Clinical applications of fetal sex determination in maternal blood in a preimplantation genetic diagnosis centre

Gérard Tachdjian1,5, Nelly Frydman1, Franciois Audibert2, Pierre Ray3, Violaine Kerbrat2, Pauline Ernault4, René Frydman2 and Jean-Marc Costa4

1 Service de Biologie et Génétique de la Reproduction, Hôpital Antoine Béclère, 92140 Clamart, 2 Service de Gynécologie Obstétrique, Hôpital Antoine Béclère, 92140 Clamart, 3 Département de Génétique, Hôpital Necker-Enfants Malades, 75015 Paris and 4 Centre de Diagnostic Prénatal, American Hospital of Paris, 92200 Neuilly, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Couples with a risk of transmitting X-linked diseases who are included in a preimplantation genetic diagnosis (PGD) programme need early and rapid fetal sex determination in two situations. The first situation is for the control of embryo sexing after PGD and the second situation is for those couples having a spontaneous pregnancy before the start of their PGD cycle. Among invasive techniques, chorionic villus sampling is the earliest procedure for fetal sex determination and molecular analysis of X-linked genetic disorders during the first trimester but it is associated with a risk of fetal loss. Non-invasive procedures such as ultrasound examination allow reliable fetal sex determination only during the second trimester. Reliable fetal sex determination can now be realised by using SRY gene amplification in maternal blood. METHODS AND RESULTS: We report the use of fetal sex determination from maternal serum as a diagnostic tool for the control of embryo sexing (two cases) and to manage spontaneous pregnancies in couples included in a PGD programme for X-linked diseases (five cases). Fetal sex determination using SRY gene amplification in maternal serum were in complete concordance with fetal sex observed by cytogenetic analysis or ultrasound examination and at birth. This novel strategy allowed the PGD results to be controlled precociously and avoided the performance of invasive procedures in four cases of female fetus. CONCLUSIONS: This rapid fetal sex determination during the first trimester provides advantages to both clinicians and patients in a PGD centre.

Key words: fetal/maternal serum/preimplantation genetic diagnosis/sex/SRY


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preimplantation genetic diagnosis (PGD) for embryo sexing is currently carried out for gender determinations of X-linked disorders and usually female embryos are transferred. When PGD for embryo sexing is performed, the risk of misdiagnosis is around 1% (Staessen et al., 1999Go; Geraedts et al., 2000Go; Hanson et al., 2001Go; ESHRE PGD Consortium Steering Committee, 2002Go). Since it is not possible to completely rule out the risk of misdiagnosis, a control prenatal diagnosis is advised in case of a pregnancy (Geraedts et al., 2000Go). The earliest procedure for prenatal diagnosis of sex chromosomes is chorionic villus sampling (CVS) during the first trimester and fetal sex determination is performed by karyotyping. Nevertheless, the risk of fetal loss as a result of this procedure is estimated to be 0.5–2% (Jauniaux et al., 2000Go). To avoid this risk, non-invasive methods for prenatal diagnosis of fetal sex are increased. Ultrasound examination is inaccurate before 13 weeks gestation and determination of fetal sex is usually performed in the second trimester (Mielke et al., 1998Go; Efrat et al., 1999Go). Recovery of transcervical cells (Daryani et al., 2000Go) or isolation of fetal cells from maternal blood (Pertl and Bianchi, 1999Go) have been reported, but the sensitivity, specificity, test-failure rate and cost-effectiveness do not currently allow routine use. One potential non-invasive approach is the use of fetal DNA circulating in maternal plasma or serum blood. Lo et al. have reported the presence of male fetal DNA in the serum and plasma of pregnant women, which can be detected by polymerase chain reaction (PCR) with primers specific for the Y-chromosome (Lo et al., 1997Go). Several studies have shown that fetal DNA can be reproducibly detected in the serum of pregnant women (Zhong et al., 2000Go; Chen et al., 2001Go; Costa et al., 2001Go; Honda et al., 2001Go; Sekizawa et al., 2001Go; Wei et al., 2001Go). This possibility of determining fetal sex in maternal serum could be applied in a PGD centre in two situations. The first situation is for the control of embryo sexing after PGD during the first trimester of gestation without the risk of fetal loss. The second situation concerns those couples who are included in a PGD programme for an X-linked disease and having a spontaneous pregnancy before starting their PGD cycle. In this situation, determination of fetal sex in maternal blood would allow avoidance of an invasive procedure for molecular analysis in 50% of the fetuses.

We report on the use of this non-invasive prenatal strategy of fetal sex determination in a PGD centre as an adjunct test to manage pregnancies in patients at risk of sex linked disorders.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Seven couples at risk for X-linked diseases who were included in a PGD programme were tested. Indications of PGD were genetic risk, familial history, birth of affected children and previous termination of pregnancy.

Two women had a pregnancy after PGD for Duchenne muscular dystrophy and for mutation of 5-alpha reductase gene. In the case of Duchenne muscular dystrophy, PGD was performed using PCR to analyse both embryo gender and deletion status. Two female embryos and one male embryo were transferred. In the case of 5-alpha reductase disorder, PGD was performed using fluorescence in-situ hybridization with sex chromosome probes. Three female embryos were transferred.

Five women had a spontaneous pregnancy with a genetic risk for Hunter disease (n = 1), Duchenne muscular dystrophy (n = 2), fragile-X syndrome (n = 1), and myotubular myopathy (n = 1). These patients refused oral contraceptives prior to commencing their PGD cycle.

Samples
All the pregnant women participating in this study gave informed written consent before blood sampling. Maternal blood was obtained during the first trimester of pregnancy between 9 and 12 weeks gestation. Five ml of blood were collected into Vacutainer SST tubes (Becton Dickinson, Meylan, France). Immediately after clotting, serum was obtained after centrifugation at 4°C for 10 min at 3000 g.

DNA extraction
As tracer for the DNA extraction and amplification steps, a low amount (250 pg) of mouse DNA (Sigma, Grenoble, France) was added to each patient's sample (400 µl of serum) immediately prior to DNA extraction. Total DNA was then extracted by the PCR Template Preparation Kit (Roche Biochemicals, Meylan, France) according to the manufacter's recommendations, except that adsorbed DNA was eluted from the column with 50 µl instead of 200 µl of elution buffer, in order to increase the concentration.

Real-time PCR for the SRY gene in maternal serum
Amplification was carried out in a LightCycler instrument (Roche Biochemicals, Meylan, France). Primers for SRY gene amplification were 5'-GGCAACGTCCAGGATAGAGTGA-3' and 5'-TGCTGATCTCTGAGTTTCGATT-3'. Two hybridization probes (TibMolBiol, Berlin, Germany) were designed to recognize adjacent internal sequences within the amplified fragment. One was labelled at the 5' end with LCRed640 and phosphorylated at the 3' end to prevent probe elongation by the Taq DNA polymerase (5'LCRed640-CGATCAGAGGCGCAAGATGGCTCT-3'Ph). The other was labelled at the 3' end with fluorescein (3'FITC-CCATGAACGCATTCATCGTGTGGTCTC). PCR reactions were set up, in a final volume of 20 µl, using the Fast DNA Master Hybridization Probes Kit (Roche Biochemicals, Meylan, France), with 0.5 µmol/l of each primer, 0.25 µmol/l of each probe, 1.25 units of uracil DNA glycosylase (UDG) (Biolabs, Saint-Quentin en Yvelines, France), 5 mmol/l of magnesium chloride, and 10 µl of extracted DNA. After an initial 1 min incubation at 50°C to allow UDG to cleave putative contaminant PCR products from previous reactions, a first denaturation step of 8 min at 95°C was started. Amplification was then performed for 50 cycles of denaturation (95°C, 10 s, ramping rate 20°C/s), annealing (56°C, 10 s, ramping rate 20°C/s). Continuous fluorescence was monitored at the annealing step for each sample, which was analysed in duplicate. During each run, a low amount of male DNA (10 pg) was used as a positive control and elution buffer for DNA extraction as a negative control.

Real-time PCR for the tracer DNA
In case of negative results for SRY gene amplification, the presence and correct amplification of the tracer mouse DNA in the extracted maternal serum DNA was checked. The same extracted serum DNA used for SRY gene amplification was assayed in a second PCR targeted at the mouse galactose-1-phosphate uridyltransferase (GALT) gene. This step allowed the distinguishing of a true negative from a false negative result coming from deficient DNA extraction process of PCR inhibitors in the eluted DNA. Specific mouse DNA amplification of the GALT gene was performed according to the same protocol, with primers and probes targeted at the mouse GALT gene. Primers for GALT gene amplification were 5'-GCTTCCCGAGGTACACTATTGC-3' and 5'-GTCACATCTGCCCGAACTCC-3'. Probes sequences were 5'LCRed705-GCTTGACTGTGACCACATCAGGGC-3'Ph and 5'-GAAAGACAAGGAAACGGCAGCCAT-3'FITC. A positive result in this PCR demonstrated the efficiency and validated any negative result for the SRY gene detection.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Determination of fetal sex using SRY amplification was obtained in <24 h. Results and confirmation of fetal sex determination are summarized in Table IGo. Serum was negative for the gene amplification in five women and positive in two others.


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Table I. Results of sex determination in the seven cases of X-linked diseases included in a PGD programme
 
Fetal sex results using the SRY gene PCR assay on maternal serum were in complete concordance with fetal sex observed by cytogenetic analysis (n = 3) or ultrasound examinations (n = 6) and at birth (n = 5).

In the two cases of male fetus, CVS was performed for molecular analysis. In one case, a fragile-X mutation was diagnosed leading to a termination of pregnancy. In the other case an unaffected fetus for Duchenne dystrophy was diagnosed.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we have carried out fetal sex determination in maternal serum using SRY gene amplification in couples included in PGD programme for X-linked diseases. Non-invasive procedures for prenatal diagnosis to avoid CVS, amniocentesis or blood cord sampling are in development (Lo et al., 1997Go; Pertl and Bianchi, 1999Go; Daryani et al., 2000Go). Isolation of fetal cells from maternal blood circulation is still under development and cannot be used as a reliable test diagnostic. Several authors have recently demonstrated that reliable fetal sex determination can be achieved by analysis of maternal serum during the first trimester of pregnancy (Costa et al., 2001Go; Sekizawa et al., 2001Go). The possibility of non-invasive prenatal diagnosis of fetal sex during the first trimester of gestation enables the development of new strategies in the management of pregnancies for X-linked diseases. Most reports on fetal sex determination in maternal serum are only evaluation studies (Honda et al., 2001Go; Sekizawa et al., 2001Go). Fetal sex determination in maternal serum has been recently used as a prenatal test for the clinical management in a pregnancy at risk for congenital adrenal hyperplasia (Rijnders et al., 2001Go). Fetal sex determination in maternal serum is a rapid (<24 h), easy and low cost test (Costa et al., 2001Go; Pertl and Bianchi, 2001Go), allowing the possibility of routine use.

Two situations in a PGD programme can profit from this non-invasive prenatal diagnosis of fetal sex. The first situation is the use of this non-invasive procedure to control PGD for embryo sexing. This novel application of fetal sex determination allows the monitoring of PGD results early during the first trimester of the pregnancy. Moreover, after an important investment in PGD treatment, the patients do not wish to risk fetal loss. One PGD for Duchenne muscular dystrophy was performed using multiplex PCR to determine embryo gender and deletion status (Ray et al., 2001Go) and two healthy female embryos and one male embryo were transferred. SRY amplification in maternal serum showed a female fetus, suggesting the absence of development of the male embryo. This result avoided CVS for molecular genetic control. In the case of embryo sexing for 5-alpha reductase disorder, the female fetal sex determined in maternal serum confirmed the PGD results.

The second situation corresponds to couples who are included in a PGD programme for an X-linked disease and then have a spontaneous pregnancy. In PGD for embryo sexing, the majority of patients are fertile. Spontaneous pregnancies can occur in those patients who refuse oral contraceptives prior to commencing their PGD cycle. In these situations, early non-invasive prenatal diagnosis of fetal sex in maternal serum represents a useful adjunct to PGD, avoiding an unnecessary invasive procedure if the fetus was found to be female. For these couples, a fetal sex determination in maternal serum was proposed at 10 weeks gestation. In the case of a male fetus, conventional prenatal diagnosis was then performed by CVS. If the SRY gene amplification in maternal serum was negative, ultrasound examination was suggested to test the accuracy of the fetal sex prediction. Thus, prenatal diagnosis using invasive procedures was only performed for male fetuses, avoiding the risk of fetal loss in the case of a female fetus. Although less efficient in case of multiple pregnancies because the probability for having only female fetuses is lower, this strategy can be offered as it can detect if at least one fetus is male. In our series, three fetuses were predicted as female and two as male. CVS was performed in cases of male fetus. In one case a fragile-X mutation was diagnosed leading to a termination of pregnancy and in the other case an unaffected fetus for Duchenne dystrophy was diagnosed. The fragile-X syndrome is an X-linked disorder which may cause mental retardation in both males and females. Nevertheless, the phenotype in females with fragile-X chromosomes is usually less distinct than in males, as would be expected from the influence of lyonization (De Vries et al., 1999Go). After non-directive genetic counselling, the couple was included in the PGD programme for embryo sexing with transfer of female embryos without prenatal molecular diagnosis. For this reason, when a spontaneous pregnancy occurred in this couple, we proposed a non-invasive fetal sex diagnosis associated with a prenatal molecular diagnosis in case of a male fetus. One couple with the prediction of a female fetus insisted on having CVS to confirm the fetal sex. For the other couples, invasive procedures were not performed and fetal sex was confirmed by ultrasonography and at birth.

In conclusion, the discovery of high concentrations of fetal DNA in maternal plasma represents a promising non-invasive approach to prenatal diagnosis (Lo et al., 2001Go; Pertl and Bianchi, 2001Go). This novel strategy can complete the diagnostic service in a PGD centre. Although only paternally inherited traits can be identified by analysing fetal DNA from maternal serum, the strategy offers many advantages when compared with analysis of fetal cells in maternal blood. Because fetal DNA in maternal serum is rapidly cleared after delivery (Lo et al., 1999Go), it is not present in the current pregnancy, thus avoiding the possibility of misdiagnosis. On the contrary, persistence of fetal cells in maternal blood from previous pregnancies may lead to potential false results. Besides the search for Y-chromosome sequences, single gene disorders such as myotonic dystrophy and achondroplasia have also been studied using fetal DNA from maternal plasma (Amicucci et al., 2000Go; Saito et al., 2000Go). Moreover, Chen et al. have recently reported the detection of fetal-derived paternally inherited chromosomal polymorphisms in maternal serum for the non-invasive prenatal diagnosis of chromosomal disorders (Chen et al., 2001Go). Non-invasive control of PGD results for paternal translocations could also be performed by using this strategy in maternal serum during the first trimester.


    Notes
 
5 To whom correspondence should be addressed at: Service de Biologie et Génétique de la Reproduction, Hôpital Antoine Béclère, 157, rue de la Porte de Trivaux, 92140 Clamart, France. E-mail: gerard.tachdjian{at}abc.ap-hop-paris.fr Back


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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on December 14, 2001; resubmitted on April 10, 2002; accepted on May 9, 2002.





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