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BRIEF REPORT

Analysis of Cell-free Fetal DNA in Plasma and Serum of Pregnant Women

T.V. Zolotukhina, N.V. Shilova and E. Yu Voskoboeva

Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russia

Correspondence to: T.V. Zolotukhina, Research Centre for Medical Genetics, Prenatal Diagnosis, Russian Academy of Medical Sciences, Moskvorechje 1, Moscow, 115478, Russia. E-mail.tvz{at}medgen.ru


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 Summary
 Introduction
 Patients
 Cytogenetics
 DNA Extraction from Plasma...
 PCR Analysis
 Literature Cited
 
Sixty blood samples from pregnant women during gestational weeks 9–28 were investigated. Cell-free fetal DNA was extracted from maternal plasma or serum to be detected by nested PCR for determination of fetal gender. The SRY gene as a marker for fetal Y chromosome was detected in 34/36 women carrying a male fetus. In 3/24 women carrying female fetuses, the SRY sequence was also detected. Overall, fetal sex was correctly predicted in 91.7% of the cases. Therefore, the new, non-invasive method of prenatal diagnosis of fetal gender for women at risk of producing children with X-linked disorders is reliable, secure, and can substantially reduce invasive prenatal tests.

(J Histochem Cytochem 53:297–299, 2005)

Key Words: fetal DNA • maternal serum and plasma • SRY • nested PCR


    Introduction
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 Summary
 Introduction
 Patients
 Cytogenetics
 DNA Extraction from Plasma...
 PCR Analysis
 Literature Cited
 
DURING THE LAST few years, non-invasive methods for prenatal diagnosis have attracted the attention of clinicians and researchers. Modern ultrasonography or measuring the level of maternal serum markers is routinely used as the primary screening test for developmental malformations. Presently, invasive procedures based on the genetic analysis of fetal chromosomes or DNA from chorionic villus samples or amniocytes are performed. However, the application of these invasive procedures increases the risk of fetal loss (Elias and Simpson 1993Go; Jackson and Wapner 1993Go). Recent interest in nucleic acids present in maternal plasma has opened many new promising possibilities for the non-invasive detection of fetal conditions, including rhesus D status (Zhong et al. 2000Go) and sex-linked diseases (Lo et al. 1998Go). Cell-free fetal DNA in maternal plasma can be used as a diagnostic tool for diseases of pregnancy such as preeclampsia or preterm labor, or for fetal anomalies such as aneuploidies (Lo et al. 1999Go; Lee et al. 2002Go).

The aim of this investigation was the development of an efficient nested PCR-based method for the detection of Y-chromosome-specific sequence (SRY) gene in fetal DNA extracted from plasma or serum of pregnant women.


    Patients
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 Patients
 Cytogenetics
 DNA Extraction from Plasma...
 PCR Analysis
 Literature Cited
 
Sixty pregnant women at a gestational age ranging from 9 to 28 weeks and undergoing an invasive prenatal diagnostic procedure for reasons such as advanced maternal age, maternal serum, and ultrasound screening were recruited for this study.


    Cytogenetics
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 Patients
 Cytogenetics
 DNA Extraction from Plasma...
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Karyotypes of the fetuses during the first trimester were determined in chorionic villus samples using semi-direct methods. Karyotypes of the fetuses during the second or third trimester were determined by conventional banding cytogenetics method of cord blood samples obtained by cordocentesis. All fetal karyotypes were normal.


    DNA Extraction from Plasma or Serum Samples
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 Patients
 Cytogenetics
 DNA Extraction from Plasma...
 PCR Analysis
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Blood samples from the 60 women were centrifuged at 1850 x g for 10 min and the supernatants were collected and stored at –20C. DNA was extracted from 500 µl plasma or serum using the Diatom DNA Kit (IsoGene; Moscow, Russia) according to the manufacturer's instructions. DNA was eluted in 50 µl of water and 5–10 µl was used as a template for the PCR reaction.


    PCR Analysis
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 Patients
 Cytogenetics
 DNA Extraction from Plasma...
 PCR Analysis
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Two rounds of PCR were carried out to amplify the fragment of the SRY gene. The first PCR analysis was performed in a total volume of 50 µl. The PCR mixture contained 5–10 µl extracted DNA, 5 µl PCR buffer, 250 µM of dNTP mixture, 20 pM "external" SRY-specific oligonucleotide (Y1 5'-GTG TCC TCT CGT TTT GTG AC-3', Y2 5'-GTA ATC ATC GCT GTT GAA TAC-3') and 0.5 U Taq-polymerase (Sileks; Moscow, Russia). The PCR conditions were preheating at 95C for 3 min, then 30 cycles of 94C for 30 sec, 57C for 30 sec, 72C for 3 min, and then 72C for 3 min. The size of the PCR fragment was 369 bp. The second round was also performed in a total volume of 50 µl. The PCR mixture contained 5–10 µl of the product of the first PCR round, 5 µl PCR buffer, 250 µM of dNTP mixture, 20 pM "internal" SRY-specific oligonucleotide (Y3 5'-TGG CGA TTA AGT CAA ATT CGC-3', Y4 5'-CCT AGT ACC CTG ACA ATG TAT-3') and 0.5 U Taq-polymerase (Sileks). PCR conditions were preheating at 95C for 3 min, then 30 cycles of 94C for 30 sec, 57C for 30 sec, 72C for 1 min, and then 72C for 3 min. The size of the final PCR fragment was 130 bp.

The control PCR for a fragment of amylo-1,6-glucosidase gene (AGL) was carried out as standard assay in a total volume of 50 µl. The part of the AGL gene that is located on 1p21 was amplified. Oligonucleotide primers GL3.5 5'-CCG AGC TTA TTC TGT AGA AG-3' and GL3.6 5'-ACA TGC TCC TGA TGA CTT AC-3' were designed to amplify exons 33–34 of AGL. The size of the PCR product was 469 bp.

Nucleotide sequences for the SRY gene and AGL gene were obtained from the Gene Data Bank.

PCR products were analyzed in 2% agarose gel containing ethidium bromide.

PCR fragment of amylo-1,6-glucosidase gene was detected in all samples investigated, confirming the presence of DNA in the samples. Cytogenetic analysis revealed that 24 fetuses had normal female karyotype—46,XX, and 36 fetuses had normal male karyotype—46,XY.

Specificity of PCR of SRY sequence was 87.5%: specific PCR fragment for Y chromosome was amplified in three DNA samples extracted from plasma or serum of 24 women carrying female fetuses. Thus, false-positive results were 5% (3/60 samples investigated). The sensitivity of the method was 94.5%: specific PCR fragment for Y chromosome was not amplified in two DNA samples extracted from plasma or serum of 36 women carrying male fetuses. Thus, false-negative results were 3.3% (2/60 samples investigated). Detection rate of this method (the coincidence of results of cytogenetic analysis with PCR results) was 91.7% (55/60 samples investigated) (Table 1).


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Table 1

Results of total and fetal DNA detection in maternal plasma (P) and serum (S)

 
It is well known that fetal cells circulate in maternal plasma of pregnant women. It has been shown that fetal cells crossing into maternal plasma are composed of different cell types such as erythroblasts, trophoblasts, and lymphocytes. Fetal white blood cells can even accumulate in maternal spleen or liver and remain there for a long time after labor (Bianchi et al. 1996Go). Thus, using fetal white blood cells for diagnosis may cause false-positive results. Only fetal nucleated red blood cells (erythroblasts) rapidly disappear after delivery and, consequently, can be correlated with the current pregnancy.

Some authors have reported successful applications of fetal cells for prenatal diagnosis of chromosome aneuploidy (Bianchi et al. 1997Go). However, low concentration of fetal cells in the maternal circulation (on average, one fetal cell per 106 maternal cells) (Simpson and Elias 1994Go) combined with a low success rate in sorting of these cells (e.g., by monoclonal antibodies) hampers detection and isolation of fetal cells. Thus, attempts have been tested to detect free fetal nucleic acids instead of fetal cells in maternal blood because fetal cells are exposed to apoptosis (Van Wijk et al. 2000Go). As maternal cells also are destroyed, the blood of pregnant women contains different DNA fragments (=cell-free DNA) of fetal and maternal origin. The presence of nucleic acids in human plasma has already been described (Mandel and Metais 1948Go). Prior to 1997, it had not been demonstrated that Y-chromosome-specific sequences could be detected by PCR in plasma or serum of pregnant women carrying male fetuses (Lo et al. 1997Go).

To detect small amounts of fetal DNA, real-time QF-PCR is the method of choice (Zhong et al. 2000Go). In cases of known X-linked disease, the possibility to screen for Y-chromosome gene presence in maternal blood allows women to avoid invasive procedures (Rijnders et al. 2004Go).

Two rounds of PCR were performed using two pairs of primers for Y-chromosome-specific sequence in the present approach (SRY gene region). The efficiency of nested PCR assay used was 91.7%, correlating well with published data (Lo 2000Go). PCR fragment for Y chromosome was amplified in three DNA samples extracted from plasma or serum of 24 women carrying female fetuses. Two of the three women were primagravida. In these cases it was probable that there was DNA contamination of samples. A third woman had earlier termination of her pregnancy, and in this case SRY-positive result might be due to a probable previous male pregnancy. It may be that the lymphocytes from this previous pregnancy are the source of cell-free DNA. The persistence of fetal DNA in maternal plasma long after delivery has been reported (Invernizzi et al. 2002Go). A long presence of entire fetal cells in maternal circulation is well known (Bianchi et al. 1996Go), and it is likely that fetal lymphocytes being degraded by plasma nucleases are followed by the appearance of cell-free DNA.

It is likely that the use of multiplex PCR assay with few Y-chromosome-specific gene markers will allow increased sensitivity and specificity of fetal DNA detection in maternal serum and/or plasma.

In summary, the presence of fetal cell-free DNA in the maternal circulation is a good, low-cost approach for the future development of novel strategies that are expected to provide non-invasive techniques for early prenatal diagnosis.


    Footnotes
 
Presented in part at the 14th Workshop on Fetal Cells and Fetal DNA: Recent Progress in Molecular Genetic and Cytogenetic Investigations for Early Prenatal and Postnatal Diagnosis, Friedrich-Schiller-University, Jena, Germany, April 17–18, 2004.

Received for publication May 17, 2004; accepted November 12, 2004


    Literature Cited
 Top
 Summary
 Introduction
 Patients
 Cytogenetics
 DNA Extraction from Plasma...
 PCR Analysis
 Literature Cited
 

Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, DeMaria MA (1996) Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci USA 93:705–708[Abstract/Free Full Text]

Bianchi DW, Williams JM, Sullivan LM, Hanson FW, Klinger KW, Shuber AP (1997) PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet 61:822–829[Medline]

Elias S, Simpson JL (1993) Amniocentesis. In Simpson JL, Elias S, eds. Essentials of Prenatal Diagnosis. New York, Churchill Livingstone, 27–44

Invernizzi P, Biondi ML, Battezzati PM, Perego F, Selmi C, Cecchini F, Podda M, et al. (2002) Presence of fetal DNA in maternal plasma decades after pregnancy. Hum Genet 110:587–591[CrossRef][Medline]

Jackson L, Wapner RJ (1993) Chorionic villus sampling. In Simpson JL, Elias S, eds. Essentials of Prenatal Diagnosis. New York, Churchill Livingstone, 45–61

Lee T, Le Shane ES, Messerlian G, Canick JA, Farina A, Heber W (2002) Down syndrome and cell-free fetal DNA in archived maternal serum. Am J Obstet Gynecol 187:1217–1221[CrossRef][Medline]

Lo YM (2000) Fetal DNA in maternal plasma: biology and diagnostic applications. Clin Chem 46:1903–1906[Abstract/Free Full Text]

Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS (1997) Presence of fetal DNA in maternal plasma and serum. Lancet 350:485–487[CrossRef][Medline]

Lo YM, Leung TN, Tein MS, Sargent IL, Zhang J, Lau TK (1999) Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 45:184–188[Abstract/Free Full Text]

Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM (1998) Quantitative analysis of fetal DNA in maternal plasma and serum: implications for non-invasive prenatal diagnosis. Am J Hum Genet 62:768–775[CrossRef][Medline]

Mandel P, Metais P (1948) Les acides nucleiques du plasma sanguin chez l'homme. CR Acad Sci Paris 142:241–243

Rijnders RJ, Christiaens GC, Bossers B, van der Smagt JJ, van der Shoot CE, de Haas M (2004) Clinical applications of cell-free fetal DNA from maternal plasma. Obstet Gynecol 103:157–164[Abstract/Free Full Text]

Simpson JL, Elias S (1994) Isolating fetal cells in maternal circulation for prenatal diagnosis. Prenat Diagn 14:1229–1242[Medline]

Van Wijk IJ, De Hoon A, Jurhawan R, Tjoa ML, Griffioen S, Mulders MA (2000) Detection of apoptotic fetal cells in plasma of pregnant women. Clin Chem 46:729–731[Free Full Text]

Zhong XY, Holzgreve W, Hahn S (2000) Detection of fetal Rhesus D and sex using fetal DNA from maternal plasma by multiplex polymerase chain reaction. BJOG 107:766–769[Medline]





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