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1
Department of Obstetrics and Gynecology, Hiroshima University School of Medicine, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
a Author for correspondence. Fax 81-82-257-5264;
honda{at}mcai.med.hiroshima-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Peripheral blood samples were obtained from 61 pregnant women at 10–17 weeks of gestation before amniocentesis. DNA was extracted from 800 µL of each plasma or serum sample. To detect the Y-chromosome-specific sequences DYS14 and DYZ3 in the maternal plasma and serum, 40 cycles of PCR were carried out for each DNA extract. The PCR products were analyzed by 2.5% agarose gel electrophoresis and ethidium bromide staining, and the results were compared with the results of the cytogenetic analyses of amniocentesis.
Results: Cytogenetic analysis of amniocentesis revealed that 31 pregnant women had a male fetus and the remaining 30 pregnant women had a female fetus. Both DYS14 and DYZ3 were detected in 27 of the 31 plasma samples obtained from pregnant women carrying a male fetus and in all of 31 serum samples obtained from the same women. Neither DYS14 nor DYZ3 was detected in either the plasma or serum samples obtained from any of the 30 pregnant women carrying a female fetus.
Conclusion: PCR analysis of maternal serum can be used to diagnose fetal gender.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lo et al. (4) showed the presence of fetal DNA in maternal plasma and serum. These researchers also showed that fetal DNA is present in the total DNA in maternal plasma and serum at concentrations much higher than the number of NRBCs found in the maternal blood cells in whole blood (5). Lo et al. (5) documented that during early pregnancy, fetal DNA concentrations were, on average, 3.4% of the total DNA in the maternal plasma and 0.13% in serum.
We diagnosed fetal gender using conventional PCR on DNA solutions extracted from the maternal plasma and serum. This method for diagnosing fetal gender can be used as a pre-test to determine whether invasive prenatal diagnoses, such as amniocentesis and chorionic villi sampling, should be performed on a fetus having a risk of X-linked recessive inheritance. Our results also indicated that we can accurately detect fetal DNA; thus, fetal DNA in maternal plasma and serum may be considered a new material for noninvasive prenatal diagnosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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Peripheral blood samples obtained from five healthy men and five nonpregnant women were used to determine the PCR sensitivity and as negative controls, respectively. In each case, 4 mL of peripheral blood was collected into an EDTA-containing Vacutainer Tube for plasma separation, and 6 mL of peripheral blood was collected into a Vacutainer Tube containing no anticoagulant for serum separation. The blood samples were centrifuged at 3000g, and the plasma and serum were carefully removed from their respective tubes and transferred into plain polypropylene tubes. The plasma and serum samples were again centrifuged at 3000g, and these recentrifuged plasma and serum samples were transferred into fresh polypropylene tubes. The samples were stored at -20 °C until further processing.
dna extraction from plasma and serum samples
DNA was extracted from the plasma and serum samples by the QIAamp
DNA Blood Mini method (Qiagen) according to the "blood
and body fluid protocol" with minor modifications. We extracted the
DNA from 800 µL of plasma and serum in each case. The volume of the
extracted DNA solution was usually 200 µL, and the extracted DNA
solution was then concentrated to a volume of 20 µL by ethanol
precipitation. Each DNA solution extracted from the healthy men was
serially diluted from 1:10 to 1:10 000.
pcr
The Y-chromosome-specific sequences DYS14 and
DYZ3 were amplified for the detection of fetal DNA in
maternal plasma and serum. We used the primer sets Y1·7/Y1·8
(6) and Y1·1/Y2·2 (7) to amplify
DYS14 and DYZ3, respectively. For
DYS14, PCR amplification was performed in a total volume of
25 µL containing extracted DNA, 200 µM dNTPs, 20 pmol of each
primer (Y1·7/Y1·8), 1x Taq polymerase buffer (containing 1.5 mM
MgCl2), and 0.625 U of Taq polymerase (Takara
Biomedicals); the PCR product was 198 bp. For DYZ3, PCR
amplification was performed in a total volume of 25 µL containing
extracted DNA, 200 µM dNTPs, 12.5 pmol of each primer
(Y1·1/Y2·2), 1x Taq polymerase buffer (containing 1.5 mM
MgCl2), and 0.75 U of Taq polymerase; the PCR
product was 172 bp. The thermal cycling for DYS14 was as
follows: denaturation at 94 °C for 5 min, followed by 40 cycles of
94 °C for 30 s, 57 °C for 1 min, and 72 °C for 2 min,
with final incubation at 72 °C for 7 min. For DYZ3, the
thermal cycling began with denaturation at 94 °C for 5 min, followed
by 40 cycles of 94 °C for 40 s, 55 °C for 30 s, and
72 °C for 1 min, with final incubation at 72 °C for 7 min. The
PCR amplification products were separated by 2.5% agarose gel
electrophoresis and visualized by exposure to ultraviolet light after
ethidium bromide staining.
The samples were coded, and the PCR analysis was always performed before cytogenetic analysis of cultured amniocytes. The PCR results were compared with the fetal gender revealed by the cytogenetic analysis. The individuals performing or interpreting the cytogenetic analysis did not know the PCR results. Each DNA solution extracted from the plasma and serum of pregnant women, healthy men, and nonpregnant women was tested three times in different PCR sessions. Any samples that tested positive at least twice among the three amplifications were considered positive. If the result for a DNA extract from either the plasma or serum of the pregnant women, the nonpregnant women, or the healthy men was negative, the DNA extracts from the plasma and its paired serum sample or the serum and its paired plasma sample were further amplified for an autosomal locus, glyceraldehyde-3-phosphate (GAPDH), to assess the presence of DNA.
anticontamination measures
Great care was taken to prevent PCR contamination (8).
Aerosol-resistant pipette tips were used for all liquids. Separate
areas were used for the extraction of DNA, the preparation of
amplification reactions, the carrying out of amplification reactions,
and the detection of the PCR products, and all manipulations except the
detection were carried out in a laminar flow hood.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Neither DYS14 nor DYZ3 was detected in
the DNA extracted from any of the five nonpregnant women.
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Cytogenetic analysis of cultured amniocytes revealed that 31 of the
pregnant women were carrying a male fetus and the remaining 30 pregnant
women were carrying a female fetus (Table 1
). Both DYS14 and DYZ3 were detected in
all three PCR analyses in the plasma samples from 27 of the 31
male-bearing pregnant women. Of the four remaining male-bearing
pregnant women, the DYS14 sequence but not the
DYZ3 sequence was detected one time in one woman, and
neither the DYS14 nor the DYZ3 sequence was
detected in the plasma of the remaining three women. In the serum
samples from the 31 male-bearing pregnant women, both the
DYS14 and DYZ3 sequences were detected in all
three PCR analyses in all of the subjects. Thus, of the 31 women
identified as carrying a male fetus by cytogenetic analysis, our
plasma-based method indicated that 27 were carrying a male fetus,
whereas our serum-based method indicated that all 31 were carrying a
male fetus (Fig. 3
and Table 2
). The four male-bearing pregnant women for whom the plasma
samples had been negative had had their blood collected early in
the gestational period (11–13 weeks of gestation). Neither the
DYS14 nor the DYZ3 sequence was detected in
either the plasma or serum of any of the 30 female-bearing pregnant
women. We were thus able to determine by fetal gender diagnosis using
maternal plasma or serum that all 30 of these pregnant women were
carrying a female fetus (Fig. 3
and Table 2
).
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The GAPDH sequence was detected in the undiluted DNA
solutions from the plasma and serum of the 4 male-bearing pregnant
women for whom the gender of the male fetus could not be determined
from maternal plasma, the 30 female-bearing pregnant women, and the 5
nonpregnant women (Table 3
). In the 1:10 dilutions of DNA from plasma, the
GAPDH sequence was detected in 28 of the female-bearing
pregnant women and the 1 male-bearing pregnant woman in whom the
DYS14 sequence was detected in only one PCR analysis and the
DYZ3 sequence was not detected; however, in the 1:10
dilutions of DNA from serum, the GAPDH sequence was detected
in all 30 of the female-bearing pregnant women and all 4 of the
male-bearing pregnant women for whom the gender of the male fetus could
not be determined from maternal plasma. The GAPDH
sequence was also detected the 1:10 dilutions of DNA from both the
plasma and serum of the five nonpregnant women. However,
GAPDH was not detected in the plasma samples of three of the
four male-bearing pregnant women for whom the gender of the male fetus
could not be determined from maternal plasma, and in two female-bearing
pregnant women (Table 3
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The accuracy of our method for diagnosing fetal gender was
significantly higher when we used maternal serum rather than maternal
plasma (P = 0.0387, 
2
analysis). Because there theoretically is no difference between the
concentration of fetal DNA in maternal plasma and serum, the difference
in the sensitivity for detecting fetal DNA between plasma and serum
might reflect (a) a DNA extraction efficiency that is higher
for serum than for plasma, or (b) a fetal DNA concentration
in maternal plasma at early gestational ages that is below the
detection limits for the DYS14 and DYZ3
sequences.
Regarding the efficiency of DNA extraction, to estimate extraction efficiency, we analyzed the concentration of GAPDH, a housekeeping gene. In three of four male-bearing pregnant women for whom the plasma results were incorrect, GAPDH was not detected in the 1:10 dilutions of DNA extracted from their plasma samples but was detected in the 1:10 dilution of DNA extracted from their serum samples. This result indicates a lower efficiency of DNA extraction from plasma than from serum. In the present study, this lower extraction efficiency might be attributable to cryoprecipitates in the plasma. The manufacturer’s manual for the QIAamp DNA Blood Mini method indicates that cryoprecipitates may interfere with DNA extraction (14).
Regarding the second possibility, i.e., that low concentrations of fetal DNA in maternal plasma may lead to incorrect diagnosis of fetal gender, the maternal plasma samples in our study for which the gender of the male fetus was incorrectly diagnosed were collected at an early gestational age (11–13 weeks). Because the concentration of fetal DNA in maternal plasma or serum increases with gestational age (5), incorrect diagnosis of fetal gender based on maternal plasma might be attributable to the lower concentration of fetal DNA at early gestational ages compared with late gestational ages. However, fetal gender was diagnosed correctly from serum samples of pregnant women for whom the tests results from plasma had been incorrect; therefore, the higher sensitivity of our serum-based method for detecting male fetuses depends mainly on the efficiency of DNA extraction.
Our results differ from those of Lo et al. (4) and
Houfflin-Debarge et al. (15). Lo et al. (4) found
no significant difference in accuracy for diagnosing fetal gender
between their maternal plasma-based method and their maternal
serum-based method (the sensitivity of their plasma-based method for
male-bearing pregnancies was 80%, and that of their serum-based method
was 70%; P = 0.371, 2
analysis). Houfflin-Debarge et al. (15) found that the
accuracy of their method for fetal gender diagnosis was significantly
higher when they used maternal plasma rather than maternal serum (the
sensitivity of their plasma-based method for diagnosing male-bearing
pregnancies was 89%, and that of their serum-based method was 46%;
P <0.001, 2 analysis). Because the
PCR protocol of our method was not essentially different from the
methods of Lo et al. (4) and Houfflin-Debarge et al.
(15), this difference could be a result of differences in
the methods used for DNA extraction. Our method uses the QIAamp Blood
reagent set, whereas Lo et al. (4) and Houfflin-Debarge et
al. (15) used methods based on heat extraction.
The detection of viral DNA in human plasma or serum is similar to that of fetal DNA in maternal plasma or serum in regard to "foreign DNA" in host plasma or serum. Hamprecht et al. (16) indicated that viral DNA in serum could be detected with greater sensitivity than could viral DNA in plasma. Patel et al. (17) showed that the sensitivity for detecting viral DNA was similar for serum and plasma. However, Boom et al. (18) found that viral DNA could be detected in serum with less sensitivity than viral DNA in plasma. Because these three reports differed in their methods of DNA extraction, the lack of consistent superiority of plasma or serum in terms of sensitivity for detecting viral or fetal DNA suggests that the extraction efficiencies for plasma and serum might differ according to the DNA extraction method used. According to Dixon et al. (19), the QIAamp DNA Blood reagent set is one of the best methods for extracting DNA from plasma or serum. Because this DNA extraction method is more efficient for serum than for plasma, we believe that maternal serum is more suitable than plasma for fetal gender diagnosis.
Among Y-chromosome-specific sequences, including DYZ1, sex-determining region Y (SRY), zinc finger protein, Y-encoded (ZFY), and the amelogenin-like gene on Y chromosome (AMELY), we chose the DYS14 and DYZ3 sequences for diagnosis of fetal gender because DYS14 has been used frequently in the past, and DYZ3, which has tandem repeat units, was suspected to be highly sensitive in the detection of male fetal DNA. The detection limit for the DYS14 sequence was lower than that for DYZ3 in our PCR conditions; therefore, the sensitivity of DYS14 for the detection of male fetal DNA is potentially higher than that of DYZ3. However, in the present study, the sensitivity of the DYS14 sequence for the detection of fetal male DNA was the same as that of the DYZ3 sequence in both maternal plasma and serum at 10–17 weeks of gestation. These results depend on the concentration of male fetal DNA being higher than the detection limit of DYZ3 in all of the cases. Because the concentration of male fetal DNA usually increases with gestational age (5), it is likely that the sensitivity of the DYS14 sequence for detecting fetal male DNA may be higher than that of the DYZ3 sequence at an early gestational age.
Because we successfully used maternal serum to diagnose fetal gender, our diagnostic method may be useful in place of invasive prenatal diagnostic methods such as amniocentesis and chorionic villi sampling in cases of X-linked recessive inheritance. If a fetus has a risk of X-linked recessive inheritance and is found to be female by our maternal serum-based method, use of an invasive prenatal diagnostic method may not be necessary. Our success in diagnosing fetal gender shows that we can easily detect fetal DNA extracted from plasma or serum. Therefore, we can also use fetal DNA obtained from maternal serum in the prenatal diagnosis of other paternally inherited autosomal-dominant diseases, of diseases caused by germline mutations, and of fetal RhD status (20). With quantitative analysis of fetal DNA, we may also assess the prognosis for pregnancies in which abnormal conditions such as preeclampsia and threatened premature delivery are present, as well as the presence of fetal chromosomal abnormalities (21)(22)(23). In the near future, maternal plasma and serum may play an important and powerful role in noninvasive prenatal diagnosis.
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Acknowledgments |
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References |
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The following articles in journals at HighWire Press have cited this article:
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  C. F. Wright and H. Burton The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis Hum. Reprod. Update, January 1, 2009; 15(1): 139 - 151. [Abstract] [Full Text] [PDF]
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 I. Stanghellini, R. Bertorelli, L. Capone, V. Mazza, C. Neri, A. Percesepe, and A. Forabosco Quantitation of fetal DNA in maternal serum during the first trimester of pregnancy by the use of a DAZ repetitive probe Mol. Hum. Reprod., September 1, 2006; 12(9): 587 - 591. [Abstract] [Full Text] [PDF]
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 Y.M. D. Lo Recent Advances in Fetal Nucleic Acids in Maternal Plasma J. Histochem. Cytochem., March 1, 2005; 53(3): 293 - 296. [Abstract] [Full Text] [PDF]
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 R. J.P. Rijnders, G. C.M.L. Christiaens, A. A. Soussan, C. E. van der Schoot, P. Invernizzi, G. Simoni, and M. Podda Cell-Free Fetal DNA Is Not Present in Plasma of Nonpregnant Mothers * Authors of the article cited above respond: Clin. Chem., March 1, 2004; 50(3): 679 - 681. [Full Text] [PDF]
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O. Samura, N. Miharu, M. Hyodo, H. Honda, Y. Ohashi, N. Honda, T. Hara, and K. Ohama Cell-free Fetal DNA in Maternal Circulation after Amniocentesis Clin. Chem., July 1, 2003; 49(7): 1193 - 1195. [Full Text] [PDF]
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 G. Tachdjian, N. Frydman, F. Audibert, P. Ray, V. Kerbrat, P. Ernault, R. Frydman, and J.-M. Costa Clinical applications of fetal sex determination in maternal blood in a preimplantation genetic diagnosis centre Hum. Reprod., August 1, 2002; 17(8): 2183 - 2186. [Abstract] [Full Text] [PDF]
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D. W. Swinkels, J. B. de Kok, J. C.M. Hendriks, E. Wiegerinck, P. L.M. Zusterzeel, and E. A.P. Steegers Hemolysis, Elevated Liver Enzymes, and Low Platelet Count (HELLP) Syndrome as a Complication of Preeclampsia in Pregnant Women Increases the Amount of Cell-free Fetal and Maternal DNA in Maternal Plasma and Serum Clin. Chem., April 1, 2002; 48(4): 650 - 653. [Full Text] [PDF]
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J.-M. Costa and P. Ernault Automated Assay for Fetal DNA Analysis in Maternal Serum Clin. Chem., April 1, 2002; 48(4): 679 - 680. [Full Text] [PDF]
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Y. Y.N. Lui, K.-W. Chik, R. W.K. Chiu, C.-Y. Ho, C. W.K. Lam, and Y.M. D. Lo Predominant Hematopoietic Origin of Cell-free DNA in Plasma and Serum after Sex-mismatched Bone Marrow Transplantation Clin. Chem., March 1, 2002; 48(3): 421 - 427. [Abstract] [Full Text] [PDF]
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Y. Ohashi, N. Miharu, H. Honda, O. Samura, and K. Ohama Correlation of Fetal DNA and Human Chorionic Gonadotropin Concentrations in Second-Trimester Maternal Serum Clin. Chem., February 1, 2002; 48(2): 386 - 388. [Full Text] [PDF]
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 G. Vona, C. Beroud, A. Benachi, A. Quenette, J. P. Bonnefont, S. Romana, Y. Dumez, B. Lacour, and P. Paterlini-Brechot Enrichment, Immunomorphological, and Genetic Characterization of Fetal Cells Circulating in Maternal Blood Am. J. Pathol., January 1, 2002; 160(1): 51 - 58. [Abstract] [Full Text] [PDF]
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