Prenatal laboratory testing

Prenatal Laboratory Testing
Dr.Ekbal Mohamed Abo-Hashem . MD
Professor of Clinical Pathology
Mansoura University-Egypt

Agenda :
• Laboratory tests early in pregnancy
• Gestational diabetes mellitus :
• Risk factors-pathogenesis-maternal and neonatal risks-diagnosis-lab workup
• Hypertensive disorders in pregnancy :pre-eclampsia and eclampsia
• Prenatal diagnosis of congenital malformations and genetic disorders
• Indications-benefits-first and second trimester screening and diagnostic tests :
• Screening for neural tube defects
• Screening for Down Syndrome
• Non-invasive and invasive tests
• Cytogenetic investigations
• Molecular genetic investigations

3LLLLL
First trimester routine tests in the mother
 full blood count
 glucose challenges for diabetes
 ultrasound to check for dates, number of fetuses and development
 blood group and antibodies
 midstream urine analysis ,culture
 Syphilis, rubella,hepatitis B ,hepatitis C ,HIV antibodies
 chlamydia screening
 sickle cell and Thalassaemia (Haemoglobinopathy) screening for at risk women
(Ethnic groups at high risk – Mediterranean, Middle Eastern, African, Asian,
Pacific Islander, South American, New Zealand Maori)L
LABORATORY TESTS IN EARLY PREGNANCY

Testing for health conditions in the baby
Prenatal tests are also available to check the health of the baby.
What conditions can be found?
 Chromosome conditions such as Down syndrome, Trisomy 13 and Trisomy 18.
 Neural tube defects such as spina bifida and anencephaly.
 Some birth defects such as congenital heart conditions and malformed kidneys.

Background
Gestational diabetes mellitus (GDM), defined as any degree of glucose intolerance
with onset or first recognition during pregnancy, affects 2-10% pregnancies . Of these
cases, 80-88% are related to abnormal glucose control of pregnancy or gestational
diabetes mellitus. Of mothers with preexisting diabetes, 35% have been found to have
type 1 diabetes mellitus, and 65% have been found to have type 2 diabetes mellitus.
Infants of diabetic mothers (IDMs) have experienced a nearly 30-
fold decrease in morbidity and mortality rates since the development
of specialized maternal, fetal, and neonatal care for women with
diabetes and their offspring. Before then, fetal and neonatal
mortality rates were as high as 65%.
Gestational Diabetes Mellitus

Women with gestational diabetes have a 35-60% chance of developing
diabetes mellitus over 10-20 years after pregnancy. Hyperglycemia in
pregnancy results in both maternal and fetal complications. Maternal
complications consist of hypertension, preeclampsia, increased risk of
cesarean delivery, and development of diabetes mellitus after
pregnancy. Fetal complications include macrosomia, neonatal
hypoglycemia, polycythemia, increased perinatal mortality, congenital
malformation, hyperbilirubinemia, respiratory distress syndrome, and
hypocalcaemia. Long-term consequences of macrosomia include
increased risk of glucose intolerance, diabetes, and obesity in
childhood.

Women with underlying insulin resistance are at risk for developing
GDM. This progression to GDM is thought to be due to physiologic
changes of late pregnancy. In pregnancy, human placental lactogen,
which is structurally similar to growth hormone, and tumor-necrosis
factor-alpha induce changes in the insulin receptor and in post-
receptor signaling. Changes in the beta-subunit of the insulin
receptor, decreased phosphorylation of tyrosine kinase on the
insulin receptor, and alterations in insulin receptor substrate-1 (IRS-
1) and the intracytoplasmic phosphatidylinositol 3-kinase (PI3K)
appear to be involved in reducing glucose uptake in skeletal muscle
tissue .
Pathogenesis:

Infants born to mothers with glucose intolerance are at an
increased risk of morbidity and mortality related to the
following:
 Respiratory distress
 Growth abnormalities (large for gestational age [LGA], small for
gestational age [SGA])
 Hyperviscosity secondary to polycythemia
 Hypoglycemia
 Congenital malformations
 Hypocalcemia, hypomagnesemia, and iron abnormalities

Fetal congenital malformations are most common when maternal
glucose control has been poor during the first trimester of pregnancy.
As such, the need for preconceptional glycemic control in women with
diabetes cannot be overstated.
Communication between members of the perinatal team is of crucial
importance to identify infants who are at the highest risk for
complications from maternal diabetes.

Fetal macrosomia
Fetal macrosomia (>90th percentile for gestational age or >4000 g in the term
infant) occurs in 15-45% of diabetic pregnancies. It is most commonly observed
as a consequence of maternal hyperglycemia. When macrosomia is present, the
infant appears puffy, fat, ruddy, and often hypotonic.
Maternal hyperglycemia during late pregnancy is commonly followed by
excessive fetal growth.
LGA infants should be routinely screened for hypoglycemia. This is particularly
important if the mother has received glucose-containing fluids during her labor.

Impaired fetal growth
Infants whose birthweight is below the 10th percentile, when plotted
against gestational age on a standard growth curve, are considered
small for gestational age (SGA).
Impaired fetal growth may occur in as many as 20% of diabetic
pregnancies, compared with a 10% incidence (by definition) for infants
born to mothers without diabetes. Maternal renovascular disease is the
common cause of impaired fetal growth in pregnancies complicated by
maternal diabetes.

Pulmonary disease
These infants are at an increased risk of respiratory distress
syndrome and may present within the first few hours after birth
with tachypnea, nasal flaring, intercostal retractions, and hypoxia.
Operative delivery due to macrosomia also increases the risk for
transient tachypnea of the newborn, whereas polycythemia
predisposes the infant to persistent pulmonary hypertension of
the newborn.

May present within the first few hours of life. Although the infant is generally
asymptomatic, symptoms may include jitteriness, irritability, apathy, poor
feeding, high-pitched or weak cry, hypotonia, or frank seizure activity.
Hypoglycemia that requires intervention may persist for as long as 1 week.
Hypoglycemia is caused by hyperinsulinemia due to hyperplasia of fetal
pancreatic beta cells consequent to maternal-fetal hyperglycemia.
Hypoglycemia

Because the continuous supply of glucose is stopped after
birth, the neonate develops hypoglycemia because of
insufficient substrate. Stimulation of fetal insulin release by
maternal hyperglycemia during labor significantly increases
the risk of early hypoglycemia in these infants. Perinatal
stress may have an additive effect on hypoglycemia due to
catecholamine release and glycogen depletion. The overall
risk of hypoglycemia is anywhere from 25-40%, with LGA and
preterm infants at highest risk

Hypocalcemia or hypomagnesemia may also be apparent in the first few
hours after birth. Symptoms may include jitteriness or seizure activity.
Hypocalcemia (levels < 7 mg/dL) is believed to be associated with a delay in
parathyroid hormone synthesis after birth.
Sixty-five percent of all infants of diabetic mothers (IDMs) demonstrate
abnormalities of iron metabolism at birth. Iron deficiency increases the
infant's risk for neurodevelopmental abnormalities. Iron is redistributed to
the iron-deficient tissues after birth, as the red blood cell (RBC) mass is
postnatally broken down.
Electrolyte abnormalities

Caused by increased erythropoiesis triggered by chronic fetal hypoxia, may
present as a clinically "ruddy" appearance, sluggish capillary refill, or
respiratory distress. Hyperviscosity due to polycythemia increases the IDM’s
risk for stroke, seizure, necrotizing enterocolitis, and renal vein thrombosis.
Thrombocytopenia
Thrombopoiesis may be inhibited because of an excess of RBC precursors
within the bone marrow as a result of chronic in utero hypoxia and increased
erythropoietin concentration.
Hyperbilirubinemia
This is common, especially in association with polycythemia. The increased
red cell mass results in increased number of RBCs that are taken out of
circulation each day and increase the bilirubin burden presented to the liver.
Polycythemia

Cardiovascular anomalies
Cardiomyopathy with ventricular hypertrophy and outflow tract
obstruction may occur in as many as 30% of IDMs. The
cardiomyopathy may be associated with congestive failure with a
weakly functioning myocardium or may be related to a hypertrophic
myocardium with significant septal hypertrophy and outflow tract
obstruction. When cardiomegaly or poor perfusion and hypotension
are present, performing echocardiography to differentiate between
these processes is important.
These infants are also at an increased risk of congenital heart defects,
including (most commonly) ventricular septal defect (VSD) and
transposition of the great arteries (TGA).

Congenital malformations
Central nervous system (CNS) malformations are 16 times more likely in IDMs. In particular,
the risk of anencephaly is 13 times higher, whereas the risk of spina bifida is 20 times
higher. The risk of caudal dysplasia is up to 600 times higher in these infants.
Neurologic immaturity, demonstrated by immature sucking patterns, has been found in
infants born to insulin-managed mothers with diabetes. This may be due to abnormal brain
metabolism and electroencephalogram (EEG) findings as a result of the fetal hyperglycemia.
Renal (eg, hydronephrosis, renal agenesis, ureteral duplication), ear, gastrointestinal (eg,
duodenal or anorectal atresia, small left colon syndrome), and cardiovascular (eg, single
umbilical artery, VSDs, atrial septal defects, TGA, coarctation of the aorta, cardiomegaly)
anomalies are more frequent in these infants.

Indications for glucose testing in pregnancy :
All pregnant women need to be screened for gestational diabetes. The
timing of the screening depends on risk factor assessment. Pregnant
women with no known history of diabetes are screened at 24-28
weeks gestation. Women at high risk for GDM are screened at the first
prenatal visit. A 75-g 2-hour OGTT is the test of choice in both groups.

The risk factors for GDM :
Increased weight (ie, BMI greater than or equal to 25)
 Decreased physical activity
 First degree relative with diabetes
 Member of ethnic group with high prevalence of diabetes (African American,
Latino, Native American, Asian American, Pacific Islander)
 Prior history of GDM or delivery of a baby greater than 9 pounds
 Metabolic abnormalities - Hypertension, HDL less than 35 mg/dL, triglyceride
level greater than 250 mg/dL
 Polycystic ovarian syndrome
 HbA1C 5.7% or higher
 Impaired glucose tolerance or impaired fasting glucose testing in the past
 Evidence of insulin resistance (acanthosis nigricans or severe obesity)
 History of cardiovascular disease

The standard criteria for the diagnosis of diabetes in the general
population is as follows:
 HbA1c of 6.5% or higher
 Fasting plasma glucose of 126 mg/dL or higher or
 2-h plasma glucose of 200 mg/dL or higher during an 75-g OGTT
or
 A symptomatic patient with random plasma glucose of 200 or
higher (all plasma glucose values are recorded as mg/dL).

The 75-g Glucola OGTT is performed after an overnight fast;
the diagnosis is made if :
 fasting plasma glucose is documented at 92mg/dL or higher,
 a 1-hr plasma glucose of 180 mg/dL or higher,
 or a 2-hr plasma glucose of 153 mg/dL or higher.
The diagnosis of gestational diabetes is confirmed with a
minimum of one abnormal value. The cut-offs were based on the
prior HAPO study outcomes.( The HAPO trial : Hyperglycemia
and Adverse Pregnancy Outcomes-2008) .
Blood glucose testing during pregnancy:

Technical Considerations
Two major changes from the previous diagnostic guidelines
are :
 lower diagnostic cut points for the fasting, 1-hour and 2-
hour plasma glucose measurements
 and needing only one abnormal value to make the diagnosis
.

In September 2011, , the American College of Obstetricians and
Gynecologists (ACOG ) recommended screening for GDM at initial
prenatal visit by history, risk factors or 50 gram/1-hour OGTT. The
diagnosis of GDM continues to be based on the 100 gram/3-hour
tolerance test using the Carpenter and Coustan cutoffs of :
 fasting less than 95mg/dL,
1-hr less than 180 mg/dl,
2-hr less than 155 mg/dL,
 and 3-hr less than 140 mg/dL,
 with 2 or more abnormal values to confirm diagnosis.

Glucose concentration (serum or whole-blood)
Seizures, coma, and long-term brain damage may occur if neonatal
hypoglycemia is unrecognized and untreated. Most centers recognize
levels lower than 20-40 mg/dL within the first 24 hours after birth as
abnormal, but the precise level remains controversial.
An infant with compromised metabolic adaptation (ie, IDM) undergo
blood glucose measurements (1) as soon as possible after birth, (2)
within 2-3 hours after birth and before feeding, and (3) at any time
abnormal clinical signs are observed.
Laboratory workup for infants of diabetic mothers :

CBC count
Polycythemia, commonly defined as a central hematocrit level
higher than 65%, is a potential concern. Maternal-fetal
hyperglycemia and fetal hypoxia is a strong stimulus for fetal
erythropoietin production and subsequent increase in fetal
hemoglobin concentration. Thrombocytopenia may occur because
of impaired thrombopoiesis due to "crowding-out" of
thrombocytes by the excess of erythroid precursors in the bone
marrow.

Bilirubin level (serum, total and unconjugated)
Hyperbilirubinemia is more common in IDMs than in the general
population of neonates. Causative factors include prematurity, hepatic
enzyme immaturity, polycythemia, and reduced RBC half-life.
Arterial blood gas
Assessing oxygenation and ventilation is essential in infants with clinical
evidence of respiratory distress. Although noninvasive methods (eg,
transcutaneous oxygen and carbon dioxide electrodes, oximeters) have
gained wide acceptance at many centers, comparison of results with those
from arterial blood is intermittently required.

Magnesium concentration (serum)
Hypomagnesemia is related to younger maternal age, severity of
maternal diabetes, and prematurity. Neonatal magnesium levels are also
related to maternal serum magnesium, neonatal calcium and
phosphorus levels, and neonatal parathyroid function.
Calcium concentration (serum, ionized or total levels)
Low serum calcium levels in IDMs are common. They are speculated to
be caused by a functional hypoparathyroidism .

Hypertensive Disorders in Pregnancy

Introduction
Hypertensive disorders affect up to 10% of pregnancies .Elevated
blood pressure (BP) in pregnancy may represent :
 Chronic hypertension (occurring before 20 weeks’ gestation or
persisting longer than 12 weeks after delivery),
 Gestational hypertension (occurring after 20 weeks’ gestation),
 preeclampsia, or
 Preeclampsia superimposed on chronic hypertension.

Preeclampsia is becoming an increasingly common diagnosis in the
developed world . It is one of the most common and life threatening
conditions occurring in pregnancy.
It remains a high cause of maternal and fetal morbidity and mortality in
the developing world. Delay in childbearing in the developed world
feeds into the risk factors associated with preeclampsia, which include
older maternal age, obesity, and/or vascular diseases. Inadequate
prenatal care partially explains the persistent high prevalence in the
developing world. Early screening allows for early intervention, which
can protect baby’s health

Screening during the first trimester can identify
women at increased risk for early-onset pre-
eclampsia. Interventions include low-dose aspirin
and early delivery depending on gestation. Without
treatment, pre-eclampsia may affect the normal
growth of the baby.

RISK FACTORS FOR PRE-ECLAMPSIA
The risk factors for PE include:
 Maternal and paternal family history
 Previous pregnancy with PE
 Multiple pregnancy (e.g. twins)
 Maternal age (over 40 years)
 Body Mass Index (BMI over 30)
 Pre-existing high blood pressure,
diabetes, smoking or kidney disease
 Systemic inflammation
 Ethnic origin

. AT1-AA, autoantibodies to angiotensin receptor 1;
 COMT, catechol-O-methyltransferase;
 HTN, hypertension; LFT, liver function test;
 PlGF1, placental growth factor 1;
 PRES, posterior reversible encephalopathy syndrome;
 sEng, soluble endoglin;
 sFlt-1, soluble fms–like tyrosine kinase 1;
 sVEGFR1, soluble vascular endothelial growth factor receptor 1;
 VEGF, vascular endothelial growth factor.
These factors are implicated in the pathogenesis of PE :
Pathogenesis of preeclampsia: two-stage model.

Severe features of preeclampsia include a systolic
blood pressure of at least 160 mm Hg or a diastolic
blood pressure of at least 110 mm Hg, platelet count
less than 100 × 103 per μL, liver transaminase levels
two times the upper limit of normal, a doubling of the
serum creatinine level or level greater than 1.1 mg per
dL, severe persistent right upper-quadrant pain,
pulmonary edema, or new-onset cerebral or visual
disturbances.

•Hematocrit. A high hematocrit value can be a sign of
preeclampsia.A hematocrit value of 42 means that red blood
cells make up 42% of the blood volume. A normal hematocrit
value for a nonpregnant woman is between 36% and 44%.
During pregnancy, the hematocrit value normally decreases—
due to increase in plasma volume., making red blood cells less
concentrated. But preeclampsia often causes the body's tissues
to absorb blood plasma. The blood becomes more concentrated,
resulting in an abnormally high hematocrit value.
Laboratory investigations in pre-eclampsia

•Platelets. . Preeclampsia may cause an abnormally low
platelet count.
•Partial thromboplastin time (PTT).. Preeclampsia can
affect the coagulation system with an increase the partial
thromboplastin time.
•Electrolytes. Examples of important electrolytes include
sodium, potassium, magnesium, calcium, and chloride. This
is due to affection of kidney or fluid distribution (edema).

 Liver function tests. Elevation of aminotransferase
levels due to liver parynchemal affection
 Uric acid. Increased uric acid in the blood is often the
earliest laboratory finding related to preeclampsia.
 Kidney function tests. Increase in blood urea
nitrogen and creatinine,.

Prenatal diagnosis of congenital malformations
and genetic disorders

Indications of Prenatal Diagnosis
Prenatal testing and/or diagnosis is offered to all couples, whether it
involves prenatal serum screening, cf-DNA, ultrasound or invasive
procedures. Some common indications include:
 Advanced maternal age defined as age 35 at time of delivery
 A previous child with a chromosomal abnormality.
 The couple is known to be carriers of a chromosomal translocation.
 The pregnant woman is affected with type 1 diabetes mellitus, epilepsy,
or myotonic dystrophy.
 Exposure to viral infections, such as rubella or cytomegalovirus.

 The mother is exposed to excessive medication or to environmental
hazards.
 In her or her spouse's family, a history of Down syndrome or some
other chromosomal abnormality is present.
 A history of a relatives of single gene disorder is present in her or her
spouse's family.
 Her male has Duchenne muscular dystrophy, severe hemophilia or
intellectual deficiency (Fragile X).
 She is suspected of an X-linked chromosomal abnormality
 The fetus is diagnosed in utero to have some hereditary error of
metabolism.
 The fetus is detected to be at increased risk for a NTD or other
structural or multiple structural abnormalities.
Indications of Prenatal Diagnosis (cont. ) :

Benefits of Prenatal Diagnosis
An offer of prenatal screening or diagnosis provides prospective
mothers and couples the option of choosing or declining to receive
genetic information pertinent to their personal situation prior to
conception.
After conception, prenatal diagnosis provides various benefits.
 Prenatal diagnosis determines the outcome of pregnancy and identifies possible
complications that can arise during pregnancy and birth.
 It can be helpful in improving the outcome of pregnancy using fetal treatment.
 Screening can help couples determine whether to continue the pregnancy and
prepares couples for the birth of a child with an abnormality.

Screening for Neural Tube Defects
The prevalence of neural tube defects varies worldwide which reflects the
differences in genetic and environmental factors. ACOG recommends screening
for open neural tube defects by ultrasound, maternal serum alpha-fetal protein or
both ,if the following is present :
 Ultrasound findings suspicious for NTD
 A previous child with NTDs
 A family history of NTDs exists, especially a mother with NTDs.
 Type 1 diabetes mellitus during pregnancy.
 Maternal exposure to drugs, such as valproic acid
 Elevated level of MSAFP.
 Obesity
 Race: MSAFP level is 10 to 20 percent higher in black women

Measuring Maternal Serum Alpha-Fetoprotein
The developing fetus has 2 major blood proteins, albumin and
alpha-fetoprotein (AFP), while adults have only albumin in
their blood. The MSAFP level can be used to determine the
AFP levels from the fetus. AFP is produced by the yolk sac and
later by the liver; it enters the amniotic fluid and then the
maternal serum via fetal urine.
In conditions such as open NTD (eg, anencephaly, spina bifida)
and abdominal wall defects in the fetus, AFP diffuses rapidly
from exposed fetal tissues into amniotic fluid, and the MSAFP
level rises.

However, the MSAFP levels also increase with gestational age, as
such, incorrect dating or fetal demise are a cause of elevated
MSAFP. Other causes of elevated MSAFP include maternal
diabetes, multiple gestations, pregnancies complicated by
bleeding, abnormal placentation or function (accreta or
intrauterine growth restriction) as well as other fetal
malformations (fetal sacral teratoma) and rarely maternal liver
tumors.

The MSAFP test can be performed between 15-22 weeks' gestation. A
combination of the MSAFP test and ultrasonography detects almost all
cases of anencephaly and most cases of spina bifida. When 2.0 or 2.5 MoM
is used, the American College of Medical Genetics and Genomics reported
the detection rate for anencephaly is ≥95%. NTD can also be distinguished
from other fetal defects, such as abdominal wall defects, by the use of an
acetylcholinesterase test carried out on amniotic fluid obtained by
amniocentesis.
.

• In cases where a low level of MSAFP is reported, it
may be associated with Down syndrome, other
chromosomal aneuploidy or failing pregnancies.
AFAFP and amniotic fluid acetylcholinesterase (AChE) are
the primary biochemical tests performed on amniotic fluid
for detection of NTDs. AChE is an enzyme contained in
blood cells, muscle, and nerve tissue. An elevation
of both AFAFP and AChE values suggests a fetal NTD with
96% accuracy;

Screening for Fetal Down Syndrome
The quadruple test is usually performed at 15 to 18 weeks of gestation but can
be done as late as 22 weeks. In 2012, the quadruple test was the most common
Down syndrome screening test performed in the United States. However, there
are several advantages to earlier assessment including maximum time for
decision making and safer methods of termination. Early risk assessment (first
trimester screening) includes 3 markers: one ultrasound and 2 biochemical:
 The nuchal translucency measurement
 Maternal serum pregnancy associated plasma protein (PAPP-A)
 Maternal serum free- beta human chorionic gonadotropin (beta hCG)

The nuchal translucency (NT) is the normal fluid filled space behind the fetal
neck. Fetal NT thickness is measured between 10 3/7 and 13 6/7 weeks
gestation and is increased in fetuses with Down syndrome. As a screening
test, it is weaker as gestational age increases from 10 to 13 weeks and the cut-
off for abnormal values are in the 95-99th percentile based on gestational
age. Without biochemical markers, the sensitivity of NT is lower.
PAPP-A is a complex, high molecular weight glycoprotein with levels that
are lower in pregnancies affected with fetal Down syndrome and as a
marker, decreases with increasing gestational age between 9 and 13 weeks. In
contrast ,free B-hCG levels on average will double in pregnancies at risk
for Down syndrome.

There have been several ways to offer screening including the full integrated
test which consists of nuchal translucency and PAPP-A measured at 10 to 13
weeks followed by measurement of AFP, uE3, hCG, and inhibin at 15 to 18
weeks as well as serum integrated test which includes all of the tests of
the full integrated test (PAPP-A, AFP, uE3, beta-hCG, inhibin), but no
ultrasound marker (ie, no nuchal translucency).
There are also different ways to offer testing including contingency
(sequential )testing, where if the first trimester screen is positive, women are
offered invasive testing whereas if the first trimester test is negative, they can
opt to complete second trimester screening or not have any other testing.

Measuring maternal unconjugated estriol
The amount of estriol in maternal serum depends upon viable fetus, a
properly functioning placenta, and on maternal well-being. Fetal adrenal
glands produce dehydroepiandrosterone (DHEA) that gets metabolized
to estriol in the placenta. Estriol crosses to the maternal circulation and
is excreted either by maternal kidney in urine or by maternal liver in the
bile. A low level of estriol can be an indicator of Down syndrome, adrenal
hyperplasia with anencephaly, The low uE3 level is noted because the
steroid precursors required for estriol synthesis in the fetus are defective.

Measuring maternal serum beta-human chorionic gonadotropin
Following conception and implantation of the developing embryo into the uterus,
the trophoblasts produce enough beta-HCG, which is an indication for pregnancy.
In the middle to late second trimester, the level of beta-HCG also can be used in
conjunction with other biomarker level to screen for chromosomal abnormalities.
An increased beta-HCG level coupled with a decreased MSAFP level suggests
Down syndrome.
The beta-HCG level also can be quantified in serum from maternal blood, and, if
its amount is found to be lower than expected, it indicates abortion or ectopic
pregnancy. If the level of HCG is estimated to be considerably high, then it
indicates the possibility of trophoblastic diseases.

Measuring maternal inhibin-A levels
The hormone 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 linked with an
increased risk for trisomy 21. A high inhibin-A level may
also be associated with a risk for adverse perinatal outcome
including preterm delivery and fetal growth restriction.

Maternal serum Cell-free fetal DNA (cfDNA) :
Non-invasive prenatal screening uses next-generation sequencing of cell-free DNA
(cfDNA) in the maternal circulation. Circulating cfDNA is derived from both the
mother and the fetal-placenta unit and is highly fragmented. Cell-free fetal DNA and
RNA can be extracted from maternal blood around 7 weeks’ gestation, which can be
used to screen for Down syndrome, as well as other trisomies (18 and 13), and
common sex chromosome aneuploidies (45,X0; 47,XXX; 47,XXY; 47,XYY). The test
can identify 98-99% of affected pregnancies.
Sex determination for families with inherited sex-linked diseases, diagnosis of certain
single gene disorders, and blood Rhesus factor status (in the case of Rhesus D-
negative mothers) can also be performed using cell-free fetal nucleic acids from the
placenta.

The test is best drawn after 10 weeks to allow the cell fraction to increase to at least 4%
of the total fetal cell fraction. As such in obesity, as maternal weight increases, the fetal
fraction may be low leading to a non-result. Factors that may increase the risk of false
positive include; demise of one fetus, confined placental mosaicism, maternal
mosaicism, or maternal cancer. If results are not reported, indeterminate, or
uninterpretable from cell-free DNA screening, women should receive further genetic
counseling, ultrasound evaluation and diagnostic testing..
This test does not replace invasive testing like CVS or amniocentesis as it is limited in its
ability to identify all chromosome abnormalities. Women should also be counseled that
cell free DNA testing does not eliminate the risk of a structural congenital abnormality
and these patients should still be offered ultrasound and MSAFP.

Fetal tissue sampling - Chorionic villus sampling
The choice of chorionic villi sampling versus amniocentesis is personal
as they essentially provide the same genetic information. CVS is
performed very early in gestation between 9-12 weeks, ideally at 10
weeks' gestation. A catheter is passed through the cervix or through the
abdominal wall into the uterus under ultrasound guidance, usually at a
tertiary care facility and a sample of chorionic villi surrounding the sac
is obtained. The villi are dissected from the decidual tissue, and
chromosome analysis is carried out on these cells to determine the
karyotype of the fetus

• Chorionic Villus Sampling through the abdomen

Chorionic Villus Sampling - through the cervix

DNA can be extracted from these cells for molecular analysis. DNA analysis
of CVS specimens is helpful for early diagnosis of diseases such as
hemoglobinopathies. In addition, tissue culture can be initiated on these
cells for further studies. Fetal DNA from both villi or amniocytes can also
be tested for specific genetic conditions.
Single gene testing and other genetic conditions in the prenatal period often
relies on a positive family history or a previously identified mutation, thus
parental blood samples are often required for confirmatory testing.

The major advantage of CVS over amniocentesis is its use in earlier in pregnancy.
Abnormalities can be identified at an early stage, and more acceptable decisions about
termination of the pregnancy can be taken. Abortion is also much safer at this early
stage.
A disadvantage of CVS as compared to amniocentesis is :
 A 1-2% risk of miscarriage, and, rarely, CVS can result with limb defects in the
fetus.
 Maternal sensitization is possible, and known maternal alloimmunization is a
relative contraindication as it may result in more severe disease.
 A higher rate of maternal cell contamination and confined placental mosaicism with
CVS may result in diagnostic ambiguity, leading to the need for additional invasive
diagnostic tests

Fetal tissue sampling - Amniocentesis
Amniocentesis is an invasive, well-established, safe, reliable, and
accurate procedure usually performed at 15-18 weeks, but can be
performed any time in gestation after 15 weeks. Prior to 15 weeks,
there has been an increased risk of loss and fetal clubbed foot. It is
performed under ultrasound guidance. A 22-gauge needle is passed
through the mother's abdomen through the uterus, into the
amniotic cavity. About 10-20 mL of amniotic fluid that contains
cells from amnion, fetal skin, fetal lungs, and urinary tract
epithelium are collected. These cells are grown in culture for
chromosomal, biochemical, and molecular biologic analyses.
Supernatant amniotic fluid is used for the measurement of
substances, such as amniotic fluid AFP, hormones, and enzymes..

• The results of cytogenetic and biochemical studies on amniotic cell
cultures are more than 90% accurate. In the third trimester of
pregnancy, the amniotic fluid can be analyzed for determination
of fetal lung maturity. Risks with amniocentesis are rare but
include 0.5% fetal loss and maternal Rh sensitization, as such
women with Rh negative blood type should receive RhoGAM post
procedure.

• . Karyotype can detect chromosomal changes as small as 5-10 Mb
as well as balanced translocations or inversions. The use of
chromosomal microarray (CMA) has increased as it can detect
smaller (10-100 Kb) gains and losses of genetic material that
would not be detected by traditional karyotype. Another
advantage of CMA is that it does not require cell culture, allowing
availability of results in shorter time frames.

Fetal tissue sampling - Percutaneous skin biopsy
To prenatally diagnose a number of serious skin disorders, percutaneous fetal skin biopsies are
taken under ultrasonic guidance between 17-20 weeks' gestation.
Fetal tissue sampling - Other organ biopsies, including liver and muscle biopsy
Case reports have described fetal liver biopsy to diagnose an inborn error of metabolism, such
as ornithine transcarbamylase deficiency, glucose-6-phosphatase deficiency, glycogen storage
disease type IA, etc…. Fetal muscle biopsy has also been described to analyze the muscle fibers
histochemically for prenatal diagnosis of Becker-Duchenne muscular dystrophy. . These are not
routinely used nor readily available outside specialized centers.
Fetal tissue sampling - Preimplantation biopsy of blastocysts obtained by in vitro
fertilization
Techniques are being developed to test cells obtained from biopsy of early cleavage stages or
blastocysts of pregnancies conceived through in vitro fertilization. These techniques will be
helpful for selective transfer and implantation of those pregnancies into the uterus that are not
affected by a specific genetic disorder.

Cytogenetic Investigations
Detection of chromosomal aberrations
Chromosomal aberrations, such as deletions, duplications,
translocations, and inversions diagnosed in affected parents
or siblings, can be detected prenatally in a fetus by
chromosomal analysis This analysis can be undertaken on fetal
cells obtained through such techniques as amniocentesis and
CVS ,then subjected to any of the following techniques :

Fluorescent in situ hybridization(FISH)
FISH uses different fluorescent-labeled probes, which are single-stranded DNA
conjugated with fluorescent dyes and are specific to regions of individual chromosomes.
These probes hybridize with complementary target DNA sequences in the genome and
can detect chromosomal abnormalities, such as trisomies, monosomies, and
duplications. This technique allows counting of the number and location of large pieces
of chromosomes and increased the sensitivity, specificity, and resolution of chromosome
analyses. FISH can be performed on metaphase chromosomes or interphase nuclei and is
technically straightforward.

Microarray comparative genomic hybridization
Array-CGH (microarray comparative genomic hybridization) also
referred to as chromosomal microarray analysis (CMA), is considered to
be useful in detecting genomic imbalance in the fetus
(duplications/deletions ).
CMA is also useful in evaluation of stillbirth (pregnancy loss ≥20 weeks
of gestation) because both chromosomal abnormalities and culture failure
are common in these cases. Culture failure is common when the fetus has
died, and thus prevents the accurate diagnosis of a karyotypic
abnormality in these cases.

Molecular Genetic Techniques
Molecular genetic techniques are being used for prenatal diagnosis. These
techniques are based upon the fact that DNA complement is generally identical in
every cell of the body; therefore, any hereditary defect diagnosed at the DNA level
will be present in nucleated cells from that individual.
For molecular analysis, DNA is extracted from amniocytes, chorionic villi, or fetal
blood cells. Then, it is amplified by PCR and is used for the diagnosis of genetic
mutations or deletions within a gene that causes a specific genetic disease. The
following molecular biologic techniques can be used for prenatal diagnosis of
different diseases.
Linkage analysis by microsatellite markers
Single nucleotide polymorphisms
Restriction fragment length polymorphism
DNA chip
Dynamic allele-specific hybridization

Prenatal laboratory testing

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