Sonographic diagnosis of fetal abnormalities is based on the recognition of sonographic patterns associated with structural abnormalities. Although diagnosis in some situations, such as neural tube defects, gastroschisis, and omphalocoele, can be straightforward, in many situations, the constellation of fetal abnormalities suggest an underlying chromosomal or genetic cause. In these situations, invasive testing is needed to provide the information required to make a definitive diagnosis, and thus accurately counsel parents. Since the identification of cell-free fetal DNA in maternal plasma, the potential for non-invasive prenatal diagnosis is increasingly becoming possible. In this chapter, the current role and future potential of non-invasive prenatal diagnosis, combined with new molecular techniques as an aid to sonographic diagnosis, will be discussed.
Properties of cell-free fetal DNA
Cell-free fetal DNA (cffDNA) was first identified in maternal plasma in the late 1990s . It originates from the placenta and comprises fragments of DNA that are shorter on average than maternal cell-free DNA , but generally constitutes around 10% of total cell-free DNA (cfDNA) in maternal plasma, the majority being maternal in origin . Cell-free fetal DNA can be detected in the maternal blood from 4 weeks’ gestation . As it is rapidly cleared from the maternal circulation, it is undetectable 2 h after delivery . It, therefore, makes a potentially ideal source of fetal genetic material for non-invasive prenatal diagnosis (NIPD). This approach is safer than current invasive prenatal diagnosis, as it is based on a maternal blood sample, and thus avoids the small but significant risk of miscarriage associated with chorionic villus sampling and amniocentesis . Key properties of cffDNA are listed in Table 1 .
| Placental in origin |
| Minority of cell-free DNA in maternal plasma |
| Detectable from 4 weeks gestation |
| Cell-free fetal DNA is shorter on average than maternal cell-free DNA |
| Cleared from the maternal circulation within hours of delivery |
Current clinical applications of cell-free fetal DNA
The earliest uses of cffDNA were for the detection of alleles not present in the mother but present in maternal blood because they had been inherited from the father. These include the two most widely used applications, namely fetal sex determination and fetal Rhesus D status in Rhesus D-negative mothers . More recently, some developments have allowed for NIPD of some single gene disorders, mainly those that have arisen de-novo at conception or are inherited from the father . Aneuploidy testing for the major trisomies is also available through commercial providers in the USA, Asia, and much of Europe, but is not yet available in the public sector . Analysis of cfDNA in the maternal plasma for fetal sex determination, some single gene disorders, and aneuploidy can aid sonographic diagnosis. These aspects will be discussed in more detail here.
Current clinical applications of cell-free fetal DNA
The earliest uses of cffDNA were for the detection of alleles not present in the mother but present in maternal blood because they had been inherited from the father. These include the two most widely used applications, namely fetal sex determination and fetal Rhesus D status in Rhesus D-negative mothers . More recently, some developments have allowed for NIPD of some single gene disorders, mainly those that have arisen de-novo at conception or are inherited from the father . Aneuploidy testing for the major trisomies is also available through commercial providers in the USA, Asia, and much of Europe, but is not yet available in the public sector . Analysis of cfDNA in the maternal plasma for fetal sex determination, some single gene disorders, and aneuploidy can aid sonographic diagnosis. These aspects will be discussed in more detail here.
Fetal sex determination using cell-free fetal DNA in maternal plasma
Fetal sex determination using cffDNA is based on the detection of Y-chromosome sequences, either SRY or DYS14 , in maternal plasma. If Y-chromosome sequences are detected, the fetus is predicted to be male and, if they are absent, the fetus is predicted to be female. The exact laboratory methodology used can vary, and a recent meta-analysis of 57 reports detailing more than 6000 pregnancies tested suggested that before 7 weeks’ gestation cffDNA testing is unreliable, with the best results reported after 20 weeks’ gestation . In the UK, an audit of fetal sex determination using cffDNA reported that it was highly accurate when carried out after 7 weeks’ gestation, but that it should be used in conjunction with fetal ultrasound to confirm the gestational age before testing . Ultrasound should also be used to detect multiple pregnancies, and the presence of an empty gestation sac, as it is known that the placenta can continue to shed cffDNA after demise of the fetal pole p2]. Non-invasive prenatal diagnosis for fetal sex determination is now increasingly used to determine fetal sex in pregnancies at increased risk of X-linked genetic disorders and congenital adrenal hyperplasia. In the UK and some other European countries, it is now the standard of care in these situations . Both women and health professionals value this approach to sex determination in these high-risk pregnancies , as it is a test that can be done early in pregnancy and is highly accurate (>99%) when delivered by accredited molecular genetic laboratories, reducing the rate of invasive testing by around 45%, and thereby avoiding unnecessary exposure to miscarriage risk . Furthermore, in pregnancies at risk of congenital adrenal hyperplasia, it allows early cessation of steroid treatment where the fetus is predicted to be male . Indeed, in some European countries, dexamethasone treatment is delayed until NIPD carried out at 7 weeks’ gestation indicates the presence of a female fetus. This allows avoidance of any unnecessary steroid treatment in male-bearing pregnancies. Finally, a detailed health economic analysis has shown that, when used in pregnancies at high risk of serious X-linked conditions (where parents might elect to terminate an affected pregnancy) and those at risk of congenital adrenal hyperplasia, the savings generated by avoiding invasive testing and unnecessary steroid treatment mean that NIPD for fetal sex determination is no more expensive than invasive diagnostic testing .
Fetal sex determination using cell-free fetal DNA in the investigation of genital ambiguity
Sonographic detection of an isolated abnormality of the external genitalia occurs infrequently. More common is the identification of a genital anomaly or ambiguity when detailed examination is carried out after detecting another structural abnormality. Whether an isolated finding, or in association with other structural anomalies, determination of genetic sex can be useful in defining the underlying diagnosis and directing parental counselling. In the past, this required invasive testing, but first-line testing in many cases should now be analysis of cffDNA after a maternal blood sample, although invasive testing may ultimately be required for definitive diagnosis.
Isolated genital anomalies
It can be difficult to differentiate cliteromegaly from hypospadias using ultrasound alone ( Fig. 1 ), and the use of cffDNA testing to confirm the genetic sex is useful in this situation ( Fig. 1 ), as in the absence of other anomalies or intrauterine fetal growth restriction (IUFGR), further testing for chromosomal abnormalities is not indicated. When seen in isolation in a fetus confirmed to be male, the diagnosis is most likely to be hypospadias ( Table 2 ), and the parents should be referred to a paediatric urologist for further discussion of management and prognosis. A small risk of an underlying abnormality of steroid biosynthesis remains ( Table 2 ), but these are rare and difficult to diagnose in cases arising de-novo in the prenatal period as, although molecular testing is available, it has a relatively low yield in the absence of a family history . If cffDNA testing suggests that the fetus is female, there is a greater risk of an underlying genetic abnormality of steroid biosynthesis, and the parents are best referred to the disorders of sexual development team for further investigation and management ( Table 2 ). In this situation, invasive testing may be required for more accurate evaluation of steroid profiles .
| Genital anomaly | Other sonographic findings | cffDNA testing | Differential diagnosis | Other aids to prenatal diagnosis | Management |
|---|---|---|---|---|---|
| Ambiguous genitalia | None. | Male. | Isolated hypospadias. Inadequate production of testosterone caused by Leydig cell hypoplasia or biosynthetic defects.
5α-reductase deficiency. True hermaphrodite. | Steroid profile. a Consider sequencing of the androgen receptor gene. a | Refer to DSD team for investigation and counselling. |
| Ambiguous genitalia | None. | Female. | Isolated cliteromegaly. Congenital adrenal hyperplasia
Maternally derived androgens (e.g. luteoma of pregnancy). Placental aromatase deficiency. | Amniotic steroid levels. a Maternal serum androgen levels. a Maternal urinary oestrogen levels. Maternal ovarian scan for multicystic change. | Refer to DSD team for investigation and counselling. Refer to gynaecology or oncology if luteoma. |
| Ambiguous genitalia or hypospadias | Biometry ≤3rd percentile. Abnormal maternal and fetal Dopplers | Male. | Isolated hypospadias with intrauterine growth restriction. Aneuploidy. Confined placental mosaicism. | Fetal biometry. cfDNA or invasive testing to exclude aneuploidy | Serial monitoring by high-risk pregnancy team. Urology team for management of hypospadias counselling. |
| No penis seen, splayed glans and micropenis | No intra-abdomina; bladder. Low cord insertion. | Male. | Bladder exstrophy. Cloacal exstrophy. | Detailed anomaly scan. | Refer to urologists and paediatric surgeons for counselling. |
| Abnormal genitalia | Absent bladder, intra-abdominal cystic mass, dilated bowel, and abnormal spine. | Female. | Cloacal exstrophy. Other cloacal abnormality. Aneuploidy. | Detailed anomaly scan. cfDNA snf invasive testing to exclude aneuploidy. | Refer to combined fetal-urology team for counselling. |
| Abnormal genitalia | Omphalocoele and gastroschisis, abnormal spine, absent bladder, and hydronephrosis | Male or female. | OEIS complex (omphalocoele, bladder exstrophy, imperforate anus, spinal defects). Cloacal abnormality. Aneuploidy. | cfDNA and invasive testing to exclude aneuploidy. | Refer to paediatric surgeons and urologists. |
| Abnormal genitalia or micropenis | Echogenic kidneys, with or without polydactyly | Male. | Bardet–Biedl syndrome. Trisomy 13. | cfDNA and invasive testing to exclude aneuploidy. | Autosomal recessive: family history for affected members and consanguinity. Refer to clinical genetics. Refer to urology and nephrology teams for counselling. |
| Ambiguous or female appearing genitalia | Multiple anomalies, cardiac anomaly, polysyndactyly, intrauterine fetal growth restriction, oedema, cleft lip, central nervous system anomalies, microcephaly, and short limbs. | Male. | Smith–Lemli–Opitz syndrome. Cranio–cerebellar–cardiac syndrome. Short-ribbed polydactyly syndromes. Other genetic syndrome. Aneuploidy. | cfDNA and invasive testing to exclude aneuploidy. Detailed scan. Maternal urinary steroids levels. Mostly autosomal recessive, take family history for affected members and consanguinity. | Refer to clinical geneticist. Refer to all relevant paediatric teams for discussion of prognosis. |
| Ambiguous or female appearing genitalia | Bowing of femora, with or without tibia and fibula. Micrognathia, cardiac anomalies. | Male. | Campomelic dysplasia. | Detailed scan. | Refer clinical geneticist and skeletal dysplasia clinic. |
| Genotype discordant with phenotype | None. | Male. | Laboratory or clerical error. Androgen insensitivity syndrome. | cfDNA and invasive testing to confirm discordance between genotype and phenotype. b | Refer specialist DSD team. |
a Investigations such as steroid profiling, sequencing of androgen receptor gene, or both, are best managed by a DSD team.
b In situations where there is an abnormality in the SRY gene (i.e. as androgen insensitivity syndrome), cffDNA using SRY may give misleading results. cffDNA, cell-free fetal DNA; DSD, disorders of sex development.
Genital anomalies and intrauterine fetal growth restriction
Sonographically detected genital anomalies and ambiguities in a fetus with intrauterine fetal growth restriction (IUFGR), as shown by biometry on or below the third percentile, with or without abnormal maternal and fetal Doppler measurements, is not uncommon . Determination of fetal sex using cffDNA can be a useful aid to diagnosis, and adds weight to the diagnosis of IUFGR. The non-invasive approach can be particularly useful in situations where parents are keen to avoid the risks associated with invasive testing. Uniparental disomy or confined placental mosaicism have been implicated as possible causes, but aneuploidy-related or idiopathic IUFGR are more common. In view of the risk of chromosomal abnormalities, analysis of cfDNA could include testing for aneuploidy (see section on non-invasive prenatal testing for aneuploidy), but currently this will only detect the major trisomies. Therefore, invasive testing and microarray analysis may be more appropriate, as this will detect a wider range of chromosomal rearrangements .
Genital anomalies in association with urogenital tract abnormalities
Genital abnormalities are most commonly identified in association with other fetal abnormalities . In this situation, the underlying pathology can be broad, with the two main categories being a urogenital tract anomaly or genetic syndrome ( Table 2 ). Probably the most common association is with abnormalities of development of the urogenital tract, including bladder and cloacal exstrophy. Here, knowledge of genetic sex aids prenatal counselling, as the prognosis for these conditions, and hence prenatal counselling, varies for affected males and females . Accurate sonographic gender assignment in these conditions is rarely achieved because of the involvement of external genitalia . As an association with aneuploidy is uncommon in the absence of extra-renal anomalies, fetal sex determination using cffDNA is appropriate in these situations, facilitating accurate prenatal counselling without recourse to unnecessary invasive testing.
Genital anomalies in association with other fetal abnormalities
The association of genital anomalies with genetic syndromes is broad. There are more than 350 syndromes with hypospadias or micro-penis and around 40 with cliteromegaly . In many instances, exclusion of aneuploidy may be the most appropriate first-line investigation in fetuses with multiple abnormalities; however, in some cases, accurate determination of fetal genetic sex using cffDNA can be instrumental in arriving at a definitive diagnosis without recourse to invasive testing ( Table 2 ). Examples include Smith–Lemli–Opitz (SLO) syndrome, Bardet–Biedl syndrome, and campomelic dysplasia.
Smith Lemli Opitz syndrome is a genetic syndrome, inherited in an autosomal recessive fashion, which results in an abnormality in cholesterol biosynthesis owing to a mutation in the 7-dehydrocholesterol reductase ( 7DHC ) gene. The clinical spectrum of SLO is wide, varying from developmental delay and mild dysmorphic features to those with major structural defects with early or prenatal lethality . The severe form usually presents prenatally with a wide variety of fetal abnormalities , including postaxial polydactyly, facial clefts, anomalies of the brain, heart and renal tract, hydrops, and genital anomalies, which are reported to occur in around 90% of affected males . Fetal sex determination in the presence of this spectrum of abnormalities in association with genital ambiguity can increase suspicion of SLO and direct investigations towards analysis of maternal urine for measurement of dehydro-oestriol and dehydropregnanetriol in maternal urine rather than testing for aneuploidy.
Another genetic syndrome where fetal sex determination using cffDNA may be of value in coming to a definitive diagnosis is Bardet–Biedl syndrome. This is a rare condition, usually inherited in an autosomal recessive fashion and characterised by obesity, developmental delay, polydactyly, genital anomalies, and renal failure in some cases . In our unit, we have seen cases presenting with large echogenic kidneys, polydactyly, and abnormal external genitalia ( Fig. 2 ), features compatible with trisomy 13, except that fetal growth was normal. Determination of male sex after analysis of cffDNA helped arrive at a probable diagnosis of Bardet–Biedl syndrome. Definitive diagnosis in cases at low prior risk with no family history currently requires molecular genetic analysis. Although it is becoming possible using next-generation sequencing to screen for mutations in the multiple genes responsible for this condition , it remains difficult in low-risk cases presenting de-novo in pregnancy. The development of comprehensive gene panels, however, may make rapid, definitive prenatal diagnosis possible in the near future. Furthermore, given the advances in NIPD, this may become possible by analysing cell-free DNA in maternal plasma .
