Cytogenetic analysis in couples with recurrent miscarriages: a retrospective study from Punjab,north India

Human reproduction is considered as the most inefficient event as ～15–20% of human pregnancies end in miscarriage and in the product of miscarriages, chromosomal anomalies are a common occurrence. The aim of the present retrospective study was to assess the frequency of chromosomal aberrations in couples with recurrent miscarriages in the region of Punjab and to compare with worldwide frequencies. In this study, a total of 440 cases were referred between the period 1995–2015. After lymphocyte culturing, giemsa–trypsin banding was done for each case to assess the chromosomal anomalies. The frequency of chromosomal aberrations among couples was found to be 3.41% in our study. Among these aberrations, balanced reciprocal translocations formed the largest group with 60% anomalies. We would conclude that clinicians should understand the importance of chromosomal analysis in these couples and refer them for karyotyping after two miscarriages to rule out the possible genetic cause of recurrent miscarriages.     

Cytogenetic study of recurrent miscarriages and their parents

There is substantial evidence that genetic alterations are contributing factors to the risk for recurrent miscarriages. This study was conducted to determine the frequency and contribution of chromosomal abnormalities in miscarriages and in couples with recurrent miscarriages. We studied a total of 41 miscarriages and their parents with a history of 2–11 recurrent miscarriages. Chromosomal analysis from chorionic villus sampling (CVS) and fetal tissues were performed according to standard cytogenetic methods using G-banding technique. Major chromosomal aberrations and polymorphic variants were found in 51 and 4.8%, respectively. The chromosomal abnormalities were structural (34.4%) and numerical (65.1%) of which 26.1, 21.7, 8.7 and 8.7% were fetal sex aneuploid, triploid, mosaics and trisomic, respectively. Unbalanced and balanced rearrangements were found in 17.2 and 8.6% of all abnormalities, respectively. Major chromosomal abnormalities in couples were seen in 4.9%. The chromosomal abnormalities associated with pregnancy losses and recurrent miscarriages are mostly numerical ones. The incidence of balanced translocations found here is 4.9% which is near to the mode (about 3–6%) observed in the previous studies. Those frequencies are greater than in the general population (0.3%). This indicates that balanced translocations, seen in parents, have some importance in causing miscarriage. The major parental chromosomal aberrations are significantly associated with fetal wastage. Mosaicism should be taken into account for cytogenetic analyses of pregnancy losses. Thus, cytogenetic analyses should be recommended in couples with recurrent miscarriages, when clinical data fail to clarify the cause. The text was submitted by the authors in English.     

Cytogenetic study of recurrent miscarriages and their parents

There are substantial evidences that genetic alterations are contributing factors to the risk for recurrent miscarriages. This study was conducted to determine the frequency and contribution of chromosomal abnormalities in miscarriages and in couples with recurrent miscarriages. We studied a total of 41 miscarriages and their parents with a history of 2-11 recurrent miscarriages. Chromosomal analysis from chorionic villus sampling (CVS) and fetal tissues were performed according to standard cytogenetic methods using G-banding technique. Major chromosomal aberrations and polymorphic variants were found in 51 and 4.8%, respectively. The chromosomal abnormalities were structural (34.4%) and numerical (65.1%) of which 26.1, 21.7, 8.7 and 8.7% were fetal sex aneuploid, triploid, mosaics and trisomic, respectively. Unbalanced and balanced rearrangements were found in 17.2% and 8.6% of all abnormalities, respectively. Major chromosomal abnormalities in couples were seen in 4.9%. The chromosomal abnormalities associated with pregnancy losses and recurrent miscarriages are mostly numerical ones. The incidence of balanced translocations found here is 4.9% which is near to the mode (about 3-6%) observed in the previous studies. Those frequencies are greater than in the general population (0.3%). This indicates that balanced translocations, seen in parents, have some importance in causing miscarriage. The major parental chromosomal aberrations are significantly associated with fetal wastage. Mosaicism should be taken into account for cytogenetic analyses of pregnancy losses. Thus, cytogenetic analyses should be recommended in couples with recurrent miscarriages, when clinical data fail to clarify the cause.     

The most frequent chromosomal abnormalities in karyotypes of patients with reproductive disorders

Cytogenetic and molecular cytogenetic characteristics have been studied in 210 couples with fertility problems. The patients’ karyotypes contained various chromosomal rearrangements in 46 cases (10.95%). The structural chromosomal rearrangements such as pericentric inversions, Robertsonian translocations, balanced reciprocal translocations, and marker chromosomes were more frequent than numerical chromosome aberrations (89.13 and 10.87% of cases, respectively). We have found 19 (4.52%) karyotypes with “hidden’ low mosaicism in X and Y chromosomes. We believe that the patients with chromosomal anomalies in the karyotype need differentiated treatment.     

Results of cytogenetic examinations in 255 married couples with a history of infertility]

The results of cytogenetic examination carried out in 255 couples with a history of infertility have been analysed. Chromosomal aberrations have been detected in 15 examinations (6.7%) with predominance of translocations (mutual - 3.6%; Robertson' - 1.3%). Identified aberrations have been noted in 9 women and 6 men. The obtained results have been analysed in relation to the number of abortions, dead and live newborn babies with inherited abnormalities. It was found that the incidence of chromosomal aberrations increases with the number of the spontaneous abortions in anamnesis. Such aberrations have also been more frequent in the families in which spontaneous abortions have been accompanied by other fertility disorders.     

Influence of different occupations with possible mutagenic effects on reproduction and level of induced chromosomal aberrations in peripheral blood.

In a group of 200 dysfertile couples (400 persons), the possible role of different occupations in failures of reproduction was assessed. These couples were examined from different points of view, classical genetic examination (pedigree, kayrotype, etc.) included. The suspected genotoxic effects in the personal history were checked also by testing the level of induced chromosomal aberrations. A significantly increased level of induced chromosomal aberrations was detected in 37 persons, i.e., 9.3% of the whole group under study. The average level of induced aberration in these subjects was 6.8%, as opposed to the control group (fertile and dysfertile persons without any unusual exposure to mutagens) with a mean of 1.58% aberrant cells in peripheral blood. Most of the occupations with demonstrated genotoxic effects involve daily contact with chemicals of different types. In some persons also intensive therapy in the recent past had genotoxic effects.     

Influence of different occupations with possible mutagenic effects on reproduction and level of induced chromosomal aberrations in peripheral blood

In a group of 200 dysfertile couples (400 persons), the possible role of different occupations in failures of reproduction was assessed. These couples were examined from different points of view, classical genetic examination (pedigree, kayrotype, etc.) included. The suspected genotoxic effects in the personal history were checked also by testing the level of induced chromosomal aberrations. A significantly increased level of induced chromosomal aberrations was detected in 37 persons, i.e., 9.3% of the whole group under study. The average level of induced aberration in these subjects was 6.8%, as opposed to the control group (fertile and dysfertile persons without any unusual exposure to mutagens) with a mean of 1.58% aberrant cells in peripheral blood.Most of the occupations with demonstrated genotoxic effects involve daily contact with chemicals of different types. In some persons also intensive therapy in the recent past had genotoxic effects.     

Prenatal diagnosis and characteristics of chromosome aberrations in families with genetic risk

Characteristics of the chromosomal aberrations diagnosed in 959 prenatal tests in the II trimester of pregnancy is presented. Chromosomal aberrations were diagnosed in 33 tests (3.4%). Twenty one out of these aberrations (2.2%) were of labile character. Six aberrations resulted from the parental segregation, translocation or chromosomal inversion. In 12 cases fetus inherited stable aberration from one of parents. It amounted to 1.2% of all tested cases. Chromosomal aberrations were diagnosed in 2.7% cases tested due to the risk related to the mother's age. Half of them was trisomy of chromosome 21. Chromosomal aneuploidy in the progeny of families with a child with the same abnormality was diagnosed in 1.6% of cases. Chromosomal mosaicism was diagnosed in 2.2% of cases including 0.2% of cases with true mosaicism and 1.98% of cases with pseudomosaicism. Incidence and type of the diagnosed chromosomal aberrations coincided with foreseen aberrations for each group of the genetic risk.     

Value of chromosome tests in genetic counseling of infertile couples

A cytogenetic analysis of more than 7000 subjects has been performed in the aetiological screening of infertile couples. This study revealed that infertile couples can be divided into three groups: those with one or more spontaneous abortions before 13 weeks of gestations, those with primary sterility (no gestation for at least 24 months) or those with secondary sterility (after one or more children, no further gestation for at least 24 months). The frequency of chromosomal abnormalities observed in this population is 2,9%, i.e. 1/17 couple. The results, according to sex of subjects bearing the chromosomal abnormality, were then analysed in each of the three groups. From this study, the authors insist on the advantage of systematically performing a chromosome analysis in the case of infertile couples for two main reasons: frequency of chromosomal abnormalities is relatively high in this population, detection of such abnormalities enables some couple to be rapidly directed to other solutions (Artificial Insemination), and others, due to prenatal chromosomal diagnosis, to have offspring in safety.     

The effect of age and low-dose irradiation on the chromosomal aberration frequency in human lymphocytes

The effect of age and of low-dose irradiation on the base level of chromosomal aberrations in lymphocytes was studied in two human groups: control one (128 people) and exposed in past to uncontrolled low-dose irradiation (283 people). In exposed group the frequencies of all types of chromosome aberrations were higher comparing to control group. For the investigation of the age response of the number of chromosomal aberrations three statistical approaches were used: correlation analysis of individual data, correlation analysis of mean frequencies of chromosomal aberrations for 10 years intervals, comparison of 3 age groups (young, middle age and old). In control group the significant increase of the level of chromosomal aberrations with age was found only when six 10-year intervals were analysed. In exposed group significant age increase of chromosomal aberration frequency (particularly due to double fragments) was observed with all 3 approaches. Low-dose irradiation of people is supposed to cause the functional defects of repair systems, controlling the level of genetic damages and they accumulate more intensive through age.     

Comparative analysis of the clastogenicity and cytotoxicity of airborne particulate matter generated during the fire at Schweizerhalle on November 1, 1986

Methanol extracts from 4 pairs of airconditioner filters (one fire-exposed and one control) from various locations (A, B, C and D) at various distances from the site of the fire were examined for their capacity to induce structural chromosomal aberrations and/or cytotoxicity in Chinese hamster V79 cells. Extracts from 2 additional sets of 3 filters which were exposed to urban air for 3 consecutive periods of 10 or 11 days some 4 months after the fire were also tested. Chromosomal aberrations were induced by all filter extracts from location B, as well as by an unused (non-exposed) filter, in a dose-dependent manner. Without the addition of metabolizing enzymes, aberrations were induced only at concentrations which caused more than 95% cell killing. This was not taken as an indication for clastogenic activity of the filter extracts, but was assumed to represent the chromosomal expression of metabolic changes in dying cells. Upon the addition of S9, chromosomal aberrations were induced at biologically relevant survival rates. Under metabolizing conditions, the ranking of the potential of the filter extracts from location B to induce chromosomal aberrations and to cause cell killing was identical. The remaining extracts (locations A, C and D) were therefore tested for cytotoxicity only. The toxicity data indicated that, of 3 pairs of filters, the fire-exposed one was not different from the control. Of the fourth pair (location B), the fire-exposed filter was 2.0-2.5 times more cytotoxic and clastogenic than the control. However, extracts of urban air-exposed filters from this location (exposed in March and April 1987) showed a large variation in toxicity and clastogenicity as well. One was clearly more active than the control (but less than the fire-exposed filter), while the other 2 were either somewhat more or less clastogenic than the control filter. In addition, 4 out of 5 filters from this location were more polluted (as indicated by cytotoxicity) than all the filters from the other locations, irrespective of whether they were fire-exposed or not. It is concluded that the results of this V79 cytotoxicity/clastogenicity test did not confirm the hypothesis that the fire at Schweizerhalle produced clastogenic material at quantities detectable under the conditions employed.     

Bilan des examens chromosomiques de 277 couples candidats à l’injection intracytoplasmique de spermatozoïde

In these study the frequency of congenital chromosomal aberrations was assessed in a sample of 277 couples included in a intracytoplasmic sperm injection (ICSI) program. A total of 7 aberrant karyotypes were diagnosed, corresponding to an abnormality rate of 1.3% per individual and 2.6% per couple. The following types of abnormalities were observed: reciprocal translocations (n=2), Robertsonian translocations (n=2), pericentral inversion (n=1), sex chromosomal mosaicism (n=1), marker chromosome (n=1). Surprisingly, 71% (5/7) of all abnormal karyotypes were contributed by the female partner of ICSI patients. These data confirm the higher incidence of chromosomal aberration in infertile couples compared with the baseline population risk. It also confirms that not only the male, but also the female should be routinely karyotyped prior to ICSI, and then receive appropriate genetic counselling.     

Sister chromatid exchange and chromosome aberrations induced by curcumin and tartrazine on mammalian cells in vivo

Sister chromatid exchanges (SCEs) and chromosomal aberrations induced by curcumin (a natural dye) and tartrazine (a synthetic dye) were studied on bone marrow cells of mice and rats following acute and chronic exposure via the diet. Except for two low concentrations in the curcumin and one low concentration in the tartrazine treated series a significant increase in SCEs was observed in all the concentrations of the two dyes tested. Except for two high concentrations during the 9 months treatment no significant increase in chromosomal aberrations was observed in the curcumin treated series, whereas tartrazine showed a significant increase in chromosomal aberrations in some of the higher concentrations in all the series tested. The results indicate that tartrazine is more clastogenic than curcumin.     

Induction of chromosomal aberrations in cultured Chinese hamster cells by short-term treatment with cadmium chloride

Inducibility of chromosomal aberrations and cytotoxicity in cultured Chinese hamster cells by cadmium chloride (CdCl2) was investigated under 3 different treatment conditions: (i) 2-h treatment in MEM medium supplemented with 10% fetal bovine serum (MEM + 10% FBS) or (ii) in HEPES-buffered Hanks' solution (HEPES-Hanks), and (iii) continuous treatment for 24 h in MEM + 10% FBS. Two-h treatment with CdCl2 in HEPES-Hanks or continuous treatment for 24 h in MEM + 10% FBS was respectively 2 or 3 times more cytotoxic than 2-h treatment with the metal in MEM + 10% FBS. Continuous treatment for 24 h with a CdCl2 concentration in excess of 5 X 10(-6) M was too toxic to the cells to allow chromosomal analysis, and moreover, only a slight increase in incidence of chromosomal aberrations was observed at a concentration of 5 X 10(-6) M CdCl2. In contrast, a marked and concentration-dependent increase in incidence of chromosomal aberrations was observed after post-treatment culture for 22 h follows 2-h treatment with 1 X 10(-6) M to 5 X 10(-5) M of CdCl2 in both MEM + 10% FBS and HEPES-Hanks. Two-h treatment with cadmium in HEPES-Hanks was approximately 3 times more potent for the induction of chromosomal aberrations than that in MEM + 10% FBS. Types of aberrations induced by CdCl2 mainly consisted of chromatid gaps and breaks, although a few exchanges, dicentrics and fragmentations were observed at high concentrations of cadmium. Increase in incidence of tetraploidy was also observed with a concentration dependency after 2-h treatment with CdCl2. Potency of CdCl2 to induce chromosomal aberrations after 2-h exposure was comparable to that of benzo[a]pyrene activated with S9 at equitoxic concentrations. Two-h treatment with cadmium markedly inhibited incorporation of [3H]thymidine, even at concentrations at which incorporation of [3H]uridine or [3H]leucine was less inhibited. However, the inhibition of [3H]thymidine incorporation by cadmium was reversible and the incorporation restored to the control level during 2-6 h of post-treatment incubation. These findings suggest that restoration of DNA synthesis after cadmium exposure is required for the efficient detection of chromosomal aberrations induced by the metal.     

Proliferative kinetics and mitomycin C-induced chromosome damage in Fanconi's anemia lymphocytes

Lymphocytes from two sisters with Fanconi's anemia (FA) were studied for cell cycle kinetics, sister chromatid exchanges (SCEs), and chromosomal aberrations when they had undergone one, two, or three or more divisions in mitomycin C (MMC)-treated cultures. Lymphocytes from the parents, another sister of the probands, and a healthy unrelated adult were examined as controls. Analyses of cell cycle kinetics by the sister chromatid differential staining method revealed that the relative frequency of metaphase cells at their third or subsequent divisions was much smaller in untreated FA cultures than in normal cultures fixed at 96 h after phytohemagglutinin stimulation. These data indicate that FA cells proliferate much more slowly than normal cells. MMC treatments of FA and normal cells led to a clearly dose-related delay in cell turnover times, the duration of delay being much longer in FA than in normal cells. FA cells had about 1.4 times higher frequencies of SCEs than normal cells in both MMC-treated and untreated cultures. FA cells also showed several times higher frequencies of chromosomal aberrations than normal cells, and the frequency of chromosomal aberrations decreased through subsequent mitoses by approximately 60% in both FA and normal cells.     

Inducibility of chromosomal aberrations by metal compounds in cultured mammalian cells.

Metal compounds were tested for their ability to induce chromosomal aberrations in cultured mammalian cells. Chromosomal aberrations were induced by the application of some Cr, Mn and Ni compounds. Among 6-valent Cr compounds, K2Cr2O7 and CrO3 induced high levels of aberrations, at rates which were similar for Cr-equivalent doses. The perchromate compounds were more efficient in producing chromosomal aberrations than was a chromate compound, K2CrO4. A 3-valent Cr compound, Cr2(SO4)3, was less toxic and failed to induce a demonstrable increase in chromosomal aberrations. KMnO4 induced aberrations, but at a low rate. As to Ni compounds, NiCl2 and (CH3COO)2Ni induced few aberrations. Administration of K2Ni(CN)4 induced only gaps. NiS induced a low but definite increase in chromosomal aberrations. The rate of these aberrations increased with an increase in treatment time from 24 to 48 h, indicating a time-dependent increase in the hereditable toxicity of metal compounds. CdCl2 and HgCl2 were somewhat toxic, but failed to induce chromosomal aberrations in the present study.     

Molecular mechanisms involved in the production of chromosomal aberrations

Chinese hamster ovary cells (CHO cells) and mouse fibroblasts (PG 19) were permeabilized with inactivated Sendai virus, treated with different types of restriction endonucleases (Eco RV, Pvu II, Bam HI, Sma I, Asu III, Nun II), and studied for the occurrence of chromosomal aberrations at different times following treatment. The pattern of chromosomal aberrations observed was similar to that induced by ionizing radiations. Restriction endonucleases that induce blunt double-strand breaks (Eco RV, Pvu II) were more efficient in inducing chromosomal aberrations than those that induce breaks with cohesive ends (Bam HI, Nun II, Asu III). Ring types were very frequent among the aberrations induced by restriction enzymes. Cytosine arabinoside, an inhibitor of DNA repair, was found to increase the frequencies of aberrations induced by restriction enzymes, indicating its effect on ligation of double-strand breaks. The relevance of these results to the understanding of the mechanisms of chromosomal aberration formation following treatment with ionizing radiations is discussed.     

Induction of chromosomal aberrations in active oxygen-generating systems. I. Effects of paraquat in Chinese hamster cells in culture

A possible role for the superoxide anion radical (O2-) in the clastogenicity of paraquat (PQ) was investigated in cultured Chinese hamster cells. When cells were treated with 0.8 mg/ml of PQ for 3 h followed by 21 h of recovery time, structural chromosome aberrations were induced in about 50% of the metaphases examined. Almost all aberrations were of the chromatid-type and involved exclusively gaps and breaks. The induction of chromosomal aberrations by PQ was enhanced by a 1-h pretreatment with diethyldithiocarbamate, an inhibitor of superoxide dismutase. Diethyl maleate, a glutathione scavenger, also enhanced the induction of chromosomal aberrations, but 3-aminotriazole, an inhibitor of catalase, showed no such effects. Enhanced induction of chromosomal aberrations was also observed when PQ-treated cells were cultured at a high oxygen concentration (80%). The present results suggest that the production of chromosomal aberrations by PQ may be directly or indirectly related to the generation of O2-, but not to the formation of hydrogen peroxide by the dismutation reaction of O2- or of other active oxygen species including the hydroxyl radical and singlet oxygen.     

Repair of single-strand DNA breaks and recovery of chromosomal and chromatid aberrations after treatment of plant seeds with propyl methanesulfonate in vivo.

Repair of single-strand breaks of DNA and simultaneous recovery of chromosomal aberrations were studied after treatment of barley seeds with the monofunctional alkylating chemical mutagen, propyl methanesulfonate in vivo. In soaked seeds the diminution of single-strand breaks of DNA induced by PMS was correlated with the decrease of chromosomal aberrations, whereas in dried seeds the repair of DNA breaks was depressed and, in accord with this, the frequency of chromosomal aberrations increased. The prolonged storage of seeds led to a more delayed repair of chromosomal aberrations in dry seeds and a more delayed accelerated repair in soaked seeds.     

Chromosomal aberrations and sister-chromatid exchanges in tool and die workers

Because tool and die workers are exposed to a number of potentially genotoxic agents, including mutagenic metals, polyaromatic hydrocarbons, and nitrosamines, and may be at increased cancer risk, the present study was undertaken to test whether chromosomal damage in peripheral blood cells is associated with work in the tool and die industry. Lymphocyte cultures were established from 27 tool and die fabrication workers from one manufacturing plant who had worked in the trade for more than 15 years. 15 of these workers also had some form of malignancy at the time of the study, but had not been treated with radiation or chemotherapies that could themselves induce chromosomal damage. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCEs) were measured in workers and the data compared with those of a control group consisting of 7 non-fabrication workers from the same plant and 8 age-matched community controls. In addition, the relative rates of lymphocyte proliferation were estimated for each group by analyzing the percentages of first-, second- and third-division mitotic cells after 72 h of culture. The results of the chromosomal studies show that tool and die workers have significantly increased frequencies of aberrations whether engaged in fabrication work or not, compared to control subjects. The frequency of SCEs and the frequencies of 1st, 2nd and 3rd division figures are not different among the study groups. Among workers who are engaged in fabrication, including those who are cancer patients, the frequency of more complex aberrations (i.e., interstitial deletions and small acentric fragments) is increased. In a five-year follow-up of these workers, 2 of the 13 workers with these aberrations developed some form of colon cancer. Whether the presence of interstitial deletions and small acentric fragments is related to the occupation of these workers, or is tangentially related to the development of cancer, is presently under consideration.