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Objectives Preeclampsia (PE) is a pregnancy-related syndrome characterized by the onset of hypertension and proteinuria that can lead to end-organ dysfunction. Galectin-3 (Gal-3) is involved in cell growth, differentiation, inflammation and fibrosis. Thioredoxin (TXN) acts as antioxidant enzyme in several cellular processes, regulating inflammation and inhibiting apoptosis. TXNIP is an endogenous inhibitor of TXN. We evaluated changes in the inflammatory response of Gal-3, TXN, and TXNIP at the level of maternal blood, placenta, and umbilical cord blood of women with PE. Study design Ten women with PE and 20 with normal pregnancy (NP) were recruited during admission for delivery. Blood samples were obtained from parturients and umbilical cords, and placental tissue for analysis. Results Gal-3 and TXNIP mRNA expression were higher in maternal plasma in PE group compared to NP and were lower in cord blood plasma and placentas in the PE group. In the PE group, TXN/TXNIP mRNA ratio was higher in cord blood plasma (2.07) compared to maternal plasma (1.09). TXN/TXNIP placental protein ratio was similar between PE (0.89) and NP (0.79). ELISA demonstrated that Gal-3 levels in maternal serum were significantly higher in the PE vs. the NP group. Conclusions Pro-inflammatory changes were expressed by high Gal-3 and TXNIP mRNA in maternal blood of PE women, but not in their placental and cord blood samples. These findings may imply that the placenta has a role in protecting the fetus from the damages of inflammatory response, which is more common in PE than in NP.

Telomeres are a specific base sequence of DNA, responsible for chromosome stability and DNA protection. We aimed to investigate the association between telomere systems and IVF outcomes according to patients' BMI. For all telomere characteristics, there was a distinct trend towards shorter telomeres and activation of telomere shortening compensatory mechanisms in the BMI group >25 kg/m2, reaching statistical significance for senescence only (r = 0.7, p value

A ground glass opacity (GGO) lung lesion may represent early stage adenocarcinoma, which has an excellent prognosis upon prompt surgical resection. However, GGO lesions have broad differential diagnoses, including both benign and malignant lesions. Our objective was to study telomere length and telomerase activity in patients with suspected lung cancer in which GGO was the predominant radiographic feature. Knowledge of telomere biology may help distinguish malignant from benign radiographic lesions and guide risk assessment of these lesions. Peripheral blood samples were taken from 22 patients with suspected adenocarcinoma with the GGO radiographic presentation. Multidisciplinary discussion confirmed the need for surgery in all cases. We used an age and gender‐matched group without known lung disease as a control. Telomere length and aggregates were assessed by quantitative fluorescence in situ hybridization (QFISH) and quantitative PCR. Cell senescence was evaluated by senescence‐associated heterochromatin foci. Subjects with GGO lesions had a higher percentage of lymphocytes with shorter telomeres (Q‐FISH, P = 0.003). Furthermore, relative telomere length was also reduced among the GGO cases (qPCR, P < 0.05). Increased senescence was observed in the GGO group compared to controls (P < 0.001), with significant correlation between the senescence‐associated heterochromatin foci and aggregate formation (r = −0.7 and r = −0.44 for cases and controls, respectively). In conclusion, patients with resectable early adenocarcinoma demonstrate abnormal telomere length and cell senescence in peripheral blood leukocytes compared to control subjects. Abnormal telomere biology in the peripheral blood may increase suspicion of early adenocarcinoma among patients with GGO lesions.

Early-onset preeclampsia (EOPE;34 weeks' gestation) usually has more severe morbidity for the mother and fetus compared to late-onset preeclampsia (LOPE). Telomere homeostasis is disrupted in preeclampsia (PE) and senescence markers are increased. The pathophysiologic differences between early and LOPE are not fully unraveled yet.We studied placental biopsies from 7 pregnancies with EOPE, 6 pregnancies with LOPE, and 13 healthy gestational age-matched controls. Telomere length and aggregate formation were assessed using qualitative fluorescence in situ hybridization and electronic quantitative methods. Senescence markers were evaluated including senescence-associated heterochromatin foci, β-galactosidase (SAβ-Gal), and P16 staining, as was the expression of P16 complementary DNA (cDNA) using real-time quantitative polymerase chain reaction (RT-qPCR).There were no differences in maternal age, gravidity, parity, body mass index, and mode of conception between the study and the control groups. The percentage of trophoblasts with short telomeres was higher in placental samples from EOPE (52.61% [12.27%]) versus LOPE (28.72% [10.14%]); both were higher compared to controls (7.53% [5.14%],Impaired telomere homeostasis and senescence markers are more prominent in EOPE versus LOPE. These findings may contribute to our understanding of the pathophysiology and explain their different clinical presentations and outcomes.

Background and aims Primary sclerosing cholangitis and inflammatory bowel disease are two associated, chronic inflammatory, pre-malignant conditions. We hypothesized that patients with these disorders may harbour telomere dysfunction as a marker of chromosomal instability. The aim of our study was to compare parameters of the telomere-telomerase system in these cohorts. Methods In this prospective study, peripheral blood was withdrawn from patients with primary sclerosing cholangitis (N = 20), inflammatory bowel disease (N = 20) and healthy controls (N = 20), and lymphocytes were isolated. Telomere length was quantified as a function of the signal intensity and telomere number. Random aneuploidy and telomere capture were determined by fluorescence in situ hybridization technique with specific probes. Results Patients with inflammatory bowel disease had higher measures of intestinal disease activity than patients with primary sclerosing cholangitis. Despite this, shorter telomere length and telomere aggregates, especially the fusion of 2–5 telomeres, were observed at significantly higher rate in patients with primary sclerosing cholangitis relative to inflammatory bowel disease or healthy controls. Rates of aneuploidy and telomere capture were higher in the two probes in both diseases compared to controls (p Conclusion Dysfunction of telomeres was demonstrated in primary sclerosing cholangitis patients more than inflammatory bowel disease and healthy controls patients, which attests to genetic instability and immunosenescence. Trial registration number NCT02247622 .

Telomeres are nucleoprotein structures located at the termini of chromosomes. They are essential for chromosome stability. Telomeres become shorter due to mitotic cycles and environmental factors. When telomeres are shortened and therefore dysfunctional, cellular senescence occurs and organ dysfunction might develop. During pregnancy, fetal growth restriction secondary to placental insufficiency has been linked to impaired telomere homeostasis in which telomeres are shorter, telomerase is decreased, and compensatory mechanisms of telomere capture are enhanced. These characteristics, along with increased signs of senescence, indicate telomere dysfunction in trophoblasts from placentas affected by intrauterine growth restriction (IUGR). This review summarizes the information currently available regarding telomere homeostasis in trophoblasts from human pregnancies affected by IUGR. Improved understanding of placental physiology might help in the development of treatment options for fetuses with IUGR.

Introduction The intrauterine environment, including the placenta, is influenced by a variety of factors, among which is diabetes during pregnancy. These factors can affect lifetime morbidity. Senescence is a state of cellular metabolic arrest, known to be correlated with age-related diseases and is usually accompanied by short telomeres. This study evaluated telomere characteristics in placentas and in cord blood from term pregnancies complicated by uncontrolled diabetes mellitus. Methods Placental biopsies and cord blood were collected from 16 pregnancies with poorly controlled diabetes and from 16 healthy controls. Senescence-associated heterochromatin foci (SAHF) and senescence-associated β-galactosidase (SAβ-Gal) staining were evaluated. Apoptosis was evaluated using tunel staining. Telomere length and aggregate formation were assessed in placentas and in cord blood using Q-FISH. Results Increased SAHF (19.28% ± 7.93 vs. 7.78% ± 5.31, P

Primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD) are associated chronic inflammatory diseases with malignant potential. Loss of replication synchrony during the S-phase of the cell cycle has been shown to be linked to several malignant and premalignant states. This study evaluated temporal differences in replication timing between these diseases. The replication pattern of peripheral blood lymphocytes obtained from patients with PSC and IBD and healthy individuals was analyzed by fluorescence in situ hybridization (FISH) in 2 pairs of alleles, in 15qter and 13qter. Asynchrony was determined by the presence of 1 single and 1 set of double dots in the same cell. Samples from subjects with PSC showed significantly greater temporal differences in replication timing, in contrast to the high level of synchrony observed in samples from healthy individuals (p = 0.045). Samples from IBD patients exhibited a nonsignificant increase in replication asynchrony. We believe that these results reflect impairment in the replication control of structural homologous loci in PSC, and that this phenomenon may be correlated with the inflammation-induced malignant potential of this condition.

Placentas from pregnancies complicated with IUGR (intrauterine growth restriction) express altered telomere homeostasis. In the current study, we examined mechanisms of telomere shortening in these placentas.Placental biopsies from 15 IUGR and 15 healthy control pregnancies were examined. The percentage of trophoblasts with fragmented nuclei: senescence-associated heterochromatin foci (SAHF), was calculated using DAPI staining. The amount of human telomerase reverse transcriptase (hTERT) mRNA was evaluated using RtPCR levels of telomere capture using FISH in those samples were estimated.The percentage of trophoblasts with SAHF was higher in IUGR compared to control samples, (25±13.4% vs. 1.6±1.6%, P0.0001), hTERT mRNA was decreased (0.5±0.2 vs. 0.9±0.1, P0.0001) and telomere capture was increased (13.2±9.7% vs.1.3±2.5%, P0.001).We suggest that IUGR placentas express increased signs of senescence as part of the impaired telomere homeostasis. One factor that mediates telomere shortening in these placentas is decreased hTERT mRNA, leading to decreased protein expression and therefore, reduced telomere elongation. Telomere capture, which is a healing process, is increased in IUGR trophoblasts as a compensatory mechanism.

Senescence has been described as a stable cell proliferation arrest resulting from the progression of primary human fibroblasts through a finite number of population doublings in vitro. Accelerated telomere shortening was observed in pregnancies complicated by intrauterine growth restriction, in placentas of diabetic mothers and trisomy 21 amniocytes. We hypothesized that under conditions of stress, telomeres in placentas will be shorter and there will be more cells with the senescence phenotype.The two study groups included placental biopsies from 7 cases of trisomy 21 and amniocytes from 10 cases of trisomy 21. The control groups consisted of placental biopsies from 6 cases and amniocytes from 10 pregnancies with a normal karyotype. The samples were analyzed for the presence of senescent cells based on the number of fragments in each cell.A significantly higher percentage of cells in the senescent state, based on a higher percentage of cells with more fragmentations, were found in the amniocytes (20.8%) and in trophoblasts (94.3%) from placentas with trisomy 21 compared to the control groups.Among other genetic instability parameters, trisomy 21 amniocytes and trophoblasts express a higher prevalence of senescent cells than were previously reported.

Nonalcoholic fatty liver disease (NAFLD) and cryptogenic cirrhosis (CC) are considered preneoplastic conditions that might progress to hepatocellular carcinoma. We evaluated parameters of telomere dysfunction in these patient groups to study the correlation between telomere length and the progression of NAFLD. We analyzed peripheral lymphocytes from 22 patients with NAFLD, 20 patients with CC, and 20 healthy, age-matched controls. Telomere length was analyzed using quantitative fluorescence in situ hybridization, and cellular senescence was evaluated by the percentage of cells with senescence-associated heterochromatin foci. The expression of telomerase reverse transcriptase (hTERT) mRNA was measured using polymerase chain reaction, and telomere capture (TC) was assessed with 2 Cytocell probes, 15qter and 13qter. Shorter telomere length and increased cellular senescence was demonstrated in patients with NAFLD, compared to the CC patients and healthy controls. While hTERT mRNA was significantly decreased, TC was increased in CC patients, compared to the NAFLD group and healthy individuals. Thus, there is a correlation between hTERT mRNA expression and telomere length in patients with NAFLD, which might be related to associated metabolic disorders and the risk of malignant transformation. Patients with CC, on the contrary, elongate their telomeres through the TC mechanism.

Objective Individuals with chromosomal aneuploidies tend to develop malignancies. Telomerase is an enzyme complex that lengthens telomeres and has enhanced expression in numerous malignancies; one of its components is encoded by the TERC gene. In this study, we evaluated the TERC gene copy number in amniocytes from fetuses with aneuploidy, other than trisomy-21. Methods In this prospective, basic research study, fluorescence in situ hybridization (FISH) for the TERC gene (3q26) was applied to amniocytes retrieved from 14 fetuses with various aneuploidies and from a control group of 6 fetuses with a normal karyotype, to determine the TERC gene copy number. Results The percentage of cells with more than two copies of the TERC gene was lowest in the control group (x3 = 1.2 ± 0.4%; x4 = 0 ± 0%), higher in the sex chromosome aneuploidies (x3 = 4 ± 3%; x4 = 0.7 ± 0.95%) and even higher in trisomy 18 (x3 = 10.6 ± 2.3; x4 = 4.6 ± 1.8). The differences were statistically significant (P Conclusion The TERC gene copy number is increased in aneuploid amniocytes, which demonstrates their genetic instability and is presumably related to their tendency to develop malignancies.

Objective Telomeres are nucleoprotein structures located at the termini of chromosomes, and protect them from fusion and degradation. Telomeres are progressively shortened with each mitotic cycle and by environmental factors. We hypothesized that antepartum stress can lead to accelerated telomere shortening in placental trophoblasts, and plays a role in intrauterine growth restriction (IUGR). Methods Placental biopsies were derived from 16 pregnancies complicated with IUGR and from 13 uncomplicated pregnancies. Fluorescence-in-situ protocol was used to determine telomere length. Immunohistochemistry for hTERT was performed to assess telomerase activity. Clinical and histopathological characteristics were collected to ensure that IUGR was secondary to placental insufficiency. Fluorescence-in-situ-hybridization was used to rule out aneuploidy as a reason for shortened telomeres. Results The number and intensity of telomeres staining and telomerase activity were significantly lower in the IUGR placentas. No aneuploidy was detected for the chromosomes checked in the placental biopsies. Conclusions Telomeres are shorter in trophoblasts of IUGR placentas.

Trisomy 21 is the most common chromosomal abnormality among persons with intellectual disability, with a live birth rate of 1 in 800-1,000. As such, this abnormality may serve as a model for human disorders that result from supernumerary copies of a genomic region. Down syndrome carries an increased risk of developing acute leukemia and other malignancies. Telomeres of tumor cells nuclei tend to form aggregates (TA). This study evaluated TA formation in amniocytes from trisomy 21 pregnancies, compared with amniocytes from normal euploid pregnancies. A commercially available peptide nucleic acid telomere kit was used to evaluate TA formation, using two-dimensional fluorescence microscopy. Significantly higher frequencies of TA were found in trisomy 21 amniocytes than in amniocytes from normal pregnancies. The TAs found in trisomy 21 amniocytes apparently represent an additional parameter that reflects the high genetic instability of this syndrome and its recognized predisposition to develop leukemia and other malignancies.

Telomeres are specific repetitive DNA sequences that cap and stabilize the ends of chromosomes. Functional telomeres are essential for the normal segregation and maintenance of chromosomes during mitotic and meiotic division. Pre-eclampsia, a pregnancy-specific syndrome of increased blood pressure accompanied by proteinuria, is often associated with growth deficiency in the fetus. Oxidative stress is a major component in the pathophysiology of pre-eclampsia. In contrast to the nonoverlapping nature of telomeres in normal nuclei, telomeres of tumor nuclei tend to form aggregates (TAs) in various numbers and sizes. The formation of TAs represents a stress-related process and is independent of telomere length and telomerase activity. The aim of this study was to evaluate TA formation in paraffin-embedded placentas from pregnancies complicated with pre-eclampsia (study group), compared with placentas from normal pregnancies (control group). There were significantly more TAs in the study group (mean, 8.00 TAs per case) than in the control group (mean, 2.36 TAs per case) (P < 0.01). Pre-eclampsia-related stress may accelerate apoptosis and cell death and lead to placental dysfunction. TAs formation, which has been linked to stress and tumorgenesis is increased in placentas of pre-eclamptic patients.

Telomeres of tumor nuclei tend to form aggregates (TA). The aim of this study was to estimate the TA formation in leukocytes of patients with chronic hepatitis C (HCV) which is considered to be premalignant disease, in patients of HCV who eradicated the virus. PNA Telomere kit (Dako) was used to evaluate the TA formation with the utilization of 2D fluorescence microscopy. A higher rate of TA was found in both HCV groups as compared to controls. Our results indicate that HCV patients have some of the components that create the cascade of events leading to malignancies.

Background/aims Non-alcoholic fatty liver disease (NAFLD) and cryptogenic cirrhosis (CC), which is largely a late sequela of NAFLD, are considered pre-neoplastic conditions that might progress to hepatocellular carcinoma. Aneuploidy, telomere aggregates and synchronization of replication were evaluated as markers of genetic instability in these patients. Methodology Peripheral blood lymphocytes from 22 patients with NAFLD, 20 patients with CC and 20 age-matched healthy controls were analyzed. To determine random aneuploidy, we used the fluorescence in situ hybridization (FISH) with probes for chromosomes 9 and 18. The rate of aneuploidy was inferred from the fraction of cells revealing one, three or more hybridization signals per cell. Aggregate size was divided into three fusion groups of 2–5, 6–10 and 11–15 telomeres, relative to the size of a single telomere. The replication pattern was determined by FISH in two pairs of alleles, 15qter and 13qter. Asynchrony was determined by the presence of one single and one set of double dots in the same cell. Results Significantly higher random aneuploidy rate was found in the CC patients than in the control group, and to a lesser degree in NAFLD patients. Telomere aggregates were insignificantly higher in both groups. Only patients with CC showed significantly higher rate of asynchronous replication with proportionately more cells with two single dots among the normal cells (p Conclusions These results likely reflect changes in gene replication and cell cycle progression in these conditions, possibly correlating with their malignant potential.

Objective Diabetes during pregnancy causes an intrauterine environment that influences lifetime sickness of the mother and the fetus. There is a correlation between diabetes and telomere shortening; however, very little is known about telomere homeostasis in the placenta. We aimed to study the telomerase complex in placentas and in cord blood leukocytes from patients with poorly controlled diabetes. Methods Biopsies from 16 third-trimester placentas and cord blood samples from pregnancies complicated with uncontrolled diabetes and from 16 gestational age-matched controls from uncomplicated pregnancies were examined. The expression of hTERT (human telomerase reverse transcriptase) was evaluated by immunohistochemistry and by RT-RCR. TERC gene copy number and telomere capture were evaluated by FISH. Results Telomerase expression was significantly lower in the diabetic placentas, both the protein (17.8 ± 2.8% cellular staining vs. 37 ± 5.32%, P = 0.012) and the mRNA levels (0.42 ± 0.03 folds, P = 0.022). Lower expression of TERC gene copy number were shown in the diabetic placentas compared to the healthy controls (1.7 ± 0.8% vs. 3.7 ± 1.6%, P = 0.035). We also detected higher percentage of cells with telomere capture among the diabetic trophoblasts compared to the healthy controls (19.8 ± 5.12% vs. 9.6 ± 3.65%, P = 0.038). Those differences were not observed in cord blood leukocytes from the same samples. Conclusions Uncontrolled diabetes during pregnancy disrupts telomere-telomerase homeostasis in the trophoblasts. These changes may increase the risk for metabolic diseases in adulthood among offspring of pregnancies complicated by gestational diabetes mellitus as part of intrauterine programming. These variations were not observed in cord blood leukocytes, which imply different telomere homeostasis mechanisms in fetal cord blood.

Pericentric inversion of one chromosome 9 [inv(9)] is considered a polymorphic variation and is one of the most common forms of autosomal inversion diagnosed prenatally in amniocytes. Yet its clinical significance remains uncertain. Most publications suggest that this finding is insignificant. However, some articles report on abnormal ultrasonic findings in association, such as hydramnios, anhydramnios, hydroureter, hydronephrosis, encephalocele, and prune belly syndrome. Other reports suggest that inv(9) might be one of the etiologies of psychiatric disorders. The homozygote state, on the other hand, is rarely encountered. We report two cases of pericentric inversion of the two homologues of chromosome 9. Two similar cases were previously reported. One affected fetus was had intrauterine growth restriction and the other had Walker-Warburg syndrome as opposed to the normal outcome of our patients. Finally, a workup of this finding is suggested.

Fetal growth restriction (FGR) secondary to placental insufficiency and preeclampsia (PE) are associated with substantially increased childhood and adult morbidity and mortality. The long-term outcomes are related to placental aberrations and intrauterine programming. Advances in microarray technology allow high-resolution, genome-wide evaluation for DNA copy number variations - deletions and duplications. The aim of our study was to demonstrate the usefulness of microarray testing in FGR placentas. Using Affymetrix GeneChip for chromosomal microarray (CMA), we analyzed 10 placentas from pregnancies with FGR attributed to placental insufficiency; 5 with FGR below the 5th percentile and 5 from the 5th to

In this study we evaluated the aneuploidy rate of cells from patients considered to have a premalignant condition (monoclonal gammopathy or MGUS) and patients with multiple myeloma, as well as healthy controls. By applying a fluorescence situ hybridization technique, we estimated the random aneuploidy rate of alpha-satellite (centromeres) probes from chromosomes 9 and 18. The monosomy and total aneuploidy rates were higher in the two study groups compared to the control group. The monosomy rate was significantly higher in the MGUS group compared to the group with chromosome 18 alpha-satellite probes, a finding that was reported before in preneoplastic conditions. Our results support the cancer aneuploidy theory that carcinogenesis is initiated by a random aneuploidy, which is induced either spontaneously or by a carcinogen. The resulting karyotype instability sets a chain reaction of aneuploidization, which generates even more abnormal and eventually cancer-specific combinations and rearrangements of chromosomes.

Objective. In this study, we applied the fluorescent in situ hybridization (FISH) technique and compared the common numerical abnormalities with chromosomes 13, 16, 18, 21, X, and Y in spontaneous to artificial abortion. This would cover about 75% of the common aneuploidy in spontaneous abortion. Methods. Placentas were taken from 59 patients with a first trimester spontaneous abortion and 61 patients who underwent an elective first trimester pregnancy termination. The range of growth was from 5 to 12 gestational weeks. Placentas were processed according to direct chorionic villi preparation. Direct dual color FISH was performed according to Vysis protocol with the probes for the following chromosomes: 13, 16, 18, 21, X, and Y. Results. The aneuploidy rate in spontaneous abortion was 55.9% and in artificial abortion 8.2%. There was a significant difference between the two groups in the aneuploidy rate ( P = 6 × 10 −9 ). Conclusion. FISH is a rapid, efficient, and relatively inexpensive tool in detecting a...

Our objective was to compare the genetic abnormalities in the primary tumors of epithelial ovarian cancer and their associated secondary peritoneal implants using comparative genomic hybridization (CGH). CGH was performed on seven apparent stage III ovarian serous cancer cases. Dissected tissue samples from the primary tumor and from the metastatic peritoneal implant were obtained at initial surgical staging and analyzed in each case. We used CGH as this technique allows the entire genome of the tumor to be examined simultaneously for chromosomal imbalances without the need for tissue culture or targeting of specific loci. The chromosomal abnormalities detected in the distinct sites were then reviewed and compared. CGH studies were successful in all 14 samples from the seven patients. The analysis revealed chromosomal aberrations in six patients with certain repeated changes as amplification of 1q, 2p, 2q, 3q, 6q, 8q, and 12p and underrepresentation of 18q and X chromosomes. Comparing the genomes of the primary tumors with the metastatic samples showed four cases with a balanced metastatic CGH profile while the primary site was aberrant. Greater chromosomal complexity associated with the primary site was detected in two other patients. In one case, both primary and secondary sites had no detectable chromosomal imbalances. The cytogenetic patterns in six of the seven primary tumors showed complex karyotypic changes, unlike the inconsistent findings that were associated with the secondary sites. The chromosomes of the secondary sites expressed either normal genomes or fewer genetic aberrations. Such genomic heterogeneity between the primary and secondary sites may indicate that the secondary peritoneal implants are de novo carcinogenesis occurrences. The results may support the concept that at least part of advanced ovarian cancer is a multicentric disease in the early stages. Further genetic studies are needed to reassess this assumption.

Ataxia telangiectasia (AT) is a pleiotropic and rare (1:40,000 to 1:100,000) recessive disease. Laboratory investigations have failed to detect any consistent anomaly in cells from AT heterozygotes. To estimate random aneuploidy, we applied a fluorescence in situ hybridization technique with alpha-satellite probes for chromosomes 8 and 9 and replication pattern for RB-1, HER-2/neu, and the imprinted SNRPN loci on primary AT carrier fibroblasts. Higher random aneuploidy was not found in the carrier fibroblasts compared to control amniocytic cells. The asynchrony pattern was higher in the AT carrier cells with the RB-1 locus (P=0.057) and significantly higher with the HER-2/neu locus (P < 0.001) compared to control cells. As for the imprinted locus SNRPN, there was a significantly lower asynchrony rate in the AT carriers (P < 10(-5)) compared to the control group. Molecular cytogenetic parameters of random aneuploidy and replication pattern may reflect predisposition for the development of cancer. It is possible that in some AT carriers the genetic instability phenomena associated with the abnormal replication pattern may represent their potential for developing malignancies.

Second trimester maternal serum biochemical markers, introduced between 1990 and 1995, were supplemented with new ultrasound methods at 14-16 weeks and first trimester biochemical markers between 1995 and 2000. This study evaluated the effectiveness of a Down syndrome (DS) prevention program among the Israeli Jewish population between 1990 and 2000. We collected data on the total number of prenatal tests performed on Israeli Jewish women, DS cases detected prenatally and DS livebirths in Israel during these years. We also studied the use of the newer screening tests in 1990, 1992, and 2000. Between 1990 and 1995, use of chromosomal studies for DS in this population increased from 11.3% to 21.6% and the percentage of cases detected prenatally from 53% to 70%. However, between 1996 and 2000, even with the new screening methods, the utilization rate remained similar (20.7% and 19.8%, respectively) and the percentage detected prenatally decreased to 61% in 2000. The total cost per case detected increased from $47,971 US dollars in 1990 to $75,229 US dollars in 1992, and to $190,171 US dollars in 2000. Between 1990 and 1995, improvement in the percentage of cases detected prenatally was associated with a significant increase in the amniocentesis rate-both are attributed to the introduction of second trimester maternal serum biochemical marker tests. Unexpectedly, the introduction between 1995 and 2000 of new genetic methods to assess the DS risk did not improve the percentage detected or reduce the amniocentesis rate, and was accompanied by an increased cost per case detected.

We applied a comparative genomic hybridization (CGH) technique to paraffin-embedded tissue samples taken from fibroadenomas, benign breast tumors, to detect possible numerical and unbalanced genetic changes. We compared the results to those from previous cytogenetic studies of fibroadenomas. In concurrence with previous cytogenetic studies of fibroadenomas, we detected genetic aberrations in chromosomes 4–6, 8–13, 16, 18, 19, 20, and 22. In addition, with the CGH technique we were able to find two new aberrations, 15q+ and 16p−. Because these aberrations have also been reported to be present in breast cancer, the importance of this finding is yet to be determined.

To evaluate possible genomic instability and possible random aneuploidy, we applied comparative genomic hybridization and fluorescence in situ techniques, and evaluated telomerase activity in 16 cases of Ewing sarcoma (EWS) and compared the results to 7 controls. Common secondary aberrations (gains of chromosomes 8 and 12) were found in the study group. There was a direct correlation between the detection of random aneuploidy and development of tumor relapse (P = 0.0047). Other detectable abnormal parameters (secondary) and high telomerase activity were also more common among the cases with relapse but did not reach a statistical significance (probably because of the small sample size). In EWS, the detection of random aneuploidy seems to be a sensitive parameter in the prediction of tumor relapse.

Purpose: Transcriptional activity of genes is related to their replication timing; alleles showing the common biallelic mode of expression replicate synchronously, whereas those with a monoallelic mode of expression replicate asynchronously. Here the level of synchronization in replication timing of alleles was determined in subjects with Turner syndrome. Methods: Fluorescence in situ hybridization was used for three loci not linked to X chromosome, in lymphocytes derived from 12 controls, 3 individuals with Turner, and 4 with mosaic Turner syndrome. Results: In cells derived from controls, each pair of alleles replicated synchronously; yet these same alleles replicated asynchronously in cells monosomic for X chromosome derived from Turner and mosaic Turner patients. When the level of 45,X was low in the mosaic samples, the replication pattern of the 46,XX cells was normal. However, in samples with a high level of mosaicism, a significantly increased asynchronous replication was detected in the 46,XX cells. Conclusion: An altered temporal replication control in Turner syndrome affecting the aneuploid and euploid cells is shown. This alteration may potentially be involved in the determination of the syndrome.

The replication status of malignant cells is usually asynchronous. However, to date the pattern of replication has not been studied in myeloproliferative disorders nor has the effect of chemotherapy been systematically evaluated. Therefore, we used fluorescence in situ hybridization to interphase nuclei in PHA-stimulated peripheral blood lymphocytes to examine replication timing of three alleles associated with the malignant process. The study group comprised hydroxyurea treated and untreated patients with essential thrombocytosis (ET) or polycythemia vera (PV). A significantly higher rate of the asynchronous pattern of replication in both treated and untreated patients was found as compared to healthy controls. The highest rate of asynchronous replication was observed in untreated patients. Also, the frequency of the two doublets pattern was significantly higher in the untreated group compared to the treated patients and to the control groups. In conclusion, patients with PV and ET have a higher rate of asynchronous pattern of replication. A possible correlation between disease activity and the pattern of replication is suggested. The effect of hydroxyurea on the pattern of replication is variable.

Genomic aneuploidy is a common cause of human genetic disorders. Individuals with aneuploidy tend to develop malignancies. Recent studies correlated aneuploidy with early aging, senescence and organ dysfunction. This study investigated potential explanations for these increased risks by evaluating random aneuploidy and senescence rates in amniocytes from fetuses with aneuploidy.The rates of random aneuploidy in amniocytes from normal pregnancies were evaluated and compared to amniocytes from fetuses with trisomies 21, 18 and 47,XXY using a FISH technique with X+Y, 9 and 18 probes. Senescence was evaluated by calculating the percentage of amniocytes with fragmented nuclei: senescence associated heterochromatin foci (SAHF), using DAPI staining.Significantly increased rates of cells with aneuploidy were observed in trisomies 18 and 21, and 47,XXY (p0.001) compared to the control group for the somatic and sex chromosomes. Increased rates of amniocytes with SAHFs were observed among the trisomy samples compared to the control group.Higher incidence of random aneuploidy and senescence were observed in amniocytes from fetuses with trisomy. These findings might explain the greater lifetime tendency to develop malignancies and diseases related to early aging in these individuals.

Polycythemia vera (PV) and essential thrombocytosis (ET) are clonal chronic myeloproliferative disorders originating from a multipotent stem cell. Bone marrow examinations reveal chromosomal abnormalities in 15-43% of PV patients and 5% of ET patients, but no specific recurring abnormality has been found to date. We aimed to find cytogenetic aberrations in PV and ET by comparative genomic hybridization (CGH), a relatively new molecular cytogenetic technique. In this study, CGH analysis was performed on peripheral blood leukocytes of 12 PV patients and 8 ET patients. One patient (8.3%) with PV had an abnormal karyotype with a deletion in 7q11.2 and one patient with ET (12.5%) had a gain in 18p. Peripheral blood analysis by CGH revealed a low frequency of cytogenetic abnormalities in PV and ET patients. However, using CGH we were able to detect two cytogenetic aberrations that were not reported previously in these disorders.

We describe a man with pericentric inversion 9 and constitutive heterochromatin, and a high disomy rate in his sperm cells (with all probes analyzed). The disomy rate was estimated with the following probes: 8, 9, 18, X, and Y, and was significantly higher than that in control sperm cells, while chromosome 9 showed the highest disomy frequency. The probes of X and Y together showed the same disomy frequency as X and Y alone, which indicates the same nondisjunction rate in the first meiotic division. We suggest that the interchromosomal effect found in this man differed from other findings in sperm cells of men carrying an inversion in terms of the difference in the length of the heterochromatin between the two chromosomes 9. Also, it is well known that the effect of inversion 9 with increased heterochromatin is highly variable and may even vary in members of the same family.

Background Fetal cells exfoliate in the uterine cavity during early pregnancy and are a potential source of material for NIPD. Aims This study was designed to test the hypothesis that fetal cells obtained from the uterine cervix during the first trimester of pregnancy could be utilized for prenatal diagnosis of chromosomal aneuploidy. Study design Fetal cells retrieved from the distal endocervical canal during the first trimester of pregnancy were hybridized with chromosome 21 specific FISH probes and analyzed with an automated fluorescence microscope. Subjects and outcome measures Cells with 3 copies of chromosome 21 were detected in 5 out of 5 trisomy 21 pregnancies. Results The number of trisomic cells detected ranged from 1 to 27 with a median value of 5. Conclusions FISH-based scanning can identify trisomy 21 pregnancies by analysis of routine cervical brushings. The approach offers the potential for non-invasive prenatal diagnosis as early as 5 weeks gestation.

Objective. The aim of this study was to report the clinical features, management, and outcome of two cases of complete hydatidiform mole with a coexisting viable fetus and to review the literature. Patients. In this article, we report on the well-documented follow-up of 2 cases of twin pregnancies with complete hydatidiform mole and a viable fetus, both of which ended with the delivery of a normal infant at 41 and 26 weeks of gestation. It is of interest that both pregnancies were achieved following induction of ovulation with hMG/hCG. Since 1977, the year in which complete and partial moles were characterized as distinct pathologic entities, 15 cases (including our 2) have been reported. Results. Persistent GTT developed in eight patients (53.3%) and four patients (27.7%) developed metastatic disease. Seventy-five percent patients with persistent GTT were treated with single-agent chemotherapy. The median gestational age of the patients with subsequent persistent GTT was 34.5 weeks compared to 38 weeks in the patients without persistent GTT. Conclusion. Complete hydatidiform mole and coexistent fetus is a rare occurrence and is associated with an increased risk of persistent gestational trophoblastic tumor. Based on currently available information, it seems that in the presence of a stable pregnancy, normal karyotype, and a normal sonogram it is reasonable to allow the pregnancy to continue.

Recently, a working-model of a stepwise malignant transformation in the molecular pathogenesis of multiple myeloma (MM) was proposed, involving the tumor suppressor gene TP53 and retinoblastoma gene (RB1) as prominent components of cell cycle control. To further define the role of TP53 and RB1 in disease progression, we retrospectively analyzed by fluorescence in situ hybridization (FISH) cytological material from 16 patients who underwent sequential bone marrow biopsies during the course of their disease. For TP53, no deletions were detected at presentation or during follow-up. It is possible that the patients reported here represent a subset with relatively long survival, and therefore did not demonstrate the TP53 deletions that had been reported in patients with a very poor prognosis. For RB1, monoallelic deletion was demonstrated in nine patients. In each case, the deletion appeared already in the first biopsy analyzed. The presence of a deletion did not affect the rate of tumor progression or the length of follow-up, and thus prognosis. Monoallelic deletions of RB1 appear to be a frequent and early event in the pathogenesis of MM, without obvious relevance for disease progression.

In this study we evaluated the replication pattern and cell-cycle dynamics of cells from patients considered to have a premalignant condition (monoclonal gammopathy, or MGUS) and patients with multiple myeloma (MM), as well as healthy controls. We applied the fluorescence in situ hybridization (FISH) technique with the TP53, RB-1 and 21q22 loci on the patient's cells. Asynchrony was determined by the presence of one single and one set of double dots in the same cell. The rate of asynchronic replication was significantly higher in the cells from MM patients, with intermediate value in the cells from MGUS, while the lowest rate was in cells from controls. We suggest that these results may reflect the changes in gene replication and cell-cycle progression that occur in premalignant and malignant cells.

BACKGROUND Loss of replication synchrony during the S-phase of the cell cycle has been shown to be associated with aneuploidy in malignant cells. METHODS The replication pattern of cervical cells obtained from normal patients and from patients with preinvasive and invasive carcinoma of the cervix was evaluated. The fluorescence in situ hybridization technique was applied to Papanicolaou smear slides using p53 and 21q22 loci. Asynchrony was determined by the presence of one single and one set of double dots in the same cell. RESULTS The rate of asynchrony in low grade squamous intraepithelial lesions was not significantly different from that of normal controls. However, the rate of asynchrony was significantly higher in the high grade squamous intraepithelial lesions and even higher in the invasive carcinoma. CONCLUSIONS The authors believe these results reflect impairment of the replication control of homologous loci and that this phenomenon may be correlated to the phenotype of the tumor. Cancer 1998;83:1966-1971. © 1998 American Cancer Society.

We have used FISH to determine the level of synchronisation in replication timing of four pairs of alleles, unrelated to chromosome 21 (p53, HER2, RB1, and c-myc), in foetal (amniotic fluid) cell samples of Down syndrome and in normal foetuses. All samples derived from the Down syndrome subjects showed large temporal differences in replication timing, in contrast to the high level of synchrony shown in all samples of normal individuals. Thus, as judged by four independent loci which are not associated with chromosome 21, the additional chromosome in the Down syndrome genome induces changes in the replication pattern of an allelic pair: from a synchronous pattern characteristic to concomitantly expressed alleles to an unsynchronised one shown by alleles displaying an allele-specific mode of expression.

The clinical significance of mosaicism trisomy 20 detected prenatally following amniocentesis remains uncertain, due to the rarity of liveborn cases with inconsistent clinical findings, the short postnatal follow-up, and failure in evaluating other fetal tissues for the presence of the trisomy. We report on a 15 month-old 46,XX chromosome constitution in white blood cells, while skin fibroblasts demonstrated trisomy 20 mosaicism (54%) by fluorescence in situ hybridization (FISH) analysis. Clinical examination of the baby showed only minor phenotypic signs (bilateral epicanthal folds, delayed closure of fontanel with no other gross anomalies), but demonstrated a considerable developmental delay in gross and fine motor skills along with hypotonicity. This is the second oldest described liveborn with trisomy 20 mosaicism confirmed in skin fibroblasts. This cytogenetic aberration along with her developmental delay suggests that the two findings are related and that aberration affects various fetal tissues and is not confined to extra-embryonic tissue as suggested previously. Yet, an undiagnosed condition may be the cause of the child's developmental delay. Based on this case and following a review of the literature we suggest that when mosaic trisomy 20 is identified in amniocytes, further evaluation is required. Cord blood should be analyzed preferably by FISH. During counseling the parents should be advised of an additional risk, such as developmental delay, even when fetal cord karyotype and detailed ultrasonic scan are normal.

This study was undertaken in an attempt to determine the significance of elevated maternal serum human chorionic gonadotropin (MShCG), in the presence of an otherwise normal screen with respect to fetal malformations, chromosomal aberrations, and pregnancy outcome. Targeted ultrasound findings and perinatal outcome of 298 women in whom serum hCG was > or = 2.5 MOM and who were screen-negative for Down syndrome (the study group) were compared with a control group of 229 women in whom serum hCG as well as the other parameters were within the normal range. Genetic amniocentesis was performed in 125 women from the study group. Ultrasonically detected malformations were significantly more frequent among the study group (12 vs. 1, P = 0.01). Pregnancy complications were similar in the two groups, with the exception of pre-eclampsia-toxaemia, which was significantly more frequent in the study group (5 vs. 0, P = 0.02). There was one case of an abnormal karyotype (47,XXY). Although genetic amniocentesis does not appear warranted, isolated elevated MShCG levels during the second trimester screening was associated with an increased risk of fetal anomalies detected by ultrasound and of toxaemia of pregnancy.

Chromosomal aberrations can be detected in 50% of patients with chronic lymphocytic leukemia (CLL). A role for tumor suppressor genes in the genesis of lymphoid tumors has been reported. In B-CLL, p53 gene mutations were found in 10-15% of the patients. We used fluorescence in situ hybridization (FISH) to detect p53 deletion in B-CLL. We also correlated the cytogenetic findings with the clinical course. In situ hybridization to interphase nuclei showed monallelic p53 deletion in 6 of 23 patients (26%). The percentage of cells with one p53 signal ranged from 12 to 100. A statistically significant correlation between p53 deletion and progression of CLL was demonstrated. We conclude that FISH is a sensitive and reliable method to detect deletion of specific genes (i.e., p53) in CLL. The finding of p53 deletion is associated with disease progression.

Essential thrombocytosis (ET) is a clonal, chronic myeloproliferative disorder (MPD) originating from a multipotent stem cell. To date no specific cytogenetic marker has been found in ET. It was recently reported that chromosomal aberrations have been detected by fluorescence in situ hybridization (FISH) in patients with normal karyotypes or nonanalyzable metaphases. Therefore, we evaluated whether trisomies 8 and 9, which are commonly found in MPDs, can be detected in ET by FISH and compared the results with chromosome analysis. Peripheral blood mononuclear cells of patients with essential thrombocytosis were studied by classical chromosome banding and by FISH. We used biotin labeled α satellite of chromsome 8 and biotin labeled β satellite of chromosome 9 as probes for the FISH studies. FISH detected 5 patients with trisomy 8 and 5 with trisomy 9 of the 18 patients evaluated. No trisomy was found by cytogenetic studies. The trisomies were detected by FISH in only a minority of the cells. No correlation was found between the presence of a trisomy and clinical characteristics. FISH is a sensitive method for the detection of trisomes 8 and 9 in patients with ET. The common finding of these chromosomal aberrations in MPD suggests that genes associated with myeloid proliferation are located on these chromosomes. Standardization of interphase cytogenetics is needed before this technique can be accepted for routine use in the clinic.

Cytogenetic and fluorescent in situ hybridization (FISH) studies were performed on several formalin-fixed tissues obtained from four fetuses diagnosed at amniocentesis as 45,XO-Turner syndrome. Three of the four were phenotypically normal and one had malformations. The three phenotypically normal cases were found to have an additional normal cell line, which may explain their ability to survive, at least to the time of pregnancy termination well into the second trimester. The abnormal 45,XO fetus was not found to be mosaic in all of the tissues examined. In 45,XO cases in which no malformation is detected, the possibility of mosaicism should be raised and thus the counselling should be modified accordingly.

Interleukin-11 (IL-11) is a novel cytokine that has been shown to stimulate human hematopoietic progenitors including the CD34+ CD33- DR- early progenitors. IL-11 has little effect on its own but it synergizes with other hematopoietic growth factors. We investigated the recovery of human myeloid progenitors incubated with IL-11 alone or in combination with other cytokines, including stem cell factor (SCF), interleukin-3 (IL-3) and granulocyte macrophage colony-stimulating factor (GM-CSF) following their in vitro treatment with ARA-C (10(-9) M) or Eilatin (10(-7) M). IL-11 in combination with IL-3 and GM-CSF markedly increased CFU-C colony growth pre- and post-ARA-C or Eilatin incubation from CML and normal individual bone marrow (BM) cells. Similarly, IL-11 alone or in combination with other cytokines increased cell recovery following 7-day suspension culture. A decrease in BCR/ABL fusion product was observed (by FISH analysis) after incubation of BM cells from CML patients in liquid culture for 7 days with 10(-9) M ARA-C or 10(-7) M Eilatin in the presence of IL-11 alone or in combination with other cytokines. These results indicate that following cytoreductive therapy IL-11 may enhance to a greater extent the growth of normal myeloid progenitors than the malignant clone and may, therefore, be of clinical importance for CML patients treated with chemotherapeutic agents.

Objective Primary sclerosing cholangitis (PSC) is a chronic cholestatic disorder that involves inflammatory and fibrotic changes in the bile ducts. Up to 80% of patients have concomitant inflammatory bowel disease (IBD) with colitis. PSC patients are predisposed to develop hepatobiliary, colonic and other extrahepatic malignancies, probably related to inflammatory processes that might promote carcinogenesis. Telomerase is an enzyme complex that lengthens telomeres and has enhanced expression in numerous malignancies. In this study, we evaluated the TERC gene copy number, the proportion of cells in senescence and the amount of fragmentation in the senescent state. Methods Fluorescence in situ hybridization (FISH) for the TERC gene was applied to lymphocytes retrieved from PSC (N = 19), colitis (N = 20) and healthy control patients (N = 20) to determine the TERC copy number. On the same FISH slides, cells stained with DAPI were also analyzed for senescence-associated heterochromatin foci (SAHF) status, including the number of cells with fragments and the number of SAHF fragments in each cell. Results A higher TERC gene copy number was observed in cells from PSC patients compared to colitis and control group patients. It was also higher in the colitis than in the control group. Significantly more cells in the senescent state and more fragmentation in each cell were observed in the PSC group compared to colitis and control groups. Conclusion The TERC gene copy number and the number of cells in the senescent state were increased in PSC patients compared to the colitis and control groups. These findings are probably related to the genetic instability parameters that reflect the higher tendency of this patient group to develop malignancies.

The putative oncogenes BCL-1, BCL-2, and BCL-3 are commonly rearranged by translocations to the immunoglobulin genes in B-cell malignancies. However, Southern blotting rarely detected their involvement in chronic lymphocytic leukemia (CLL). This discrepancy could stem from some unique features of the oncogenesis of CLL or be due to shortcomings of Southern blotting. We have therefore evaluated the role of fluorescence in situ hybridization (FISH) in the detection of these oncogenes in CLL. Twenty consecutive CLL patients were studied by FISH for the detection of BCL-1, BCL-2, or BCL-3 rearrangement and for the presence of trisomy 12. Selected patients were also evaluated by classical cytogenetic techniques and by Southern blot analysis. Juxtaposition of JH and BCL-1 was demonstrated in 10 (50%), BCL-2 in three (15%), and BCL-3 in four (20%) of the patients. Trisomy 12 was detected by FISH in 11 (55%) patients. The coexistence of trisomy 12 and translocation of the BCL-1 oncogene was common. Three of the patients had chromosomal aberrations compatible with those detected by FISH. In contrast, in none of the five patients selected by their positive FISH findings was a rearrangement demonstrated by Southern blotting. We conclude that FISH is a sensitive method for the detection of oncogene involvement in CLL. Mainly BCL-1, but also BCL-2 and BCL-3, are commonly translocated to the immunoglobulin heavy chain locus on chromosome 14. These translocations are often associated with trisomy 12. These findings indicate that the BCL oncogenes are commonly involved in CLL and lend support to the multi-hit theory of cancer development.

The malignant plasma cells of multiple myeloma (MM) have a low proliferative activity and therefore cytogenetic studies of the disease have been severely limited. We evaluated the role of fluorescence in situ hybridization (FISH) in the detection of numerical chromosomal abnormalities in early stages of myeloma and the applicability of the method to stored archival slides. Old air-dried bone marrow smears from 15 myeloma patients obtained at presentation were probed with α satellite DNA sequences to chromosomes 3 and 7. Numerical chromosome aberrations were found in eight (53%) of the patients, including six (of 12) with trisomy 7, and two (of eight) with trisomy 3. This study demonstrates that FISH is a sensitive method for the detection of numerical aberrations in myeloma and for the study of old slides for retrospective analysis.

Background. Malignant thymoma is composed of neoplastic epithelial cells and small lymphocytes. Rarely, patients also may have peripheral T-lymphocytosis. These lymphocytes have been considered nonneoplastic because of their microscopic appearance and immunophenotype, as well as gene rearrangement studies. Methods. A 42-year-old man developed lymphocytosis 3 years after the completion of intensive combined chemoradiotherapy protocol for lymphocytic thymoma. These peripheral blood lymphocytes were evaluated phenotypically and genotypically. Results. Immunophenotyping established that the cells were CD3 positive, CD4 negative, CD8 negative, T-cell receptor (TCR)-alpha/beta negative, and TCR-gamma/delta positive. Gene rearrangement studies with TCR-delta probe confirmed the monoclonality of these cells. Chromosome analysis showed deletion of chromosome Y. The clinical course was progressive and had the features of malignant lymphoma. Conclusions. To the authors' knowledge, this is the first report of a patient with thymoma in whom monoclonal proliferation of T-gamma/delta peripheral blood lymphocytes was confirmed immunophenotypically and genotypically. These monoclonal TCR-gamma/delta lymphocytes may belong to the malignant clone of the thymoma; however, the possibility that they represent an evolution of a second lymphatic malignancy cannot be excluded.

Individuals with trisomy 21 have an increased risk of developing leukemia and premature dementia. They also have a higher rate of telomere loss. The aim of the study was to compare telomere length and the hTERC gene copy number, which encodes the telomerase RNA subunit, in amniocytes of trisomy 21 conceptions and normal pregnancies. A quantitative fluorescence-in-situ protocol (Q-FISH) was used to compare telomere length in amniocytes cultured from 11 trisomy 21 conceptions and from 14 normal pregnancies. Quantification was conducted using novel computer software. Fluorescence in situ hybridization (FISH) was used to assess the percentage of cells with additional copies of hTERC. We found that the immunofluorescence intensity, which represents telomere length, was significantly lower in amniocytes from trisomy 21 conceptions compared to the control group. The trisomy 21 group had a higher number of cells with additional copies of hTERC. This observation could be one of the cytogenetic parameters that represent a state of genetic instability and might play a role in the pathomechanism of typical features of Down syndrome, such as dementia and malignancy.