Invasive techniques such as chorionic villus sampling or amniocentesis have been used for early fetal sex determination in women carrying fetuses at risk for ambiguous genitalia and X-linked disorders. However, such invasive diagnostic tests are expensive, require expertise technique and carry a risk for miscarriage of approximately 1%.
Cell-free fetal DNA (cff-DNA) in the maternal circulation has been proposed as a potential material for noninvasive prenatal diagnosis, which poses no risk to mother or child. Among the various clinical applications of cff-DNA, fetal sex determination is the most investigated part and has a reported diagnostic accuracy close to 100%. However, such procedures can raise ethical concerns, as they may be used for the purpose of family balancing or due to preference for a child of a particular sex. Nevertheless, this technology may be useful in clinical applications for early sex detection of fetuses at high risk for ambiguous genitalia and X-linked disorders.
Cell-free fetal DNA
The majority of human DNA is located inside cells. However, a small amount of extracellular DNA is present in the circulation of both healthy and diseased subjects. It is thought to be a product of apoptosis, which results in the fragmentation and ejection of chromosomal DNA from the cell Citation[1].
The presence of cff-DNA in the maternal circulation was first demonstrated in 1997 by Lo et al.Citation[2]. cff-DNA originates from apoptotic and/or necrotic placental cells (trophoblasts) derived from the embryo Citation[3] and presents in the maternal circulation throughout pregnancy Citation[4]. Unlike cellular DNA, it consists predominantly of short DNA fragments rather than whole chromosomes, of which 80% are 193 base pairs in length Citation[5]. cff-DNA can be detected in the maternal circulation from 4 weeks of gestation Citation[6] and the concentration increases with gestational age Citation[4]. It is rapidly cleared from the maternal circulation with a half-life of 16 min, and is undetectable from 2 h after delivery Citation[7]. Therefore, cff-DNA has been proposed as an effective material for noninvasive prenatal diagnosis to monitor fetal status during pregnancy.
The applications of cff-DNA can be broadly divided into two categories: first, detection of specific paternally inherited single-gene disorders in families with high genetic risk; and second, routine antenatal care offered to all pregnant women, including prenatal screening/diagnosis of aneuploidy, particularly Down syndrome. Among these applications, the most common clinical application of cff-DNA is fetal sex determination. Such procedures have already transitioned into clinical practice for high-risk individuals with X-linked disorders or ambiguous genitalia Citation[8].
Fetal sex detection using cff-DNA
To date, fetal sex detection using cff-DNA focuses on the detection of paternally inherited Y-chromosome sequences that are entirely absent from the maternal genotype. The majority of studies using cff-DNA for fetal sex determination have used relatively simple techniques such as real-time PCR to detect genes on the Y chromosome of the male fetus; the sex-determining region Y, a single-copy gene, and other Y-chromosome-specific sequences present in multiple copies per male genome, including DYS, DYZ and DAZ.
Although sensitivity and specificity for fetal sex determination using cff-DNA range from 31 to 100%, depending on the protocols and methods used, the majority of studies demonstrate diagnostic accuracy close to 100%. In UK testing laboratories, an accuracy rate of 97.6% was achieved for tests performed at 7 weeks of gestation Citation[9]. In a recent meta-analysis of 57 selected studies, Devaney et al. reported that quantitative real-time PCR analysis of the sex-determining region Y sequence achieved the greatest sensitivity (99.0%) and specificity (99.6%) after 20 weeks of gestation Citation[10]. However, such techniques have potential problems such as false-negative results due to undetectable low concentrations of cff-DNA (e.g., calling a male fetus female) or false-positive results due to unspecific PCR amplification (e.g., calling a female fetus male). A number of studies have tried to address these problems. First, protocols such as the addition of formaldehyde and extraction system of virus DNA have been used to increase the yield of cff-DNA Citation[11,12]. A recent workshop was held to evaluate a number of different protocols, and a standardization process for the extraction of cff-DNA from maternal plasma has been initiated Citation[12]. Second, differences between the sizes of circulating fetal and maternal DNA have been applied. Modified extraction methods, such as size fractionation, have been used to increase the purity of cff-DNA Citation[5]. Third, fetal-specific markers, such as biallelic insertion/deletion polymorphisms, short tandem repeats originating in paternally inherited sequences, and differently methylated regions (DMR) between maternal blood and placenta, have been investigated to confirm the presence of cff-DNA Citation[13–15]. In fetal sex determination using cff-DNA, a female fetus is not detected directly but only inferred by a negative result for Y-chromosome-specific sequences. Therefore, the presence of fetal DNA must be verified to validate the determination of female fetal sex. Confirming the presence of fetal DNA is of the utmost importance when negative results for Y-chromosome-specific sequences are found.
Recently, Scheffer et al. reported a method of noninvasive fetal sex determination using biallelic insertion/deletion polymorphisms to confirm the presence of fetal DNA Citation[13]. The sensitivity and specificity of the test were 100%. Although indel markers are clinically applicable and led to conclusive results, the use of such markers is labor intensive. Moreover, they do not represent true internal controls and do not provide enough data to detect fetal sex unless a large number of polymorphisms are used. We have investigated differently methylated regions in order to overcome these problems, and recently developed an effective method for fetal sex detection using the DYS14/GAPDH ratio Citation[15]. This method can achieve 100% diagnostic accuracy in fetal sex detection during the first trimester. These promising results may be related to the correct interpretation of false-negative results by confirmation of cff-DNA using a DMR, such as unmethylated PDE9A (U-PDE9A). Therefore, confirming the presence of cff-DNA is essential for accurate fetal sex detection using cff-DNA.
Implications
Early prenatal determination of fetal sex is required for pregnant women at risk of X-linked diseases such as hemophilia, Duchenne muscular dystrophy, adrenoleukodystrophy, Alport’s syndrome, anhidrotic ectodermal dysplasia, Hunter’s syndrome, Menke’s syndrome or Lesch–Nyhan syndrome, because a male fetus could indicate potential diseases. Although individual incidence for each disease is low, it has been estimated that their cumulative incidence is approximately five in every 10,000 live births. Fetal sex determination is also important in cases in which the development of external genitalia is ambiguous and in some endocrine disorders such as congenital adrenal hyperplasia, where there is masculinization of the female fetus that is preventable with antenatal treatment. Generally, early fetal sex determination has been performed by ultrasonography and invasive procedures, such as chorionic villus sampling or amniocentesis. However, reliable determination of fetal sex using ultrasonography cannot be performed in the first trimester because the development of the external genitalia is not complete Citation[16]. Invasive procedures carry a 1–2% risk of miscarriage and cannot be performed until 11 weeks of gestation Citation[17].
Previous studies using cff-DNA for fetal sex determination have confirmed that this technique is not only feasible, but also more accurate than ultrasound in the first trimester Citation[18]. In the Netherlands, the UK, France and Spain, this testing has already transitioned to routine clinical care Citation[8]. More recently, companies have begun offering this technology directly to the consumer over the Internet Citation[19]. Such tests have been marketed for nonmedical use to curious parents-to-be with promises of accuracy as high as 95–99% at as early as 5–7 weeks of gestation Citation[19,20]. However, their performance has not been formally assessed, and stringent criteria that provide validation of negative results are necessary. Before identifying a sample as female, the laboratory should confirm that fetal DNA is present and can be amplified. Clinical laboratories should also consider the reason for the test when setting the threshold for positive determinations. For example, in cases of X-linked disease, it is more important to eliminate false-negative results, because all male-bearing pregnancies will require invasive follow-up testing. Therefore, the threshold should be set to favor sensitivity at the deficit of specificity. In the case of congenital adrenal hyperplasia, which affects female genitalia in utero, specificity should be favored Citation[10].
Fetal sex determination using cff-DNA is not available at point of care, is not approved by the Clinical Laboratory Improvement Amendments and is not reimbursed by insurers. Furthermore, the fact that fetal sex detection using cff-DNA requires only a small sample of blood raises numerous ethical, social and legal implications, owing to the ease with which the test can be performed. In particular, a much broader potential application for fetal sex detection is family balancing or choice of a particular sex, which poses ethical concerns. Therefore, the application of such a test should consider cultural factors due to the extreme preference for male babies shown in some parts of the world, such as India and China Citation[21].
Nevertheless, fetal sex detection using cff-DNA demonstrates advantages over conventional techniques, since the sampling method is noninvasive and therefore poses no risk to mother or child. Furthermore, such tests can be performed early during the first trimester and would be cheaper than current invasive tests. Therefore, this technology may be useful in clinical settings for the early detection of fetuses at high risk for X-linked disorders or congenital adrenal hyperplasia requiring follow-up testing or antenatal treatment. Eventually, the availability of a reliable noninvasive test to determine fetal sex would reduce unintended fetal losses and would presumably be welcomed by pregnant women carrying fetuses at risk for genetic disorders.
Financial & competing interests disclosure
This work was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A111550). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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