Double trisomy in spontaneous abortions

Cytogenetic data on products of conception from spontaneous abortions studied over a 10-year period have been reviewed for double trisomies. A total of 3034 spontaneous abortions were karyotyped between 1986 and 1997. Twenty-two cases with double trisomy, one case with triple trisomy, and a case with a trisomy and monosomy were found. The tissues studied were mostly sac, villi, or placenta. The gestational age ranged from 6 to 11 weeks and the mean age was 8.2 ± 1.7 (SD) weeks. The mean maternal age in years was 35.9 ± 5.3. Of the twenty-two cases, four were mosaics. All but two of the cases involved autosomal aneuploidies. The double trisomies included chromosomes 2, 4, 5, 7, 8, 12, 13, 14, 15, 16, 17, 18, 20, 21, and 22. The chromosomes that were trisomic in more than one double trisomy case were numbers 16 (8 cases), 8 (5 cases), 15 (4 cases), 2, 13, and 21 (3 cases each), and 5, 7, 14, 18, 20, 22, and X (2 cases). The triple trisomy involved chromosomes 18, 21, and X. The monosomy and trisomy case was a mosaic, with a monosomy 21 in all cells and some cells also with a trisomy 5. The double trisomies cited for the first time in this study were 4/13, 5/16, 8/14, 8/15, 14/21, 15/20, and 7/12. The pooled mean maternal age for double trisomy cases (34.1 ± 5.7 years) was higher than that for single trisomy cases (31 ± 6.1 years). The difference was statistically significant at P = < 0.001. The pooled mean gestational age of spontaneous abortions was lower for double trisomy (8.7 ± 2.2 weeks) than for reported single trisomy cases (10.1 ± 2.9 weeks). This difference is also statistically significant at P = < 0.001. The sex ratio among double trisomies was 15 females to 13 males. This difference was not statistically significant from the expected 1 : 1. Received: 27 June 1997 / Accepted: 4 September 1997     

Tissue-specific placental mosaicism for autosomal trisomies in spontaneous human abortuses: mechanisms of formation and phenotypic effects]

The frequencies of autosomal trisomies in extraembryonic human tissues were estimated in the cases of different abnormalities of prenatal development, from the confined placental mosaicism (CPM) with either relatively normal embryogenesis or restricted intrauterine growth to spontaneous abortion. A tissue-specific compartmentalization was found to be characteristic of cell lines with trisomies for individual autosomes. Analysis of various phenotypical effects of chromosomal aberrations associated with mosaicism is necessarily required to understand the mechanisms and factors responsible for tissue chromosomal mosaicism. Based on analysis of the cell karyotype during prenatal diagnosing of chromosome aberrations in tissues of both extraembryonic and embryonic origin, in 1996, Wolstenholme proposed a model of CPM for individual chromosomes. According to the model, the distribution of cell lines with autosomal trisomies between extraembryonic tissues depends on the ratio between meiotic and mitotic mutations early in embryonic development. However, the model cannot be used to study tissue chromosomal mosaicism in spontaneous abortions, because little information is available on cell karyotype in embryonic tissues themselves after intrauterine fetal death. In this work, a model of tissue-specific chromosomal mosaicism was suggested based on the data on cell karyotype determined in extraembryonic tissues alone, which can be helpful in evaluating the contribution of tissue chromosomal differences into the etiology of early intrauterine death. Along with the experimental evidence, comparative analysis of the two models indicated that the meiotic chromosome nondisjunction plays the major role in trisomy formation and the resultant spontaneous arrest of embryonic development. Other factors responsible for tissue-specific distribution of chromosomal aberrations are also discussed. These are differences in cell proliferative activity, as well as changes in compartmentalization and migration of cells with abnormal karyotypes.     

Tissue-Specific Placental Mosaicism for Autosomal Trisomies in Human Spontaneous Abortuses: Mechanisms of Formation and Phenotypical Effects

The frequencies of autosomal trisomies in extraembryonic human tissues were estimated in the cases of different abnormalities of prenatal development, from the confined placental mosaicism (CPM) with either relatively normal embryogenesis or restricted intrauterine growth to spontaneous abortion. A tissue-specific compartmentalization was found to be characteristic of cell lines with trisomies for individual autosomes. Analysis of various phenotypical effects of chromosomal aberrations associated with mosaicism is necessarily required to understand the mechanisms and factors responsible for tissue chromosomal mosaicism. Based on analysis of the cell karyotype during prenatal diagnosing of chromosome aberrations in tissues of both extraembryonic and embryonic origin, in 1996, Wolstenholme proposed a model of CPM for individual chromosomes. According to the model, the distribution of cell lines with autosomal trisomies between extraembryonic tissues depends on the ratio between meiotic and mitotic mutations early in embryonic development. However, the model cannot be used to study tissue chromosomal mosaicism in spontaneous abortions, because little information is available on cell karyotype in embryonic tissues themselves after intrauterine fetal death. In this work, a model of tissue-specific chromosomal mosaicism was suggested based on the data on cell karyotype determined in extraembryonic tissues alone, which can be helpful in evaluating the contribution of tissue chromosomal differences into the etiology of early intrauterine death. Along with the experimental evidence, comparative analysis of the two models indicated that the meiotic chromosome nondisjunction plays the major role in trisomy formation and the resultant spontaneous arrest of embryonic development. Other factors responsible for tissue-specific distribution of chromosomal aberrations are also discussed. These are differences in cell proliferative activity, as well as changes in compartmentalization and migration of cells with abnormal karyotypes.     

Cytogenetic analysis of 750 spontaneous abortions with the direct-preparation method of chorionic villi and its implications for studying genetic causes of pregnancy wastage

Altogether, 750 cases of spontaneous abortion between the fifth and 25th week of gestation were analyzed cytogenetically by the direct-preparation method using chorionic villi. The majority of cases (68%) were derived from early abortions before the 12th week of gestation. The frequency of abnormal karyotypes was 50.1%; trisomy was predominant (62.1%), followed by triploidy (12.4%), monosomy X (10.5%), tetraploidy (9.2%), and structural chromosome anomalies (4.7%). Among trisomies, chromosomes 16 (21.8%), 22 (17.9%), and 21 (10.0%) were prevalent. The frequency of chromosomally abnormal abortions increased with maternal age but only because of an increase of trisomy. Polyploidy and monosomy X, however, decreased. Mean maternal age was significantly increased for trisomies 16, 21, and 22 and was highest for trisomies 18 and 20. The results obtained are within the range of variability reported earlier from tissue culture-type studies. A consistent feature during our study is the excess of females in chromosomally normal abortions (male:female sex ratio 0.71). According to the methodology applied, maternal cell contamination and undetected 46,XX molar samples cannot have influenced the sex ratio. However, a bias introduced by social status or maternal age cannot be excluded. With the more rapid and convenient direct preparation of chorionic villi, reliable cytogenetic data on causes of spontaneous abortions can be obtained.     

Molecular Cytogenetic Characteristics of Chromosome Imbalance in Spontaneous Human Abortion Cells with Low Proliferative Activity in Vitro

Karyotyping of noncultivated cells of 60 first-trimester spontaneous abortions (blighted ovum and missed abortions) was carried out using fluorescence in situ hybridization (FISH) with centromere-specific DNA probes for all chromosomes of the karyotype. Conventional cytogenetic study of these abortions was impossible because of cell culture failures. The algorithm is proposed for molecular cytogenetic FISH analysis of interphase karyotypes. Chromosome abnormalities were found in 32 fetuses (53.3%). In groups of missed abortions and blighted ovum, the frequency of numerical chromosome abnormalities was 50 and 60%, respectively. Both the numerical chromosome abnormalities typical of spontaneous human abortions (autosomal trisomies, sex chromosome aneuploidy, and polyploidy) and a relatively rare type of genomic imbalance unidentifiable by standard cytogenetic analysis (autosomal monosomies 7, 15, 21, and 22 in mosaic state) were observed. The frequency of these type of chromosome abnormalities comprised 19% of all known karyotype abnormalities determined in spontaneously aborted embryos. Note that the level of confined placental mosaicism in embryos with low cell proliferative activity was 25%, which is substantially higher than the corresponding parameter (1–2%) determined by prenatal diagnosis of chromosome abnormalities in developing embryos. The results of interphase FISH analysis of cells with low proliferative activity in vitro suggest that the pathology of early fetal development and missed abortion in humans are associated with a wider spectrum of chromosome abnormalities.     

Molecular cytogenetic characteristics of chromosome imbalance in cells of spontaneous human abortion fetuses with low proliferative activity in vitro

Karyotyping of noncultivated cells of 60 first-trimester spontaneous abortions (blighted ova and missed abortions) was carried out using fluorescence in situ hybridization (FISH) with centromere-specific DNA probes for all chromosomes of the karyotype. Conventional cytogenetic study of these abortions was impossible because of cell culture failures. The algorithm is proposed for molecular cytogenetic FISH analysis of interphase karyotypes. Chromosome abnormalities were found in 32 fetuses (53.3%). In groups of missed abortions and blighted ova, the frequency of numerical chromosome abnormalities was 50 and 60%, respectively. Both the numerical chromosome abnormalities typical of spontaneous human abortions (autosomal trisomies, sex chromosome aneuploidy, and polyploidy) and a relatively rare type of genomic imbalance unidentifiable by standard cytogenetic analysis (autosomal monosomies 7, 15, 21, and 22 in mosaic state) were observed. The frequency of these type of chromosome abnormalities comprised 19% of all known karyotype abnormalities determined in spontaneously perished embryos. Note that the level of confined placental mosaicism in embryos with low cell proliferative activity was 25%, which is substantially higher than the corresponding parameter (1-2%) determined by prenatal diagnosis of chromosome abnormalities in developing embryos. The results of interphase FISH analysis of cells with low proliferative activity in vitro suggest that the pathology of early fetal development and missed abortion in humans are associated with a wider spectrum of chromosome abnormalities.     

Q-banding of chromosomes in human spontaneous abortions

Chromosome studies of 242 spontaneous abortions were carried out by Q-banding technique. The abortuses were selected for study because they were phenotypically abnormal, had not progressed beyond 12 weeks development or were from women with repeated abortions. Chromosome anomalies were found in 126 (52%) of the abortuses. Of these, 71 (56%) were trisomies. Trisomies were found for all the autosomes except Nos. 1, 3, 5, 11, 17 and 18. Triploidy was the second commonest anomaly in this series, making up 26 (21%) of the total anomalies. About 70% of these had an XXY sex chromosome complement. Only 16 (13%) of the abortuses had X monosomy, a lower frequency than would be expected in an unselected study. Tetraploidy was found in 8 abortuses and the 5 remaining specimens had various anomalies. These included 3 translocations, a trisomy 21,X monosomy and a ring chromosome 13. Except for the greater frequency of XXY than XXX sex chromosomes in the triploids, there was no evidence of a distortion of the sex ratio, either among the trisomic or among the chromosomally normal abortuses.     

Karyological study of human spontaneous and medical abortions

80 spontaneous abortions (40 at 6-12 weeks and 40 at 13-32 weeks of pregnancy) and 50 induced abortions (49 at 6-12 weeks and 1 at 18 weeks) were karyotyped. Among spontaneous abortions of the first trimester were found 14 different types of chromosome anomalies. The chromosome complex 48,XY, D+, F+, has not yet been described in the literature and belongs to the eempty embryonic sac 1.0:1.5 cm with a weakly developed trophoblast. Among late-term spontaneous abortions no chromosome anomalies were found. An aberrant karyotype of a 7-8 week induced abortion was revealed as 46, XX/47, XXX (5% of the nuclei with double sex chromatin and 47% with single sex chromatin). Normal karyotypes were distributed according to sex chromosome complex as follows: spontaneous abortions of 6-12 weeks, 18(XY), 8(XX); spontaneous abortions 13-32 weeks, 19(XY), 21(XX); induced abortions, 16(XY), 32(XX). The data revealed a random character in the series of investigations. The absence of the XO monosomies was noted.     

Pathology of amniogenesis in the early prenatal period of human development

Morphological and, in a number of cases, cytogenetical investigation has been performed in 420 intact embryonal sacs and in embryos 7-8-week-old, obtained at spontaneous abortions (272) and at tubal pregnancy (148). Among these cases 202 (48.1%) intact empty embryonal sacs, 75 (17.9%) embryos with panorganodysplasia, 25 (6%) embryos with isolated developmental defects and 118 (28%) phenotypically normal embryos have been revealed. Pathology of amniogenesis such as aplasia or hypoplasia of the amniotic cavity is noted in 136 (32.4%) cases. Among 75 embryos with panorganodysplasia anomalies such as hypoplasia of the amniotic cavity in combination with a partial extra-amniotic++ position of the embryos in exocelom (10.7%), aplasia (5.3%) or hypoplasia (17.3%) amniotic peduncle is present in 43 (57.3%) observations. Out of 40 such cases at spontaneous abortions, cytogenetically investigated, in 27 (67.5%) chromosomal disorders (tetraploidy, triploidy, autosomal trisomy and monosomy) are revealed. Aplasia and hypoplasia of the amniotic cavity are considered as pathology of histogenesis at the tissue stage of the early human ontogenesis, that most evidently occurs as a result of asplasia, destruction or anomaly of embryoblast during the first phase of gastrulation on the 7th-11th day of the intrauterine development.     

Sex ratio in early embryonal mortality in man

The problem of the functioning specificity of sex chromosomes during the early stages of embryogenesis in man and the associated problem of the sex ratio in spontaneous and induced abortions, as well as in newborns, remains open. We have conducted a cytogenetic examination of 342 spontaneous abortions divided into three clinical groups on the basis of the severity of the developmental disturbances of the embryo: spontaneous abortions sensu stricto with a developed embryo without any significant intrauterine delay of development (n = 100), nondeveloping pregnancies (n = 176), and anembryonic fetuses (n = 66). The frequency of chromosomal mutations in these groups was 22.0, 48.3, and 48.5%, respectively. Statistical analysis has demonstrated significant differences between the studied groups in the frequencies of the normal and abnormal karyotypes: the major contributions to these differences were associated with autosomal trisomy, triploidy, and 46,XY karyotype. The presence of 46,XY may reflect specific genetic mechanisms of prenatal mortality of embryos with normal karyotype, associated with sex and/or with the imprinting of X-chromosomes. The sex ratio in spontaneous abortions with normal karyotype was as follows: 0.77 for spontaneous abortions with well-developed embryos without any significant intrauterine delay of development; 0.60 for non-developing pregnancies; and 0.31 for anembryonic fetuses. An analysis of DNA from the embryos and their parents has demonstrated a low probability of contamination of cell cultures with mother cells as a possible source of prevalence of embryos with 46,XX karyotype among spontaneous abortions. Nondeveloping pregnancies and anembryonic fetuses showed statistically significant differences in the sex ratio (1.11) from the control group consisting of medical abortions. Differences in the sex ratio were due to an increasingly lower proportion of embryos with karyotype 46,XY (relative to the expected one) among the fetuses with an increased severity of developmental disturbances. The statistical "chances ratio" index also provided evidence that embryos with 46,XY karyotype had a higher propensity to produce a well-formed fetus as compared with the female embryos. We propose that the expression of genes of the maternal X-chromosome in XY embryos supports a more stable development during early embryogenesis as compared with XX embryos. In the latter case, normal development is coupled with the operation of an additional mechanism for compensation of the dose of X-linked genes. Operation of this mechanism increases the probability of disturbances in female embryos. A higher viability of XY embryos during the early stages of ontogenesis in man appears to explain their underrepresentation in samples of spontaneously aborted embryos and appears to be the major factor responsible for the deviation of the sex ratio from the theoretically expected value.     

Holoprosencephaly in human embryos: epidemiologic studies of 150 cases

One hundred fifty embryos with holoprosencephaly were found among the total of 36,380 conceptuses obtained through induced abortion in the period from 1962 to 1974, giving an overall incidence of 0.4 percent. The occurrence was period from 1962 to 1974, giving an overall incidence of 0.4 percent. The occurrence was largely at random through time, and no "epidemic" was noted in particular years or months, but there appeared more cases derived from conceptions in winter than in summer months. The mean maternal age did not differ significantly from that of the general embryonic population, indicating that, although none of our cases were karyotyped, chromosome aberrations such as trisomies 13 and 18 that are closely associated with maternal age may not constitute a major part as causes of holoprosencephaly in human embryos. Materal age did not differ by the presence or absence of associated external anomalies. No association was found with paternal age, parental consanguinity nor with maternal medical history, including irregularity of menstrual cycles, and smoking and drinking habits. There was an indication that the mothers were prone to have repeated miscarriages, supporting the view that some kind of maternal predisposition is responsible for the causation of holoprosencephaly. Argument was made that, apart from various chromosome aberrations well documented as causes of this malformation, polygenic mechanism probably accounts for the majority of the cases with normal karyotype.     

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.     

About the sex ratio in connection with early embryonic mortality in man

The problem of the functioning specificity of sex chromosomes during the early stages of embryogenesis in man and the associated problem of the sex ratio in spontaneous and induced abortions, as well as in newborns, remains open. We have conducted a cytogenetic examination of 342 spontaneous abortions divided into three clinical groups on the basis of the severity of the developmental disturbances of the embryo: spontaneous abortionssensu stricto with a developed embryo without any significant intrauterine delay of development (n=100), nondeveloping pregnancies (n=176), and anembryonic fetuses (n=66). The frequency of chromosomal mutations in these groups was 22.0, 48.3, and 48.5%, respectively. Statistical analysis has demonstrated significant differences between the studied groups in the frequencies of the normal and abnormal karyotypes: the major contributions to these differences were associated with autosomal trisomy, triploidy, and the 46.XY karyotype. The presence of 46.XY may reflect the specific genetic mechanisms of the prenatal mortality of embryos with the normal karyotype, associated with sex and/or with the imprinting of X-chromosomes. The sex ratio in spontaneous abortions with the normal karyotype was as follows: 0.77 for spontaneous abortions with well-developed embryos without any significant intrauterine delay of development; 0.60 for nondeveloping pregnancies; and 0.31 for anembryonic fetuses. An analysis of DNA from the embryos and their parents has demonstrated a low probability of contamination of cell cultures with mother cells as a possible source of the prevalence of embryos with the 46.XX karyotype among spontaneous abortions. Nondeveloping pregnancies and anembryonic fetuses showed statistically significant differences in the sex ratio from the control group consisting of medical abortions (1,11). Differences in the sex ratio were due to an increasingly lower proportion of embryos with karyotype 46.XY (relative to the expected one) among the fetuses with an increased severity of developmental disturbances. The statistical “chances ratio” index also provided evidence that embryos with the 46.XY karyotype had a higher propensity to produce a well-formed fetus as compared with the female embryos. We propose that the expression of genes of the maternal X-chromosome in XY embryos supports a more stable development during early embryogenesis as compared with XX embryos. In the latter case, normal development is coupled with the operation of an additional mechanism for compensation of the dose of X-linked genes. Operation of this mechanism increases the probability of disturbances in female embryos. A higher viability of XY embryos during the early stages of ontogenesis in man appears to explain their underrepresentation in samples of spontaneously aborted embryos and appears to be the major factor responsible for the deviation of the sex ratio from the theoretically expected value.     

Cytogenetic studies in spontaneous abortion: the Calgary experience

In a series of 493 apparently consecutive products of spontaneous abortions obtained for cytogenetic studies, tissue culture was attempted in 428 cases; chromosome analysis using the Q-banding technique was completed in 215 cases (50.2%). Abnormal karyotypes were identified in 80 cases (37.2%). Maternal tissue contamination was apparent and the actual frequency of karyotypic abnormal abortuses could be as high as 50%. Comparison of the frequency of a specific type of chromosome abnormalities with nine other series of studies showed the lowest frequency of autosomal trisomies and the highest frequency of triploidies and structural aberrations in the Calgary series. In addition, a significantly lower gestational age was observed for triploidies 69, XXX as compared to the 69, XXY.     

A cytogenetic study directly from chorionic villi of 140 spontaneous abortions

Summary Spontaneous abortions were studied by analyzing chromosomes directly from chorionic villi. The frequency and the type of anomalies detected among 140 abortuses are in good agreement with those observed by others using conventional tissue cultures. Abnormal karyotypes were found in 48.6% of the cases. Trisomy predominated (66.2%), followed by polyploidy (22.1%), monosomy X (7.4%), and structural anomalies (4.4%). Among the trisomies, the most prevalent were of chromosome 22 (22.2%), 16(22.2%), and 13 (9.5%). The relative frequencies of trisomies, monosomy X, and the different chromosomes involved in trisomies seem to differ between our study and those in which tissue cultures were analyzed. Our low frequency of 45,XO karyotypes and the shift to trisomies of chromosomes whose involvement increases steeply with maternal age are considered due to the approximately 3 year higher mean maternal age in our sample. The sex ratio (male to female) in chromosomally abnormal abortuses was 1.28, which is nearly identical to the 1.2 found in earlier studies. Surprisingly, in chromosomally normal abortions males were significantly outnumbered by females (sex ratio 0.76). Since maternal cell contamination cannot have influenced the sex ratio in our study, we consider it worthwhile to investigate whether failures associated with X inactivation are responsible for pregnancy wastage of some euploid female conceptuses. Knowledge of the karyotypes may serve as a prerequisite for the investigation of non-chromosomal genetic causes of pregnancy wastage.     

Skewed X Chromosome Inactivation and Trisomic Spontaneous Abortion: No Association

Several studies suggest that highly skewed X chromosome inactivation (HSXI) is associated with recurrent spontaneous abortion. We hypothesized that this association reflects an increased rate of trisomic conceptions due to anomalies on the X chromosome that lead both to HSXI and to a diminished oocyte pool. We compared the distribution of X chromosome inactivation (XCI) skewing percentages (range: 50%–100%) among women with spontaneous abortions in four karyotype groups—trisomy (n = 154), chromosomally normal male (n = 43), chromosomally normal female (n = 38), nontrisomic chromosomally abnormal (n = 61)—to the distribution for age-matched controls with chromosomally normal births (n = 388). In secondary analyses, we subdivided the nontrisomic chromosomally abnormal group, divided trisomies by chromosome, and classified women by reproductive history. Our data support neither an association of HSXI with all trisomies nor an association of HSXI with chromosomally normal male spontaneous abortions. We also find no association between HSXI and recurrent abortion (n = 45).     

Cytogenetics and mechanisms of spontaneous abortions: increased apoptosis and decreased cell proliferation in chromosomally abnormal villi

Genetic defects of the zygote, such as chromosome aberrations, are the most frequent causes of abnormal embryonic development and spontaneous abortion. However, the underlying mechanisms remain unknown. Chromosome aberrations likely cause changes in placental morphology and function (such as size, shape, vascularity, and the presence of trophoblastic inclusion). We postulated that chromosome aberrations may affect rates of cell proliferation or programmed cell death (apoptosis) during the differentiation of chorionic villi. To address these questions, we evaluated cell proliferation using a monoclonal antibody to Ki-67 (a cell-cycle marker) and apoptosis using the in situ end-labeling method (TUNEL) on paraffin-embedded placental tissues. Tissues were obtained from spontaneous abortions in early gestational periods with normal (11 cases) and abnormal karyotypes (15 cases), as well as eight normal control placentas from elective abortions. Apoptotic cells were found in the stroma of all cases, but were significantly higher in number in the stroma of chromosomally abnormal versus chromosomally normal spontaneous abortions. The apoptotic index of the trophoblasts was not significantly different between groups. Cell proliferation was higher in muscularized blood vessels in chromosomally normal placentas (both elective and spontaneous abortions) versus chromosomally abnormal spontaneous abortions. Cell proliferation was different in the trophoblast and stroma between the groups but to a lesser degree than in blood vessels. The morphological and biological data presented here suggest that: (1) chromosomally abnormal spontaneous abortions may occur because of different mechanisms than chromosomally normal spontaneous abortions, (2) apoptosis of the stromal cells and cell proliferation in blood vessels and stroma play an important role in the differentiation and functioning of villi, and (3) these changes could explain the etiology of spontaneous abortion and growth retardation of chromosomally abnormal embryos.     

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 log-growth phase serves as marker of maternal cell contamination. We developed a mathematical 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 accuracy of prenatal cytogenetic diagnosis of chromosomal abnormalities.     

Chromosome studies in 500 induced abortions.

A survey of the chromosome constitution in 500 induced abortions (5-12 menstrual weeks) was undertaken over a period of 1 1/2 years. There were 34 cases (6.8%) of gross chromosome anomalies: 2 cases of trisomy A; 5 of trisomy C (including XXX and XXY); 1 of mosaic trisomy C; 4 of trisomy D; 2 of trisomy E; 2 of trisomy G; 1 of double trisomy E and G; 1 of XYY; 4 of monosmy C (including XO); 2 of mosaic monosomy C; 1 of mosaicism of ring D chromosome; 1 of extra small metacentric chromosome; 3 of triploidy (including triploidy with double trisomy C and G); and 5 of tetraploidy and its mosaicism. An increased risk for the occurrence of trisomic anomalies was found with advancing age of the mothers. In contrast, the production of monosomies was not age-related. Trisomies were the most common type of anomalies and were found almost at random, regardless of the characteristics of chromosomes. Neither satellited nor small chromosomes were predominantly involved in the formation of chromosome anomalies.     

False Negative NIPT Results: Risk Figures for Chromosomes 13, 18 and 21 Based on Chorionic Villi Results in 5967 Cases and Literature Review

Non-invasive prenatal testing (NIPT) demonstrated a small chance for a false negative result. Since the “fetal” DNA in maternal blood originates from the cytotrophoblast of chorionic villi (CV), some false negative results will have a biological origin. Based on our experience with cytogenetic studies of CV, we tried to estimate this risk. 5967 CV samples of pregnancies at high risk for common aneuplodies were cytogenetically investigated in our centre between January 2000 and December 2011. All cases of fetal trisomy 13, 18 and 21 were retrospectively studied for the presence of a normal karyotype or mosaicism < 30% in short-term cultured (STC-) villi. 404 cases of trisomies 13, 18 and 21 were found amongst 5967 samples (6,8%). Of these 404 cases, 14 (3,7%) had a normal or low mosaic karyotype in STC-villi and therefore would potentially be missed with NIPT. It involved 2% (5/242) of all trisomy 21 cases and 7.3% (9/123) of all trisomy 18 cases. In 1:426 (14/5967) NIPT samples of patients at high risk for common aneuploidies, a trisomy 18 or 21 will potentially be missed due to the biological phenomenon of absence of the chromosome aberration in the cytotrophoblast.