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.
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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Figure. No caption available.