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Fetal DNA in Maternal Circulation of First-Trimester Spontaneous Abortions

SKINNER, JULIET MRCOG, MRCPI; LUETTICH, KARSTA PHD; RING, MARTINA; O'LEARY, JOHN J. MD, DPHIL, MSC, MRCPATH; TURNER, MICHAEL J. FRCOG, FRCPI

ORIGINAL RESEARCH
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Objective To establish whether fetal DNA can be identified in the maternal circulation in first-trimester spontaneous abortions.

Methods Women with confirmed spontaneous abortions and no histories of previous pregnancy were recruited. Peripheral venous blood samples were obtained and DNA extracted. Real-time quantitative polymerase chain reaction was done using SRY and β-actin systems for calculating fetal and total DNA, respectively.

Results Of 25 women, SRY-specific signals were detected in 11 indicating that the abortions were male. The remaining 14 were negative for the SRY gene. Women with positive results were of similar gestational age to those who were negative (mean 68.4 and 69.0 days). Fetal:total DNA ratio was calculated for positive samples and ranged from 15.8 to 360.1 × 10+3. Mean ratio was 99.4 × 10+3 and median was 67.5 × 10+3.

Conclusion Fetal DNA is present in the maternal circulation of first-trimester spontaneous abortions.

Fetal DNA is present in the maternal circulation of first-trimester spontaneous abortions.

Coombe Women's Hospital and Trinity College, Dublin, Ireland.

Michael J. Turner, FRCOG, FRCPI, Coombe Women's Hospital, Dublin, 8, Ireland; E-mail: mturner@coombe.ie

Received May 24, 2000. Received in revised form September 14, 2000. Accepted October 5, 2000.

Spontaneous abortion is the most common complication of pregnancy. One in four women will experience at least one spontaneous abortion during their reproductive years.1 Spontaneous abortion may have a serious psychologic impact on a couple.2 The grief experienced by couples after spontaneous abortion can be as intense as that after perinatal death.3–5 Feelings of self-blame and personal responsibility are significantly associated with elevated levels of anxiety and depression.6,7 Self-blame decreases significantly when a cause for the spontaneous abortion can be identified.8

The most common cause of sporadic spontaneous abortion is genetic abnormality. Offering genetic testing of the conceptus to all couples after spontaneous abortion is not generally done, in part because of technical difficulties in karyotyping the aborted specimen. First, collection of the specimen can be difficult because many women abort at home or outside of standard laboratory hours. Second, standard cytogenetic techniques are associated with high culture failure rates and thus a low yield of results.9–11

In the prenatal diagnosis of ongoing pregnancy, interest has focused on an alternative source of fetal DNA, which is present in maternal venous blood. The relative scarcity of this DNA requires specialized molecular biology technology, but it is noninvasive and therefore has potential major advantages in terms of pregnancy risk. A model developed using a male genetic sequence assessed the quantity of fetal DNA present in the maternal circulation of pregnancies in which the fetus was male.12 Application of molecular diagnostic techniques has been successful in identifying genetic abnormalities of some fetuses.13,14 To date, research has been limited predominantly to ongoing pregnancies in the second trimester, and the quantity of fetal DNA present in the first trimester of pregnancy has not been extensively studied. Based on menstrual dates, Y-chromosome-specific DNA sequences have been found from 6 weeks' gestation.12,15 A small study on pregnancies conceived with in vitro fertilization detected fetal DNA from as early as 19 days after embryo transfer.16

Fetal DNA in the maternal circulation could be a valuable source for genetic investigations of spontaneous abortion. We wanted to identify whether or not fetal DNA is present in the maternal circulation of women with first-trimester spontaneous abortions.

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Materials and Methods

Women with confirmed ultrasound diagnoses of spontaneous abortion attending the Coombe Women's Hospital, Dublin, were recruited between November 1998 and April 1999 and gave informed consent. The Hospital Research Ethics Committee approved the protocol. The study was confined to women with no histories of previous pregnancies. Women with suspected multiple pregnancies on ultrasound were excluded. Gestations according to menstrual dates on days of diagnosis were recorded.

Nonpregnant negative controls, included to calibrate the threshold of the polmerase chain reaction (PCR) system, consisted of five newborn female infants and three nonpregnant female adults with no known histories of previous pregnancy. Control male genomic DNA of a known concentration was used to titrate experiments.

Peripheral blood from subjects and adult controls was obtained and stored in 2.5-mL monovettes (Sarstedt, Wexford, Ireland) containing ethylenediaminetetra-acetic acid at −20C until further processing. In the case of the newborn negative controls, umbilical cord blood was collected using a sterile technique. All specimens were then assigned a code number to ensure anonymous analysis.

DNA was extracted from 1 mL of the collected blood sample using a standard phenol/chloroform extraction protocol.17 A spectrophotometer (Beckman Du 530) was used to measure total DNA extracted concentration.

Real-time quantitative PCR based on a 5′ nuclease assay was done using the ABI prism 7700 sequence detection system (PE Biosystems, Foster City, CA). The SRY TaqMan system has been described.18 The primer pair used was SRY-109F, 5′ TGG CGA TTA AGT CAA ATT CGC-3′, and SRY-254R, 5′ CCC CCT AGT ACC CTG ACA ATG TAT T-3′ (PE Biosystems, Warrington, Cheshire, UK). The dual-labeled fluorescent TaqMan probe was SRY-142T, 5′ (FAM) AGC AGT AGA GCA GTC AGG GAG GCA GA (TAMRA) (PE Biosystems, Warrington, Cheshire, UK). Primers and probe were identical to those described. A second control PCR system using human endogenous control β-actin primer and probe sequence was used to calculate the total number of DNA copies per milliliter (PE Biosystems, Warrington, Cheshire, UK).

Polymerase chain reaction amplification was performed in a 25-μL volume containing 1× universal mastermix (PE Biosystems, Warrington, Cheshire, UK), 250 μL of each primer, 150 μL of the TaqMan probe, and 100 μL of DNA. Standards and negative controls were analyzed in triplicate, patient samples in duplicate. Amplification and detection were done with the ABI 7700 system: incubation at 50C for 2 minutes, followed by denaturation at 95C for 10 minutes and 40 cycles at 95C for 15 seconds and 60C for 1 minute.

Data were collected by the 7700 sequence detector and analyzed with Sequence Detection 1.6 (PE Biosystems, Foster City, CA). Mean quantities for each duplicate of SRY and β-actin were recorded. Based on evidence that one cell contains 20 copies of the β-actin gene and two copies of the SRY gene (if male), the fetal:total ratio was calculated with this equation: fetal/total = (n SRY copies/2)/(n β-actin copies/20).

Strict precautions against PCR contamination were taken. Aerosol-resistant pipette tips were used to handle all liquids. Separate areas were used for extraction of DNA, preparation of amplification reactions, addition of the DNA template, and amplification and detection. The ABI 7700 system offers an extra level of protection in that its optical detection system does not need to reopen reaction tubes, thus minimizing the possibility of carryover contamination.

Statistical analysis was done with Excel (Microsoft, Redmond, WA). Values for continuous variables with normal distributions are presented as means ± standard deviations. Subjects with a positive SRY signal were compared with those with a negative SRY signal. P less than .05 was considered significant.

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Results

Male genomic DNA was diluted serially to check the sensitivity of the PCR system. Positive signals were obtained for dilutions up to 5 pg/μL, corresponding to the equivalent found in a single male cell. The signal detected in the negative controls was used to calculate the threshold level, above which a sample was called positive for the SRY gene.

Of 25 women recruited, positive SRY signals were identified in 11, suggesting that fetuses were male. In the remaining 14 women, their maternal blood was negative for the SRY gene. The fetal:total ratio was calculated for positive samples and ranged from 15.8 to 360.1 × 10+3. Mean ratio was 99.4 × 10+3 and the median was 67.5 × 10+3.

Table 1 shows comparative maternal ages, gestations at time of miscarriage based on menstrual dates, and types of miscarriage between women with positive SRY signals and those with negative SRY signals.

Table 1

Table 1

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Discussion

This study shows that fetal DNA is present in the maternal circulation of first-trimester spontaneous abortions. The relative scarcity of fetal DNA necessitates a sensitive PCR-based technique to verify its presence. We used the same model, the SRY gene present on the Y chromosome, as that used to demonstrate fetal DNA in the maternal circulation of ongoing pregnancies.18

In spontaneous abortion, pregnancy failure can occur at a very early stage of development, eg, before formation of the fetal pole as in an anembryonic pregnancy. Alternatively, it can occur at a later stage when fetal development occurs but subsequently ceases, eg, missed abortion. The degree of fetal DNA shedding into the maternal circulation might be insufficient in anembryonic pregnancy and so may be associated with a false-negative result. However, in this study, positive SRY sequences were found in women with anembryonic abortions, suggesting that sufficient DNA crosses into the maternal circulation even if a visible fetus is not seen on ultrasound.

To date, studies on normal pregnancies have shown that the quantity of fetal cell or DNA traffic into the maternal circulation increases with increasing gestation.18,19 Thus, if the mean gestation of women negative for the SRY gene was less than that of women positive for that gene, DNA quantity might have been a limiting factor and created a false result. However, there was no significant difference in the mean gestation of women positive (68.4 days) or negative (69.0 days) for the gene.

Critics of previous studies evaluating fetomaternal DNA traffic suggested that persistence of DNA from previous pregnancies might account for a false-positive result.20,21 This cannot account for the finding of male DNA sequences in maternal blood in miscarriages in this study because those women did not have histories of pregnancy.

Previous work using cytogenetic techniques in miscarriage found that female excess occurs when chromosomes are normal, with a male:female ratio of 0.7.22 Others found a sex ratio closer to unity when the chromosome complement is abnormal.23 If it is assumed that the fetus was male in SRY-positive women and female in SRY-negative women, the male:female ratio in this small study was 0.78. However, a larger study would be required to evaluate this fully.

The benefit of fetal sex determination in miscarriage is to provide an individual answer to the commonly asked question, “Was it a boy or girl?” In first-trimester spontaneous abortion, the answer is usually not clinically obvious. In many circumstances, tissue is unavailable or unsuitable for sex determination. Our study suggests that fetal sex may be determined from maternal venous blood samples. Using this method, the samples can be stored and need not be analyzed immediately, unlike conventional genetic analysis. This would allow analysis in batches or from a centralized laboratory and would be more cost effective.

This small but important study showed that fetal DNA is present in maternal circulation in first-trimester spontaneous abortion. Therefore, fetal DNA in the maternal circulation might be used for genetic testing in spontaneous abortion. In so doing, we might provide couples with individual genetic results for their miscarriage.

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