Hum Genet (2003) 112 : 617–618 DOI 10.1007/s00439-003-0919-3
S H O RT R E P O RT
Maddalena Smid · Silvia Galbiati · Antonia Vassallo · Dania Gambini · Augusto Ferrari · Elsa Viora · Marco Pagliano · Gabriella Restagno · Maurizio Ferrari · Laura Cremonesi
No evidence of fetal DNA persistence in maternal plasma after pregnancy Received: 5 November 2002 / Accepted: 14 January 2003 / Published online: 27 February 2003 © Springer-Verlag 2003
Abstract Short- and long-term persistence of fetal DNA in maternal plasma has been investigated. Short-term persistence at very low concentration was detected in 47 out of 105 women within two days after delivery. Twelve out of 13 samples re-tested within three days scored negative. No long-term persistence was detected in 172 women who had previous sons or abortions. Molecular microchimerism due to circulating fetal DNA persisting from previous pregnancies should not hamper non-invasive plasma-based prenatal testing. Fetal DNA circulating in maternal plasma may represent an accessible source of fetal genetic material for noninvasive prenatal diagnosis (Lo et al. 1998). For a clinical application, an essential prerequisite to avoid false positive results is the complete clearance of fetal DNA molecules after pregnancy completion. Absence of short-term fetal DNA persistence had already been reported (Lo et al. 1999). However, in a recent report fetal male DNA was detected in the plasma of 22% healthy women with previous sons for up to several decades after delivery (Invernizzi et al. 2002).
M. Smid · D. Gambini · A. Ferrari Department of Obstetrics and Gynecology, H. San Raffaele, Milano, Italy S. Galbiati · A. Vassallo · L. Cremonesi (✉) Unit of Genomics for Diagnosis of Human Pathologies, IRCCS H. San Raffaele, 58 via Olgettina, 20132 Milano, Italy Tel.: +39-02-26434779, Fax: +39-02-26434767, e-mail:
[email protected] E. Viora Centro di Ecografia e Diagnosi Prenatale, Ospedale S. Anna, Torino, Italy M. Pagliano Cattedra A, Università di Torino, Italy G. Restagno Dipartimento di Patologia Clinica, A.O.O.I.R.M.-S. Anna, Torino, Italy M. Ferrari Diagnostica e Ricerca S. Raffaele S.p.A., H. San Raffaele, Milano, Italy
In an attempt to further explore this controversial issue we carried out male DNA quantification on 172 femalebearing pregnant and non-pregnant women with previous sons or abortions (Table 1). Additionally, we analysed short-term postpartum persistence in 105 women 1–2 days after they had delivered male babies. Detailed written informed consent, as approved by the local ethical committee, was obtained for each woman. Ten millilitres of maternal blood were collected in EDTA. After centrifuging at 1,600 g, plasma was carefully removed and re-centrifuged at full speed (16,000 g) in a microcentrifuge (Chiu et al. 2001). DNA extraction was carried out using the QIAamp Blood Kit (Qiagen, Milano, Italy) protocol; 400 µl of plasma per column for DNA extraction was used, according to instructions recommended by the manufacturer (Lo et al. 1998). SRY-specific DNA quantification was carried out by the real-time TaqMan method originally described by Lo et al. (1998). Real-time PCR analysis was performed by use of a PE Applied Biosystems 7700 Sequence Detector (PE Applied Biosystem, Foster City, Calif., USA) using the TaqMan PCR Core Reagent Kit (PE Applied Biosystems) (Lo et al. 1998). Ten microlitres of DNA was used for amplification. Each reaction was done in three independent aliquots; results were expressed as mean values. The sensitivity of the assay had been previously established by serial dilution of known amounts of male DNA from 1,000 to one male cell. The assay is able to detect one SRY genome equivalent (ge) per sample, in agreement with data reported by Lo et al. (1998). Remarkably, concerning long-term persistence of fetal DNA, we could not detect any fetal DNA sequence in any of the pregnant or non-pregnant women with previous sons or abortions. A total of 143 non-pregnant and female-bearing pregnant women either primigravidas or with no previous son or abortion were used as negative controls and gave no SRY amplification (Table 1). SRY amplification was obtained only 1–2 days following delivery in 47 out of 105 (39%) samples (range: 0.8– 106.8 ge/ml of maternal plasma; mean: 10.2 ge/ml; median: 4.6 ge/ml). Thirteen women, among who five were positive at day 1–2 postpartum (range 2.6–27.8 ge/ml)
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Table 1 Long-term fetal DNA persistence in maternal plasma No. of cases 47 55 70 117 26 a
Category of women
Age (years)a
Gravidity b
No. of previous sons
No. of previous abortions
Age of last son (years)
Years from last abortion
Female-bearing pregnant women with previous sons Female-bearing pregnant women with previous abortions Non-pregnant women with previous sons Female-bearing pregnant women with no previous sons or abortions Non-pregnant women with no previous sons or abortions
24–44
1–5
1–3
–
2–24
–
28–43
1–5
–
1–5
–
0–22
29–80 22–43
1–6 0–2
1–5 –
– –
1–56 –
– –
19–88
0–2
–
–
–
–
Age of women at sampling Number of previous male- and female-bearing pregnancies and abortions
b
were re-sampled blindly within three days after the first blood collection. Among them, only one who was positive at the first sampling (10.5 ge/ml) remained weakly positive (0.6 ge/ml). The proportion of women who scored positive in the second analysis (one out of 13) was significantly decreased when compared with the proportion observed in women who were sampled within 48 h since delivery (47/105, P=0.0140). Fetal DNA levels detected in plasma by Invernizzi et al. (2002) are surprisingly high compared with not only to those we obtained in the short-term postpartum but also in the 685 physiological male-bearing pregnancies sampled at different gestational ages. Indeed, in these women, mean and median fetal DNA ranged from 21.6 to 9.3 ge/ml in the first trimester to 36.9–19.3 ge/ml in the second trimester and to 79.8–54.1 ge/ml in the third trimester of gestation, respectively, in agreement with Lo et al. (1998) and Sekizawa et al. (2001). We found high fetal DNA concentration comparable with those reported by Invernizzi et al. (2002) only in overt placental pathological conditions such as pre-eclampsia (24 samples; fetal DNA range: 4.9–990.2 ge/ml; mean 255.06 ge/ml; median 135.05 ge/ml) and intra-uterine growth retardation (IUGR) (19 samples; fetal DNA range: 7.1– 1834.6 ge/ml; mean 294.04 ge/ml; median 122.7 ge/ml). In our opinion, the discrepant results between our study and that reported by Invernizzi et al. (2002) might be ascribed to different centrifugation protocols for blood processing, since the volume of extracted plasma, the amount of extracted DNA used for amplification and the amplification protocol are the same as reported by Lo and co-workers for both groups (Lo et al. 1998). This also implies that the sensitivity of the assays is comparable. Concerning the centrifugation protocol, the paper by Invernizzi and co-workers does not mention a second centrifugation step. Correct blood processing based on re-centrifuging at maximum speed the first aliquot of supernatant is essen-
tial to generate cell-free plasma samples (Chiu et al. 2001). Long-term persistence of fetal cells from previous pregnancy has been extensively demonstrated (Bianchi et al. 1996) and could affect the results if no cell-free plasma samples are analysed. Our data clearly demonstrate that fetal DNA is rapidly cleared from maternal plasma and no long-term persistence is observed after delivery. These findings further confirm that maternal plasma fetal DNA is a suitable source for non-invasive prenatal diagnosis. Acknowledgements The financial support of Telethon-Italy (Grant no. E.1110) to L. C. is gratefully acknowledged. A.F. was funded by MIUR Cofin 2002.
References 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 Chiu RWK, Poon LLM, Lau TK, Leung TN, Wong EMC, Lo YMD (2001) Effects of blood processing protocols on fetal and total DNA quantification in maternal plasma. Clin Chem 47: 1607–1613 Invernizzi P, Biondi ML, Battezzati PM, Perego F, Selmi C, Cecchini F, Podda M, Simoni G (2002) Presence of fetal DNA in maternal plasma decades after pregnancy. Hum Genet 110: 587–591 Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, Wainscoat JS, Johnson PJ, Chang AM, Hjelm NM (1998) Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 62:768–775 Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM (1999) Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 64:218–224 Sekizawa A, Kondo T, Iwasaki M, Watanabe A, Jimbo M, Saito H, Okai T (2001) Accuracy of fetal gender determination by analysis of DNA in maternal plasma. Clin Chem 47:1856–1858
