Assessment of QF-PCR as the First Approach in Prenatal Diagnosis

Abstract

Quantitative fluorescent PCR (QF-PCR) has been used by many laboratories for prenatal diagnosis of the most common aneuploidies. QF-PCR is rapid, cost-effective, and suitable for automation and can detect most abnormalities diagnosed by conventional karyotyping. Whether QF-PCR should be used alone in most of the samples and in which karyotyping should also be offered is currently a topic of debate. We evaluated and compared the results obtained from 7679 prenatal samples in which conventional karyotype and QF-PCR had been performed, including 1243 chorionic villi and 6436 amniotic fluid samples. Concordant QF-PCR and karyotype results were obtained in 98.75% of the samples. An abnormal karyotype associated with adverse clinical outcome undetected by QF-PCR was found in 0.05% of samples. Therefore, QF-PCR can be used alone in a large number of samples studied in a prenatal laboratory, thereby reducing both the workload in cytogenetic laboratories and parental anxiety when awaiting results.

Conventional karyotyping has been the usual technique of prenatal diagnosis since the 1970s. The reporting time for the majority of tests is around 2 to 3 weeks. In the last years, alternative methods have been developed to reduce the reporting time, the work load and to allow the introduction of automatic methods. Three rapid aneuploidy tests (RATs) are used to detect the commonest aneuploidies (trisomies 13, 18, 21, and sex chromosomes): fluorescence in situ hybridization,¹ quantitative fluorescent PCR (QF-PCR),² and multiplex ligation-dependent probe amplification.³ The reporting time of these RAT techniques is reduced to 24 to 48 hours, allowing decisions on pregnancy management to be made earlier. The risk of abnormal karyotype is low in cases with negative RAT results. All these tests have been used and validated for prenatal diagnosis (reviewed in 4, 5). On the other hand, another technique has also been developed: array-based comparative genomic hybridization, which has higher resolution, and also provides information about the DNA copy number of the whole genome with an average reporting time of 6 to 16 days (depending on whether fresh or cultured material is used).⁶ Array-based comparative genomic hybridization is also rapid, less labor-intensive than karyotyping and is suitable for automation. The main drawback is the cost, and it is sometimes difficult to interpret.⁷ However, future reductions in the cost of this technique may lead in most cases to the replacement of karyotyping in the future.

Nowadays, most prenatal diagnosis centers offer karyotype or RAT combined with karyotype. Nevertheless, these studies are expensive, and in systems with public funding, cost is important.⁸ In fact, QF-PCR has been introduced as the only test in some cases in at least two countries. Since January 2005, in Stockholm (Sweden) women can choose between QF-PCR alone or full karyotype when the indications for the prenatal test are advanced maternal age, increased risk for a specific monogenic disorder, or parental anxiety. Seventy percent of the women choose QF-PCR alone.⁹ Similarly, since May 2007 a QF-PCR-alone strategy (testing only chromosomes 13, 18, and 21) has been introduced in the London and South-East England region in the UK for amniotic fluid (AF) and chorionic villi (CV) samples. Karyotype is only performed in cases of ultrasound (US) detection of a structural abnormality, the presence of two or more soft markers for trisomy 21, a nuchal measurement >3 mm at less than 14 weeks of gestation or >6 mm for gestations ≥14 weeks, or a family history of chromosome rearrangement.¹⁰

The aim of the present study was to compare the results obtained in 7679 prenatal samples in which QF-PCR and karyotype were simultaneously performed to evaluate the feasibility of QF-PCR as the only approach in prenatal diagnosis.

Materials and Methods

Samples

We performed conventional karyotype and QF-PCR in 7679 prenatal samples: 1243 CV and 6436 AF, sent from three different hospitals from Barcelona (Hospital Clínic, Hospital Vall d'Hebron, and Hospital Sant Joan de Deu) from September 2004 to May 2008. When the AF was hematic, a sample from the mother was also studied (whole blood or buccal wash).

Written informed consent was obtained in all cases.

Cytogenetic Analysis

CV samples were collected in RPMI1640 medium (BioWhittaker, Cambrex, Belgium) supplemented with antibiotics and delivered immediately to the laboratory. Processing for cytogenetic analysis was performed with a semidirect method after 20 to 24 hours incubation to obtain G-banded metaphase chromosome preparations.¹¹ The number of cells examined varied between 15 and 20. The results were reported within the first week.

For AF samples, 20 G-banded metaphases from two independent primary cultures were analyzed. The number of cells examined varied between 15 and 20. The results were reported within 14 to 20 days.

QF-PCR

Genomic DNA was extracted from CV samples and whole blood using the standard extraction protocol from QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). For AF and maternal buccal washes between 0.5 and 2 ml of sample were used, and DNA extraction was performed using the Instagene Matrix (Bio-Rad Laboratories Inc., Hercules CA), following the manufacturer's instructions. Before use, Instagene-obtained samples were vortexed and centrifuged (2 minutes at maximum speed).

QF-PCR was performed using a set of STR markers for chromosomes 13, 18, 21, X, and Y. Initially, 17 markers were used, but these were incremented to 21 to reduce the number of noninformative samples to which additional studies should be performed. A total of five STRs from chromosomes 13 (D13S256, D13S303, D13S618, D13S631, D13634), five from chromosome 18 (D18S386, D18S391, D18S535, D18S858, D18S976), six for chromosome 21 (D21S11, D21S1411, D21S1412, D21S1413, D21S1435, D21S1444), and three STRs from chromosome X (DXS996, DXS1283, P39) and two from the pseudoautosomal regions PAR1 and PAR2 (X22 and DXYS218) were selected. AMXY and SRY were also included in the study to determine fetal sex.

All forward primers were fluorescently labeled with different fluorochromes (FAM, VIC, NED, and PET). PCR products were analyzed with an ABI3100 (Applied Biosystems, Foster City, CA) and GeneMapper v3.5 software was used to analyze the results. Peak area ratios between 0.8 and 1.4 were considered normal, whereas ratios above and below these were interpreted as trisomy, and the presence of three alleles of equal peak area was also considered trisomy. The presence of a single peak was considered uninformative and a minimum of two concordant informative markers was required to give a result. In samples with insufficient informative markers or discordant results, a set of additional markers selected from panels from the ABI PRISM Linkage Mapping Set v.2.5 (Applied Biosystems) was used.

Results

We included 7679 prenatal samples in this comparative study. In all samples conventional cytogenetic analysis and QF-PCR were performed. The reasons for prenatal diagnosis were: advanced maternal age (≥38 years) in 2171 (28.2%), abnormal biochemical screening in 2416 (31.4%), increased nuchal translucency in 527 (6.8%), maternal anxiety in 1034 (13.4%), other reasons in 1411 (mainly abnormal sonographic findings, family history of chromosomal rearrangements) (18.3%), and unknown in 120 (1.5%).

In 7611 samples (6374 AF and 1237 CV) a result was obtained with both methods and 98.75% of samples had concordant QF-PCR and karyotype results. Abnormal concordant results were detected in 336 cases (4.4%): 185 (2.9%) of AF and 151 (12.1%) of CV samples. The reason for prenatal diagnosis in the 7611 samples is summarized in Table 1, the cases with concordant abnormal result by QF-PCR and karyotype are also indicated. Table 2 summarizes results for AF and CV together and reason for referral grouped in three classes: i) advanced maternal age, altered biochemical screening and anxiety; ii) increased nuchal translucency, other (this group includes all abnormal US findings and family history of chromosomal rearrangement); and iii) unknown.

Table 1.

Samples and Indications

	Amniotic fluid		Chorionic villi
	Samples	Abnormal	Samples	Abnormal
Advanced maternal age	1776	27 (1.5%)	395	15 (3.8%)
Abnormal screening	2239	27 (1.2%)	177	15 (8.5%)
Increased nuchal translucency	303	39 (12.9%)	224	58 (25.8%)
Anxiety	881	7 (0.79%)	153	4 (2.6%)
Other (mainly U.S. abnormalities)	1138	78 (6.8%)	273	55 (20.1%)
Unknown	99	7 (7.1%)	21	4 (19%)
Total	6436	185 (2.87%)	1243	151 (12.1)

Abnormal QF-PCR results for each group are also shown.

Table 2.

Samples with QF-PCR and Karyotype Results and Indications

	Samples	Abnormal QF-PCR
Advanced maternal age + abnormal screening + anxiety	5575	95 (28.3%)
Increased nuchal translucency + other	1917	230 (68.4%)
Unknown	119	11 (3.3%)
Total	7611	336 (100%)

Abnormal QF-PCR results are shown (percentage refers to the total of abnormal QF-PCR samples).

QF-PCR and karyotype had discordant results in 94 cases: 59 (0.9%) in AF and 35 (2.8%) in CV. Table 3 summarizes the discrepancies and their likely clinical outcome. Remarkably, 48 of these discrepancies corresponded to balanced rearrangements, with no presumed effect on the fetal phenotype and the current pregnancy.

Table 3.

Discrepancies between QF-PCR and Karyotype Results

	Number	QF-PCR	Karyotype
Amniotic fluid		60 discrepancies
No expected clinical effect	36	Normal	Balanced rearrangement
Possible clinical effect	17	Normal	Unbalanced rearrangement
	5	Abnormal	Normal
	2	Normal	Abnormal (mosaicism)
Chorionic villi		35 discrepancies
No expected clinical effect	13	Normal*	Balanced rearrangement
Possible clinical effect	7	Normal	Unbalanced rearrangement
	5	Normal	Abnormal (mosaicism)
	3	Abnormal	Abnormal (mosaicism)
	3	Abnormal	Normal
	4**	Abnormal	Abnormal

^*One case was a trisomy 21 that also carried a balanced translocation. This case was considered Down Syndrome by QF-PCR.

^**Abnormal results were different between QF-PCR and karyotype.

There were 21 pregnancies in which results from karyotype or QF-PCR varied the pregnancy management (these cases are indicated in bold in Table 4 for AF and Table 5 for CV). In three of them, the abnormal result was only detected by QF-PCR (cases 23 and 24 in AF and case 21 in CV). Six of the 18 remaining discrepancies with normal QF-PCR result were studied because of abnormal US findings, two due to increased nuchal translucency and one due to a family history of chromosomal rearrangement. In two samples the referral reason was unknown and therefore we have not considered them in the following statistics. To summarize, only seven samples of 7611 (0.09%) from all cases would not have been correctly identified if the recommendations given by Ogilvie et al¹⁰ had been followed (karyotype only performed in cases with US structural abnormality, presence of two or more soft markers for trisomy 21, increased nuchal measurement, or a family history of chromosome rearrangement). Three samples (0.04%) were wrongly interpreted by QF-PCR as having abnormal number of sexual chromosomes, while the remaining four (0.05%) were associated with an adverse clinical outcome. If we consider only those samples in which karyotype would not have been performed, then the cases not detected by this approach would be 0.07% (4/5473). Figure 1 shows a suggested flow-chart for prenatal diagnosis if the following recommendations were followed: karyotype only performed to samples with normal QF-PCR result and referred due to abnormal US findings, increased nuchal translucency or presence of chromosomal rearrangement in the family. The numbers for the cases presented in this study are indicated in brackets; also the discrepancies between QF-PCR and karyotype are shown.

Table 4.

Discrepancies for Amniotic Fluid Samples with Different Clinical Management between QF-PCR and Karyotype

	Abnormal karyotype for non QF-PCR chromosomes
	Karyotype	QF-PCR	Reason for referral	Comments	Result
1	46,XY,del(3)(p24.3)	Normal	Increased nuchal translucency		TOP
2	46,XX,idic(17)(p11.2)[6]/46,XX[18]	Normal	Abnormal US findings		TOP
3	mos47,XY,+mar[12]/46,XY[28]	Normal	92,XXYY in CV		Continue pregnancy
4	46,XY,rec(14)dup(14p)inv(14) (p11.1;q24)	Normal	Abnormal US findings	Balanced rearrangement in the family	TOP
5	46,XX,del(15)(q13q15)	Normal	Abnormal US findings		TOP
6	46,XX,t(2;9)(q2.1;p2.2),ins(18)(?)	Normal	Maternal anxiety		TOP
7	47,XY,+20[4]/46,XY[36]	Normal	Advanced maternal age		Continue pregnancy
8	46,XX,dup(1)(q43-qter)	Normal	Advanced maternal age		TOP
9	47,XY,+20/46,XY	Normal	Increased nuchal translucency		Continue pregnancy
10	46,XX,del(7p)	Normal	Abnormal US findings	de novo abnormality	TOP
11	46,XX,der(2)t(2;11)mat	Normal	Mother carrier of balanced translocation		TOP
12	46,XY,add(17)(q25)	Normal	Abnormal US findings		TOP
13	47,XX,+mar	Normal	Altered biochemical screening	Marker in the family	Continue pregnancy
14	46,XX,22p+	Normal	Altered biochemical screening	Maternal polymorphism	Continue pregnancy
15	46,XX,dup(16q)	Normal	Maternal anxiety	Inherited abnormality	Continue pregnancy
			QF-PCR chromosomes
			Abnormal karyotype
16	46,XX,der(18)add(18q21)	Normal	Abnormal US findings		TOP
17	46,X,add(Xq26)	Normal	Altered biochemical screening	de novo abnormality	TOP
18	45,X/46,XX	Normal	Advanced maternal age		Continue pregnancy
19	47,XXX/46,XX	Normal	Unknown		Continue pregnancy?
		Abnormal QF-PCR
20	46,XX	69,XXX	Abnormal US findings		Spontaneous fetal demise
21	46,XX	45,X	Advanced maternal age	46,XX when more X-markers were used in QF-PCR	Continue pregnancy
22	46,XY	47,XYY	Increased nuchal translucency	Copy number variant	Continue pregnancy
23	46,XX	46,XX,SRY+	Discrepancy between karyotype and sex on US	Male on US examination	Continue pregnancy
24	46,XX	46,XX,SRY+	Maternal anxiety	Male on US examination	Continue pregnancy

AF, amniotic fluid; US, ultrasound; TOP, termination of pregnancy.

Samples with different clinical outcomes are shown in bold.

Table 5.

Discrepancies for Chorionic Villi Samples with Different Clinical Management between QF-PCR and Karyotype

	Abnormal karyotype for non-QF-PCR chromosomes
	CV karyotype	QF-PCR	AF karyotype	Reason for referral	Comments	Outcome
1	mos46,XX,i(14q)[7]/46,XX[29]	Normal	46,XX	Altered 1st trimester screening	Normal. CPM	Continue pregnancy
2	mos47,XX+3[14]/46,XX[3]	Normal	46,XX	Advanced maternal age	Normal. CPM	Continue pregnancy
3	mos48,XX+2+15[14]/46,XX[9]	Normal	Not done	Abnormal US finding		Spontaneous abortion
4	47,XX,+inv dup(15)	Normal	Not done	Maternal chromosomal rearrangement	Normal. Molecular study performed for UPD	Continue pregnancy
5	47,XX,+7	Normal	46,XX	Previous T21 gestation	Normal. CPM. Molecular study performed for uniparental disomy	Continue pregnancy
6	46,XX,4p+	Normal	Not done	Increased nuchal translucency	de novo abnormality	TOP
7*	46,XX,15ph+	Normal	Not done	Altered 1st trimester screening	Normal. Inherited abnormality	Continue pregnancy
8	92,XXXX	Normal	46,XX	Altered 1st trimester screening	Normal. CPM	Continue pregnancy
9	92,XXXX	Normal	FISH: normal	Altered 1st trimester screening	Normal. CPM	Continue pregnancy
10	92,XXYY	Normal	46,XY	Advanced maternal age	Normal. CPM	Continue pregnancy
		Mosaics of QF-PCR chromosomes with complex rearrangements
11	mos 47,XXY[5]/46,XY[20]	Normal	46,XY	Altered 1st trimester screening	Normal. CPM	Continue pregnancy
12	mos47,XY,+21[4]/46,XY[20]	Normal	46,XY	Altered 1st trimester screening	Normal. CPM	Continue pregnancy
13	mos46,XX,der(13;13)(q10;q10) [20]/46,XX[10]	Trisomy 13 (mosaic, ratio 2:1)	46,XX	Altered 1st trimester screening	Normal. CPM	Continue pregnancy
14	mos46,XX,18p+[15]/r(18)[14]/i(18q)[1]/45,XX,−18[3]	18p monosomy	46,XX,del(18p)	Increased nuchal translucency		TOP
15	mos46,XX,i(13)(q10)[17]/46,XX[3]	trisomy 13 (ratio 2:1)	Not done	Chromosomal abnormality in previous gestation	Unknown	TOP?
16	mos46,XY,der(21;21) (q10;q10)/47,XY,+21	Trisomy 21	47,XY,+21	Abnormal US finding		TOP
17	mos 46,X,+mar/45,X	45,X	Not done	Increased nuchal translucency		TOP
18	mos46,X,+mar[22]/45,X[13]	45,X	Not done	Increased nuchal translucency		TOP
19	mos45,X[15]/46XX[6]	45,X	46,XX	Increased nuchal translucency	Normal. CPM	Continue pregnancy
		Abnormal QF-PCR
20	46,XX	46,XX,SRY+	46,XX, SRY−	Chromosomal abnormality in previous gestation: anxiety		Continue pregnancy
21	46,XY	69,XXY	46,XY	Abnormal US findings	Normal. Mesenchymal dysplasia	Continue pregnancy
22	46,XY	47,XYY	46,XY	Unknown		Continue pregnancy

CV, chorionic villi; US, ultrasound; TOP, termination of pregnancy; CPM, confined placental mosaicism.

Samples with different clinical outcomes are shown in bold.

^*Trizygotic gestation.

Figure 1.

Flow chart to evaluate prenatal diagnosis results. Number of samples are in parentheses. The suggested protocol is shown in the gray upper area, and the results obtained in the present study are summarized below. Light gray boxes include discordant results that could affect pregnancy management. Dark gray boxes indicate discordant results that would be missed if the proposed protocol had been followed. Note that 0.07% of cases with different clinical management would be missed following the proposed protocol.

Amniotic Fluid Samples with Discordant Result by QF-PCR and Karyotype

Among the 6374 AF samples, discordant results, which could affect the clinical management of the pregnancy, were found in 24 samples (Table 4). Fifteen of these discrepancies corresponded to chromosomes not tested by QF-PCR, and in 9 of them termination of pregnancy (TOP) was carried out due to severe congenital malformations.

Regarding chromosomes studied by QF-PCR, there were two cases in which QF-PCR failed to detect the alteration because they were partial duplications (cases 16 and 17), and in both cases TOP procedure was selected. In two cases (18 and 19) QF-PCR failed to detect a sexual chromosome mosaicism, in cases 21 and 22 the karyotype did not confirm the abnormal QF-PCR result while in two additional (cases 23 and 24) QF-PCR detected 46, XX, SRY positive samples.

Interestingly, one case with a normal karyotype had a triploidy compatible pattern by QF-PCR (case 20). The sample was referred for abnormal US findings at gestational week 26. A second sample was obtained at week 30 and QF-PCR detected a female with markers within the limits of the normal range (all informative markers had an almost 1:1 ratio). A fetal blood sample was obtained just before labor induction after spontaneous fetal death and QF-PCR resulted in a compatible homozygous pattern for all markers tested (inherited from the father) with no allele inherited from the mother (reported in 12).

In summary, a discrepancy between a normal QF-PCR result and abnormal karyotyping, in which karyotype result ended in TOP, was observed in 11 cases (0.17%).

Chorionic Villi Samples with Discordant Result by QF-PCR and Karyotype

We studied 1237 CV samples and detected 35 discrepant results. In 13 of them cytogenetic analysis revealed an apparently balanced chromosomal rearrangement.

Although the remaining 22 could have a potential clinical effect (Table 5), 10 (45.5%) of them were diagnosed as confined placental mosaicisms (CPM) after performing karyotype on an AF sample.

Although the abnormalities detected by both procedures were different in five cases (cases 14 to 18) (22.7%), there was no effect on clinical management.

Of the remaining seven discordant cases, one resulted in a spontaneous fetal loss after CV procedure (case 3) and in six more studies were required to achieve a final diagnosis (cases 4 to 7, and 20 and 22). Of these latter cases, only one (case 6) implicated a change in clinical outcome (TOP). However, there were 2 cases (cases 20 and 22) in which the discrepancy affected sexual chromosomes that could have an impact on pregnancy management.

Interestingly, in one sample referred for abnormal US findings (case 21), QF-PCR detected a triploidy (69, XXY) while karyotyping disclosed a normal male. Amniocentesis was performed at 15 weeks confirming the normal karyotype (both by karyotyping and QF-PCR). After delivery, different areas of placenta were studied and co-existence of two different cell lines was confirmed by different studies, thereby explaining the triploidy-like pattern detected by QF-PCR (described in 12).

In summary, of the 22 CV cases with discrepancies only one (case 6) had an adverse clinical effect and was not detected by QF-PCR (1/1237: 0.08%) after referral for increased nuchal translucency.

Single Markers with Trisomic Patterns

We detected some markers (D13S631, D21S11, D21S1435, DXS1283, DXS996, P39 DXYS218, AMXY, and X22) with three alleles, while the remaining markers showed a diallelic pattern in 18 samples. These markers represent submicroscopic duplications with no previously reported clinical effect. Only markers D13S631, AMXY, X22, and DXYS218 were duplicated in more than one sample. X22 was detected with three doses in three samples; karyotype located heterochromatic Y material in chromosome 15p in all these samples.

Discussion

The application of RAT methods such as QF-PCR alone approach in prenatal diagnosis is much debated. The major criticism is that RAT tests only detect numerical abnormalities of the chromosomes tested. Nevertheless, the low frequency of severe abnormalities not detected by RAT, but revealed by conventional karyotyping has been of note.⁸ Moreover, it has also been questioned which prenatal samples should be studied only by RAT methods and which by karyotyping (with or without the RAT method).

In the present study, the results obtained by QF-PCR and karyotype are compared in 7611 prenatal cases. QF-PCR and karyotyping yielded a concordant result (either normal or abnormal) in 98.75% of samples. Discordant results with different implications in fetal phenotype were detected in 0.24% (18 cases) of prenatal samples. In four cases, abnormal sexual chromosome results detected by QF-PCR led to wait for karyotype. Nevertheless, the introduction of new markers for X and Y chromosomes will obviate these discrepancies.

It is important to highlight that if the selection of indications used in London and the South-East England region in the UK had been followed (karyotype only performed in cases with US structural abnormality, presence of two or more soft markers for trisomy 21, increased nuchal measurement, or a family history of chromosome rearrangement),¹⁰ ten of the 18 cases misdiagnosed by QF-PCR would have been detected, as karyotype would have been performed (Figure 1). These results reinforce the policy of performing karyotype when samples with normal QF-PCR result are referred for abnormal US findings, increased nuchal translucency or a family history of chromosome rearrangement. If this procedure had been followed in our cohort, karyotype would have been performed only in 22% samples (20.5% of AF and 30.7% of CV); and 0.05% of all samples with adverse clinical outcome would not have been detected or 0.07% of samples in which no karyotype would be performed (Figure 1). This result is in agreement with the data provided by other authors.¹³,¹⁴,¹⁵,¹⁶,¹⁷,¹⁸,¹⁹

One of the drawbacks that has been argued against QF-PCR is that it does not detect low levels of mosaicism. It is difficult to detect 46XX/45X and other mosaics when the aneuploidy is present in less than 15 to 20% of cells.^20,21 In our study, QF-PCR did not detect five samples in which mosaicism was detected by karyotyping and may affect the management of the pregnancy (5/7681 samples: 0.06%). This percentage is in concordance with previously reported data.^20,21 On the other hand, as stated by Donaghue et al,²¹ QF-PCR can help to differentiate if trisomies have arisen from meiotic or mitotic nondisjunctions. Tri-allelic patterns are detected when the trisomy is meiotic in origin, while di-allelic patterns are likely to be mitotic in origin. The identification of tri- or di-allelic patterns has implications on pregnancy as meiotically derived errors are more likely to indicate the presence of a trisomic fetus than a mitotically derived error.²¹

Finally, this study has detected duplications of only one marker with no clinical implication in 18 samples. Except for the X22 marker, such cases represent submicroscopic duplications. In the case of X22, heterochromatic Y chromosome material was detected in chromosome 15 when karyotyping was performed. These submicroscopic duplications have already been described by some authors.^20,22,23 However, when a trisomic pattern is detected in only one marker, parents should be tested to detect the presence of such a duplication, thus ruling out the possibility of partial trisomy.

In conclusion, the results provided herein favor the use of QF-PCR alone in a large number of prenatal samples. Karyotyping should be carried out in pregnancies with normal QF-PCR results and abnormal US findings, increased nuchal translucency or a family history of chromosome rearrangement. However, full information should be provided to pregnant women to allow them to decide between QF-PCR or karyotype. This policy would greatly reduce the workload in cytogenetic laboratories and the cost of prenatal studies, as well as anxiety for pregnant women when waiting for a result.