Prenatal Diagnosis
Prenatal diagnosis employs a variety of techniques to determine the health and condition of an unborn fetus.
Without knowledge gained by prenatal diagnosis, there could be an untoward outcome for the fetus or the mother or
both. Congenital anomalies account for 20 to 25% of perinatal deaths. Specifically, prenatal diagnosis is helpful for:
Managing the remaining weeks of the pregnancy
Determining the outcome of the pregnancy
Planning for possible complications with the birth process
Planning for problems that may occur in the newborn infant
Deciding whether to continue the pregnancy
Finding conditions that may affect future pregnancies
There are a variety of non-invasive and invasive techniques available for prenatal diagnosis. Each of them
can be applied only during specific time periods during the pregnancy for greatest utility. The techniques
employed for prenatal diagnosis include:
Ultrasonography
Amniocentesis
Chorionic villus sampling
Fetal blood cells in maternal blood
Maternal serum alpha-fetoprotein
Maternal serum beta-HCG
Maternal serum estriol
Ultrasonography
This is a non-invasive procedure that is harmless to both the fetus and the mother. High frequency sound
waves are utilized to produce visible images from the pattern of the echos made by different tissues and
organs, including the baby in the amniotic cavity. The developing embryo can first be visualized at about 6
weeks gestation. Recognition of the major internal organs and extremities to determine if any are abnormal
can best be accomplished between 16 to 20 weeks gestation.
Although an ultrasound examination can be quite useful to determine the size and position of the fetus, the
size and position of the placenta, the amount of amniotic fluid, and the appearance of fetal anatomy, there
are limitations to this procedure. Subtle abnormalities may not be detected until later in pregnancy, or may
not be detected at all. A good example of this is Down syndrome (trisomy 21) where the morphologic
abnormalities are often not marked, but only subtle, such as nuchal thickening.
Amniocentesis
This is an invasive procedure in which a needle is passed through the mother's lower abdomen into the
amniotic cavity inside the uterus. Enough amniotic fluid is present for this to be accomplished starting
about 14 weeks gestation. For prenatal diagnosis, most amniocenteses are performed between 14 and 20
weeks gestation. However, an ultrasound examination always proceeds amniocentesis in order to
determine gestational age, the position of the fetus and placenta, and determine if enough amniotic fluid is
present. Within the amniotic fluid are fetal cells (mostly derived from fetal skin) which can be grown in
culture for chromosome analysis, biochemical analysis, and molecular biologic analysis.
In the third trimester of pregnancy, the amniotic fluid can be analyzed for determination of fetal lung
maturity. This is important when the fetus is below 35 to 36 weeks gestation, because the lungs may not be
mature enough to sustain life. This is because the lungs are not producing enough surfactant. After birth,
the infant will develop respiratory distress syndrome from hyaline membrane disease. The amniotic fluid
can be analyzed by fluorescence polarization (fpol), for lecithin:sphingomyelin (LS) ration, and/or for
phosphatidyl glycerol (PG).
Risks with amniocentesis are uncommon, but include fetal loss and maternal Rh sensitization. The
increased risk for fetal mortality following amniocentesis is about 0.5% above what would normally be
expected. Rh negative mothers can be treated with RhoGam. Contamination of fluid from amniocentesis
by maternal cells is highly unlikely. If oligohydramnios is present, then amniotic fluid cannot be obtained.
It is sometimes possible to instill saline into the amniotic cavity and then remove fluid for analysis.
Chorionic Villus Sampling (CVS)
In this procedure, a catheter is passed via the vagina through the cervix and into the uterus to the
developing placenta under ultrasound guidance. Alternative approaches are transvaginal and
transabdominal. The introduction of the catheter allows sampling of cells from the placental chorionic
villi. These cells can then be analyzed by a variety of techniques. The most common test employed on
cells obtained by CVS is chromosome analysis to determine the karyotype of the fetus. The cells can also
be grown in culture for biochemical or molecular biologic analysis. CVS can be safely performed between
9.5 and 12.5 weeks gestation.
CVS has the disadvantage of being an invasive procedure, and it has a small but significant rate of
morbidity for the fetus; this loss rate is about 0.5 to 1% higher than for women undergoing amniocentesis.
Rarely, CVS can be associated with limb defects in the fetus. The possibility of maternal Rh sensitization
is present. There is also the possibility that maternal blood cells in the developing placenta will be sampled
instead of fetal cells and confound chromosome analysis.
Maternal blood sampling for fetal DNA
This technique makes use of the phenomenon of fetal blood cells gaining access to maternal circulation
through the placental villi. Ordinarily, only a very small number of fetal cells or cell free DNA enter the
maternal circulation in this fashion (not enough to produce a positive Kleihauer-Betke test for fetal-
maternal hemorrhage). The sequencing of maternal plasma cell-free DNA (cfDNA testing) can detect fetal
autosomal aneuploidy, but without the risks that invasive procedures inherently have. Fluorescence in-situ
hybridization (FISH) is another technique that can be applied to identify particular chromosomes of the
fetal cells recovered from maternal blood and diagnose aneuploid conditions such as the trisomies and
monosomy X.
The problem with this technique is that it is difficult to get large amounts of fetal DNA. There may not be
enough to reliably determine anomalies of the fetal karyotype or assay for other abnormalities.
Maternal serum alpha-fetoprotein (MSAFP)
The developing fetus has two major blood proteins--albumin and alpha-fetoprotein (AFP). Since adults
typically have only albumin in their blood, the MSAFP test can be utilized to determine the levels of AFP
from the fetus. Ordinarily, only a small amount of AFP gains access to the amniotic fluid and crosses the
placenta to mother's blood. However, when there is a neural tube defect in the fetus, from failure of part of
the embryologic neural tube to close, then there is a means for escape of more AFP into the amniotic fluid.
Neural tube defects include anencephaly (failure of closure at the cranial end of the neural tube) and spina
bifida (failure of closure at the caudal end of the neural tube). The incidence of such defects is abbout 1 to
2 births per 1000 in the United States. Also, if there is an omphalocele or gastroschisis (both are defects in
the fetal abdominal wall), the AFP from the fetus will end up in maternal blood in higher amounts.
In order for the MSAFP test to have the greates utility, the gestational age must be known with certainty.
This is because the amount of MSAFP increasses with gestational age (as the fetus and the amount of AFP
produced increase in size). Also, the race of the mother and presence of gestational diabetes are important
to know, because the MSAFP can be affected by these factors. The MSAFP is typically reported as
multiples of the mean (MoM). The greater the MoM, the more likely a defect is present. The MSAFP has
the greatest sensitivity between 16 and 18 weeks gestation, but can still be useful between 15 and 22
weeks gestation.
However, the MSAFP can be elevated for a variety of reasons which are not related to fetal neural tube or
abdominal wall defects, so this test is not 100% specific. The most common cause for an elevated MSAFP
is a wrong estimation of the gestational age of the fetus.
Using a combination of MSAFP screening and ultrasonography, almost all cases of anencephaly can be
found and most cases of spina bifida. Neural tube defects can be distinguished from other fetal defects
(such as abdominal wall defects) by use of the acetylcholinesterase test performed on amniotic fluid
obtained by amniocentesis--if the acetylcholinesterase is elevated along with MSAFP then a neural tube
defect is likely. If the acetylcholinesterase is not detectable, then some other fetal defect is suggested.
NOTE: The genetic polymorphisms due to mutations in the methylene tetrahydrofolate reductase gene
may increase the risk for NTDs. Folate is a cofactor for this enzyme, which is part of the pathway of
homocysteine metabolism in cells. The C677T and the A1298C mutations are associated with elevated
maternal homocysteine concentrations and an increased risk for NTDs in fetuses. Prevention of many
neural tube defects can be accomplished by supplementation of the maternal diet with only 4 mg of folic
acid per day, but this vitamin supplement must be taken a month before conception and through the first
trimester.
The MSAFP can also be useful in screening for Down syndrome and other trisomies. The MSAFP tends to
be lower when Down syndrome or other chromosomal abnormalities is present.
Maternal serum beta-HCG
This test is most commonly used as a test for pregnancy. Beginning at about a week following conception
and implantation of the developing embryo into the uterus, the trophoblast will produce enough detectable
beta-HCG (the beta subunit of human chorionic gonadotropin) to diagnose pregnancy. Thus, by the time
the first menstrual period is missed, the beta-HCG will virtually always be elevated enough to provide a
positive pregnancy test. The beta-HCG can also be quantified in serum from maternal blood, and this can
be useful early in pregnancy when threatened abortion or ectopic pregnancy is suspected, because the
amount of beta-HCG will be lower than expected.
Later in pregnancy, in the middle to late second trimester, the beta-HCG can be used in conjunction with
the MSAFP to screen for chromosomal abnormalities, and Down syndrome in particular. An elevated
beta-HCG coupled with a decreased MSAFP suggests Down syndrome.
Very high levels of HCG suggest trophoblastic disease (molar pregnancy). The absence of a fetus on
ultrasonography along with an elevated HCG suggests a hydatidiform mole. The HCG level can be used to
follow up treatment for molar pregnancy to make sure that no trophoblastic disease, such as a
choriocarcinoma, persists.
Maternal serum estriol
The amount of estriol in maternal serum is dependent upon a viable fetus, a properly functioning placenta,
and maternal well-being. The substrate for estriol begins as dehydroepiandrosterone (DHEA) made by the
fetal adrenal glands. This is further metabolized in the placenta to estriol. The estriol crosses to the
maternal circulation and is excreted by the maternal kidney in urine or by the maternal liver in the bile.
The measurement of serial estriol levels in the third trimester will give an indication of general well-being
of the fetus. If the estriol level drops, then the fetus is threatened and delivery may be necessary
emergently. Estriol tends to be lower when Down syndrome is present and when there is adrenal
hypoplasia with anencephaly.
Inhibin-A
Inhibin is secreted by the placenta and the corpus luteum. Inhibin-A can be measured in maternal serum.
An increased level of inhibin-A is associated with an increased risk for trisomy 21. A high inhibin-A may
be associated with a risk for preterm delivery.
Pregnancy-associated plasma protein A (PAPP-A)
Low levels of PAPP-A as measured in maternal serum during the first trimester may be associated with
fetal chromosomal anomalies including trisomies 13, 18, and 21. In addition, low PAPP-A levels in the
first trimester may predict an adverse pregnancy outcome, including a small for gestational age (SGA)
baby or stillbirth. A high PAPP-A level may predict a large for gestational age (LGA) baby.
"Triple" or "Quadruple" screen
Combining the maternal serum assays may aid in increasing the sensitivity and specificity of detection for
fetal abnormalities. The classic test is the "triple screen" for alpha-fetoprotein (MSAFP), beta-HCG, and
estriol (uE3). The "quadruple screen" adds inhibin-A.
Condition
MSAFP
uE3
HCG
Neural tube defect
Increased
Normal
Normal
Trisomy 21
Low
Low
Increased
Trisomy 18
Low
Low
Low
Molar pregnancy
Low
Low
Very High
Multiple gestation
Increased
Normal
Increased
Fetal death (stillbirth)
Increased
Low
Low
Note: the levels of these analytes change markedly during pregnancy, so interpretation of the
measurements depends greatly upon knowing the proper gestational age. Otherwise, results can be
misinterpreted.
Techniques for Pathologic Examination
A variety of methods can be employed for analysis of fetal and placental tissues:
Gross Examination
The most important procedure to perform is simply to look at the fetus or fetal parts. Obviously,
examination of an intact fetus is most useful, though information can still be gained from
examination of fetal parts.
The pattern of gross abnormalities can often suggest a possible chromosomal abnormality or a
syndrome. Abnormalities can often be quite subtle, particularly the earlier the gestational age.
Consultations are obtained with clinical geneticists to review the findings. A description of the
findings is put into a report (surgical pathology or autopsy).
Examination of the placenta is very important, because the reason for the fetal loss may be a
placental problem.
Microscopic Examination
Microscopic findings are generally less useful than gross examination for the fetus, but
microscopic examination of the placenta is important. Microscopy can aid in determination of
gestational age (lung, kidney maturity), presence of infection, presence of neoplasia, or presence of
"dysplasia" (abnormal organogenesis).
Radiography
Standard anterior-posterior and lateral radiographic views are essential for analysis of the fetal
skeleton. Radiographs are useful for comparison with prenatal ultrasound, and help define
anomalies when autopsy consent is limited, or can help to determine sites to be examined
microscopically. Conditions diagnosed by postmortem radiography may include:
Skeletal anomalies (dwarfism, dysplasia, sirenomelia, etc.)
Neural tube defects (anencephaly, iniencephaly, spina bifida, etc.)
Osteogenesis imperfecta (osteopenia, fractures)
Soft tissue changes (hydrops, hygroma, etc.)
Teratomas or other neoplasms
Growth retardation
Orientation and audit of fetal parts (with D&E specimens)
Assessment of catheter or therapeutic device placement
Microbiologic Culture
Culture can aid in diagnosis or confirmation of congenital infections. Examples of congenital
infection include:
T - toxoplasmosis
O - other, such as Listeria monocytogenes, group B streptococcus, syphilis
R - rubella
C - cytomegalovirus
H - herpes simplex or human immunodeficiency virus (HIV)
Cultures have to be appropriately obtained with the proper media and sent with the proper
requisitions ("routine" includes aerobic and anaerobic bacteria; fungal and viral cultures must be
separately ordered).
Viral cultures are difficult and expensive. Separate media and collection procedures may be
necessary depending upon what virus is being sought.
Bacterial contamination can be a problem.
Karyotyping
Tissues must be obtained as fresh as possible for culture and without contamination.
A useful procedure is to wash the tissue samples in sterile saline prior to placing them into cell
culture media.
Tissues with the best chance for growth are those with the least maceration: placenta, lung,
diaphragm.
Obtaining tissue from more than one site can increase the yield by avoiding contamination or by
detection of mosaicism.
FISH (performed on fresh tissue or paraffin blocks)
In addition to karyotyping, fluorescence in situ hybridization (FISH) can be useful. A wide variety
of probes are available. It is useful for detecting aneuploid conditions (trisomies, monosomies).
Fresh cells are desirable, but the method can be applied even to fixed tissues stored in paraffin
blocks, though working with paraffin blocks is much more time consuming and interpretation can
be difficult. The ability to use FISH on paraffin blocks means that archival tissues can be examined
in cases where karyotyping was not performed, or cells didn't grow in culture.
DNA Probes
Fetal cells obtained via amniocentesis or CVS can be analyzed by probes specific for DNA
sequences. One method employs restriction fragment length polymorphism (RFLP) analysis. This
method is useful for detection of mutations involving genes that are closely linked to the DNA
restriction fragments generated by the action of an endonuclease. The DNA of family members is
analyzed to determine differences by RFLP analysis.
In some cases, if the DNA sequence of a gene is known, a probe to a DNA sequence specific for a
genetic marker is available, and the polymerase chain reaction (PCR) technique can be applied for
diagnosis.
There are many genetic diseases, but only in a minority have particular genes been identified, and
tests to detect them have been developed in some of these. Thus, it is not possible to detect all
genetic diseases. Moreover, testing is confounded by the presence of different mutations in the
same gene, making testing more complex.
Biochemical Analysis
Tissues can be obtained for cell culture or for extraction of compounds that can aid in identification
of inborn errors of metabolism. Examples include:
long-chain fatty acids (adrenoleukodystrophy)
amino acids (aminoacidurias)
Overview of Fetal-Placental Abnormalities
Chromosomal Abnormalities
The risk for chromosomal abnormalities increases with increasing maternal age, mainly because
non-dysjunctional events in meiosis are more likely, and result in trisomies. The table below
indicates the relative risk of having a baby with various trisomies based upon maternal age:
Maternal Age Trisomy 21 Trisomy 18 Trisomy 13
15 - 19
1:1600
1:17000
1:33000
20 - 24
1:1400
1:14000
1:25000
25 - 29
1:1100
1:11000
1:20000
30 - 34
1:700
1:7100
1:14000
35 - 39
1:240
1:2400
1:4800
40 - 44
1:70
1:700
1:1600
45 - 49
1:20
1:650
1:1500
Listed below are some of the more common chromosomal abnormalities that can occur. The
descriptions are for the completely abnormal condition in which all fetal cells contain the abnormal
karyotype.
Bear in mind that "mosaicism" can occur. A "mosaic" is a person with a combination of two cell
lines with different karyotypes (normal and abnormal). When karyotyping is performed, multiple
cells are analyzed to rule out this possibility. An example would be a Turner's mosaic, with a
45,X/46,XX karyotype, with some cells having the abnormal karyotype and some cells having a
normal karyotype. The mosaic condition is not as severe as the completely abnormal karyotype,
and the features may not be as marked, and livebirths may be possible. Sometimes the mosaicism
is confined to the placenta ("confined placental mosaicism").
A placenta with an abnormal karyotype (confined placental mosaicism) may lead to stillbirth, even
though the fetus has a normal karyotype; conversely, a placenta with a normal karyotype may
allow longer survival for a fetus with a chromosomal abnormality. Rarely, a translocation of part of
one chromosome to another in the parent will be passed on to the child as a partial trisomy (such as
6p+ or 16p+) which may not be as severe as a complete trisomy.
Trisomy 21: Down syndrome; incidence based upon maternal age, though translocation type is
familial; features can include: epicanthal folds, simian crease, brachycephaly, cardiac defects.
Trisomy 18: Features include micrognathia, overlapping fingers, horseshoe kidney, rocker bottom
feet, cardiac defects, diapragmatic hernia, omphalocele.
Trisomy 13: Features include microcephaly, cleft lip and/or palate, polydactyly, cardiac defects,
holoprosencephaly.
Trisomy 16: Seen in abortuses from first trimester. Never liveborn.
Monosomy X: Turner's syndrome; can survive to adulthood; features include short stature, cystic
hygroma of neck (leading to webbing), infertility, coarctation.
Monosomy X, or Turner's syndrome (45, X) karyotype, diagram
Monosomy X, or Turner's syndrome, streak ovaries in adult, gross
Massive fetal hydrops with monosomy X, or Turner's syndrome, gross
Cystic hygroma with monosomy X, or Turner's syndrome, gross
XXY: Klinefelter's syndrome; features include elongated lower body, gynecomastia, testicular
atrophy (incidence: 1/500 males)
Klinefelters' syndrome karyotype, diagram
Triploidy: There is often a partial hydatidiform mole of placenta. Fetal features include 3-4
syndactyly, indented nasal bridge, small size.
Partial hydatidiform mole, gross
3-4 syndactyly with triploidy, gross
A host of other chromosomal abnormalites are possible. In general, fetal loss earlier in gestation,
and multiple fetal losses, more strongly suggests a possible chromosomal abnormality.
Neural Tube Defects
The maternal serum alpha-fetoprotein (MSAFP) is useful for screening for neural tube defects, but
the gestational age must be known for proper interpretation. The frequency of neural tube defects
has been shown to be reduced if women supplement their diet with folic acid (before and during
pregnancy).
Anencephaly: There is absence of the fetal cranial vault, so no cerebral hemispheres develop.
Anencephaly is the most common congenital malformation--about 0.5 to 2/1000 live births. Other
neural tube defects are as frequent, but the incidence varies with geography.
Iniencephaly: Imperfect formation of the base of the skull, with rachischisis and exaggerated
lordosis of the spine.
Exencephaly: Incomplete cranial vault, but the brain is present.
Meningomyelocele: Defect in the vertebral column allowing herniation of meniges and spinal cord;
location and size determine severity.
Encephalocele: Herniation of brain through a skull defect.
Occipital encephalocele, radiograph
Occipital encephalocele with iniencephaly, gross
Spina bifida: A defective closure of the posterior vertebral column. It may not be open (spina bifida
occulta).
Hydrops Fetalis
There are many causes for fetal hydrops, and in about 25 to 30% of cases, no specific cause for
hydrops can be identified. Multiple congenital anomalies can also be associated with hydrops,
though the mechanism is obscure for everything except cardiac anomalies that produce heart
failure.
Hydrops can be classified as immune and non-immune. Immune causes such as Rh incompatibility
between mother and fetus are now uncommon. Non-immune causes can include:
Congenital infections
Cardiac anomalies
Chromosomal abnormalities
Fetal neoplasms
Twin pregnancy
Fetal anemia
Other anomalies (pulmonary, renal, gastrointestinal)
Congenital Infections
The hallmark of congenital infections is fetal hydrops along with organomegaly. Diagnosis can
depend upon:
TORCH titers
Tissue culture
Congenital Neoplasms
Such tumors are uncommon, but those that are seen most frequently include:
Teratoma. These tumors occur in midline regions (sacrococcygeal, cerebral, nasopharyngeal).
Nasopharyngeal teratoma, gross
Teratoma, low power microscopic
Immature teratoma, medium power microscopic
Skeletal Abnormalities
Ultrasound may reveal long bones that are shortened. There are several possibilities, including
short-limbed dwarfism, osteogenesis imperfecta, and short rib-polydactyly syndrome
Achondroplasia is a form of short-limbed dwarfism that is inherited in an autosomal dominant
fashion, though in most cases there is no affected parent and the disease is due to a new mutation.
The homozygous form of the disease is lethal. The heterozygous form is not lethal, and affected
persons can live a normal life. They have short extremities, but a relatively normal sized thorax and
normal sized head.
Osteogenesis imperfecta occurs in several forms. There is a lethal perinatal form in which fractures
appear in long bones even in utero. This condition is due to an abnormal synthesis of type 1
collagen that forms connective tissues, including bone matrix.
Placental Abnormalities
Abruptio placenta: Premature separation of the placenta near term, with retroplacental blood clot.
Placenta previa: Low-lying implantation site can lead to hemorrhage during delivery.
Velamenous insertion: Cord vessels splay out in the membranes before reaching the placental disk
and predispose to traumatic rupture.
Long - short cord: Umbilical cord length is determined by the amount of fetal movement. More
movement increases cord length. A long cord can become entangled with the baby or more easily
prolapse.
True knot of umbilical cord, gross
Twin placenta: Monozygous twinning is associated with increased risk for both abnormalities and
accidents. A twin-twin transfusion syndrome can occur when a vascular anastomosis is present
Vascular anastomosis in placenta, gross
Vascular anastomosis in placenta, gross
Hypertension: Vascular changes can be associated with pregnancy-induced hypertension (PIH) and
the more severe complications of eclampsia and pre-eclampsia.