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Fetal DNA in maternal plasma in twin pregnancies.

Twin pregnancies have increased in recent years as assisted reproductive technologies have been adapted. The maternal age of women undergoing assisted reproduction is generally higher than that of women with spontaneous pregnancies. This also implies a higher risk of fetal aneuploidies. Furthermore, twin pregnancies are known to have a higher risk of developing complications such as preeclampsia, intrauterine growth retardation, and pre-term labor [for a review, see Ref. (1)].

Of note is that women who have been trying for years to become pregnant and have finally undergone cumbersome therapeutic interventions could be particularly reluctant to expose their pregnancy to any invasive prenatal diagnostic procedure. Thus, noninvasive screening or diagnostic tests could be particularly welcome in this subset of pregnant women.

Noncellular fetal DNA circulating in maternal plasma may represent a suitable source of fetal genetic material that can be obtained noninvasively. Several studies have shown a significantly higher concentration of fetal DNA in maternal plasma in some fetal aneuploidies and placental pathologies (2-6). Nevertheless, existing data about fetal DNA concentrations in maternal blood under physiologic and pathologic conditions refer only to singleton pregnancies, and reference values for twins are still missing.

The origin of the fetal DNA released into maternal plasma is still unclear. Although there is evidence that some of the cell-free fetal DNA in the maternal plasma is derived from blood elements, several authors favor the hypothesis that the majority is derived from the placenta (7,8). Interestingly, twin pregnancies may present with one or two placentas, and although dizygotic twins must have two placentas, monozygotic twins can be either mono- or bichorionic. It has been reported that monozygotic twins exhibit smaller uteroplacental junctional areas than do dizygotic twins (9).

The influence of the presence of more than one fetus and placenta on fetal DNA content in maternal plasma has never been reported; we therefore aimed to quantify fetal DNA in different subsets of twins.

We quantified fetal DNA by real-time PCR of SRY (10) in maternal plasma in a cohort of 73 multiple pregnancies (68 twin and 5 triplet), among which 55 carried at least one male fetus (53 twins and 2 triplets). Of the 55 male-bearing multiple pregnancies, 29 women carried one male and one female fetus (MF), 24 carried two male fetuses (MM), and the women carrying the 2 triplet fetuses had only one male baby (MFF). The MM twin pregnancies included 17 bichorionic and 7 monochorionic pregnancies. Samples were collected from the 10th week of gestation onward, mainly between 10 and 21 weeks and between 25 and 38 weeks. Placental weight measured at birth was available for 17 of 46 bichorial twin pregnancies (mean, 964 g; median, 985 g; range, 600-1270 g) and for 5 of 7 monochorial twin pregnancies (mean, 953 g; median, 735; range, 600-1530 g).

All of the women enrolled in this study gave written informed consent, and the study was approved by the Institutional Ethical Committee.

We collected 5 mL of maternal peripheral blood in EDTA. After centrifugation at 1600 g, plasma was carefully removed and recentrifuged at full speed (16 000g) on a microcentrifuge (11). DNA extraction was carried out with the QIAamp Blood Kit (Qiagen) protocol; 400 [micro]L of plasma per column was used for DNA extraction according to the instructions from the manufacturer (10).

Fetal SRY-specific and (3-globin (for total DNA) DNA quantification was carried out by a real-time TagMan method (10). Real-time PCR analysis was performed by use of a 7700 Sequence Detector (PE Applied Biosystems) (10). Reactions were carried out in 50-[micro]L volumes with the TagMan PCR Core Reagent Kit (PE Applied Biosystems) with 10 [micro]L of extracted plasma DNA. The conversion factor of 6.6 pg of DNA/cell was used for expression of results as genome-equivalents (GE). Thermal cycling for both SRY and [beta]-globin included 40 cycles of 95[degrees]C for 15 s and 60[degrees]C for 1 min (10). To reduce sample-to-sample variability, for all samples plasma separation was performed immediately after blood collection. All samples were stored at -20[degrees]C and analyzed within 2 weeks after sampling. Thus, exactly the same analytical conditions have been applied to both singleton and multiple pregnancies.

Because a dilutional effect in maternal plasma volume in multiple gestations has been reported (12), we measured the [beta]-globin concentration to determine how total DNA is affected by the presence of multiple fetuses. We analyzed 19 multiple pregnancies vs 81 singleton pregnancies, matched for gestational age, at a maximum of 5 singleton pregnancies per multiple pregnancy. Samples were collected from 10 to 35 weeks of gestation. The mean, median, and range were 1484, 1070, and 246-7064 GE/mL of maternal plasma, respectively, in singleton pregnancies and 2307, 2528, and 549-4346 GE/mL, respectively, in multiple pregnancies, suggesting that total DNA in maternal plasma was not diluted by the presence of multiple fetuses.

The mean, median, and range of fetal SRY DNA concentration were 39.3, 19.4, and 0-286 GE /mL of plasma in 29 twin MF pregnancies and 111.5, 60.2. and 1.5-580 GE/mL in 24 twin MM pregnancies. Of five triplet pregnancies, two bore a male fetus (MFF); the fetal DNA concentrations were 40.3 and 9.9 GE/mL, respectively.

Because of the highly asymmetric distribution of the DNA concentration and the presence of 0 values in three cases (MF), we used logarithmic transformation of DNA concentration plus 1 [i.e., log(DNA + 1)].

To evaluate whether any difference in the mean DNA content exists between MF and singleton male (M) pregnancies, we compared each of the 29 twin MF pregnancies with, at maximum, 5 M pregnancies (for a total of 128), matched for gestational week, randomly sampled from a pool of 832 physiologic singleton M pregnancies analyzed previously with the same protocol within 2 weeks from collection. In a linear regression model, there was no significant difference between MF and M pregnancies after correction for week of gestation (P = 0.34). Because the presence of a female fetus does not apparently affect the concentration of male fetal DNA in the maternal circulation, we grouped the two MFF pregnancies with the MF ones.

A linear regression model was then fitted to evaluate the difference between MM pregnancies, distinguished as monochorial and bichorial, and MF ones, correcting for effect of week of gestation (Table 1). The DNA content was significantly higher in MM bichorial than in M singleton pregnancies, whereas we found no significant difference between monochorial MM and both MF and MM bichorial pregnancies (linear contrast MM monochorial vs MM bichorial = 0.29; P = 0.61). These results are shown in the boxplot of the residuals of the regression of log (DNA + 1) on week of gestation, grouped by pregnancy type (Fig. 1). Such findings must be considered carefully because of the small number of individuals compared in this study. All analyses were performed with the R package (Ver. 1.6.2) (13).

Our data show that SRY quantification correlates with the number of male fetuses. This suggests that physicians performing physiopathologic monitoring of twin pregnancies with one male fetus should refer to fetal DNA normative values already established for singleton pregnancies with one male fetus. Conversely, normative reference values for twin-bearing pregnancies with two or more male fetuses should be assessed on a sample population displaying the same number of male fetuses.


We found no statistically significant association between the concentration of fetal DNA in the maternal circulation and chorionicity in MM pregnancies, which does not help in elucidating the origin of cell-free fetal DNA in maternal plasma. Nevertheless, the number of monochorionic twin pregnancies was too small (reflecting the lower frequency of spontaneous monochorionic vs bichorionic twin pregnancies) to allow reliable conclusions. The placental weight data available to us seem to be similar between mono- and bichorionic pregnancies. Data obtained for placental weight may vary considerably; depending on how the placenta is prepared, the weights may differ by nearly 50% (11). Thus, because of the small proportion of twin pregnancies with respect to singleton ones, collecting multicentric data could be useful to reach an adequate sample size to further elucidate the origin of cell-free fetal DNA in maternal plasma.

L.C. was funded by Telethon (Project GGP02015). A.F. was funded by Ministero dell' Istruzione, dell' Universita e della Ricerca (Coffin 2002).


(1.) Keith L, Papiernik E, Kiely J, Oleszczuk J, Cervantes A, Dommergues M, et al. Clinical obstetrics and gynecology. In: Multiple gestation, Vol. 41. Philadelphia: Lippincott-Raven, 1998:1-139.

(2.) Farina A, LeShane ES, Lambert-Messerlian GM, Canick JA, Lee T, Neveux LM, et al. Evaluation of cell-free fetal DNA as a second-trimester maternal serum marker of Down syndrome pregnancy. Clin Chem 2003;49:239-42.

(3.) Wataganara T, LeShane ES, Farina A, Messerlian GM, Lee T, Canick JA, et al. Maternal serum cell-free fetal DNA levels are increased in cases of trisomy 13 but not trisomy 18. Hum Genet 2003;112:204-8.

(4.) Zhong XY, Holzgreve W, Hahn S. The levels of circulatory cell free fetal DNA in maternal plasma are elevated prior to the onset of preeclampsia. Hypertens Pregnancy 2002;21:77-83.

(5.) Hahn S, Holzgreve W. Fetal cells and cell-free fetal DNA in maternal blood: new insights into pre-eclampsia. Hum Reprod Update 2002;8:501-8.

(6.) Smid M, Vassallo A, Lagona F, Valsecchi L, Maniscalco L, Danti L, et al. Quantitative analysis of fetal DNA in maternal plasma in pathological conditions associated with placental abnormalities. Ann N Y Acad Sci 2001;945:132-7.

(7.) Bianchi DW, Lo YM. Fetomaternal cellular and plasma DNA trafficking: the Yin and the Yang. Ann N Y Acad Sci 2001;945:119-31.

(8.) Zhong XY, Holzgreve W, Hahn S. Cell-free fetal DNA in the maternal circulation does not stem from the transplacental passage of fetal erythroblasts. Mol Hum Reprod 2002;8:864-70.

(9.) Steinmann G, Valderrama E. Mechanisms of twinning. III. Placentation, calcium reduction and modified compaction. J Reprod Med 2001;46:9951002.

(10.) Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for non-invasive prenatal diagnosis. Am J Hum Genet 1998;62:768-75.

(11.) Chu RWK, Poon LLM, Lau TK, Leung TN, Wong EMC, Lo YMD. Effects of blood processing protocols on fetal and total DNA quantification in maternal plasma. Clin Chem 2001;47:1607-13.

(12.) Wiilliams J. The placenta and fetal membranes. In: Cunnignam FG, MacDonald PC, Gant NF, Leveno KJ, Gilstrap LC, Hankins GDV, Clark SL, eds. Obstetrics, 20th ed. Stamford: Prentice-Hall International, Inc., 1997:95123.

(13.) Ross I, Gentleman RA. Language for data analysis and graphics. J Comput Graph Stat 1996;5:299-314.

Maddalena Smid, [1] Silvia Galbiati, [2] Antonia Vassallo, [2] Dania Gambini, [1] Augusto Ferrari, [1] Gabriella Restagno, [1] Elsa Viora, [4] Marco Pagliano, [5] Stefano Calza, [6] Maurizio Ferrari, [7] and Laura Cremonesi [2] * ([1] Department of Obstetrics and Gynecology and [2] Unit of Genomics for Diagnosis of Human Pathologies, IRCCS, H. San Raffaele, Via Olgettina 60, 20132 Milan, Italy; [3] Struttura Complessa Genetica Molecolare, A.O.O.I.R.M.-S. Anna, Piazza Polonia 94, 10126 Torino, Italy; [4] Centro di Ecografia e Diagnosi Prenatale, Ospedale S. Anna, Corso Spezia 60, 10126 Torino, Italy; [5] Cattedra A, Universita di Torino, Corso Spezia 60, 10126 Torino, Italy; [6] Sezione di Statistica Medica e Biometria, Dipartimento di Scienze Biomediche e Biotecnologie, Universita di Brescia, Viale Europa 11, 25123 Brescia, Italy; [7] Diagnostica e Ricerca, S. Raffaele S.p.A., H. San Raffaele, Via Olgettina 60, 20132 Milan, Italy; * address correspondence to this author at: Unit of Genomics for Diagnosis of Human Pathologies, H. San Raffaele, Via Olgettina 58, 20132 Milan, Italy; fax 39-02-2643-4767, e-mail cremonesi.
Table 1. Summary of the regression model of log(DNA + 1)
on week of gestation and type of pregnancy.

 Estimate (a) SE P

Week of gestation 0.071 0.02 0.001
MM monochorial vs MF 0.89 0.53 0.095
MM bichorial vs MF 1.18 0.38 0.003

(a) Estimate is the difference in mean log(DNA + 1) for an
increase of 1 week and for MM monochorial vs MF and MM
bichorial vs MF pregnancy.
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Title Annotation:Technical Briefs
Author:Smid, Maddalena; Galbiati, Silvia; Vassallo, Antonia; Gambini, Dania; Ferrari, Augusto; Restagno, Ga
Publication:Clinical Chemistry
Date:Sep 1, 2003
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