Pregnancy and antenatal screening
Pregnancy is associated with many hormonal, physiological and metabolic changes. This topic considers how the results of biochemical tests may be affected, how they may help in the diagnosis and management of some complications of pregnancy, and how biochemical tests are applied in antenatal screening programmes for the identification of pregnancies at risk of foetal NTDs and trisomy 21.
The foetoplacental unit
The placenta produces several proteins, including hCG and (human) placental lactogen (hPL). It also produces large amounts of steroid hormones and is the main source of progesterone during pregnancy.
Human chorionic gonadotropin
There are several pregnancy-specific proteins, all of which normally originate in the trophoblast. The most commonly measured is the hCG. Following synthesis, hCG is secreted into the maternal circulation. There is a surge in maternal [hCG] in early pregnancy, peak blood levels being reached at 12 weeks; thereafter, production hCG rapidly declines. hCG becomes detectable in urine about 10 days after conception and this forms the basis of readily available pregnancy tests.
Trophoblastic tumours secrete hCG. These tumours can occur in males and females, and they include hydatidiform mole and choriocarcinoma, both of which may secrete hCG in very large amounts. A female who is found to be excreting hCG, and who is not pregnant, most frequently has a tumour of the trophoblast; in males, testicular teratoma is the commonest source.
Steroids in pregnancy
Oestrogens and progesterone are secreted by the corpus luteum during the first 6 weeks of pregnancy, but after this the placenta is the most important source of these steroids. Oestriol is the oestrogen produced in the greatest amounts, but oestradiol-17ß and oestrone are also produced in large amounts. The placenta cannot synthesise oestriol de novo, but it can produce oestriol from C-19 adrenal steroids that are supplied by the foetal adrenal in the form of dehydroepiandros-terone sulphate. The oestriol produced in this way is secreted into the maternal and fetal circulation. Oestriol production thus requires the involvement of both the placenta and the foetus, and recognition of this interdependence led to the concept of the foetoplacental unit.
Effect of pregnancy on biochemical tests
Plasma [prolactin], [oestrogens] and [testosterone] show a steady increase in pregnancy, as does the concentration of SHBG. The concentrations of growth hormone and the pituitary gonadotrophins are decreased. However, some less-specific methods for the measurement of LH may show cross-reaction with hCG, leading to apparent high LH levels.
There are large increases in serum [Cortisol] due to increased plasma [cortisol-binding globulin (CBG)], but the diurnal rhythm is retained. However, increased free and total Cortisol levels in pregnancy may also be related to resetting of the sensitivity of the hypothalamic-pituitary-adrenal axis and not merely to raised levels of CBG, progesterone or corticotropin-releasing hormone. There is also an increase in serum [free Cortisol] and in the 24-h urinary excretion of Cortisol. This may be related to a resetting of the HPA axis and also the production of an ACTH-like substance by the placenta that is not completely suppressible by low- or high-dose glucocorticoids such as dexa-methasone. This may help to explain why pregnant women often show intolerance of glucose and occasionally develop Cushingoid features. These changes make the diagnosis of Cushing's syndrome difficult in pregnancy, and several variations in the workup, when compared with non-pregnant women, may be required. An absence of diurnal variation is a useful clue to the diagnosis.
Thyroid function tests
During pregnancy, oestrogen production increases and TBG concentrations rise, leading to an increase in total T4 and total T3. There is also a large increase in the concentration of hCG, a hormone that has a mild stimulatory effect on thyroid hormone production. As a consequence, free T4 and free T3 concentration may increase slightly during the early part of the first trimester which, through the normal negative feedback loop, leads to a fall in serum TSH sometimes to undetectable concentrations. In the second and third trimesters,
Figure 1 Changes in TSH, thyroid hormones, hCG and TBG in normal pregnancy. For TSH and thyroid hormones it is important to use gestational or trimester-related reference ranges. In some pregnancies TSH may fall to <0.1 mU/L in the first trimester. Total T3 and free T3 follow the same pattern as total T4 and free T4 respectively.
the serum free T4 and free T3 concentrations decrease and may fall below the reference range derived from non-pregnant women (Figure 1). The magnitude of this fall in free thyroid hormones is method-dependent. After delivery, levels of thyroid hormones and TSH normally return to the pre-pregnant state. Trimester-related reference ranges should be applied for TSH and for total and free thyroid hormones if these are available; for free hormones, these ranges are also method-dependent
Plasma volume and renal function
During pregnancy, the plasma volume and GFR increases, sometimes by as much as 50%. This is accompanied by decreases in, for example, plasma [Na+], [urea] and [creatinine].
Plasma lipids and proteins
Plasma [triglyceride] may increase as much as 3-fold in pregnancy; plasma [cholesterol], LDL and high-density lipoprotein (HDL) increase to a lesser extent. Plasma [albumin] and [prealbumin] fall because of the increase in plasma volume. Plasma [fibrinogen] and [ceruloplasmin] increase.
Alkaline phosphatase (ALP)
In pregnancy, the placental isoenzyme is released, and total ALP activity may rise to as much as three times non-pregnant levels.
Iron and ferritin
During pregnancy, increased maternal red cell synthesis and transfer of iron to the developing foetus cause a greater demand for iron. Unless iron supplements are given, iron stores generally fall, with accompanying falls in plasma [ferritin], plasma [iron] and rises in plasma [transferrin] and TIBC.
Complications in pregnancy
In ectopic pregnancy, plasma [hCG] fails to rise at the normal rate (approximately doubling every 2-3 days). If levels have failed to rise by 66% in 2 days, there is a 90% chance of an abnormal pregnancy. In practice, the diagnosis is made on a high index of clinical suspicion, qualitative pregnancy tests, ultrasound and, if indicated, laparoscopy.
Women with Type I diabetes are at greater risk from both diabetic and obstetric complications during pregnancy. Rates of foetal and neonatal complications including late intrauterine death, foetal distress, congenital malformation, hypoglycaemia, respiratory distress syndrome and jaundice are also increased. To minimise these risks, it is essential that maternal glucose control and HbAlc is optimised prior to conception and that tight control is maintained throughout pregnancy. Particular emphasis is placed on the need for careful home glucose monitoring (4-6 times a day) and intensive insulin regimens. Women should aim to maintain blood glucose and HbAlc concentrations as near to the non-diabetic range as possible without excessive risk of hypoglycaemia. Type II diabetes is less common during the reproductive years, but its management during pregnancy should follow the same intensive pattern.
'Gestational diabetes mellitus' is the term used to describe the abnormal glucose tolerance or diabetes mellitus that may develop during pregnancy. It is particularly important to identify women with undiagnosed Type I or Type II diabetes mellitus as urgent action is required to normalise metabolism. The diagnosis of gestational diabetes mellitus is made on the basis of an oral GTT. Glucosuria detected at routine antenatal testing may suggest the need for an oral GTT, but may have no significance, since the renal threshold for glucose tends to be lowered in pregnancy. One approach is to screen women with appropriate risk factors, such as a family history of diabetes mellitus, or a previous large baby. Also, glucosuria is more significant if detected on the second specimen of urine passed after an overnight fast (i.e. the first specimen passed is discarded). Mild abnormalities should be reassessed not less than 6 weeks after delivery. In the majority of cases of gestational diabetes, the response to a GTT reverts to normal after the pregnancy, but about 50% of patients go on to develop diabetes mellitus within the next 7 years.
Maintenance of a euthyroid state in the mother is very important during pregnancy. In the first trimester the developing foetal thyroid has little function and maternal thyroid hormone is required for normal foetal neurological development. Increased foetal loss as well as IQ deficits have been reported in infants born to mothers with either undiagnosed or inadequately treated hypothyroidism. There is an increased requirement for T4 in pregnancy, and mothers with hypothyroidism are required to have the dose of T4 increased by 25-50 jxg/day when pregnancy occurs. Adequacy of T4 therapy should be assessed using measurements of both TSH and free T4 during each trimester, and the dose of thyroxine should be adjusted to ensure that TSH lies between 0.4-2.0 mU/L and FT4 concentrations are within the appropriate trimester-related reference ranges. The TSH should be checked 4 weeks post-partum, at which time the dose of thyroxine can usually be reduced back to the pre-pregnancy dose.
Patients treated with anti-thyroid drugs will require the dose to be revised at the diagnosis of pregnancy, as these drugs cross the placenta and may induce foetal hypothyroidism. Frequent monitoring is important and the dose of antithyroid drug should be kept to the minimum consistent with maintaining euthyroidism. Patients on carbimazole may be switched to propylthiouracil which is claimed to have some advantages in the pregnant patient and is preferable during breastfeeding. The aim of therapy should be to maintain free T4 at the upper end of the trimester-related reference range. This is particularly important in the first trimester, when even mild hypothyroidism must be avoided because of the risk to the foetus.
Measurement of TRAbs at antenatal booking can be useful in women who have had a thyoidec-tomy or radioiodine treatment for Graves' disease. Such women may have high titres of TRAbs that can cross the placenta and induce intrauterine or neonatal thyrotoxicosis.
Patients with hyperemesis gravidarum may have thyroid function tests suggestive of hyperthyroidism with a suppressed TSH and increased free T4. This is due to the very high levels of hCG that have a stimulatory action on the maternal thyroid. It is important to exclude Graves' disease in patients with hyperemesis gravidarum; this can be done by measurement of TRAbs which are negative in patients with hyperemesis.
Post-partum thyroiditis occurs in approximately 5% of the population in iodine-replete areas, within 2-6 months after delivery or miscarriage. It gives rise to transient thyroid dysfunction, which is most frequently characterised by a brief thyrotoxic phase followed by hypothyroidism, usually with spontaneous resolution. Women who exhibit symptoms suggestive of post-partum thyroiditis should have TSH and FT4 measured at 6-8 weeks post-partum or post-abortus. If the TSH and free T4 results suggest hyperthyroidism, further tests may be required to differentiate post-partum thyroiditis from Graves' disease (e.g. TRAbs or isotope uptake and scan). If the thyrotoxicosis is secondary to post-partum thyroiditis, treatment is not required but the TFT should be monitored to detect onset of hypothyroidism. If the initial tests indicate hypothyroidism, thyroxine treatment may be started in a symptomatic patient but can be discontinued after about 6 months if the thyroiditis has resolved.
Pre-eclampsia is a major cause of maternal and foetal morbidity and mortality affecting approximately 3% of primagravidae. It usually develops during the third trimester, often after 32 weeks. The biochemical abnormalities that are most commonly of value in the diagnosis of pre-eclampsia are proteinuria, raised plasma creatinine, abnormal liver function tests and a raised plasma urate. These are usually found in association with hypertension. At the antenatal clinic, urine specimens should also be routinely tested for protein. Proteinuria, if detected, may be the first evidence of pre-eclampsia and, as the condition worsens, proteinuria in excess of 1 g/24 h may occur. Patients with preeclampsia may develop impaired renal function with increasing plasma [creatinine] and [urea] as the renal impairment worsens, or as a result of vomiting and dehydration. A plasma [urea] of 7.0 mmol/L should be regarded as definitely abnormal, since plasma [urea] is normally reduced in pregnancy due to the increase in plasma volume.
Impaired renal function causes reduced tubular clearance of urate. Plasma [urate] may be measured to assess the severity of pre-eclampsia and to provide an index of prognosis. A plasma [urate] greater than 0.35 mmol/L before 32 weeks' gestation, or greater than 0.40 mmol/L after 32 weeks, is significantly raised.
Intravascular coagulation and hepatic ischaemia can result in the HELLP (haemolysis, elevated /iver enzymes and tow platelets) syndrome which is seen in 4-12% of women with preeclampsia. Plasma [LDH] may also increase as a result of haemolysis and renal function tests may be abnormal.
Obstetric cholestasis usually occurs in the
third trimester of pregnancy and affects approximately 0.5% of all pregnancies
While a prominent clinical feature is generalised pruritus, itching is common in pregnancy and it is important to distinguish obstetric cholestasis from other forms of liver disease. The most sensitive and important biochemical test is the measurement of serum bile acids which may be elevated by up to 100 times normal. Modest elevations (2-3-fold) in transaminase levels are also observed. There is no correlation between serum bile acid concentrations and foetal outcome.
Pre-natal diagnosis of foetal abnormalities
Fetal chromosomal abnormality
About 2-3% of couples are at high risk of producing offspring with genetic disorders and 5% of the population will have displayed some form of genetic disorder by the age of 25 years.
Particular risk factors are:
•Advanced maternal age (e.g. Down's syndrome)
• Family history of inherited diseases (e.g. fragile X syndrome, Huntington's chorea)
• Previous child with genetic disorder (e.g. Tay-Sachs disease, congenital adrenal hyperplasia).
The techniques for prenatal diagnosis that can be used and the appropriate timings are given in Table 1.
Table 1. Techniques for prenatal diagnosis
Chorionic villus sampling
Chromosomal abnormalities, fetal sexing in X-linked
Inborn errors of metabolism
Haemoglobinopathies, Duchenne muscular dystrophy
AFP, Triple test screening for
High incidence of neural tube defects
Spina bifida, anencephaly, hydrocephaly, cystic renal
disease, renal tract dilatation, exomphalos, gastroschisis,
duodenal atresia, limb abnormalities, cardiac
Heamoglobin studies, fetal viral infection, rhesus disease,
unexplained hydrops and fetal anaemia pH
Here we will focus on screening for Down's syndrome which is characterized by an extra chromosome 21. The overall incidence is 1: 600 live births, but depends on maternal age, being 1:2000 at age 20 and 1:100 atage
Gastrointestinal atresias are common and there is early dementia with similarities to Alzheimer's disease.
Twenty percent die before age 1 but 45% reach age 60.
Antenatal screening for Down's syndrome is possible by measuring levels of serum markers at 15+ weeks - low levels of alpha -fetoprotein (AFP) ± high levels of unconjugated oestriol and human chorionic gonadotrophin (hCG) are corrected for maternal weight and age. This allows ~ 60% of cases of Down's syndrome to be picked up, with amniocentesis required on ~ 4% of the screened population. The pick-up rate is higher in older women, but the chance of being recalled with an elevated risk is also higher. It is therefore not essential
to advise women over the age of 35 years to have an amniocentesis as serum screening is more sensitive in this age group. Fluorescent in situ hybridization (FISH) techniques may be used to exclude the commoner aneuploidies within 72 hours. Routine karyotyping does take up to 3 weeks because of the need to culture cells
first. Screening for open neural tube defects is also carried out by measuring the maternal serum AFP at16 weeks.
Screening for aneuploidy is also possible by measuring the fetal nuchal thickness on first trimester ultrasound.
Sensitivities of 70-90% have been quoted for detecting Down's syndrome, particularly when combined with first trimester serum levels of specific fetal proteins. Increased nuchal translucency is also a marker for structural defects (4% of those >
Both these tests are screening tests for chromosomal problems. This allows selection of a group of mothers who can then be considered for an invasive diagnostic test.
Methods of obtaining tissue
1. Chorionic villus sampling (CVS):
Samples of mesenchymal cells of the chorionic villi are obtained for chromosomal and DNA analysis. The transabdominal technique is now more favoured, as the transcervical technique may give a higher infection and fetal loss rate. Chorionic villus sampling is performed at 11-14 weeks' gestation. A needle is introduced through the maternal abdomen under ultrasound guidance, into the placenta and along the chorionic plate. A sample of the villi is aspirated. Cells from the direct preparation allow preliminary karyotype and DNA analysis within 24 hours, but this is usually confirmed with a cultured preparation as well. Chorionic villus sampling only rarely leads to erroneous results, due to placental mosaicism (placental tissue of different cell lines can be identified from one placenta, e.g. XO, XX) but errors from this can be virtually eliminated providing decisions are deferred until both the direct and culture results are available. Karyotypic discrepancy between fetus and placenta increases with increasing gestation and if rapid results are required over 20 weeks fetal blood sampling or amniocentesis with FISH is preferable
The advantage of chorionic villus sampling is that there is no breach of the amniotic cavity and that it allows an early diagnosis with the option of a suction termination of pregnancy. There is, however, good evidence to suggest that psychological parental morbidity is independent of whether a diagnosis is made in the first or second trimester and indeed medical termination of pregnancy may carry less psychological morbidity than surgical (even if medical complicationsare higher).
Amniocentesis involves withdrawing a sample of amniotic fluid containing fetal cells by passing a needle (using direct ultrasound control) through the maternal abdomen. A karyotype of the fetal cells is obtained. In approximately 98% of cases cell culture will be successful, enabling karyotypic analysis. This is performed from 15 weeks' gestation so that sufficient viable fetal cells can be obtained but at a fetal loss rate of about 1%. Amniocentesis performed in the presence of a raised maternal AFP level appears to be associated with a significant increase in miscarriage rates.
This technique may be used later in pregnancy when a rapid result is required. Often this will be at a later gestation after an ultrasound scan has shown an anomaly that is strongly associated with a genetic defect.
A needle is introduced transabdominally into the umbilical artery or vein. The most stable portion of the cord suitable for this is at the point of insertion. The blood sample obtained can be used for karyotyping and for the diagnosis of other conditions such as haemoglobinopathies, viral infections and metabolic disorders. The disadvantage of cordocentesis is that it requires a highly skilled operator. Complications include fetal haemorrhage, cord haematoma and fetal bradycardia.
Diagnostic tests for chromosome abnormality
Human chromosomes can be examined directly in rapidly dividing tissue. However it is more usual to culture cells and then use colchicine to inhibit the formation of the spindle and arrest cell division at metaphase which allows the preparations that we are familiar with. Chromosomes can then be paired according to their size, position of the
centromere, and the Giemsa stain (this shows a characteristic banding pattern for each chromosome allowing individual identification).
In an increasing number of inherited diseases it is now possible to identify a single gene defect or omission that is
responsible. Fetal cells obtained by the various sampling techniques are cultured and their chromosomal DNA separated. This DNA is digested with restriction enzymes. The resulting fragments are separated by Southern blotting. A radioisotope-labelled DNA probe is then added and autoradiography allows identification of any hybridization. Specific probes are available for sickle cell disease, thalassaemia, and cystic fibrosis.
Fluorescent in situ hybridization:
In situ hybridization permits the analysis of genetic material of a single nucleus, by incubating a fixed dried cell with a specific probe, which binds to the gene of interest. The use of a fluorescent marker tagged to the gene probe leads to the acronym FISH. This technique is sensitive enough to demonstrate each allele on individual chromatids but is not yet reliable enough for single cell analysis so is applied to larger samples. It provides a rapid diagnosis of trisomy, triploidy or sex chromosome problems if appropriate markers are used.
The finding of some abnormality in pregnancy transforms what was previously an exciting and joyous event
into an extremely worrying and distressing time. This remains true even when the potential risks are small; for example being recalled with an abnormal level of a-fetoprotein (AFP), or with the finding of a choroid plexus cyst on routine ultrasound scan. The very greatest of care should be taken in explaining any findings to parents. Tact, understanding and reassurance (if appropriate) are paramount. The advice given to parents is of such importance that it will frequently be necessary to involve senior members of the obstetrics team as well as members of other specialties, particularly paediatricians, clinical geneticists and radiologists.
The aims of prenatal diagnosis are fourfold:
• the identification at an early gestation of abnormalities incompatible with survival, or likely to result in severe handicap, in order to prepare parents and offer the option of termination of pregnancy
•the identification of conditions which may influence the timing, site or mode of delivery
•the identification of fetuses who would benefit from early paediatric intervention
• the identification of fetuses who may benefit from in utero treatment (rare).
It should not be assumed that all parents are going to request termination of pregnancy even in the
presence of lethal abnormality. Many couples have opted to continue pregnancies in the face of severe defects that have resulted in either intrauterine or early neonatal death, and have expressed the view that they
found it easier to cope with grief having held their child. Others say that they were glad of the opportunity to
terminate the pregnancy at an early stage and that they could not have coped with going on. More controversial still are the problems of chronic diseases with long-term handicap and long-term suffering for both the child and its parents. The parents themselves must decide what action they wish to take - it is they who will have to live with the
consequences. It is our role to advise, guide and respect their final wishes, irrespective of our own personal views.
Screening for fetal abnormalities
Structural anomalies are best seen on ultrasound scan and many clinicians advocate that all mothers should be offered at least one detailed ultrasound at around 18-20 weeks or earlier. This has the advantage that previously unsuspected major or lethal anomalies (e.g. spina bifida, renal agenesis) can be offered termination, and it also allows planned deliveries of those conditions which may require early neonatal intervention (e.g. gastroschisis, transposition of the great arteries). It has the disadvantage, however, that many defects are not identified (it is likely that < 50% of cardiac defects are recognized) and the false reassurance provided by this scan may become a source of parental resentment. Furthermore, problems may be uncovered; for example one of the 'soft markers', the natural history of which is uncertain. This may generate unnecessary anxiety and increase the number of invasive diagnostic procedures (and thereby the loss rate) in otherwise healthy pregnancies.
Chromosomal abnormalities are much more difficult to identify on scan. While around two-thirds of fetuses with Down's syndrome will look normal at 18 weeks, most with Edwards' or Patau's syndrome do show some abnormality, even though these are often not specific or diagnostic. In the absence of routine ultrasound scans, it is possible to screen for open neural tube defects by measuring the maternal serum AFP at 16 weeks. AFP is an alpha-globulin of similar molecular weight to albumin, which is synthesized by the fetal liver. Any break in the integrity of the fetus allows the AFP to escape into the maternal circulation and therefore be elevated on serum testing. Those with levels greater than 2.0-2.5 multiples of the median should be recalled for an ultrasound scan, giving a sensitivity for picking up neural tube defects of around 85%. Raised levels are also found following first trimester bleeding, or with intrauterine death (fetal autolysis), abdominal walldefects,
or multiple pregnancy (increased synthesis). Even if the scan is normal, raised AFP is still a marker for later pre eclampsia or intrauterine growth restriction. Increased nuchal translucency (NT) is also a marker for structural defects (4% of those >
Aneuploidy — soft markers
These are structural features found on ultrasound scan which in themselves are not a problem, but which may be
pointers to chromosomal problems.
Examples include choroid plexus cysts, mild renal pelvic dilatation, an echogenic focus in the heart ('golf-ball'), or mild cerebral ventricledilatation. They are found in approximately 5% of all pregnancies in the second trimester and are the cause of a lot of parental anxiety. If isolated, the risk of chromosomal problems is low, but if more than one is found, or if there are any other structural defects, the risk is very much higher.
Congenital heart disease
This is the commonest congenital malformation in children and affects about 5-8:1000 live births. Of defects diagnosed antenatally, about 15% are associated with aneuploidy, most commonly trisomies 18 and 21. The four-chamber view of the heart can be used as a screening test and will identify 25-40% of all major abnormalities, particularly ventricular septal defect, ventricular hypoplasia, valvular incompetence and arrhythmias. In addition, viewing the aorta and pulmonary artery increases the sensitivity to 60+% by screening for Fallet's tetralogy (Fig. 2) and transposition of the great arteries. At 18 weeks most of the majorconnections can be seen, but high-risk pregnancies (e.g. those with diabetes, or taking anticonvulsants, or who have a personal or family history of congenital heart disease) should be re-scanned at 22-26 weeks for moreminor defects.
Neural tube defects
The neural tube is formed from the closing of the neural folds, with both anterior and posterior neuropores closed by 6 weeks' gestation (Fig. 3). Failure of closure of the anterior neuropore results in anencephaly or an encephalocele, and failure of posterior closure in spina bifida.
Anencephaly. The skull vault and cerebral cortex are absent The infant is either stillborn or, if liveborn, will usually die shortly after birth (although some may survive for several days).
Encephalocele. There is a bony defect in the cranial vault through which a dura mater sac ( brain tissue ) protrudes.
Spina bifida In a meningocele, dura and arachnoid mater bulge through the defect, whereas in a myelomeningocele, the central canal of the cord is exposed. Those with spinal meningoceles usually have normal lower limb neurology and 20% have hydrocephalus. Those with myelomeningoceles usually have abnormal lower limb neurology and many have hydrocephalus. In addition to immobility and mental retardation, there may be problems with urinary tract infection (UTI), bladder dysfunction, bowel dysfunction, and social and sexual isolation.
Spina bifida and anencephaly make up more than 95% of neural tube defects. There is wide geographical variation in births with a higher incidence in Scotland and Ireland 3 :1000), and a lower incidence in England (2 :1000), USA, Canada, Japan and Africa (< 1 :1000). There is good evidence that the overall incidence has fallen over the past 15 years (independently of any screening programmes). Daily folic acid taken from before conception reduces the recurrence risk of neural tube defects in those who have had a previously affected child. A pre-conceptual prophylactic dose for all pregnant women probably also offers some protection. There are, at present, no known teratogenic effects from folate. There is an increased incidence of recurrence in subsequent pregnancies.
Increased NT thickness is associated with:
Nuchal Translucency & Chromosome Abnormalities
Determining Risk for Chromosome Abnormalities
Every woman has a risk for having a fetus with a chromosomal abnormality. Three variables are taken into consideration for determining a woman’s risk for having a child with a chromosome abnormality.
The Nuchal Translucnecy normally increases with increasing CRL (crown rump length or size of the fetus from head to rump) and medians have been established for each gestational age.
Using these medians, the risk for trisomies can be calculated by multiplying the background risk based on maternal age and gestational age by a likelihood ratio (LR). The likelihood ratio depends on the degree of deviation in NT thickness from the normal median for that CRL.
When is this test performed?
To obtain risk assessment using Nuchal Translucnecy and age, it is critical that the nuchal translucecny be measure between 11 weeks, 0 days and 13 weeks, 6 days. Measurements are based on the crown rump length (CRL) of the fetus as calculated by the ultrasound machine. Measurements outside this window of time will not generate an individual specific risk.
How do you provide a risk figure?
A computer program calculates risk figures and provides your individual risk for Down syndrome, Trisomy 13 and Trisomy 18. You can take home a paper which indicates the risk of having a child with Down syndrome before and after Nuchal Translucency Screening.
Is Nuchal Translucency Screening standard of care?
No. It is still offered as a research test. It is, however, standard of care (offered to the entire population) in Europe. At present, The Prenatal Diagnosis and Medical Genetics Program offers this test to women over 35 and also to women at low risk. Call us to find out more.
It is important that you get genetic counseling for this test so that you understand what the test is about. Be sure to arrange for an appointment prior to Nuchal Translucency screening.
Nuchal Translucency & Maternal Serum Biochemistry
NT Screening and First Trimester Markers
Two maternal serum markers (chemicals found in the mother's blood) have been found to be effective in screening for fetal Down syndrome in the first trimester:
Maternal serum free-beta human chorionic gonadotropin (hCG)
Maternal serum pregnancy-associated plasma protein (PAPP-A).
When these markers are combined with the first trimester NT measurement and age, the detection efficiency for fetal Down syndrome increases to as high as 87% and a false positive rate of 5%.
Presently, the protocol for this screening test is being standardized and will be offered at The Prenatal Diagnosis and Medical Genetics Program in the near future.
NT Screening and Second Trimester Biochemical Screening
It is important to know that NT screening in the first trimester significantly reduces the positive predictive value of second trimester biochemical screening (e.g., maternal serum screening test, MSS). This is because most chromosome abnormalities would be detected in the first trimester, resulting in a decreased incidence of Down syndrome in ongoing pregnancies.
If the first screen is not "adjusted" for, the result is an increase in the rate of invasive testing without an increase in the detection rate. The value of combining the two screens that are performed at different gestational ages, is currently being investigated in many centres.
Biochemical markers of inborn errors
1. Pregnancy-associated plasma protein A: Abbreviated as PAPPA or PAPP-A.
A large zinc-binding protein that acts as an enzyme, specifically a metallopeptidase. PAPPA has been used in prenatal genetic screening and studies of atherosclerosis. Women with low blood levels of PAPPA at 8 to 14 weeks of gestation have an increased risk of intrauterine growth restriction, trisomy 21, premature delivery, preeclampsia, and stillbirth. PAPPA is present in unstable atherosclerotic plaques, and circulating levels are elevated in acute coronary syndromes which may reflect the instability of the plaques. PAPPA may be a marker of unstable angina and acute myocardial infarction (heart attack).
Pregnancy-associated plasma protein-A (PAPP-A) was first described by Lin et al. in 1974 as a high molecular weight component of serum obtained from individuals in late pregnancy. It has since been shown to be a large, dimeric, zinc-containing metalloglycoprotein with a molecular weight of 800 kDa. Each subunit consists of 1,547 amino acid residues and, in pregnancy, is derived from a larger precursor of placental origin. PAPP-A is produced by the placental syncytiotrophoblast (trophoblastic tissue that develops into the outer layer of the placenta) in an initial proform approximately 80 amino acids longer than the mature subunit.
The biological function of PAPP-A is still unclear. It has been shown to bind heparin and to be a noncompetitive inhibitor of human granulocyte elastase (a tissue-degenerative enzyme released when tissue inflammation occurs), which has led to postulation that it may have a role in modulating the maternal immune response and be associated with implantation and growth of the placenta.
PAPP-A and chromosomal
The natural frequency of chromosomal abnormalities at birth, in the absence of any prenatal screening, has been estimated at 6 per 1,000 births. The most common of these is trisomy 21 (Down syndrome), its risk increasing dramatically with maternal age and compounded by changing demographics in the pregnant population because of a common trend among couples to delay having families; the second trimester incidence of fetal trisomy 21 is now
Since the early 1990s, prenatal screening, initially instituted for the detection of trisomy 21, has become a standard part of obstetric practice—largely through the measurement of maternal serum biochemical markers in the second trimester (15 to 20 weeks gestation). These markers include a combination of two or three of the following: alphafetoprotein (AFP), total hCG, free β-hCG and unconjugated estriol.
In pregnancies with fetal trisomy 21, maternal serum levels of AFP and unconjugated estriol tend to be lower than normal (median MoM 0.7), while levels of free β -hCG or total hCG are increased (2.2 and 2.0 MoM, respectively). Using a combination of maternal age and maternal serum biochemistry, detection rates of 65 to 70 percent can be achieved when screening the entire pregnant population at a 5 to 6 percent false-positive rate.
PAPP-A, when measured in the second trimester, shows results in trisomy 21 cases that are very similar to those in normal pregnancies. This change in the clinical discrimination of PAPP-A between the first and second trimester is an example of a relatively unappreciated phenomenon of the temporality of marker levels.30 It is now clear that the clinical discrimination of all biochemical markers changes across the first and second trimester. For PAPP-A, large-scale studies have shown an increasing linear trend of the median MoM across the first and second trimester in pregnancies with trisomy 21.30 Similar temporality explains why total hCG is a poor first-trimester marker but an adequate second-trimester marker. Free b-hCG, on the other hand, has a relatively stable median MoM from 10 to 18 weeks, but prior to 10 weeks the median levels fall. Thus, while the best clinical discrimination for PAPP-A may be as early as 8 weeks, the clinical discrimination for free b-hCG during the early weeks of pregnancy is poor (median close to 1.2 MoM). Consequently, the optimum time for measuring both PAPP-A and free b-hCG together is in the first trimester between 10 and 13 weeks—approximately the time frame when NT should be measured (11 to 14 weeks).25 This type of screening can be readily accomplished in a one-stop clinic.
Low serum PAPP-A is not
just an indicator of trisomy
PAPP-A serum levels remain low into the second trimester in cases of trisomy 18. Currently, PAPP-A may be its best biochemical marker. It has been suggested that a two-stage screening program employing PAPP-A as a second-line test could identify 80 percent of trisomy 18 cases at a 0.1 percent false-positive rate.
Much of the initial work with PAPP-A in the early 1980s was based on the finding that low levels of PAPP-A were associated with poor fetal viability. This correlation is being demonstrated in prenatal screening programs, as low serum PAPP-A values in the absence of an ultrasound examination raise suspicions of fetal death. Studies have also shown that lower maternal serum PAPP-A is associated with women who subsequently miscarry, develop pregnancy induced hypertension and develop growth restriction. The clinical sensitivity and specificity of PAPP-A is low; hence, PAPP-A is unlikely to be a useful predictor of subsequent pregnancy complications.
Cornelia de Lange syndrome
Cornelia de Lange
syndrome is a developmental malformation characterized by mental and growth
developmental delay, limb reduction abnormalities, a distinct facial appearance
and congenital heart defects. The incidence is estimated at
The clinical value of PAPP-A continues to grow as new data become available. While its established utility as a risk assessment tool for fetal abnormalities is recognized throughout Europe, the latest findings suggest that PAPP-A may also be predictive of cardiovascular events. The versatility of this analyte is still in the throes of debate, but, without doubt, it has already proved deserving of the attention granted by investigators and clinicians.
2. Human chorionic gonadotropin (hCG) is a peptide hormone produced in pregnancy, that is made by the embryo soon after conception and later by the syncytiotrophoblast (part of the placenta). Its role is to prevent the disintegration of the corpus luteum of the ovary and thereby maintain progesterone production that is critical for a pregnancy in humans. hCG may have additional functions, for instance it is thought that it affects the immune tolerance of the pregnancy. Early pregnancy testing generally is based on the detection or measurement of hCG.
hCG is an oligosaccharide glycoprotein composed of 244 amino acids with a molecular mass of 36.7 kDa. Its total dimensions are 75x35x30 angstroms (7.5x3.5x3 nanometers). The α (alpha) subunit is 92 amino acids long and has dimensions 60x25x15 angstroms (6x2.5x1.5 nm). It is heterodimeric, with an α (alpha) subunit identical to that of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH) and β (beta) subunit that is unique to hCG. βhCG is encoded by six highly homologous genes which are arranged in tandem and inverted pairs on chromosome 19q13.3 - CGB(1,2,3,5,7,8).
hCG interacts with the LHCG receptor and promotes the maintenance of the corpus luteum during the beginning of pregnancy causing it to secrete the hormone progesterone. Progesterone enriches the uterus with a thick lining of blood vessels and capillaries so that it can sustain the growing fetus. Due to its highly negative charge hCG may repel the immune cells of the mother, protecting the fetus during the first trimester. It has also been suggested that hCG levels are linked to the severity of morning sickness in pregnant women.
Because of its similarity to LH, hCG can also be used clinically to induce ovulation in the ovaries as well as testosterone production in the testes. As the most abundant biological source is women who are presently pregnant, some organizations collect urine from gravidae to extract hCG for use in fertility treatment.
Pregnancy tests measure the levels of hCG in the blood or urine to indicate the presence or absence of an implanted embryo. In particular, most pregnancy tests employ an antibody that is specific to the β-subunit of hCG (βhCG). This is important so that tests do not make false positives by confusing hCG with LH and FSH. (The latter two are always present at varying levels in the body, while hCG levels are negligible except during pregnancy.) The urine test is a chromatographic immunoassay. Published detection thresholds range from 20 to 100 mIU/ml (milli International Units per milli-liter), depending on the brand of test. The urine should be the first urine of the morning when hCG levels are highest. If the specific gravity of the urine is above 1.015, the urine should be diluted. The serum test, using 2-4 mL of venous blood, is a radioimmunoassay (RIA) that can detect βhCG levels as low as 5 mIU/ml and allows quantitation of the βhCG concentration. The ability to quantitate the βhCG level is useful in the evaluation of ectopic pregnancy and in monitoring germ cell and trophoblastic tumors.
3. Alpha-fetoprotein (AFP) is a protein that is normally only produced in the fetus during its development. It is a normally produced by the liver and yolk sac of the fetus. AFP levels decrease soon after birth and probably has no function in normal adults. It binds the hormone estradiol to keep it from affecting the fetal brain. Its measurement during pregnancy has been useful to detect certain abnormalities - specifically, if high levels of AFP are found in amniotic fluid, it can indicate a developmental defect in the baby. In some patients who are not pregnant a tumor can produce AFP, thus it can be used as a tumor marker.
AFP is a glycoprotein of 590 amino acids and a carbohydrate moiety that is normally produced by the fetal yolk sac, the fetal gastrointestinal tract, and eventually by the fetal liver. Highest fetal serum levels of AFP are reached at the end of the first trimester and then fall. As AFP is excreted into the amniotic sac through fetal urination, amniotic fluid levels tend to mirror fetal serum levels. In contrast, maternal levels are much lower but continue to rise until about week 32. Levels are much higher in amniotic fluid.
Maternal serum AFP tests need to be interpreted according to the gestational age, as levels rise until about 32 weeks gestation. Typically, such measurements are done in the middle of the second trimester (14-16 weeks). Elevated levels are seen in multiple gestation as well as in a number of fetal abnormalities, such as neural tube defects including spina bifida, anencephaly, and abdominal wall defects. Other possibilities are errors in the date of the gestation or fetal demise. In contrast, low levels of maternal serum AFP are associated with Down syndrome and trisomy 18. Diabetic patients also have lower levels. Patients with abnormal levels need to undergo detailed obstetric ultrasonography. The information is then used to decide whether to proceed with amniocentesis.
Typically AFP measurements are done as part of the triple test, a screening program in pregnant women which also looks at hCG and estriol levels. Genetic counseling is usually offered when the AFP test result is screen positive.
What is an alpha-fetoprotein screening (AFP)?
Alpha-fetoprotein screening is a blood test that measures the level of alpha-fetoprotein in the mothers' blood during pregnancy. AFP is a protein normally produced by the fetal liver and is present in the fluid surrounding the fetus (amniotic fluid), and crosses the placenta into the mother's blood. The AFP blood test is also called MSAFP (maternal serum AFP).
Abnormal levels of AFP may signal the following:
· open neural tube defects (ONTD) such as spina bifida
· Down syndrome
· other chromosomal abnormalities
· defects in the abdominal wall of the fetus
· twins - more than one fetus is making the protein
· a miscalculated due date, as the levels vary throughout pregnancy
AFP screening may be included as one part of a two, three, or four-part screening, often called a multiple marker screen used. The other parts may include the following:
· hCG - human chorionic gonadotropin hormone (a hormone produced by the placenta)
· estriol - a hormone produced by the placenta.
· inhibin - a hormone produced by the placenta.
Abnormal test results of AFP and other markers may indicate the need for additional testing. Usually an ultrasound is performed to confirm the dates of the pregnancy and to look at the fetal spine and other body parts for defects. An amniocentesis may be needed for accurate diagnosis.
Multiple marker screening is not diagnostic. This means it is not 100 percent accurate, and is only a screening test to determine who in the population should be offered additional testing for their pregnancy. There can be false-positive results - indicating a problem when the fetus is actually healthy or false negative results - indicating a normal result when the fetus actually does have a health problem.
How is an alpha-fetoprotein test performed?
Although the specific details of each procedure vary slightly, generally, an alpha-fetoprotein test follows this process:
· Blood is usually drawn from a vein between the 15th and 20th weeks of pregnancy (16th to 18th is ideal).
· The blood sample is then sent off for laboratory analysis.
· Results are usually available within one to two weeks or less, depending on the laboratory.
What are the risks and benefits of alpha-fetoprotein screening?
There are no risks of having the actual test performed other than the usual risks of a blood test. Abnormal test results of AFP and other markers may indicate the need for additional testing. Usually an ultrasound is performed to confirm the dates of the pregnancy and to examine the fetal spine and other body parts for defects. An amniocentesis may be needed for accurate diagnosis.
Multiple marker screening is not diagnostic. This means it is not 100 percent accurate, and is only a screening test to determine who in the population should be offered additional testing for their pregnancy. There can be false-positive results - indicating a problem when the fetus is actually healthy, or false negative results - indicating a normal result when the fetus actually does have a health problem.
The purpose of this screening test is to identify those women in the population who are at increased risk of having a baby with a birth defect, whose pregnancies need additional testing, who otherwise would not have been offered this additional fetal testing
Inhibin contains an alpha and beta subunit linked by disulfide bonds. Two forms of inhibin differ in their beta subunits (A or B), while their alpha subunits are identical. Inhibin belongs to the transforming growth factor-β (TGF-β) family.
In women, FSH stimulates the secretion of inhibin from the granulosa cells of the ovary. In turn, inhibin suppresses FSH. Inhibin secretion is diminished by GnRH, and enhanced by insulin-like growth factor-1 (IGF-1). Inhibin B reaches a peak in the early- to mid-follicular phase, and a second peak at ovulation, in contrast to inhibin A, which reaches its peak in the mid-luteal phase. Inhibin is produced in the gonads, pituitary gland, placenta and other organs.
Neural tube defects (NTDs)
The foetal liver begins to produce a-fetoprotein (AFP) from the sixth week of gestation and the highest concentration of AFP in foetal serum occurs in the mid-trimester, after which it falls progressively until term. Amniotic fluid [AFP] increases steadily during early pregnancy, reaching maximum levels at 13-14 weeks and declining thereafter. In contrast, maternal serum [AFP] (MSAFP) continues to rise and peaks in the third trimester. If the foetus has an open NTD, abnormal amounts of AFP are present in both amniotic fluid and maternal serum. In many countries, [MSAFP] is measured as a screening test for NTDs, carried out with a view to identifying those women who should be further investigated by detailed ultrasound examination. If the diagnosis of open NTD is confirmed before the twentieth week, termination of pregnancy can be offered. Other causes of high [MSAFP] include multiple pregnancy and some rare, non-neurological foetal abnormalities (e.g. oesophageal or duodenal atresia, abdominal wall defects, renal anomalies). The optimum timing for screening is 16-18 weeks of gestation when approximately 80% of NTD-affected pregnancies can be identified. False-negative results may be obtained with closed NTDs where the lesion is covered by a membrane. Because [MSAFP] varies throughout pregnancy, it is normally expressed as multiples of the median (MOM) for the relevant gestation age. Therefore, reliable dating of the pregnancy is essential for the correct interpretation of results.
Screening for trisomy 21
The overall prevalence of trisomy 21 is
Abnormalities in a number of maternal serum analytes are associated with Down's syndrome pregnancies. These include decreased [MSAFP], [pregnancy-associated plasma protein A (PAPP-A)] and [unconjugated oestriol] and increased serum total [hCG], [free B hCG] and [inhibin A]. Each of these parameters shows overlap between trisomy 21 pregnancies and the normal population. However, if the distributions of the concentrations of these analytes for affected and normal pregnancies are known, a likelihood ratio for the risk of the foetus with trisomy 21 can be calculated. This is combined with the age-related risk for the woman in order to calculate the overall risk for the pregnancy. Women with a high risk of carrying an affected child may then be offered amniocentesis.
Second trimester screening for trisomy 21 has
now become an established part of obstetric practice. Protocols vary between
centres but generally involve the measurement of [MSAFP] and either
serum total [hCGJ or [free B hCGJ. These programmes usually achieve detection
rates of approximately 60% for a false-positive rate of about 5%. . A .small number of laboratories also includes
serum [unconjugated oestriol] and/or [inhibin A] as additional
markers. This improves screening performance by reducing the number of false positives
for a given detection rate. Screening may also be performed in the first
trimester when risks are calculated using
a combination of maternal age, biochemical measurements ('maternal' serum [free
|S hCG] and [PAPP-A]) and the ultrasonographic measurement of foetal
nuchal translucency thickness, which is increased in trisomy 21 pregnancies.
While this approach can yield detection rates of better than 80% for a false
positive rate of approximately 5%, first trimester screening is not yet widely
available in the
• The impact of physiological changes must be taken into account when interpreting biochemical data in pregnancy.
• To minimise maternal and foetal risks in pregnant patients with diabetes, it is essential that maternal glucose control is optimised prior to conception and that tight control is maintained throughout pregnancy.
• It is important to recognise hypothyroidism early in pregnancy and institute immediate therapy with thyroxine. The dose of thyroxine required for adequate control in pregnancy is usually 25-50 ^g/L higher than that required to adequately control non-pregnant patients.
• Hyperthyroid patients will also require careful monitoring and management during pregnancy. The measurement of TSH receptor antibodies may be helpful in identifying situations where there may be a risk of intrauterine or neonatal thyrotoxicosis and also in differentiating Graves' disease from hyperemesis gravidarum.
The biochemical abnormalities that are most
commonly of value in the diagnosis and monitoring
of pre-eclampsia are proteinuria, raised plasma creati
nine, abnormal liver function tests and a raised plasma
• The most sensitive biochemical test for the diagnosis of obstetric cholestasis is the measurement of serum bile acids which may be elevated by up to 100 times the normal value.
• Maternal serum AFP is used to screen for foetal NTDs,usually between 16 and 18 weeks gestation. If elevated concentrations are found, a detailed ultrasound scan is indicated.
• Second' trimester screening for trisomy 21 is performed using maternal serum AFP, hCG and sometimes unconjugated' oestriol' and/or t'nhibih A.
• First trimester screening for trisomy21 involves the measurement of free 3 hCG and PAPP-A with the ultrasonographic measurement of foetal nuchal translucency thickness.