Application of quantitative fluorescent PCR with short tandem repeat markers to the study of aneuploidies in spontaneous miscarriages

Dan Diego-Alvarez1, Maria Garcia-Hoyos, Maria Jose Trujillo, Cristina Gonzalez-Gonzalez, Marta Rodriguez de Alba, Carmen Ayuso, Carmen Ramos-Corrales and Isabel Lorda-Sanchez

Fundacion Jimenez Diaz - Human Genetics, Avda. Reyes Catolicos, 2 Madrid 28040 Spain

1 To whom correspondence should be addressed. Dan Diego-Alvarez, Email: ddiego{at}fjd.es


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Aneuploidies involve ~80% of chromosomal anomalies found in spontaneous miscarriages. Since cytogenetic studies show high rates of failure, we have incorporated the quantitative fluorescent polymerase chain reaction (QF-PCR) technique to the study of numerical chromosome anomalies in miscarriages. METHODS: Multiplex and simple QF-PCR assays have been performed on 160 miscarriage and 34 parental DNA samples analysing specific short tandem repeat (STR) markers for chromosomes 2, 7, 13, 15, 16, 18, 21, 22 and X. Cases successfully karyotyped were used as controls in our study. RESULTS: While maternal contamination could be detected in such cases, a molecular result was obtained for 94% of miscarriages without a cytogenetic one. Thirty-six per cent of them were diagnosed with numerical chromosome anomalies. Parental origin of the extra chromosome and the error stage of meiosis could be also determined. CONCLUSIONS: QF-PCR represents a useful and reliable tool to diagnose aneuploidies in spontaneous miscarriages. It provides information about parental and meiotic origin of anomaly, allowing an appropriate genetic counselling.

Key words: abortion/aneuploidy/QF-PCR/spontaneous miscarriage/STR marker


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
Embryo loss represents ~10–15% of conceptions with clinical evidence of pregnancy (Miller et al., 1980Go; Warburton et al., 1980Go) and ~65% of all gestations (Santalo et al., 1987Go). Presumptive causes of pregnancy loss include genetic, chromosomal, anatomic, endocrine and immunological factors, infections, thrombophilic disorders and other unidentified causes (Carp et al., 2001Go).

More than 50% of spontaneous miscarriages carry chromosomal disorders, up to 96% of them being numerical chromosome abnormalities (Carrera et al., 1996Go). Identification of the possible cause of fetal loss significantly reduces long-term psychological distress in women with a miscarriage (Nikcevic et al., 1999Go) and enables improved genetic counselling for those couples in future pregnancies (Carp et al., 2001Go).

Although cytogenetic study of miscarriages is highly recommended even in the case of the first spontaneous abortion (Sanchez et al., 1999Go; Silvestre et al., 2002Go), conventional cytogenetic studies (karyotyping or FISH) entail certain problems such as culture failure, infection of the sample or maternal contamination. Moreover, those techniques are usually expensive and need a moderate period of time in order to obtain results.

We have incorporated the molecular QF-PCR technique to the study of spontaneous miscarriages since it has been described as a rapid, sensitive, accurate, reproducible and reliable diagnostic method to detect aneuploidies (Ban et al., 2002Go).

The aim of the present study is to propose the QF-PCR technique as a molecular tool complementary to cytogenetic studies of spontaneous miscarriages. It could be especially useful for those cases in which karyotyping fails, or for discounting a wrong diagnosis when overgrowth of maternal cells occurs, by analysing maternal DNA. To our knowledge, this is the first report of the application of QF-PCR to the study of spontaneous miscarriages.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
Biological samples
One hundred and sixty miscarriage and 34 parental DNA samples corresponding to 17 couples have been studied. Miscarriage samples have been provided to the Department of Genetics of FJD since 1996 by the obstetrics service of the hospital and some private clinics to perform cytogenetic studies. Specimens of which tissue material was available were employed in this study for DNA extraction. Miscarriage samples consisted in fetal remains obtained after surgical evacuation from clinical abortions between the 4th and 24th weeks of gestation (mean 15 weeks of gestation). One hundred and forty-eight out of 160 miscarriages were cultured following the protocol described by Moragón and Ramos-Corrales (1978)Go and GTG-banded for karyotyping; three were not cultured because ultrasonographic examinations showed skeletal dysplasia phenotype and nine were non-viable for karyotyping because of deterioration of the sample (6/9) (advanced autolysis in macerated abortions) or because they were preserved in formalin (3/9). Remains were stored frozen both at –20 °C and in liquid nitrogen until DNA extraction was performed. Most genomic DNA samples from abortions were obtained from fresh, frozen and formalin-fixed tissues following standard or commercial protocols. Tissue Kit reagents (QIAGEN, Germany) and the BioRobot EZ1 (QIAGEN) were used after dissection of 100–150 mg of sample and 48–72 h of digestion at 56 °C with proteinase K. A small proportion of miscarriage DNA samples was isolated following a standard phenol–chloroform extraction procedure. Parental DNA samples were extracted from peripheral blood with the DNA blood 350 µl extraction Kit (QIAGEN) and the BioRobot EZ1.

The present study has been accomplished according to the 1964 ‘Declaration of Helsinki’ (World Medical Organization, 1996Go).

QF-PCR
QF-PCR is based on the assumption that within the exponential phase of PCR amplification, the amount of specific DNA produced is proportional to the quantity of the initial target. In order to achieve this the optimal number of PCR cycles must be limited to avoid reaching an amplification plateau. By amplifying highly polymorphic regions specific for a chromosome, such as short tandem repeats (STR), we should find a high rate of heterozygosity among different allelic forms and individuals. Tetra- or pentanucleotide repeats have been preferentially employed in view of their stability and suitability for amplification and analysis. By labelling primers with a fluorescent dye, we are able to detect dosage ratios of the PCR products from the analysis of the fluorescent peak areas shown by a Genetic Analyzer. Thus, in normal heterozygotes the ratio of fluorescent activity for the two peaks corresponding to the PCR products should be within the range 0.8–1.4 (disomic diallelic). Few normal subjects should be homozygotes showing one peak of activity (disomic monoallelic). Besides, in a trisomic patient the three doses of an STR marker can be detected either as three peaks of fluorescent activities with a 1:1:1 ratio (trisomic triallelic) or as a pattern of two peaks with a ratio or dosage <0.65 or >1.8 (trisomic diallelic) (Hulten et al., 2003Go) (Figure 1). Triploidy of specimens is assumed when all of the markers studied, which map to different chromosomes, show a trisomic pattern of amplification. Uniparental disomy (UPD) for a targeted region or chromosome is assumed when the pattern of amplification of various STR markers corresponds to the inherited alleles from one progenitor with the absence of the other progenitor alleles.



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Figure 1. Multiplex QF-PCR assays (PCR-C and PCR-D) with STR markers in a DNA sample of a spontaneous abortion affected of double trisomy (+15, +22). The x-axis shows length of PCR products in base pairs as determined by use of internal lane standard and the y-axis shows the fluorescence intensity in arbitrary units. Trisomy 15 is evidenced by trisomic triallelic pattern (1:1:1) for D15S1050 and trisomic diallelic (2:1) for D15S123; trisomy 22 is shown as trisomic diallelic pattern (2:1) for D22S689 and trisomic triallelic pattern (1:1:1) for D22S280.

 
A rapid screening of sex and numerical anomalies for chromosomes 13, 15, 16, 18, 21, 22 and X was carried out with the 160 miscarriage DNA samples performing four multiplex PCR assays (Table I). PCR assays A and B were performed in a total volume of 25 µl containing 40–100 µg of genomic DNA, 150 µmol/l dNTP (Invitrogen Corporation, USA), 1.26–37.5 pmol of each primer, 1 x AmpliTaq Gold polymerase buffer (15 mmol/l MgCl2) (Applied Biosystems, USA), and 1 U of AmpliTaq Gold polymerase (Applied Biosystems). After denaturation at 95 °C for 5 min, hot-start PCR was carried out in a GeneAmp PCR System 2700 (Applied Biosystems) for 25 cycles at 95 °C for 60 s, 55 °C for 60 s, and 72 °C for 90 s. Final extension was for 10 min at 72 °C. PCR assays C and D were carried out in a total volume of 15 µl containing 40–100 µg of genomic DNA, 125 µmol/l dNTP, 5–10 pmol of each primer, 1 x Taq polymerase buffer (15 mmol/l MgCl2), and 1 U of AmpliTaq Gold polymerase. After denaturation at 95 °C for 12 min, hot-start PCR was carried out for 10 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 90 s, and 15 cycles at 89 °C for 30 s, 55 °C for 30 s and 72 °C for 90 s, with a final extension time of 30 min at 72 °C. Fluorescence-labelled PCR products were electrophoresed in an ABI Prism 3100 Genetic Analyzer and analysed with the GeneMapper 3.5 software package (Applied Biosystems). For uninformative results (both markers of the same chromosome showed a monoallelic pattern of amplification) or to confirm a trisomic pattern, additional markers (Table II) were separately PCR-amplified in a total volume of 15 µl containing genomic DNA, 125 µmol/l dNTP, 10 pmol of each primer, 1 x Taq polymerase buffer (15 mmol/l MgCl2), and 0.6 U of AmpliTaq Gold polymerase. PCR cycles were the same as those for C and D PCR assays. Annealing temperature for D21S1412 was 58 °C.


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Table I. Primers used in Multiplex PCR assays. Note: F: forward; R: reverse. Italics: based on our expirience over Caucasian population. The Genome Database homepage: www.gdb.org

 

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Table II. Additional STR markers used in simple PCR assays. Note: H: Heterozygosity; F: forward; R: reverse; n.a.: not available. Based on our experience over Caucasian population

 
Various simple PCR assays were also carried out in some samples to confirm both a cytogenetic or CGH result or when a target chromosome existed. This was the case for specimens tested for chromosomes 2, 7 and 17.

Whenever possible, the same STR markers were studied in parental DNA in order to determine both parental and meiotic origin of aneuploidy and the fetal origin of the DNA sample.

Cases successfully karyotyped were used as blind controls in our study.


    Results
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
Ninety-four out of 148 miscarriage samples (63.5%) were successfully karyotyped: 27 of them as normal males, 32 as normal females and 35 (37%) as having numerical chromosomal aberrations. Fifty-four miscarriages (36.5%) failed culture due to misgrowth or to infection of the sample. One hundred and fifty-one out of 160 (94%) miscarriage DNA samples were successfully PCR amplified for all of the chromosome markers tested in this study.

Discrepancies between molecular and cytogenetic results occurred in eight out of 89 cases. Five of those karyotyped as normal females resulted in chromosomally male abortuses by QF-PCR, two of them trisomic for chromosome 13. Three cases karyotyped as affected of aneuploidy were diagnosed as normal females by QF-PCR.

A molecular result for 62 of the 66 (94%) abortions without cytogenetic result was obtained by QF-PCR. Thirty-six per cent (24/66) of cases were diagnosed as having numerical chromosome anomalies (Table III) and two cases of maternal contamination were detected.


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Table III. Number of abortions without karyotype diagnosed of having a numerical chromosome anomaly by QF-PCR

 
Parental and meiotic origin of the extra/absent chromosomal material was identified in 14 miscarriages. A complete trisomy 2 was found in a miscarriage whose father was a balanced carrier of a 2;17 translocation. Paternal origin of the extra chromosome material could be established. In a 45,X0 case it was determined that the sexual chromosome fault was of paternal origin. Two double trisomies involving chromosomes 15, 22 (Figure 1) and 8, 21 respectively were determined to be maternal in origin due to non-disjunction at meiosis I (MI) while a third double trisomy involving 18 and X chromosomes also had a maternal origin because of an error in meiosis II (MII) (Figure 2). Moreover, one case of trisomy 7 (Figure 3), two cases of trisomy 21 and 22 and four cases of trisomy 13 were determined to be maternal in origin due to non-disjunction in MI.



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Figure 2. Multiplex QF-PCR assays (PCR-A and PCR-B) with STR markers in a miscarriage affected of double trisomy (+18, + X) and its parents. The x-axis shows length of PCR products in base pairs as determined by use of internal lane standard and the y-axis shows the fluorescence intensity in arbitrary units. Electrophoretograms show miscarriage (a), maternal (b) and paternal (c) PCR products. Trisomy 18 is evidenced by trisomic diallelic pattern (2:1) for D18S535 (18q12.3) and trisomic triallelic pattern (1:1:1) for D18S386 (18q22.1-22.2); trisomy X is shown as trisomic diallelic pattern (2:1) for X22 (Xq28/Yqter) and trisomic triallelic pattern (1:1:1) for XHPRT (Xq26.1) (arrows). Maternal origin of the extra chromosome material and the meiotic stage of nondisjunction (MII) can be inferred by analyzing inherited alleles and chromosome location of STR markers.

 


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Figure 3. Electrophoretograms of two STRs located on chromosome 7 in a miscarriage and its parents. The x-axis shows length of PCR products in base pairs as determined by use of internal lane standard and the y-axis shows the fluorescence intensity in arbitrary units. Abortion (A), maternal (B) and paternal (C) DNA samples. Trisomy 7 in miscarriage is showed as trisomic diallelic pattern (2:1) for IV517bCA (7q32.1) and trisomic triallelic pattern (1:1:1) for D7S460 (7p14.2). Assuming no crossing-over have occurred between DNA markers and centromere, maternal origin due to an error in the first meiotic division is assumed for the extra chromosome material.

 
Although the QF-PCR technique is capable of detecting mosaicism, no such cases have been detected in our study.


    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
Identification of the cause of fetal loss reduces the feelings of self-blame, anxiety, depression and grief in women with a miscarriage (Nikcevic et al., 1999Go). Moreover, knowing the exact origin of fetal loss allows us to give an improved genetic counselling in further pregnancies and to predict the frequency of recurrence in each patient. Although some studies suggest that there are non-cytogenetic factors associated with recurrent miscarriages (RM), particularly in women aged <36 years (Stephenson et al., 2002Go), other researchers maintain that couples with RM (two or more previous abortions) produce chromosomally abnormal embryos in a significantly higher percentage than those not having this reproductive problem (Rubio et al., 2003Go). In the same study it was reported that in 22% of couples undergoing an IVF programme the incidence of chromosomal aberrations affected all of the embryos they produced. It is well known that >50% of spontaneous miscarriages carry chromosomal disorders. Among chromosomal causes of fetal loss, aneuploidies are responsible for >86% of them. Even though QF-PCR is currently being used for the rapid screening of aneuploidies for prenatal diagnosis in amniotic fluid and chorionic villus samples (Pertl et al., 1999Go; Carrera, 2001Go; Bili et al., 2002Go; Andonova et al., 2004Go), this is the first report of application of this technique to the study of spontaneous miscarriages.

Evaluation of pregnancy losses is usually done several days after fetal demise. The main risk when studying aborted specimens by classic cytogenetics is the deterioration of the sample. Success of culture is often reduced by the high rates of microbial or maternal contamination, obtaining no or incorrect results. This is the case for 36.5% of our abortions. QF-PCR has permitted us to obtain a molecular result for 94% of samples studied and to detect 24 cases of aneuploidy in miscarriages that had no cytogenetic diagnosis. While conventional cytogenetics needs living cells to culture, DNA analysis can provide results with a high efficiency, independently of the antiquity of the sample. High amounts of DNA are obtained from fresh and frozen tissue and can also be isolated from small pieces or sections of formalin-fixed or paraffin-embedded tissues (Bell et al., 2001Go; Shi et al., 2002Go) and be stored for several years. In the present study, 54 miscarriage tissue samples (36.5%) failed to culture due to misgrowth or to infection, and only nine out of 160 (5.6%) DNA samples were not suitable for PCR amplification. This could be due to the small amount of DNA (samples extracted from formalin-fixed tissues) or to an error in the extraction procedure.

It is well known that the incidence of fetuses with chromosomal anomalies is reduced as pregnancy progresses. The mechanism of natural selection may operate through developmental arrest and degeneration of abnormal embryos. Autosomal monosomies are rarely found in spontaneous abortions because of its high lethality. Although trisomies for all the chromosomes have been reported in spontaneous miscarriages (Hassold et al., 1980Go; Hanna et al., 1997Go), suggesting that they are capable of achieving implantation and initiating a pregnancy (Dunn et al., 2001Go), the most frequently cited in literature are those involving chromosomes 15, 16, 14 and 22 (Carrera et al., 1996Go; Stephenson et al., 2002Go) and are supposed to be the major cause of early abortions affected by chromosomal anomalies. Despite single trisomies comprising the vast majority of aneuploidies (~86% single trisomies versus ~14% monosomies), double and triple trisomies appear with a frequency of 0.21–2.8% and 0.05% respectively of karyotyped spontaneous abortions (Reddy, 1997Go, 1999Go). In spite of this lower frequency, several chromosomes have undergone molecular study in all of our samples in order to detect these kinds of anomalies. Multiplex analysis by QF-PCR permits us to test various chromosomes while a large number of samples can be handled simultaneously. PCR assays for chromosomes 13, 18, 21, X and Y have been carried out in the first screening because of their high incidence of aneuploidy among late abortions (Hassold et al., 1980Go). Work in progress consists in designing multiplex PCR to study aneuploidies of other chromosomes.

Parental and meiotic origin of aneuploidy can be inferred by QF-PCR (Figure 3), analysing pericentromeric STR markers whenever parents are informative for them. Heteromorphic regions at or near centromeres are not crossed over, and are the optimal markers for tracing the origin of aneuploidy, avoiding misdiagnosis due to recombination events (Robinson et al., 1993Go). Based on the literature (Bond and Chandley, 1983Go), non-disjunction at the first meiotic division in oogenesis seems to be the most common origin of the extra chromosome in trisomies. This is consistent with our results, in which, as expected, nine out of nine cases of simple trisomies and two out of three double trisomies were maternal in origin due to non-disjunction in MI. The paternal origin of a complete trisomy 2 due to 3:1 segregation was established in a miscarriage whose father was a balanced carrier of a 2;17 translocation (Lorda-Sanchez et al., 2005Go).

A general problem when studying early pregnancy losses is overgrowth of maternal cells in culture (Hassold et al., 1980Go). This usually leads to a wrong 46,XX normal karyotype. While neither karyotyping nor FISH can detect it (except some cases of male fetuses or when a numerical anomaly appears, as stated by Jobanputra et al., 2002Go), QF-PCR analysis of microsatellites can discard it and determine the maternal or fetal origin of the sample. In fact, those cases in which discrepancies occurred among cytogenetic and molecular results were due to maternal origin or contamination of the sample. Different origin of tissue material collected for culture and to store for molecular studies seems to be the cause of misdiagnosis in three cases amplified by QF-PCR obtained as normal females. Concordance with the rest of the results shows the reliability of the QF-PCR technique.

Despite aneuploidies involving the majority of chromosomal causes of abortions, total chromosome uniparental disomy (UPD) comprises ~3% of genetically unexplained pregnancy wastage (Fritz et al., 2001Go). Some studies have reported maternal heterodisomy (inheritance of both maternal homologues) as the most common cause of UPD, meiotic non-disjunction being followed by trisomy rescue (Kotzot, 2004Go) or monosomy complementation (somatic reduplication) considered to be the major mechanisms of formation. Molecular analysis of STR markers by QF-PCR in parental and abortion DNA samples would permit the diagnosis of iso- and heterodisomy of both maternal and paternal origin.

Although >83% of chromosomal anomalies can be diagnosed by QF-PCR, this technique poses some limitations for the detection of chromosomal rearrangements and small deletions or duplications. Triploidy and tetraploidy comprise ~13 and ~4% respectively of chromosomal anomalies found in spontaneous miscarriages (Alberman and Creasy, 1977Go). While triploidy can be diagnosed by QF-PCR, tetraploidy due to abnormal cleavage after a normal zygote has formed would not be easily detected. Based on our experiments and on published data (Lorda-Sanchez et al., 2003Go), autosomal and gonosomal mosaicism can be detected up to the presence of 1%, but in practice it is difficult to determine, and parental DNA is required to detect it.

Apart from its limitations (Hulten et al., 2003Go), QF-PCR also presents exclusive advantages over other techniques currently employed for the study of abortions, different from karyotyping such as FISH and comparative genomic hybridization (CGH) (Table IV). Jobanputra et al. (2002)Go have proposed multiplex interphase FISH on uncultured cells as a reliable screening for common aneuploidies in spontaneous miscarriages. They stated that maternal contamination can be detected in those cases of male abortions or when a numerical chromosome anomaly is revealed. Nevertheless, if the result by FISH were a normal female the fetal or maternal origin of the sample could not be determined. QF-PCR analysis of microsatellites can detect maternal contamination and determine the fetal or maternal origin of the sample by comparing the alleles from the fetal sample with those from maternal DNA. Another limitation of FISH consists of cross-hybridization of probes to different chromosomes, leading to wrong diagnoses. Since STR markers are specific for a selected region of a specific chromosome, QF-PCR avoids this problem. CGH provides a useful method to detect aneuploidies and can also detect partial gains or losses (>3 Mb), analysing the entire genome of the sample in a single experiment, but it does not detect changes in ploidy. CGH technique is also more laborious, time-consuming and expensive than QF-PCR.


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Table IV. Comparison between different techniques employed to the study of spontaneous abortions. 1: depending on the size of the rearrangement; 2: targeted regions using specific probes/primers; 3: referred to fungible costs only

 
Based on our experience, QF-PCR can be considered as a complementary and reliable tool to cytogenetic studies of spontaneous miscarriages: (i) It provides results in cases in which cytogenetic diagnosis fails. (ii) It allows the diagnosis of >83% of chromosomal anomalies commonly found in pregnancy losses. (iii) Despite the deterioration of the sample, the percentage of success using QF-PCR is higher than karyotyping. (iv) It permits determination of the fetal origin of the sample and allows us to discount maternal contamination. (v) Parental and meiotic origin of aneuploidy can both be determined. (vi) It is a rapid technique, which can take ~48 h from the reception of the sample to the time a diagnosis is established. (vii) A large number of samples can be processed simultaneously. (viii) It is a relatively low cost technique.


    Acknowledgements
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank collaborating volunteer couples. Dan Diego-Alvarez is supported by Fundación Conchita Rábago de Jiménez Díaz. This project is supported by a grant from FIS (PI 02/0068).


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
Adinolfi M and Cirigliano V (2000) Detection of fetal cells in transcervical samples using X22 marker. J Med Genet 37, E1.[CrossRef][Medline]

Adinolfi M, Pertl B and Sherlock J (1997) Rapid detection of aneuploidies by microsatellite and the quantitative fluorescent polymerase chain reaction. Prenat Diagn 17, 1299–1311.[CrossRef][ISI][Medline]

Alberman ED and Creasy MR (1977) Frequency of chromosomal abnormalities in miscarriages and perinatal deaths. J Med Genet 14, 313–315.[ISI][Medline]

Andonova S, Vazharova R, Dimitrova V, Mazneikova V, Toncheva D and Kremensky I (2004) Introduction of the QF-PCR analysis for the purposes of prenatal diagnosis in Bulgaria: estimation of applicability of 6 STR markers on chromosomes 21 and 18. Prenat Diagn 24, 202–208.[CrossRef][ISI][Medline]

Ban Z, Nagy B, Papp C, Toth-Pal E and Papp Z (2002) Rapid diagnosis of triploidy of maternal origin using fluorescent PCR and DNA fragment analysis in the third trimester of pregnancy. Prenat Diagn 22, 984–987.[CrossRef][ISI][Medline]

Bell KA, Van Deerlin PG, Feinberg RF, du Manoir S and Haddad BR (2001) Diagnosis of aneuploidy in archival, paraffin-embedded pregnancy-loss tissues by comparative genomic hybridization. Fertil Steril 75, 374–379.[CrossRef][ISI][Medline]

Bili C, Divane A, Apessos A, Konstantinos T, Apostolos A, Ioannis B, Periklis T and Florentin L (2002) Prenatal diagnosis of common aneuploidies using quantitative fluorescent PCR. Prenat Diagn 22, 360–365.[CrossRef][ISI][Medline]

Bond DJ and Chandley AC (1983) The origin and causes of aneuploidy in man. In Aneuploidy. Oxford Monographs on Medical Genetics. Oxford Medical Publications, pp. 55–76.

Carp H, Toder V, Aviram A, Daniely M, Mashiach S and Barkai G (2001) Karyotype of the abortus in recurrent miscarriage. Fertil Steril 75, 678–682.[CrossRef][ISI][Medline]

Carrera M (2001) Screening prenatal de aneuploidías: QF-PCR y FISH. Prog Diag Prenat 13, 262–266.

Carrera M, Ribas I, Torrents M, Méndez B, Serra B, de la Iglesia C, Boada M, Veiga A and Barri PN (1996) Abortos espontáneos de repetición y anomalías cromosómicas numéricas: ¿es el diagnóstico preimplantacional una alternativa diagnóstica? Prog Diagn Prenat 8, 342–347.

Cirigliano V, Sherlock J, Conway G, Quilter C, Rodeck C and Adinolfi M (1999) Rapid detection of chromosomes X and Y aneuploidies by quantitative fluorescent PCR. Prenat Diagn 19, 1099–1103.[CrossRef][ISI][Medline]

Dunn TM, Grunfeld L and Kardon NB (2001) Trisomy 1 in a clinically recognized IVF pregnancy. Am J Med Genet 99, 152–153.[CrossRef][ISI][Medline]

Fritz B, Aslan M, Kalscheuer V, Ramsing M, Saar K, Fuchs B and Rehder H (2001) Low incidence of UPD in spontaneous abortions beyond the 5th gestational week. Eur J Hum Genet 9, 910–916.[CrossRef][ISI][Medline]

Hanna JS, Shires P and Matile G (1997) Trisomy 1 in a clinically recognized pregnancy. Am J Med Genet 68, 98.[CrossRef][ISI][Medline]

Hassold T, Chen N, Funkhouser J, Jooss T, Manuel B, Matsuura J, Matsuyama A, Wilson C, Yamane JA and Jacobs PA (1980) A cytogenetic study of 1000 spontaneous abortions. Ann Hum Genet 44(Pt 2), 151–178.[ISI][Medline]

Hulten MA, Dhanjal S and Pertl B (2003) Rapid and simple prenatal diagnosis of common chromosome disorders: advantages and disadvantages of the molecular methods FISH and QF-PCR. Reproduction 126, 279–297.[Abstract/Free Full Text]

Jobanputra V, Sobrino A, Kinney A, Kline J and Warburton D (2002) Multiplex interphase FISH as a screen for common aneuploidies in spontaneous abortions. Hum Reprod 17, 1166–1170.[Abstract/Free Full Text]

Kotzot D (2004) Advanced parental age in maternal uniparental disomy (UPD): implications for the mechanism of formation. Eur J Hum Genet 12, 343–346.[CrossRef][ISI][Medline]

Lorda-Sanchez I, Trujillo MJ, Gomez-Garre P, de Alba MR, Gonzalez-Gonzalez C, Garcia-Hoyos M, Ayuso C and Ramos C (2003) Turner phenotype in a girl with a 45,X/46,XX/47,XX,+18 mosaicism. Am J Med Genet 15, 121A, 20–24.

Lorda-Sanchez I, Diego-Alvarez D, Ayuso C, Rodriguez de Alba M, Trujillo MJ and Ramos C (2005) Trisomy 2 due to a 3:1 segregation in an abortion studied by QF-PCR and CGH. Mol Hum Rep, Submitted.

Mannucci A, Sullivan KM, Ivanov PL and Gill P (1994) Forensic application of a rapid and quantitative DNA sex test by amplification of the X-Y homologous gene amelogenin. Int J Legal Med 106, 190–193.[CrossRef][ISI][Medline]

Miller JF, Williamson E, Glue J, Gordon YB, Grudzinskas JG and Sykes A (1980) Fetal loss after implantation. A prospective study. Lancet 13(8194), 554–556.

Moragón FJ and Ramos-Corrales C (1978) El huevo abortivo III. Estudio citogenético y consideración de su etiología. Acta Gin 32, 91–111.[ISI]

Nikcevic AV, Tunkel SA, Kuczmierczyk AR and Nicolaides KH (1999) Investigation of the cause of miscarriage and its influence on women's psychological distress. Br J Obstet Gynaecol 106, 808–813.[ISI][Medline]

Pertl B, Kopp S, Kroisel PM, Hausler M, Sherlock J, Winter R and Adinolfi M (1997) Quantitative fluorescence polymerase chain reaction for the rapid prenatal detection of common aneuploidies and fetal sex. Am J Obstet Gynecol 177, 899–906.[ISI][Medline]

Pertl B, Kopp S, Kroisel PM, Tului L, Brambati B and Adinolfi M (1999) Rapid detection of chromosome aneuploidies by quantitative fluorescence PCR: first application on 247 chorionic villus samples. J Med Genet 36, 300–303.[Abstract/Free Full Text]

Reddy KS (1997) Double trisomy in spontaneous abortions. Hum Genet 101, 339–345.[CrossRef][ISI][Medline]

Reddy KS (1999) Triple aneuploidy in spontaneous abortions. Clin Genet 56, 103–104.[CrossRef][ISI][Medline]

Robinson WP, Bernasconi F, Mutirangura A, Ledbetter DH, Langlois S, Malcolm S, Morris MA and Schinzel AA (1993) Nondisjunction of chromosome 15: origin and recombination. Am J Hum Genet 53, 740–751.[ISI][Medline]

Rubio C, Simon C, Vidal F, Rodrigo L, Pehlivan T, Remohi J and Pellicer A (2003) Chromosomal abnormalities and embryo development in recurrent miscarriage couples. Hum Reprod 18, 182–188.[Abstract/Free Full Text]

Sanchez JM, Franzi L, Collia F, De Diaz SL, Panal M and Dubner M (1999) Cytogenetic study of spontaneous abortions by transabdominal villus sampling and direct analysis of villi. Prenat Diagn 19, 601–603.[CrossRef][ISI][Medline]

Santalo J, Catala V and Badenas J (1987) Chromosomal abnormalities and IVF. In Egozcue J (ed.) Cellular Aspects of In Vitro Fertilization. Cell Biology Reviews. Springer, Leiola, pp. 63–72.

Shi SR, Datar R, Liu C, Wu L, Zhang Z, Cote RJ and Taylor CR (2002) DNA extraction from archival formalin-fixed, paraffin-embedded tissues: heat-induced retrieval in alkaline solution. Histochem Cell Biol 122, 211–218.

Silvestre E, Cusí V, Antich J and Caballín MR (2002) Protocolo para el estudio citogenético de los abortos espontáneos. Prog Diagn Prenat 14, 146–151.

Stephenson MD, Awartani KA and Robinson WP (2002) Cytogenetic analysis of miscarriages from couples with recurrent miscarriage: a case-control study. Hum Reprod 17, 446–451.[Abstract/Free Full Text]

Warburton D, Stein Z, Kline J, Susser M, et al. (1980) Chromosome abnormalities in spontaneous abortion: data from the New York City study. In Porter IH and Hook EB (eds) Human Embryonic and Fetal Death. Academic Press, New York, pp. 261–287.

World Medical Organization (1996) Declaration of Helsinki 1964. Br Med J 313, 1448–1449.[Free Full Text]

Submitted on November 22, 2004; resubmitted on January 7, 2005; accepted on January 12, 2005.