Obstetrics & Gynecology:
Reliability of Fetal Sex Determination Using Maternal Plasma
Scheffer, Peter G. MD; van der Schoot, C Ellen MD, PhD; Page-Christiaens, Godelieve C. M. L. MD, PhD; Bossers, Bernadette; van Erp, Femke; de Haas, Masja MD, PhD
From the Division of Perinatology and Gynaecology, University Medical Center Utrecht, The Netherlands; The Department of Experimental Immunohaematology, Sanquin Research Amsterdam and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, The Netherlands; The Department of Immunohaematology Diagnostics, Sanquin Diagnostic Services, Amsterdam, The Netherlands; and The Department of Human Genetics, Radboud University Nijmegen Medical Center, The Netherlands.
The development and validation of the polymerase chain reaction protocols were performed within the European Commission for Special Noninvasive Advances in Fetal and Neonatal Evaluation (SAFE) Network of Excellence (LSHB-CT-2004–503243).
The authors thank Lutgarde Govaerts, clinical geneticist (Erasmus Medical Center, Rotterdam), Eva Pajkrt, gynecologist (Academic Medical Center, Amsterdam), Maaike Vreeburg, clinical geneticist (Maastricht University Medical Center, Maastricht), Jiddeke van de Kamp, clinical geneticist (VU University Medical Center, Amsterdam), Nicolette den Hollander, clinical geneticist, and Jennie Verdoes, research nurse (Leiden University Medical Center, Leiden), and Katelijne Bouman, clinical geneticist (University Medical Center Groningen, Groningen), for the clinical implementation of the protocol.
Presented in abstract form at the 6th International Conference of Circulation Nucleic Acids in Plasma and Serum (CNAPS-IV), November 9–11, 2009, Hong Kong.
Corresponding author: Peter G. Scheffer, MD, Sanquin Research Amsterdam, PO Box 9190, 1006 AD Amsterdam, The Netherlands; e-mail: firstname.lastname@example.org.
Financial Disclosure: The authors did not report any potential conflicts of interest.
OBJECTIVE: To determine the diagnostic accuracy of noninvasive fetal sex determination in maternal plasma.
METHODS: All consecutive patients for whom fetal sex determination in maternal plasma was performed in our laboratory from 2003 up to 2009 were included in the study. Real-time polymerase chain reaction was performed for the SRY gene and multicopy DYS14 marker sequence. A stringent diagnostic algorithm was applied. In the case of a positive result for both Y chromosome–specific assays, a male-bearing pregnancy was reported. In the case of a negative result, the presence of fetal DNA was ascertained through the use of 24 biallelic insertion/deletion polymorphisms or paternally inherited blood group antigens. Only if the presence of fetal DNA was confirmed was a female-bearing pregnancy reported. Results were compared with the pregnancy outcomes.
RESULTS: A total of 201 women were tested. The median gestational age was 9 0/7 weeks (interquartile range 8 0/7 to 10 0/7 weeks). In 189 of 201 cases (94%), a test result was issued; in 10 cases, the presence of fetal DNA could not be confirmed; in two cases, an early miscarriage was observed. Pregnancy outcome was obtained in 197 cases, including 105 male-bearing and 81 female-bearing pregnancies and 11 miscarriages. Sensitivity and specificity of the test were 100% (95% confidence intervals 96.6–100% and 95.6–100%, respectively). In all 10 cases in which the presence of fetal DNA could not be confirmed, a female was born.
CONCLUSION: Noninvasive fetal sex determination in maternal plasma is highly reliable and clinically applicable.
LEVEL OF EVIDENCE: III
Knowledge of the fetal sex at an early gestational age is relevant for pregnant women carrying an X-linked chromosomal abnormality or for those at risk of being pregnant with a child with congenital adrenal hyperplasia (CAH). In CAH, it determines whether dexamethasone treatment, started immediately after pregnancy was established to reduce virilization of the female external genitalia in affected females, needs to be continued or not. Traditionally, early fetal sex determination has been performed by using invasive techniques, such as chorionic villus sampling or amniocentesis. These procedures, however, still carry a risk of miscarriage around 1%1 and cannot be performed until 11 weeks of gestation. Reliable determination of fetal sex by means of ultrasonography cannot be done in the first trimester, because development of the external genitalia is not complete.2
The discovery of cell-free fetal DNA in plasma of pregnant women at the end of the last century offered a new source of material for prenatal diagnosis.3 Cell-free fetal DNA originates from apoptotic syncytiotrophoblasts from the placenta.4 It can be detected in the maternal circulation from as early as 5 weeks of gestation,5 and it is cleared within several hours after birth.6 Its chief feature is that it can be obtained noninvasively. With regard to fetal sex determination, it would allow for definitive sex determination earlier in pregnancy, sparing most female fetuses from unnecessary invasive testing because of X-linked disorders or guiding clinical management in other cases, such as CAH.
Polymerase chain reaction (PCR)–based detection and identification of fetal DNA in maternal plasma is at present only possible for those sequences absent from the maternal genome (eg, sequences on the Y chromosome in male-bearing pregnancies).7 If amplification of the target sequence is not detected, the fetus is assumed not to carry that particular allele. However, undetectable low concentrations of fetal DNA can cause false-negative results. Several approaches have been explored to exclude these false-negative results,8–10 but the search for a universal fetal DNA identifier is still ongoing.11,12
Already, noninvasive prenatal diagnosis by means of cell-free fetal DNA in maternal plasma has found its way into clinical practice. Numerous groups have reported on fetal Rhesus D genotyping in D-negative mothers,13–15 as well as on other blood groups.16 Reports on diagnosing single gene disorders17,18 are promising. Several reports on fetal sex determination in maternal plasma have been published. Use of both the single copy SRY gene sequence19–21 and the multicopy DYS14 marker sequence of the TSPY gene on the Y chromosome have been reported.22,23 Although results are encouraging, diagnostic accuracy varies between protocols and methods used, with sensitivity and specificity ranging from 31% to 100%.24,25 Moreover, most data have been obtained in a research setting, rather than from actual clinical practice (for an overview, see Wright and Burton7).
As a national reference laboratory, we have been offering noninvasive fetal sex determination for clinical purposes since the beginning of 2003 using a stringent diagnostic algorithm with the inclusion of fetal DNA identifiers. As part of our protocol, we collected the outcome data of all tested pregnancies. The aim of this present study was to analyze the results for noninvasive fetal sex determination in maternal plasma, performed in a clinical setting over a 6-year period, and to determine its diagnostic accuracy.
MATERIALS AND METHODS
All consecutive patients for whom fetal sex determination in maternal plasma was performed in our laboratory from 2003 up to 2009 were included in the study. Tests were performed at the request of clinical geneticists or gynecologists throughout The Netherlands for different indications. Gestational age and indication for the test were listed on the application form by the requesting physician. We advised a minimal gestational age of 7 weeks for CAH and 9 weeks for all other indications. Before blood sampling, ultrasonography was recommended to establish vitality of a singleton pregnancy and to check for the presence of an empty second sac.
Ethylenediaminetetraacetic acid anticoagulated blood was drawn from both the mother (30 mL) and, if possible, the reporting father (10 mL) and was processed within 48 hours.21 Both parental samples were typed serologically for CDE groups C, c, D, E, e, and K to identify paternal blood group antigens that could potentially serve as a marker to confirm the presence of fetal DNA.
DNA was extracted in duplicate from 2×2 mL plasma using the QIAamp Blood Mini Kit (Qiagen Inc., Hilden, Germany), following the “Blood and Body Fluid Protocol” recommended by the manufacturer. Volumes of the used reagents were increased proportionately to accommodate the 2-mL sample size. Adsorbed DNA was eluted with 60 microliters of water.
Real-time PCR analysis was performed with the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) using Taqman chemistry. This technique allows for the amplification of a specific sequence of DNA. If the target sequence is present, exponential DNA amplification causes a proportional increase in a reporter dye fluorescence. The number of amplification cycles required to reach a fixed threshold signal intensity is termed the cycle threshold. Extracted DNA was analyzed for SRY and from 2005 on also for DYS14 to increase sensitivity. Also, part of the albumin gene was amplified to measure the quantity of total cell-free DNA in the sample. Primer and probe sequences were as previously described.5,23 The singleplex reactions were set up in a volume of 50 microliters, using 25 microliters of Taqman Universal PCR Master Mix (Applied Biosystems) and 9 microliters of extracted DNA for the SRY and DYS14 PCR and 3 microliters for the albumin PCR. Primers and probes were used at final concentrations of 900 and 150 nM for SRY and 300 and 100 nM for DYS14 and the albumin gene.
For both isolations, PCR for SRY was performed in triplicate, whereas PCR for DYS14 was performed in duplicate. As a positive control, 1 mL of pooled plasma from 120 pregnant women (at 28–30 weeks of gestation) was simultaneously tested. One milliliter of plasma from a nonpregnant female donor served as a negative control. To recognize contamination, a no-template control, containing DNA-free water, was also included. Each replicate was judged for amplification according to previously defined cycle threshold values (Table 1). The results were interpreted using the scoring model as outlined in Table 1. Interpretation of the combined results for DYS14 and SRY led to a positive (ie, male), negative (ie, female), or inconclusive test result (Table 2). Because, as a result of its multicopy sequence, the DYS14 assay is far more sensitive (with a 10-fold lower detection limit),23 a positive result for SRY with a negative result for DYS14 was considered a technical failure.
In the case of a negative result for SRY and DYS14, suggesting a female-bearing pregnancy, the presence of fetal DNA was ascertained by testing for paternal sequences absent from the maternal genome. If serologic testing of the parents indicated that a paternal blood group antigen (ie, CDE group C, c, D, E, e, or K) could serve as a potential fetal marker, real-time quantitative PCR for the relevant sequence was performed in maternal plasma.16,21 When no blood group polymorphism was found to be suitable, leukocyte-derived DNA from both parents was screened for 24 biallelic insertion/deletion polymorphisms (indels) by multiplex PCR. Primer and probe sequences of 18 indels had been described earlier for quantitative assessment of hematopoietic chimerism after bone marrow transplantation.26 In addition to this set of markers, we added six indels found online27 (for primer and probe sequences, see Table 3). Those alleles heterozygously or homozygously present on the paternal genome and absent from the maternal genome could potentially serve as a marker to confirm the presence of fetal DNA. Targeting these potential markers and again SRY as a control, we performed a second singleplex PCR, using freshly isolated DNA from the same maternal plasma sample. By dilution series, it was previously shown that the sensitivities of the indel markers and SRY PCR are similar.9 If amplification was seen, the presence of fetal DNA was confirmed, and a female-bearing pregnancy was reported. In case no paternal blood was available, maternal genomic DNA was screened for all indels. Alleles absent from the maternal genome were then tested for in maternal plasma. Only when we were able to confirm the presence of fetal DNA, a negative result for SRY and DYS14 was issued and we concluded that the fetus was female. If the presence of fetal DNA could not be confirmed, the overall test result was inconclusive.
As part of test performance quality control, when reporting the result to the requesting physician, we requested to receive follow-up of the fetal sex, whether determined by karyotyping, by ultrasonography, or after birth, with the patients’ informed consent. After collecting all pregnancy outcomes, descriptive statistics were generated using Prism 4 software (GraphPad Software, San Diego, CA). The Fisher exact test (two-sided) was used to determine sensitivity and specificity with 95% confidence intervals (CIs).
From 2003 up to 2009, 201 pregnant women were tested (Fig. 1). The median gestational age at time of blood sampling was 9 0/7 weeks (interquartile range 8 0/7 to 10 0/7 weeks). In one case, testing was performed before the 7th week of gestation (at 5 5/7 weeks), and in 19 cases, testing was performed after the 12th week of gestation (range 12–31 weeks). The majority of tests (n=156; 78%) were performed for X-linked conditions, including 51 (25%) for Duchenne/Becker muscular dystrophy and 31 (15%) for hemophilia. In 39 cases (19%), tests were performed for CAH. Five tests (2%) were performed for other than the aforementioned indications. These included a fetal intraabdominal mass on ultrasonography with an ovary cyst in the differential diagnosis (at 16 weeks of gestation), genital ambiguity on ultrasonography (at 31 weeks), intended use of antiandrogenic medication (spironolactone) because of a maternal apparent mineral corticosteroid excess and very high blood pressures (at 13 weeks), maternal carrier of breast cancer gene (at 8 weeks), and an extremely elevated level of maternal testosterone (12 weeks). In one case, the indication for testing was not traceable anymore.
In the first 48 cases (from 2003 until 2005), only a PCR for SRY was performed. On the 153 samples thereafter, PCR for both SRY and DYS14 was performed. Results were conclusive on first testing in 193 of 201 samples. PCR had to be repeated in eight cases, using the same blood sample (n=5) or requiring a second sample (n=3). These latter three cases were all from the first cohort, when only SRY was targeted.
Pregnancy outcome and fetal sex, as determined by karyotyping, by ultrasonography, or after birth, were ascertained in 197 cases (98%; Fig. 1). SRY and DYS14 PCR was positive in 105 of 105 plasma samples of women bearing a male fetus, resulting in a 100% sensitivity (95% CI 96.6–100%). In all 81 plasma samples of women bearing a female fetus, PCR for SRY and DYS14 was negative, resulting in a 100% specificity (95% CI 95.6–100%). A total of 11 women miscarried.
Y chromosome–specific sequences were found in 112 samples, 83 samples (74%) scoring 6/6 positive replicates for SRY and all but one sample scoring 4/4 replicates positive for DYS14 (Table 4). As plotted in Figure 2, the mean cycle threshold value for SRY in male-bearing pregnancies was 37.83 (standard deviation [SD] 1.41). The mean cycle threshold value for DYS14 in male-bearing pregnancies was 32.27 (SD 1.04).
Eighty-seven samples were negative for Y chromosome specific sequences. Although no positive replicates for DYS14 were found, amplification was observed in 35% of the replicates from women pregnant with a girl (mean cycle threshold value 40.00, SD 1.87; Fig. 2).
For two samples, the combined SRY and DYS14 PCR result was inconclusive. These samples had relatively high (greater than 35 and less than 38) cycle threshold values for DYS14, with only 1/6 and 2/6 positive replicates for SRY found, pointing to the presence of very low amounts of fetal DNA. When a second blood sample was requested, a miscarriage was reported in both cases.
In 9 of 87 cases in which no Y chromosome–specific sequences were detected, a paternal blood group antigen could successfully be used to confirm the presence of fetal DNA. In the remaining 78 cases, indel markers were used (Fig. 3). In four cases, no paternal blood was obtained, but maternal DNA was nevertheless screened for all indels available in the panel. Results for screening for informative indel alleles are shown in Table 5. On average, three (SD 1) possibly informative alleles were found per pregnancy. Using indel markers, we were able to confirm the presence of fetal DNA in 68 of 78 cases (87%). In five cases, only negative results were found, with the informative indel alleles only heterozygously present in the father. In three cases, the result was inconclusive because aspecific amplification with maternal, leukocyte-derived genomic DNA was also obtained. In all 10 cases in which the presence of fetal DNA could not be confirmed, a female was born.
Overall, a test result was issued in 94% (189 of 201). In general, test results were reported to the requesting physician within 2 (in the case of a male) to 4 (in the case of a female) working days after blood sampling. Invasive testing could be avoided in 64 of 156 pregnancies (41%) tested because of X-linked disorders (36% excluding hemophilia). Invasive testing could also be avoided in the patient carrying the breast cancer gene, because a male-bearing pregnancy was predicted. For CAH, the duration of high doses steroids treatment could be reduced by several weeks in 27 of 39 pregnancies (69%). In the patient with apparent mineral corticosteroid excess, the fetus seemed to be male, and the second-choice treatment of prednisone and alpha-methyldopa was prescribed. A healthy boy was born. In the case with the fetal intraabdominal mass on ultrasonography, a female bearing-pregnancy was predicted, not allowing narrowing of the differential diagnosis at that time. A girl with a cloacal malformation was born.
This study demonstrates that fetal sex determination in maternal plasma is highly reliable. We have shown noninvasive fetal sex determination performed in a clinical setting to be accurate. Through the application of two different PCR assays on two separate DNA isolations, as well as through confirmation of the presence of fetal DNA in the case of negative PCR results, we were able to report fully conclusive results and guide clinical management in 189 of 201 cases. Moreover, no false-negative or false-positive results were found.
Combining both PCR results for the Y-linked sequences SRY and DYS14 and using a stringent algorithm, we were able to issue a result on first testing in all but five cases (3%). With 3/4 positive replicates for the DYS14 PCR, we were confident to report a male pregnancy with only 4/6 positive replicates for SRY. In the earlier stages of our protocol, when only PCR for SRY was performed, a similar SRY result would have prompted us to repeat the test or even request a new sample. Indeed, several groups targeting only one Y chromosome–specific sequence reported a higher frequency of repeat testing.28,29
The DYS14 assay targets a multicopy sequence and therefore has a higher sensitivity than SRY.23 Because four of five repeated tests were performed because of discrepant results between the SRY assays (data not shown), testing for only the DYS14 sequence could be considered. Although this has been suggested by Zimmermann et al23 and Picchiassi et al,29 we do not favor this approach. In our opinion, the added value of the SRY assay is that it causes the overall test result to be less vulnerable to false-positive results due to, eg, contamination. Furthermore, the relatively high frequency of amplification signals obtained with the DYS14 assay in female-bearing pregnancies (see also Fig. 2), albeit at high cycle threshold values, is still difficult to explain. Because no amplification signals of SRY are seen in the plasma of women pregnant with a girl, the SRY assay increases the specificity of the test as a whole.
It has been shown that fetal DNA is present in maternal plasma in anembryonic pregnancies4 and even before fetal circulation is established.30 Therefore, we cannot exclude the possibility that a vanishing (male) twin could cause false-positive PCR results. We did not encounter this in the current study. We do, however, recommend ultrasonography before blood sampling, with explicit attention for the presence of a second gestational sac.
No false-negative results were found in our study. We presume this can be attributed to the fact that we used 2 mL of plasma to extract DNA and the addition of 9 microliters of eluted DNA to the reaction volume. Other groups that used much smaller volumes of plasma and extracted DNA reported a higher frequency of repeat testing,20 reported more false-negative results,29,31 or were not able to issue results before the 10th week of gestation.32
Because female fetuses are not detected directly but only inferred by a negative result for Y chromosome–specific sequences, it remains of the utmost importance to confirm the presence of fetal DNA when a negative result for SRY and DYS14 is found. We were able to confirm the presence of fetal DNA in 87% of samples tested for biallelic insertion/deletion polymorphisms. Some authors have questioned the use of indel markers,33 addressing the fact that it does not represent a true internal control, its labor-intensive character, and the lack of informativeness unless a large number of polymorphisms are used. Performed by experienced technicians, screening and repeat testing for the 24 indel markers took no longer than 1 working day. Although we show that paternal markers are clinically applicable, a truly universal fetal marker independent of paternally inherited sequences would overcome the aforementioned objections. Epigenetic differences between maternal and fetal DNA are currently being explored. Promising results have been published on sequences within the tumor suppressor genes maspin and RASSF1A, methylated differently in mother and child.11,12
However, in all 10 cases with an inconclusive test result due to failure to confirm the presence of fetal DNA, a female-bearing pregnancy was reported. This proves the robustness of the SRY and DYS14 assays, and we therefore recommend the following: If the presence of fetal DNA cannot be confirmed and the indication for fetal sexing is an X-linked disease not affecting the development of the external genitalia, a female-bearing pregnancy can be reported, to be confirmed by ultrasonography in the second trimester. When the indication is, eg, CAH or androgen insensitivity syndrome, fetal sex cannot be determined ultrasonographically and invasive testing is to be offered in the case of inconclusive results. With our approach, there is no need for ultrasonography if the presence of fetal DNA is confirmed.
Noninvasive fetal sex determination is a clinical reality. There is no longer a need for invasive procedures to determine fetal sex. Fetal sex determination in maternal plasma allows for early knowledge of the fetal sex, adding to timely clinical management. It can reduce the need for invasive procedures in pregnant women carrying an X-linked chromosomal abnormality up to 50%, decreasing the risks for iatrogenic damage.
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