Prenatal Diagnosis Including Preimplantation Genetic Diagnosis

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Why offer prenatal screening and PND?

All pregnant women are at risk of carrying a fetus with a genetic abnormality

Frequency of inherited malformations and genetic disorders in the newborn is 3%

Incidence of major abnormality at birth is 2-3%

95% of chromosomal anomalies are caused by trisomies 21, 18, 13 (majority T21)

Routine in some Countries: e.g. Down syndrome screening, anomaly scan in UK, US

Background risk of chromosomal abnormalities and genetic defects depends on maternal age and the gestational age. The risk—

Increases with maternal age
Reduces with gestational age

Frequency is higher among abortuses because most serious malformations and genetic disorders result in spontaneous abortion

Advantages of PNS and PND

Enables provision of information to affected women so they can make an informed choice about their baby’s health

Helps health care provider manage pregnancies better; holistic approach; family centered care

Provides reassurance/removes anxiety if results are normal

If results are abnormal, enables planning. Choice of

Continuation with pregnancy or termination
Prenatal treatment
Planning for the birth of an affected child

PNS and PND Can be for

Chromosomal diseases

Trisomy 21, 18, 13

Non-chromosomal diseases

Single gene disorders e.g., SCD, thalassemia Fetal defects
Pre-eclampsia
GDM
PTB

Screening test

Cheap and safe tests suitable for whole population
Selects a subgroup for diagnostic testing
Sensitivity and positive predictive value is important

Diagnostic tests

Have a high degree of accuracy
E.g. amniocentesis for chromosomal abnormalities
May be invasive, carry risk and are expensive

Ultrasound can be used for both screening and diagnosis

Measures of the performance of a test

Detection rate (DR): the proportion of individuals with a particular condition who test positive for that condition

True positive: affected (sick) people correctly identified as affected

False positive: unaffected (healthy) person incorrectly identified as affected (sick)

True negative: unaffected individuals correctly identified as unaffected

False negative: affected individual incorrectly identified as unaffected

Sensitivity: ability of a test to correctly identify those with the disease i.e. true positive rate e.g. if 100 people known to have a disease are tested and 45 test positive, it means the sensitivity of the test is 45%. Proportion of actual positives who are correctly identified

Specificity: ability of a test to correctly identify those without the disease i.e. True negative rate

Positive predictive value: probability that subjects with a positive screening test truly have the disease

Negative predictive value: probability that subjects with a negative screening test truly don’t have the disease

Perfect test should be 100% sensitive (all affected individuals are identified) and 100% specific (no healthy individual is identified as affected)

Conditions that may benefit from PNS

Chromosomal disorders e.g. T21, 18, 13

Neural tube defects

Genetic disorders e.g. SCD, thalassemia

Fetal structural anomalies

Others

Preterm birth— using cervical length, fetal fibronectin
Screening for hemolytic disease of the newborn (rhesus isoimmunization)
Pre-eclampsia— using maternal history, mean arterial pressure at 11-13 weeks, uterine artery doppler at 11-13 weeks, biomarkers e.g. pregnancy associated plasma protein A (PAPP-A)
Gestational DM— maternal BMI, race,
Screening for potential neonatal infection e.g. hepatitis B, HIV, syphilis

PNS is usually offered before PND because of the risk of miscarriage with invasive testing

Indications for cytogenetic studies (amniocentesis, CVS, FBS)

Advanced maternal age above 35 years

No unequivocal explanation for the relationship between aneuploidy and advanced maternal age; several theories viz:
With advancing maternal age, there is decreasing maternal selection against chromosomally abnormal conceptuses.
Chiasmata between homologous chromosomes decrease in aging oocytes, leading to nondisjunction and chromosomally abnormal ova.
Decline in the oocyte pool, or in the number of maturing oocytes per cycle, that accounts for the increase in trisomies with advancing maternal age.
Risk of trisomy 21 is 1:385 at 35-years, 1:137 at 39 years of age, and 1:30 at 45 years of age.
Trisomy 13, trisomy 18, 47XXX, and 47XXY also show an increased mean maternal age.
United States authorities believe that prenatal diagnosis should be offered, as an alternative to screening, to all women who will be 35 years of age or older when their infant is born

Maternal high risk screening score

Previous child with chromosomal abnormality

Parental translocation, inversion, or aneuploidy

Fetuses with intrauterine growth retardation

Fetuses with anomalies on ultrasound examination.

Trisomic fetuses, especially fetuses with trisomy 13 or 18, often show IUGR
Gross anomalies also frequently can be visualized in fetuses with chromosomal abnormalities

Antenatal chromosomal studies are appropriate if an abnormal fetus is detected on ultrasound examination.

Certain biometric findings (e.g., short femur, short humerus, pyelectasis, thickened nuchal fold, nuchal translucency, echogenic bowel, absent nasal bone) are indicative of an increased risk of fetal Down syndrome.

Potential indications for cytogenetic studies

Advanced paternal age

Previous unexplained stillbirth

Previous spontaneous miscarriage

Exposure to radiation or chemotherapeutic agent

Assisted reproduction; male factor infertility

Some men with severe oligospermia and nonobstructive azoospermia have microdeletions of the Y chromosome which will be transmitted to their male offspring if conception occurs, either naturally or via IVF/ICSI
Children born after IVF or ICSI have an increased rate of autosomal and sex chromosome anomalies (1–3%)

Methods of prenatal screening

Serum tests
Ultrasound
Combination of both

Routine prenatal screening

Offered to all women
Women should be fully informed and understand the implications of the test
Test results should be promptly provided
Referral for definitive diagnosis may be required in women with positive screen results
Screening offered depends on local protocols

Common chromosomal anomalies

Down’s syndrome

Chromosomal disorder resulting in extra copy of chromosome 21
Affects 1 in 700 babies in US
Commonest aneuploidy compatible with extra-uterine survival
Risk increases with advancing maternal age
95% cases caused by non-dysjunction during cell division
4% caused by translocation
1% caused by mosaicism

note

Down’s syndrome screening

All women are at risk of having a fetus with Down’s syndrome
In developed countries, all women are offered screening which identifies women at higher risk of having an affected child
Screen positive women are offered a diagnostic test (cytogenetic analysis) which could be an invasive test e.g. amniocentesis, Chorionic Villous Sampling, or a blood test (NIPT)
Although risk increases with maternal age, if diagnostic testing is only offered to women above 35, only 30% of affected pregnancies will be identified

Edward’s syndrome (T18)

Mostly results in IUGR, IUFD, NND
Very rarely does survival to adulthood occur
1 in 3,000-6,000 live births

Patau’s syndrome: (T13)

Most infants die within 1st week of life
Occurs in 1 in 16,000 newborns

Screening for chromosomal anomalies

Using certain markers of Aneuploidy

Ultrasound markers

Nuchal Translucency (NT) >3.5mm
Absence of Nasal bone
Soft markers e.g. echogenic foci in the heart, echogenic bowel

Serum markers

Alpha Fetoprotein (AFP)
Human Chorionic Gonadotropin (hCG)
Unconjugated estriol (uE3)
Pregnancy associated plasma protein A (PAPP-A)

When do we screen for chromosomal defects?

First trimester
Second trimester

1st trimester USS markers

Raised Nuchal Translucency (NT) > 3.5mm— increased risk of Down’s, Turner, heart defects
Absent nasal bone—T21
Hypoplastic maxilla—T21
Omphalocoele—T18
Single umbilical artery—T18

note

Nuchal translucency

Single most useful marker of aneuploidy
Chances of fetal abnormality increases as NT increases
Measures subcutaneous collection of fluid under baby’s neck using ultrasound
Measured in 1st trimester when CRL is 45-84 mm
99th centile is 3.5 mm
May be increased in

Congenital infection
Fetal anemia
Chromosomal anomaly e.g. Down’s
Structural malformations

Maternal serum screening for T21

Detects levels of serum markers: free beta hCG, PAPP-A, AFP, estriol, inhibin

May be performed in the first or second trimester

It is a non-invasive method of PNS i.e. no risk to the pregnancy

Aims at identifying women with increased risk of T13, 18, 21

Used in conjunction with USS markers gives detection rate up to 85%

Beta—hCG

hCG produced by placenta

In Down’s syndrome,

Both alpha and beta subunits are increased but free beta subunit is used for screening
Placenta is hypersecretory and immature resulting in increased secretion of beta hCG usually commencing at end of 1st trimester and continuing into the 2nd trimester
Detection rate for T21 when used alone = 33%

PAPP-A

Pregnancy associated plasma protein A

Concentration is lower in pregnancies with aneuploidy

Detection rate for T21 is 38% when used alone

Unconjugated estriol (uE3)

Produced by the placenta

Decreased in Down’s syndrome

Also low in IUGR, oligohydramnios

Biochemical markers are affected by

Smoking status: smoking can alter the PAPP-A result by up to 20%
Maternal weight at sampling: the exact weight on the day the sample is taken is essential for calculating the correct T21 risk. An error of as little as 3kg can have a dramatic effect on the final risk result.
Family origins: some biochemical markers like PAPP-A are 60% higher in Afro-Caribbean women, which will bias the results if not adjusted for.
Assisted conception: the risk of T21 increases with the mother's but not the father's age.

Various screening modalities

1st trimester screening for aneuploidy (Combined test)

Determines risk for aneuploidy (commonly Trisomy 21, 18, 13) using a combination of

Maternal age (to determine background risk)
Maternal serum screen (free beta hCG + PAPP-A) Nuchal Translucency (NT)
Serum markers are independent of NT so combination of both results in increased detection rate

>90% detection rate for T21

Done at GA of 11 weeks + 2 days to 14 weeks + 1 day (when CRL is 45- 84 mm)

2nd trimester screening

Triple test

E.g. triple test, quadruple test, genetic sonogram
Triple test measures alpha fetoprotein (AFP) + hCG + unconjugated estriol (uE3)
Performed at 15-20th week in women who did not present early enough to benefit from the 1st trimester screening
Serum markers are combined with maternal age, weight, ethnicity and GA to give a risk score
Has 69% DR for T21 (Down’s syndrome)

Quadruple test

For late bookers when NT no longer possible
Detection rate is 80% for T21
Inhibin is added to triple test markers i.e. measures AFP + hCG + uE3 + inhibin
Performed at 14-20 weeks (ideally, 14-16 weeks)
Has 80% DR for T21

Integrated screening

Combines 1st and 2nd trimester screening into a single risk

Gives the highest Down syndrome detection rate of 90- 96%

Disadvantage: test results are not available until 2nd trimester tests are complete which might lead to delay in intervention

Sequential screening

Results of 1st trimester test is disclosed to the woman enabling choice of early invasive test for women at high risk and those at low risk can go ahead with the 2nd trimester tests

Detection of anomalies in the fetus before birth/ May be

Genetic abnormalities/chromosomal anomalies
Structural defect
Combination of both

Aim is not to generate perfect babies but to help parents learn what they need to know about the health of their unborn child to help them make informed decisions for themselves and their family within the context of their own value system

PND aims at detecting

Birth defects e.g. NTD, cleft palate, anterior abdominal wall defects
Chromosomal abnormalities e.g. Down’s syndrome genetic diseases e.g. hamoglobinopathies, cystic fibrosis
Other conditions that may adversely affect the fetus when it is born

Why offer PND?

Primary aim of PND: to provide an accurate diagnosis that will allow informed decision making in couples at increased risk of having children with genetic conditions or congenital abnormalities
To enable early diagnosis and timely treatment in or ex- utero to give parents the option of aborting a fetus with a diagnosed condition (if local abortion laws permit) to enable the parents and healthcare provider to prepare for the birth and care of an affected child psychologically, socially, financially, medically or for the likelihood of a stillbirth

Who is PND offered to?

Usually, pregnant women with positive prenatal screening results in other to arrive at a definitive diagnosis

Indications for PND

High risk prenatal screening test result

Advanced maternal age e.g. screening for Down’s

Previous child with chromosomal or structural abnormality

Family history of inherited condition e.g. single gene disorders like SCD, Neurofibromatosis, achondroplasia

Family history of structural abnormality e.g. Neural Tube Defect (NTD), CHD

Other risk factors e.g. co-sanguinous marriage, poor obstetric history e.g. recurrent miscarriage, maternal DM, antenatal exposure to illnesses that could cause fetal abnormalities e.g. toxoplasmosis, rubella

Antenatal exposure to teratogens

Incidental findings of abnormalities during routine anomaly scan

Methods of PND

Can be invasive or noninvasive

Noninvasive methods

Fetal visualization e.g. Ultrasound, fetal echo, MRI
Maternal serum screening e.g. maternal serum alpha fetoprotein for NTD
Noninvasive prenatal testing (NIPT)

Invasive methods

Fetal visualization e.g. embryoscopy, fetoscopy
Tissue sampling e.g. amniocentesis, Chorionic villous sampling (CVS), percutaneous umbilical blood sampling (PUBS), skin, muscle or liver biopsy
Specimens from invasive tests are sent for cytogenetic analysis

Disadvantages of invasive tests

Risk of mother to child transmission (MTCT) of diseases eg HIV, hepatitis so screen before the test;
Risk of sensitization of Rhesus negative mother so give Rh immunoglobulin in Rh -women
Risk of pregnancy loss

Ultrasound

May be used in isolation or as an adjunct for invasive testing (e.g. amniocentesis and CVS)

Abnormalities may be picked up in the 1st trimester

Anomaly scan is usually offered at 20-22 weeks

Useful in detecting structural abnormalities e.g.

Neural tube defects: anencephaly, meningocoele
Anterior abdominal wall defect
Heart defects
Multiple defects

Amniocentesis

Commonest invasive procedure

Involves insertion of a needle into the amniotic cavity and aspiration of amniotic fluid which is sent for analysis

Usually performed at 15-18 weeks gestation though may be done later

Obtained sample is sent for cytogenetic analysis

Miscarriage; 1%
Intra-amniotic bleeding
Abruptio placentae
Rupture of membranes
Infection
Long term effects

Fetal lung development
Limb deformities

Chorionic villous sampling

Needle introduced to aspirate placental tissue;

Usually transabdominal; transvaginal if posterior placenta, retroverted uterus

Can be performed earlier than amniocentesis; usually 11-14 weeks

Complications of CVS

Miscarriage; 1-2%
Injury to surrounding structures
Confined placental mosaicism

Embryoscopy

1st trimester procedure

Rigid endoscope inserted via cervix under USS guidance to visualize structural malformations

Fetoscopy

Endoscopic visualisation of the fetus

Performed in 2nd trimester >16 weeks (usually at 18-20 weeks)

Endoscope inserted into amniotic cavity through an anterior abdominal wall incision;

3-5 % miscarriage rate

Enables

Detection of structural abnormalities
Fetal blood sampling for haemoglobinopathies
Fetal biopsy; skin, liver
Fetal surgery; ablation of anastomoses in ttts, selective feticide, division of amniotic bands

Cordoscentesis/percutaneous umbilical blood sampling

Ultrasound guided procedure to obtain fetal blood from umbilical cord

Also used for in-utero transfusion e.g. in anemia from Rhesus iso-immunization

Noninvasive Prenatal Diagnosis (NIPD)

Detects fetal DNA fragments present in maternal plasma during pregnancy i.e. cell free DNA most

cfDNA is maternal in origin—cell free maternal DNA
10-20% is fetal in origin…cell free fetal DNA (cffDNA)

Detectible as early as 6 weeks gestation

Can detect fetal sex, chromosomal abnormalities,

Useful in whole genome sequencing— determining the complete DNA sequence of every gene

Can detect about 99% of cases of trisomy 21, 97% of trisomy 18, and 92% of trisomy 13, with respective false- positive rates of 0.08%, 0.15%, and 0.2%

Advantages

Safe and easy; no risk to mother or fetus
Performed early in gestation, allowing time for decision-making/early pregnancy termination/preparation for affected child

cffDNA

First detected in maternal blood at 4-5 weeks
Cleared from maternal circulation within 1 hour of birth so is specific to the current pregnancy

Uses

Screening for Down’s syndrome
Detection of some single gene disorders e.g. achondroplasia, thanatophoric dysplasia, SCD
Fetal sex determination
Useful in pregnancies at risk of serious X linked conditions e.g. Congenital Adrenal Hyperplasia (CAH), Duchene muscular dystrophy (DMD)

Benefits of NIPT/NIPD

Reduced requirement for invasive testing
No risk of miscarriage
Can be offered earlier than traditional testing

Limitations

In multiple pregnancy, impossible to deternine which fetus the DNA is from
Women with high BMI have relatively reduced cffDNA because they have a higher proportion of their own cell free DNA resulting in higher chance of inconclusive result
Cost

Management options following PND

Continue with the pregnancy— increased risk of having an affected child, IUGR, stillborn, NND

Termination of pregnancy

In-utero therapy

Post-natal treatment

Types— 2D, 3D

Aneuploidy screening— NT scan

1st trimester screening for fetal anomalies

2nd trimester anomaly scan

20-22 weeks scan

Indications

Fetal anatomy and growth
Placenta localization
Amniotic fluid volume
Uterine artery dopplers
Cervical length

3D ultrasound

Useful for diagnoses of certain birth defects e.g. cleft lip

More easily understood by parents

Data can be stored for later evaluation by a specialist can be used to calculate tissue and fluid volumes e.g. lung volume measurements for prediction of pulmonary hypoplasia in CDH

note

Fetal MRI

Complementary to ultrasound
Particularly useful in CNS, thoracic and lung abnormalities e.g. encephalocoele, congenital diaphragmatic hernia

Anencephaly

Partial or complete failure of closure of anterior end of neural tube

NTD characterized by absence cranial vault and cerebral hemispheres

Absent skull; exposure of cerebral cortex

100% DR by Ultrasound. 1st trimester USS diagnosis possible

Absent cranium
Brain tissue seen exposed to amniotic cavity
Prominent eyes and ears

Lethal; 50% stillborn; remaining die early NND

Microcephaly

May occur in genetic syndromes

Trisomies

Congenital infections

Drugs/alcohol

Constitutional

Encephalocoele

Skin covered NTD affecting cranium resulting in midline mass overlying skull defect

75% occipital; 12% frontal, 13% parietal

Mass may be filled with fluid, brain or both

Spina Bifida

Failure of vertebral arches to close prior to 6th week due to failure of normal neural ectoderm development

Prenatal diagnosis

Raised MSAFP
USS

note

Screening for neural tube defects

Biochemistry: maternal serum alphafetoprotein at 16-18 weeks
Amniotic fluid serum alpha fetoprotein
Ultrasound to detect neural tube defects

Heart defects

Prenatal detection enables consideration of options

PND of genetic abnormality
Continuing with pregnancy
Termination if allowed

Plan delivery; place, timing, neonatal care

Counsel regarding risks in subsequent pregnancy

GI anomalies e.g. duodenal atresia

Associated with T21

Other cardiac, skeletal

Renal and GI defects may be present

Renal genitourinary anomalies

Various types

May be minor or major, lethal or non-lethal

Lethal e.g. bilateral renal agenesis: counsel; offer TOP if allowed

Non-lethal eg posterior urethral valve; consider in-utero therapy, optimize time and place of delivery

Anterior abdominal wall defects

Gastroschisis

Herniation of bowel, liver and other organs into amniotic cavity

Not covered by peritoneal membrane

Typically the hernia is on the right side of a normally inserted umbilical cord

Exomphalos

Herniation of bowel, liver and other organs into the intact umbilical cord

Tissues are covered by peritoneal membrane

Umbilical cord inserts into the hernia

Some ethical dilemmas in PGD

Late onset disorders or for cancer predisposition,

BRCA1 and 2 mutation, by 70years, 65% risk of breast cancer, 39% for ovarian in BRCA1; 45% and 11% for BRCA-2 respectively

Li Fraumeni syndrome (genetic predisposition to cancer later in life; 55% develop cancer by 40 yrs, 90% by 60 yrs)

Legality or otherwise of transferring an affected embryo (illegal in UK; HFEA act 2008)

PGD for HLA matching: welfare of present versus future child; harvesting organs, benefits for receiving child versus disadvantages for the future child; what If the treatment fails?

Informed decision making; non-directive counseling

RhD alloimmunization

Pre-eclampsia

Gestational diabetes

Fetal growth restriction

Preterm birth

Fetal defects

RhD isoimmunization

D antigen: present on surface of RBC of Rh + individual; absent in Rh –

Pathophysiology:

Feto-maternal exchange of blood may occur during pregnancy, delivery, invasive procedures, miscarriages etc.
First pregnancy: Rh + cells crossover into Rh - mother, mother produces anti-D antibodies
Subsequent pregnancy: mother’s antibodies cross the placenta and destroy fetus’s RBC- haemolysis, anemia and its sequelae e.g. CCF, hydrops
Prevented by giving mother anti-D immunoglobulin (it prevents mother from producing anti D antibodies; ideally given prophylactically at 28 and 34 weeks and following delivery of Rh + infant, invasive procedures etc.

Cell free fetal DNA or amniotic fluid PCR can be performed for PND of fetal rhesus status so that Rh immunoglobulin is only given if the fetus is actually Rh positive.

A sonographic short cervix diagnosed by transvaginal ultrasound is the most powerful predictor of preterm delivery (50% of women with a cervical length ≤15 mm will deliver prior to 32 weeks of gestation).

Cervical length at 22-24 weeks < 25mm by TVS results in > 6fold increased risk for PTB < 35 weeks

Randomized clinical trials and systematic reviews have demonstrated that vaginal progesterone reduces significantly the rate of preterm birth by 50% and reduces neonatal morbidity/mortality

Fetal fibronectin in cervico-vaginal secretions also used to screen for PTB

Various modalities for prenatal screening and prenatal diagnosis

Allows early decision making regarding pregnancy

PNS identifies fetus at high risk of a condition, PND identifies fetus with the condition

PNS and PND commonly offered for chromosomal diseases but may also be offered for genetic diseases (e.g. SCD), fetal defects (e.g. NTD), maternal conditions (e.g. PET, PTB)

Prenatal screening should be offered to all women depending on local protocol

Prenatal diagnosis should be offered to women with high risk prenatal screening results or other women with indications for PND

Ultrasound is beneficial in both screening and diagnosis

PNS: combined test, triple test, quadruple test

PND: may require invasive procedures which carry risks of miscarriage

NIPT enables PND without associated risk of miscarriage

PGD may be an option

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