no longer present.

Screening for Down's syndrome based on individual risk.

OBJECTIVE: To evaluate the effectiveness of biochemical screening of individual pregnancies for Down's syndrome risk. DESIGN: Retrospective determination of risk. SETTING: Obstetric and cytogenetic services in Tayside, Scotland. SUBJECTS: 3436 pregnant women who had screening for neural tube defects in the second trimester during November 1988 to March 1990 and whose pregnancies were dated by ultrasonography. Three women with pregnancies associated with Down's syndrome reported later in 1990. MAIN OUTCOME MEASURES: Individual risk calculated from age at estimated date of delivery; chorionic gonadotrophin and alpha fetoprotein concentrations in serum samples obtained at precisely determined gestational ages in second trimester. Results of karyotype determination and outcome of pregnancy. RESULTS: During November 1988 to March 1990 karyotypes were determined for 5% of pregnancies for reasons of maternal age and genetic history and one of the eight affected fetuses was detected. Individual risk could not be calculated for 347 pregnancies, but screening on this basis would have detected five of the cases and required screening in 194 out of 3089 (6.3%) pregnancies; all three affected pregnancies reported later in 1990 would also have been detected, giving a success rate of 73% (95% confidence interval 39% to 94%). The age distribution of women according to individual risk suggests that women over 35 would be screened effectively. CONCLUSION: Screening based on individual risk would use resources more effectively than screening based on maternal age and genetic history without affecting detection rates in older women.

Comparative genomic hybridization analysis of human oocytes and polar bodies.

BACKGROUND: Classical cytogenetic methods and fluorescent in situ hybridization (FISH) have been employed for the analysis of chromosomal abnormalities in human oocytes. However, these methods are limited by the need to spread the sample on a microscope slide, a process that risks artefactual chromosome loss. Comparative genomic hybridization (CGH) is a DNA-based method that enables the investigation of the entire chromosome complement. We optimized and evaluated a CGH protocol for the chromosomal analysis of first polar bodies (PBs) and oocytes. The protocol was then employed to obtain a detailed picture of meiosis I errors in human oogenesis. METHODS: 107 MII oocyte-PB complexes were examined using whole genome amplification (WGA) and CGH. RESULTS: Data was obtained for 100 complexes, donated from 46 patients of average age 32.5 (range 18-42). 22 complexes from 15 patients were abnormal, giving an aneuploidy rate of 22%. CONCLUSIONS: The results presented in this study more than double the quantity of CGH data from female gametes currently available. Abnormalities caused by whole chromosome non-disjunction, unbalanced chromatid predivision and chromosome breakage were reliably identified using the CGH protocol. Analysis of the data revealed a preferential participation of chromosome X and the smaller autosomes in aneuploidy and provided further evidence for the existence of age-independent factors in female aneuploidy.

Nucleolus organiser regions on mitotic and meiotic chromosomes from infertile males investigated using a specific silver stain.

Mitotic preparations from 30 subfertile males and meiotic preparations from 3 normal and 2 subfertile males were examined by means of the Ag-I technique of Bloom and Goodpasture (1976) to reveal nucleolus organiser regions (NORs). In the mitotic preparations, each subject was found to have a characteristic number of Ag-positive NORs per cell, within a range of 6--10. Analysis of satellite associations showed that the mean number of satellite associations per cell was related to the modal number of Ag-positive NORs for each subject. In the meiotic preparations, silver deposition was observed throughout meiotic prophase, but disappeared totally during diakinesis and metaphase II. It was seen again in early spermatids, and disappeared again as nuclear elongation took place. This pattern was observed in both normal and subfertile subjects, and may provide indirect evidence for the activation of rRNA genes during spermatogenesis.

Improved fragile site detection with trimethoprim.

The effect of trimethoprim, an inhibitor of dihydrofolate reductase, in demonstrating the presence of fragile sites on human chromosomes was investigated. Lymphocyte cultures with 20 mg/l trimethoprim added at the onset of incubation gave consistently higher frequencies of fragile sites than the other culture regimes tested.

In vitro growth rates of epidermal cells derived from the skin of psoriatic patients and non-psoriatic controls.

The growth of epidermal cells derived from clinically normal skin of psoriatic patients and controls has been studied in culture. Growth rates were measured in secondary cultures established with 5 x 10(4) cells/35 mm plate without feeder layers by determining the plating efficiency, and cell yield and surface area of growth at the end of a 14-day culture period. There was no significant correlation of plating efficiency, cell yield/colony or surface area/colony with the sex or age of the donor in either the normal or psoriatic groups. The morphological development of normal and psoriatic epidermal cell cultures was similar. Comparisons of plating efficiency, cell yield/colony and surface area/colony for the normal and psoriatic groups revealed no significant difference.

Sister chromatid exchanges in human lymphocytes exposed to 8-methoxypsoralen and long wave UV radiation prior to incorporation of bromodeoxyuridine.

Human lymphocytes exposed to the effects of long wave UV radiation in the presence of 8-methoxypsoralen prior to stimulation by PHA show dose related sister chromatid exchanges after 2 replication cycles in vitro. This has implications for interpreting the processes involved and for monitoring DNA damaging agents in vivo.

Cytogenetic and histological studies of testicular biopsies from subfertile men with chromosome anomaly.

Testicular biopsies from eight men with abnormal karyotypes have been examined for histological and cytogenetic evidence of disturbances of meiosis. Quantitative analysis of this material showed one, with a 13;14 Robertsonian translocation, to have apparently normal spermatogenesis. Three patients, one with a 47,XYY and two with 45,XY, inv 9 karyotypes, had an overall depression of spermatogenesis. Four others, all with major chromosomal abnormalities, had apparently normal spermatogenesis until the primary spermatocyte stage. Two of these had sex autosomal translocations. One, 45,Y,t(X;21), had a complete block at MI, the other, 46,X,t(Y;16), had a partial block at spermatid formation. One man with a reciprocal 2;10 translocation showed delay at all stages beyond spermatocyte formation and one man with an inversion of chromosome 3 showed impaired spermatid maturation.

Mechanisms of maternal aneuploidy: FISH analysis of oocytes and polar bodies in patients undergoing assisted conception.

We have examined unfertilised oocytes and their first polar bodies (PBs) to determine the way in which the frequency of whole chromosome imbalance compares with that involving single chromatids and whether the precocious separation of chromatids prior to anaphase I affects all pairs of chromosomes. We have applied the technique of fluorescent in situ hybridisation in a three-stage method by using locus-specific probes for chromosomes 13 and 21 and alpha-satellite probes for chromosomes 1, 9, 16, 18 and X to determine the chromosome status of oocytes and their PBs. We obtained analysable results from 127 oocytes and 57 PBs from 72 patients of average age 33 years. Six oocytes and three PBs had extra signals but, of these, three were derived from a single patient, aged 26. Anomalies were seen in chromosomes 13, 16, 18, X and, notably, 21 but none were observed in chromosomes 1 and 9. Half of the anomalies involved additional chromatids rather than whole chromosomes. Since particular chromatids were found to be prematurely separated in the metaphase II oocyte, this may provide further evidence for an additional mechanism of maternal aneuploidy that operates at anaphase II. Detailed analyses of both oocytes and PBs have elucidated possible mechanisms leading to aneuploid gametes in this group of patients with fertility problems.

Persistence of two Y chromosomes through meiotic prophase and metaphase I in an XYY man.

Studies of spermatogenesis in an XYY male, presenting at a subfertility clinic, confirm the tendency for the germ cells to lose the second Y chromosome but for some XYY cells to reach metaphase I (MI). Light microscope studies of MI revealed the presence of YY bivalents and EM studies of microspread, silver-stained pachytene stages showed 30% of the cells to have two Y chromosomes; 13 out of 16 of these showing a YY synaptonemal complex. Strikingly, the Y axes show only partial synapsis; in no case was synapsis of the long arm heterochromatic regions apparent.

45,X/46,X dic (Y) mosaicism in a phenotypic male.

Cytogenetic analysis, confirmed by in situ hybridisation studies, showed a mosaic 45,X/46,X dic (Y) (q12) karyotype in a 14 year old boy who was initially diagnosed as having Noonan's syndrome. He made an early response to recombinant growth hormone; this suggests that this treatment may improve final height.

Amylo-1,6-glucosidase activity in cultured cells: a deficiency in type III glycogenosis with prenatal studies.

Deficiency of amylo-1,6-glucosidase activity was expressed in parallel in liver and skin fibroblasts from a patient with type III glycogenosis. In crude extracts of control liver and muscle, amylo-1,6-glucosidase (M.W. 164000) was identified by immunoprecipitation; no cross-reacting material was found in the patient's liver. Assay of amylo-1,6-glucosidase activity in cultured skin fibroblasts from the affected family revealed less than 10 per cent of control value in mutant homozygous cells whereas in cells from the parents, activity was reduced to 40-60 per cent of the control value. Activity in cultured amniotic fluid cells was similar to that of control fibroblasts. In cultured amniotic fluid cells obtained during the mother's subsequent pregnancy, the normal amylo-1,6-glucosidase activity measured, predicted correctly the outcome of this pregnancy prior to the 20th week of gestation.

Chromosome abnormality in couples with histories of multiple abortions. The outcome of pregnancies subsequent to ascertainment and a study of familial translocation carriers.

A series of couples with histories of recurrent abortions and in whom one partner had been shown to have a major chromosomal anomaly were investigated with respect to the karyotypes of conceptions subsequent to ascertainment. The reproductive histories of translocation carriers amongst relatives were also studied. Results were compatible with previous reports of the behaviour of translocation chromosomes at meiosis with an additional previously undescribed outcome as a result of a maternal 13/14 Robertsonian translocation.

Incidence of familial Hodgkin's disease.

The family histories of 131 patients with histologically defined Hodgkin's disease (HD) were studied and 2,517 first and second degree relatives and spouses were identified and followed-up. The causes of death in deceased relatives were ascertained from death certificates. The numbers of deaths from selected causes were compared with the numbers that would be expected if the relatives had suffered the same mortality rates as the Scottish national population. A 4-fold increase in deaths due to HD was found among first and second degree relatives of patients with the disease (6 cases observed compared with 1.4 expected). Five of the 6 familial cases were related to index patients with the mixed cellularity form of the disease, the remaining case was the brother of a patient with the lymphocyte-depleted form of the disease. The increased risk was seen among relatives of both young and older patients and there was no consistent intrafamilial similarity in age of onset or time of onset of disease.