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.

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.

Sequential FISH analysis of oocytes and polar bodies reveals aneuploidy mechanisms.

OBJECTIVES: Constitutional aneuploidy occurs in at least 5% of recognised pregnancies, with apparent preferential involvement of the X chromosome and the smaller autosomes. Molecular cytogenetic investigations of cleavage-stage embryos have revealed anomalies affecting all sizes of chromosomes. The aim was to investigate the variety of anomalies arising during maternal meiosis I by analysis of unfertilised oocytes and polar bodies to gain insight into aneuploidy mechanisms. METHODS: Sequential FISH analysis was carried out with specific probes derived from eight chromosomes, representing all sizes. Only imbalance due to a gain of a whole chromosome or chromatid, represented by extra signals, was counted to avoid artefact. RESULTS: Data were obtained on 236 eggs from 124 patients of average age 32.5 years (range 22-44). Ten patients (average 32.6 years) had abnormal eggs. The abnormality rate for oocytes and for polar bodies was close to 4% for each. Fourteen hyperploidies were found, seven involving additional single chromatids. The abnormalities affected chromosomes 13,16,18, 21 and X but not chromosomes 1, 9 or 12. CONCLUSION: The data provide evidence for several mechanisms leading to aneuploidy, including classical non-disjunction of whole univalents; pre-division of chromatids prior to anaphase I, leading to imbalance detected at metaphase II; gonadal mosaicism for a trisomic cell line and preferential involvement of the smaller chromosomes. Monosomy for the large autosomes is not uncommon in cleavage-stage embryos and may additionally arise from anaphase lag preferentially affecting such chromosomes.

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.

Trends in prenatal diagnosis of Down syndrome and other autosomal trisomies in Scotland 1990 to 1994, with associated cytogenetic and epidemiological findings.

The present report summarizes findings on 670 cases of autosomal trisomy diagnosed in Scotland, with actual or expected dates of delivery in 1990 to 1994 inclusive. Cases were notified by cytogenetic service laboratories. There were 277 prenatal and 369 postnatal diagnoses and 24 spontaneous losses. Excluding the latter, numbers diagnosed with trisomy 21, trisomy 18, trisomy 13, and other trisomies were, respectively, 470 (72.8%), 108 (16.7%), 36 (5.6%), and 32 (5.0%). Estimated maternal age-specific birth rates for trisomy 21 were close to published values from other jurisdictions. However, comparisons with a clinically based national register of congenital anomalies suggested that 3-4% of Down syndrome births were never karyotyped, most being early neonatal deaths. There was a striking increase over the period in the proportion of cases detected prenatally, associated with increased maternal serum screening in mothers <35 years old. Over the 3 final years (1992-1994), prenatal screening followed by elective termination was estimated to reduce the birth rate in trisomy 21 by 24% in mothers aged <35 years, by 57% in older mothers, and by 35% in all mothers. The crude incidence per 1,000 births fell from 1.08 in 1990-1991 to 0.77 in 1992-1994, in spite of an upward shift in the overall maternal age distribution. For trisomies 18 and 13, the estimated overall reductions in the birth rate over the whole 5-year period were respectively, 26 and 17%. In free trisomy 18, there was a significant reduction in the sex ratio (male/female) to 0.65, in line with earlier studies.

Ascertainment and severity of Marfan syndrome in a Scottish population.

This study in north east Scotland has shown that Marfan syndrome has a minimal birth incidence of 1:9802 live births, a minimal prevalence of 1:14217, and that 8/30 (26.7%) of cases in our series are new mutations. The calculated mutation rate is 15 +/- 6.7 x 10(-6) and there is evidence of reduced reproductive fitness.

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.

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.

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.

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.

Prenatal exclusion testing for Huntington's disease: a problem of too much information.

At eight weeks of pregnancy a couple were informed that the prospective father's mother had died of Huntington's disease (HD). There were no living affected members in the immediate family to confirm the diagnosis. By inspection of the local genetic register, it was established that it was indeed HD segregating in the extended family. Genotyping of the prospective mother and father, the father's unaffected father, and his unaffected maternal grandmother was carried out using a battery of polymorphic DNA markers, including a new probe which has a very low recombination rate with the HD locus. Analysis of DNA from a chorionic villus sample taken at 10 weeks of pregnancy showed that the fetus must have inherited a chromosome from its father's affected mother. Its risk of developing HD was 47%. If the genotype of the unaffected maternal grandmother was taken into account, the risk was reduced to 42%. Neither risk was considered acceptable by the prospective parents and the pregnancy was terminated at 12 weeks' gestation. Prospects for future pregnancies are good, with a 50% chance of having a child whose risk of inheriting the HD gene is less than 1.5%. In retrospect it was noted that although genotyping of the maternal grandmother had refined the fetal risk, it had also nearly contributed to an inadvertent and unwanted predictive test for HD on the father. This case makes the point that in prenatal exclusion testing, linkage information must be generated with considerable care.

Features of di George syndrome in a child with 45,XX,-3,-22,+der(3),t(3;22)(p25;q11).

A child with an unbalanced translocation resulting in monosomy for chromosomes 22 (q11----pter) and 3(p25----pter) is described. Although no immunological dysfunction could be demonstrated, the abnormalities found are similar to those seen in the di George syndrome which has been associated with monosomy for the same region of chromosome 22.

Wiedemann-Beckwith syndrome in one of monozygotic twins.

A pair of monozygotic twins discordant for Wiedemann-Beckwith syndrome is described. The probability of monozygosity is 0.995. This observation suggests that the syndrome is unlikely to be under single gene control and genetic counselling should be based on multifactorial inheritance.

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.

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.

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.

Isodicentric X chromosome in a moderately tall patient with gonadal dysgenesis: lack of effect of functional centromere on inactivation pattern.

An isodicentric X chromosome (46, X idic (X)(pter leads to qter::qter leads to pter)) with a single functioning centromere was found in all lymphocytes and fibroblasts examined from a female patient 171.5 cm in height presenting with primary amenorrhoea. Replication of the abnormal chromosome was consistently late. In some cells the pattern was asymmetrical but the asymmetry did not appear to relate to the position of the active centromere.