Evaluation of chromosomal risk following intracytoplasmic sperm injection in the mouse

To investigate whether cytogenetic risks occur using the mouse intracytoplasmic sperm injection (ICSI) technique, the incidence of chromosome aberrations was compared in one-cell embryos produced by ICSI technique and those by conventional in vitro fertilization (IVF) technique. Spermatozoa were incubated in TYH medium for 1.5-2 h before IVF insemination. For the ICSI technique, spermatozoa were incubated in five different media: TYH, Hepes-buffered TYH (H-TYH), modified CZB (mCZB), Hepes-buffered mCZB (H-mCZB), and PB1 for 0.5 h, 2-2.5 h, and 6 h before injection into metaphase II oocytes. The incidence of IVF embryos with structural chromosome aberrations was 2%, whereas the occurrence of structural chromosome aberrations in ICSI embryos was dependent on the kind of medium and sperm incubation time. When spermatozoa were incubated in TYH medium for 2 h or more, the aberration rates in the resultant ICSI embryos (4%) were not significantly different from that of IVF embryos. However, there was a significant increase in aberration rates in ICSI embryos derived from spermatozoa that were incubated in other culture conditions (6%-28%). In addition, a time-dependent increase in aberration rates was found in ICSI embryos when H-TYH, H-mCZB, and PB1 were used for sperm incubation. There was no significant difference in incidence of aneuploidy between IVF and ICSI embryos. The chromosome analysis results of one-cell embryos were reflected by the performance of postimplantation embryo development. The causal mechanism of chromosome damage in ICSI embryos was discussed in relation to the plasma membrane cholesterol, the acrosome, and in vitro aging of spermatozoa.     

An unexpected high frequency of trisomic fetuses in 229 pregnancies monitored for advanced maternal age

Summary In 229 pregnancies monitored because of advanced maternal age, 16 (7%) abnormal fetal karyotypes were detected. We found 13 cases of trisomy 21, twice a trisomy 18, and once an additional marker chromosome. The frequency of abnormal fetal karyotypes in different maternal age groups was found to increase from 1:20 at 38–40 years, to 1:16 at 41–43 years, and finally to 1:45 in women of 44–46 years. The overall incidence of chromosomal aberrations and specifically of trisomy 21 is considerably higher than that described in retrospective studies.     

Cytogenetic results of amniocentesis materials: incidence of abnormal karyotypes in the Turkish collaborative study

The experience on prenatal chromosome diagnosis of four Turkish centers participating in a collaborative study on 6041 genetic amniocentesis performed during a 4-8 years period were reviewed. 5887 (97.5%) patients had strong clinical indications for prenatal chromosome studies and 154 (2.5%) were referred because of maternal anxiety and a bad history of previous gestations. The main indication groups were: advanced maternal age (3197 cases), positive serum screening (2011 cases), ultrasound-identified anomaly (492 cases), previous fetus/child with chromosomal aberrations (103 cases), a history of a previous abnormal and/or mentally handicapped child (70 cases) and a parental chromosome rearrangement (14 cases). The average maternal age was 33.9 years and average gestational age was 18 weeks. A total of 179 affected fetuses were detected in this collaborative study (3%) of which 133 were unbalanced (74.3%). Among the 124 (69%) numerical aberrations, 102 (82.3%) were autosomal aneuploidies, 20 (16.1%) were gonosomal aneuploidies and 2 (1.6%) were poliploidies. Among the 55 (31%) structural aberrations, balanced translocation was the most common (63.6%) and 11 cases of inversion, four cases of unbalanced translocation, two cases of marker chromosome and three cases of other abnormalities were found. The overall culture success rate was 99.7%. Pregnancy termination that is permitted by legal authorities was accepted by 94.7% (126/133) with parents at unbalanced cytogenetic result announcement.     

Detection of structural and numerical chromosome abnormalities in interphase cells using spectral imaging.

Chromosome abnormalities are common causes of congenital malformations and spontaneous abortions. They include structural abnormalities, polyploidy, trisomy, and mosaicism. In in vitro fertilization (IVF) programs, preimplantation genetic diagnosis (PGD) of oocytes and embryos has become the technique of choice to select against abnormal embryos before embryo transfer. For diagnosis of structural abnormalities, we developed case-specific breakpoint-spanning DNA probes. Screening of an in-house yeast artificial chromosome (YAC) library is facilitated by information from publicly available databases and published articles. Most numerical chromosome abnormalities, on the other hand, are detrimental to early embryonic development and increase with maternal age. We therefore developed a multichromosome screening technique based on spectral imaging to simultaneously detect and score as many as 10 different chromosome types. The probe set was chosen to detect more than 70% of all numerical chromosome aberrations responsible for spontaneous abortions. Detecting structural and numerical abnormalities in single interphase cells using spectral imaging is a powerful technique for multilocus genetic screening.     

Clonal structural chromosomal rearrangements in primary fibroblast cultures and in lymphocytes of patients with Werner's syndrome

Summary The cytogenetics of six cases of adult progeria (Werner's syndrome) from three Sardinian families were investigated. The overall increased incidence of chromosome breakage found in cultured lymphocytes and fibroblasts seems to be age-dependent. The occurrence of clonal variegated translocation mosaicism, previously found by other authors in fibroblast cell lines derived from Werner patients was demonstrated also in fibroblasts analyzed in situ on the outgrowth halos from primary skin explants; a strong indication that these aberrations are present in the in vivo precursors. The same type of clonal structural aberration was found for the first time also in 72h-cultured lymphocytes. These findings demonstrate that Werner's syndrome is indeed a further example of a chromosome rearrangement syndrome.     

Chromosome abnormalities in early chicken (Gallus domesticus) embryos

Chromosome studies were undertaken to determine if early embryonic mortality in chicken (Gallus domesticus) embryos is associated with chromosome aberrations. A rapid cytological technique was developed for screening large numbers of embryos for euploidy and aneuploidy. — Of 115 embryos examined, 6 or 5.2% had aberrant chromosome complements. All of these chromosome aberrations occurred in embryos that were phenotypically abnormal. Of 45 macroscopically abnormal embryos, 13.3 % were chromosomally aberrant. These included two cases of haploidy (A-Z), one case of trisomy-1, a case of trisomy-2 and two cases of triploidy (3A-ZZW and 3A-ZWW). — Possible modes of origin for euploid and aneuploid embryos are discussed and consideration given to the significance of these aberrations in relation to embryo viability, constancy of chromosome numbers and nucleolar organization.     

Chromosome abnormalities in chicken (Gallus domesticus) embryos: Types,frequencies and phenotypic effects

Cytological screening of 4182 chick embryos from 10 strains and 5 strain crosses was performed to determine the types and frequencies of chromosome abnormalities. Gross phenotypic effects, such as growth retardation and malformation, were noted. Clues to the etiology of such chromosome aberrations were also sought. The following euploid series was observed: Haploid mosaics (A-Z/2A-ZZ, A-Z/2A-ZZ/3A-ZZZ, A-Z/A-W/2A-ZW/2A-ZZ, A-Z/A-W/2A-ZW/ 3A-Z?), diploid (2A-ZZ and 2A-ZW), triploid (3A-ZWW, 3A-ZZW, 3A-ZZZ, 3A-ZZZW) and tetraploid (4A-ZZWW and 4A-ZZZZ). Aneuploidy was observed as follows: Trisomy for chromosome numbers 1, 2, 3, 4 and double trisomy 2/5. Trisomy-4 with deletion of 50% of the long arm of one member of the trisomic triplet was observed. A 3A-ZWW embryo was found with two cell populations: one, disomic for chromosome 2 and 6; the other, tetrasomic for 2 and 6. Of the 4182 embryos sampled 1.4% were haploids, 97.5% diploids, 0.8% triploids, 0.1% tetraploids and 0.2% trisomics. On the average 10.8% of the early dead embryos were euploid (excluding diploid) or aneuploid. However, the range for euploidy and aneuploidy among strains was 2.3–23.7% of early deads. Haploid embryos were consistently underdeveloped at 4 days of incubation (D.I.), and died by 5–7 D.I. About 90% of (36) triploid embryos died at or before 4 D.I. The remaining 10% (normal embryos) died prior to hatching. Trisomic embryos were dead or underdeveloped at 4 D.I. Tetraploidy appeared to be lethal at a very early stage. The various strains examined had different overall rates of chromosome aberrations (0.4–8.9%), and also showed different varieties of such aberrations. The modes and possible causes of meiotic, mitotic and fertilization errors are considered. Genetic control of chromosome abnormalities, particularly haploidy, is postulated.     

Chromosome aberrations in mouse embryos and fetuses produced by assisted reproductive technology

The occurrence of structural chromosome aberrations in mouse one-cell embryos produced by intracytoplasmic sperm injection (ICSI) with mature spermatozoa was dependent on the type of sperm incubation medium and sperm incubation time. When cauda epididymal spermatozoa were used following incubation in bicarbonate-buffered TYH medium for 0h (no incubation) and 0.5h, the chromosome aberration rates (6.9% and 7.4%, respectively) in the resultant embryos were significantly higher than that (2.3%) in the IVF embryos. However, when the spermatozoa were incubated for 2-2.5h and 6h in the same medium, the chromosome aberration rates were reduced to the IVF embryo level (3.8% and 4.3%, respectively). When spermatozoa incubated in Hepes-buffered H-mCZB and phosphate-buffered PB1 media were used for ICSI, chromosome aberration rates in embryos were significantly high (8.6-28.1%) and increased in a time-dependent manner. On the other hand, when immature testicular spermatozoa were incubated in those three media for 0.5h and 6h, the incidences of resultant embryos with structural chromosome aberrations ranged between 7.4% and 11.7%, and there was no medium- and time-dependent change in these aberration rates. To evaluate transmissible risk of chromosome aberrations to offspring, two- or four-cell embryos derived from cauda epididymal spermatozoa were transferred into the oviducts of pseudopregnant females and chromosomes of live fetuses were examined on gestational day 16. One (2.0%) mosaic fetus was found when spermatozoa were incubated in TYH for 2-2.5h, and there were four (6.7%) fetuses displaying a structurally abnormal karyotype when spermatozoa were incubated in H-mCZB for 2-2.5h, indicating that structural chromosome aberrations generated in ICSI one-cell embryos are transmissible to offspring. The causal mechanism of structural chromosome aberrations in ICSI one-cell embryos is discussed in relation to the acrosomal plasma membrane cholesterol and the acrosome.     

Down's syndrome in Wallonia (South Belgium), 1971–1978: Cytogenetics and incidence

Summary During the 8 year-period 1971–1978 inclusive, 268 newborn with Down's syndrome (DS) were ascertained in Wallonia (South Belgium). The chromosomes of all patients were analyzed. A standard trisomy 21 was observed in 259 cases (96.6%) and translocations in seven (2.6%). One mosaic (0.4%) and one case with a 47,XX,+21,5 p-karyotype (0.4%) were also found. The overall incidence at birth for the 8 year-period was of 0.123%. However, the comparison of the 2 four-year periods 1971–1974 and 1975–1978 showed an increased incidence in mothers below 35 during the second period, reaching a statistical significant level in Charleroi, and industrial and densely populated area of the region (Fisher's exact test: P=0.016<0.025). Young women have now become the principal source of DS patients in Wallonia. This change from the previous pattern happened abruptly in 1975, and strongly suggests that one or more persisting factors relating tomeiotic non-disjunction of chromosome 21 are operating in this region since 1974, and affecting DS births from 1975 onwards.     

Incidence of chromosome abnormalities in newborn children. Comparison between incidences in 1969–1974 and 1980–1982 in the same area

Summary As part of an ongoing study of the influence of environmental factors on pregnancy, childbirth, and fetuses, comparisons have been made between incidences in 1969–1974 and in 1980–1982 of chromosome aberrations in liverborn children in the same area of Denmark. The incidence of chromosome aberrations in the first period was 2.6 per 1000, compared with 4.1 per 1000 during the latter period. However, the difference was mainly due to an increase in inversions, and this in turn was due to a difference in chromosome staining methods between the two periods.It is concluded that the Danish study and similar studies in the United States, Canada, and Scotland indicate that early detection of chromosome aberrations by chromosome examination at birth is indicated in order to be able to inform and counsel parents of children with chromosome aberrations. Chromosome examination at birth is also of importance in the diagnosis of structural inheritable chromosome aberrations and consequent family investigation and genetic counseling.     

Mental development of unselected children with sex chromosome abnormalities

Summary Twenty-five children with sex chromosome aberrations found among 11000 consecutively newborn children from 1969 to 1974 have been followed with psychologicalpsychiatric examinations at four different times from the age of 1 year till the ages of 7 to 11. The results of the follow-up studies are presented, and it is concluded that diagnosis of sex chromosome aberrations at birth or during early childhood with full information and guidance to parents and educators are of great importance for the development of the children, especially concerning learning and behavior.     

Chromosome breakage in human preimplantation embryos from carriers of structural chromosomal abnormalities in relation to fragile sites, maternal age, and poor sperm factors

Chromosome breakage is a fairly widespread phenomenon in preimplantation embryos affecting at least 10% of day 3 cleavage stage embryos. It may be detected during preimplantation genetic diagnosis (PGD). For carriers of structural chromosomal abnormalities, PGD involves the removal and testing of single blastomeres from cleavage stage embryos, aiming towards an unaffected pregnancy. Twenty-two such couples were referred for PGD, and biopsied blastomeres on day 3 and untransferred embryos (day 5/6) were tested using fluorescence in situ hybridisation (FISH) with appropriate probes. This study investigated whether chromosome breakage (a) was detected more frequently in cases where the breakpoint of the aberration was in the same chromosomal band as a fragile site and (b) was influenced by maternal age, sperm parameters, reproductive history, or the sex of the carrier parent. The frequency of breakage seemed to be independent of fragile sites, maternal age, reproductive history, and sex of the carrier parent. However, chromosome breakage was very significantly higher in embryos from male carriers with poor sperm parameters versus embryos from male carriers with normal sperm parameters. Consequently, embryos from certain couples were more prone to chromosome breakage, fragment loss, and hence chromosomally unbalanced embryos, independently of meiotic segregation.     

Chromosome breakage in low birth weight newborns.

The frequency of chromosome aberrations in cells cultured from umbilical cord blood was determined for 50 low birth weight (LBW) and 50 normal birth weight (NBW) euploid newborns matched for sex, race, and maternal age. The metaphase spreads had been prepared in the course of an earlier study of frequency of aneuploidy and results are from 72-h cultures, i.e., presumably, at the second division in vitro. There were no significant differences between the two groups in the frequency of cells with chromosome breakage, chromosome gaps, or hyperdiploid cells. There was, however, a significantly higher frequency of hypodiploid cells in the LBW group. The present findings differ from those of others who have reported an increase in chromosome breakage in premature newborns.     

Maternal age effect: the enigma of Down syndrome and other trisomic conditions.

Aneuploidy is the most frequently observed chromosome abnormality in human liveborn, abortuses and oocytes. The only etiological factor that has been established is advanced maternal age for the occurrence of trisomies, particularly trisomy 21 which causes Down syndrome. The maternal age effect remains an enigma. Recent molecular data bearing on this question are reviewed as are the hypotheses that have been proposed linking nondisjunction and maternal age. Rationale is presented for a compromised microcirculation hypothesis that explains the cause of nondisjunction and why its occurrence changes with maternal age from menarche to menopause. It takes into account two facts: (1) 95% of Down syndrome children receive their extra chromosome from their mother, and in 80% or more of these the nondisjunction occurred in the first meiotic division, which is completed in the ovary. (2) The ovarian follicle containing the primary oocyte has no internal circulation. The hypothesis proposes that aneuploid oocytes arise from a concatenation of events. It begins with hormonal imbalance that causes a less-than-optimal microvasculature to develop around the maturing and mature follicles. The resulting decrease in the size of the perifollicular capillary bed reduces the volume of blood flow through the area, leading to an oxygen deficit and a concomitant increase inside the follicle of carbon dioxide and anaerobic products, such as lactic acid. This in turn causes a decrease in the intracellular pH of the oocyte that diminishes the size of the spindle, with consequent displacement and nondisjunction of a chromosome. The compromised microcirculation hypothesis explains the occurrence of aneuploidy in primary and secondary oocytes, sperm precursor cells, tumor and embryonic cells. It also explains why women of all reproductive ages may have a Down syndrome child.     

Maternal cell contamination of cultures of spontaneous abortion fibroblasts: importance for cytogenetic analysis of embryonic lethality

The results of standard cytogenetic analysis of the long-term cultures of embryonic fibroblasts of 478 first-trimester spontaneous abortions were retrospectively reviewed. In 16% of embryos with cytogenetically confirmed karyotype 46,XX, the Y chromosome was found by molecular genetic methods. Prior to obtaining the chromosome preparations, the cell cultures of Y chromosome-carrying embryos were maintained for a longer period than the cultures of embryos without the Y chromosome. Thus, a late entry of a culture into the logarithmic growth phase serves as marker of maternal cell contamination. We developed a mathematic model for assessment of karyotype incidence and the "sex ratio" of spontaneous abortions, taking into account risk of maternal cell contamination in extraembryonic tissue cultures. Thus estimated, the incidence of chromosomal abnormalities in the studied sample increased from 54.6 to 60.3% and the expected sex ratio increased from 0.66 to 1.02 in abortions with normal karyotype. Using molecular analysis of inheritance of polymorphic DNA markers of six autosomes (2, 11, 16, 19, 20, and 21), the proposed model was tested on 60 embryos with karyotype 46,XX and their parents. Numerical chromosome abnormalities were revealed in uncultured tissues of seven abortions (11.7%), including four without the Y chromosome, which is in a good agreement with the expected incidence of karyotype abnormalities (8.3%) predicted by our model. In view of this, estimating risk of maternal cell contamination in embryonic cell cultures seems necessary for correctly assessing the effect of natural selection in humans, for understanding the mechanisms that determine the sex ratio, and for evaluating the precision of prenatal cytogenetic diagnosis of chromosomal abnormalities.     

Cytogenetic and histologic analyses of spontaneous abortions

Summary In a study of spontaneous abortions the correlations between karyotype (166 cases), anamnestic data, and macroscopic and histologic findings in placentas (107 cases) and embryos (73 cases) were analyzed. The main results were: 1. The rate of chromosomal aberrations was 39%. Trisomies predominated (60%), followed by monosomy X (20%), triploidies (14%), and structural aberrations (6%). 2. In trisomies a clear prevalence of female sex constitution (2:1) was observed. In normal karyotypes a slight prevalence of females was seen (1.2:1). 3. With increasing maternal age, more trisomies were found in the abortions. 4. Women whose index abortion had a normal karyotype had a history of fewer births but more abortions. 5. Trisomies of acrocentric chromosomes were mainly chorionic sacs with an embryo, while trisomies of the other autosomes resulted in intact empty sacs. 6. The average developmental stage of the embryos was 5 weeks, with a mean gestational age of 14 weeks. Gross malformations were found in 58% of the embryos.     

Genetic changes in human fetuses from spontaneous abortion after in vitro fertilization detected by comparative genomic hybridization

The in vitro fertilization (IVF) technique is becoming a very important approach for infertile disease therapy, but approximately 30% of pregnancies are spontaneously aborted in the first trimester. It is believed that chromosomal abnormality is the major reason for early spontaneous abortion. Although some reports have mentioned cytogenetic changes in spontaneously aborted embryos after IVF, little is known about the comprehensive cytogenetic alterations in these aborted embryos. Here we use the comparative genomic hybridization (CGH) technique to analyze the genetic alterations in 41 spontaneously aborted human specimens after IVF. In this study, 25 of 41 cases (61%) showed chromosomal changes. Among them, autosomes and sex chromosomes were involved in 16 and 11 cases, respectively. Several nonrandom chromosomal changes were identified, including loss of one sex chromosome (six cases) and gains of 22 (four cases), Y (four cases), 21 (three cases), 4 (two cases), and 13 (two cases). Our data support the opinion that chromosome abnormality is one of the major causes of early spontaneous abortion after IVF. The association between chromosome changes in these spontaneously aborted fetuses and maternal age, infertility patterns, infertility causes, and IVF patterns (routine IVF and other methods, including intracytoplasmic sperm injection, egg donation, and embryo donation) were also studied. No significant correlation was found.     

Micronucleus and cytogenetic disorders in mouse embryos before implantation

Studies of mice embryos of different genotypes revealed 4.1% of polyploids, 8.5% of aneuploids and 7.9% of embryos with structural chromosome aberrations. Embryos of lines BAIB and CBA were reliably different in micronuclei occurrence (9.8 and 4.6%). A relation is shown to exist between the presence of micronuclei and structural aberrations. Normalization of the embryo genotype at early stages of embryogeny might occur by means of elimination of cells with disturbances or due to the death of anomalous embryos in the period of implantation.     

染色体畸形变与膀胱癌发生发展的相关性研究

目的：通过荧光原位杂交技术（FISH）结合病理分级,探讨染色体畸形变与膀胱癌发生和发展的关系。方法：采用3、7、9、17号染色体着丝粒探针和9P16区带探针对105例膀胱癌复发患者尿液脱落细胞进行荧光原位杂交．观察膀胱癌复发患者中3、7、9、17号染色体的畸形变情况并分析其与患者临床和病理特征之间的关系。结果：105例膀胱癌复发患者中,3、7、9和17号染色体的非整数倍突变率分别是21．9％、29．5％、12．4％、和36．2％,与患者的性别、年龄无显著相关性（P〉0．05）。仅7号染色体畸变与膀胱癌的病理分级具有显著相关性[（P〈0．05）。结论：7号染色体畸形变与复发膀胱癌的病理分级显著相关。     

Maternal Mortality in British Columbia: A Study of 145 Deaths from 1955 to 1962

Between 1955 and 1962, 145 maternal deaths were reported in the Province of British Columbia. One hundred of them were due to obstetrical causes. Of these deaths, hemorrhage was by far the commonest cause (40 cases), followed in frequency by vascular accidents (23 cases), infections (17 cases), toxemia (10 cases), anesthetic deaths (five cases), and other causes (five cases). Significant avoidable factors were noted in 80%. Indirect obstetrical deaths accounted for 29 cases, or 20% of all maternal mortalities. The most frequently encountered causes of indirect obstetrical deaths were cardiac (nine cases) and vascular accidents (six cases). Avoidable factors were considered to be present in 19 of the 29, an incidence of 65%.When all deaths were considered together, 72% were felt to have significant avoidable factors when judged against an academic standard. It was also apparent that some 40% to 50% of deaths were intimately involved with social factors.