5

WHAT IS ANEUPLOIDY?

 Unbalanced chromosome number- having one or more extra or missing chromosomes

 Tri 21 is the MC - 1 in 800 live births

 Tri 18 - 1 in 6,000

 Tri 13 - 1 in 10,000
 EVERY WOMAN HAS A RISK

 That her fetus/baby has a chromosomal defect

 Background or a priori risk depends on maternal age and gestation

 Individual, patient specific risk is calculated by multiplying the a priori risk with a series of likelihood ratios,

which depend on the results of a series of screening tests

 The LR for a given sonographic or biochemical measurement

= % of chromosomally abnormal fetuses / % of normal fetuses with that measurement

 Every time a test is carried out, the ‘a priori risk’ is multiplied by the LR of the test to calculate a new risk,

which then becomes the a priori risk for the next test.
 WHO SHOULD BE OFFERED SCREENING FOR ANEUPLOIDY?

 All women should be offered screening OR diagnostic testing for genetic

disorders regardless of maternal age

 Although risk increases with advancing mat age, most children with Down sy

are born to younger women because a larger proportion of all children are
born to young women.

 Screening or Diagnostic testing should be discussed and offered to all women

early in pregnancy ideally at the 1st prenatal visit.

 ASSESS BEFORE CHOOSING THE TEST

 Choice of Screen vs diagnostic depends on woman’s goals and values and her desire for

informational accuracy

 Prior history, family history, number of fetuses, gest age, availability of NT, Cost, option

for termination for abnormal result

 Pre-test counselling and communication about the sensitivity of test, FPs, risks and

benefits of test should be provided

 After counselling, patient may decline these tests for any reason

 Screening tests are available in all trimesters

 MATERNAL AGE
 Less accurate
 If only >35 only had diagnostic test then detection rate would be only 21%
 SERUM BIOCHEMISTRY

 Patterns of change in maternal serum marker levels are well characterized during the

second trimester

 The presence, level and rate of increase or decrease varies with gestational age

 Effect of gestational age is normalized by establishing day-­specific medians for each of

the markers and by dividing each woman’s value by the appropriate median to calculate
the MoM.

 The distribution of MoM values can then be compared in unaffected and Down’s

syndrome pregnancies
 RELATIVE CHANGES IN SERUM MARKERS AND NT IN TRISOMY 21, TRISOMY 18, AND OPEN NTDS

 Trisomic pregnancies Parameter Trisomy 21 Trisomy 18 Open NTD

(MOM)
 Associated with altered maternal serum
PAPP-A ↓(0.4 – 0.6) ↓
concentrations of various feto-placental
Free b-hCG ↑ (2.66) ↓
products
NT ↑ ↑
 Free bhCG has greater discriminatory value
Total b-hCG ↑ (2.06) ↓
than total hCG levels uE3 ↓ (0.73) ↓

AFP ↓ (0.73) ↓ ↑

Inhibin ↑ (1.77) -
Multiple markers, likelihood ratios and risk calculation
• These markers are largely independent of each other
• LRs for each marker can be combined by multiplying and correlationg
between each marker pair in affected and unaffected pregnancies.
• Composite LR is multiplied by the maternal age related risk to give a final
patient-specific risk.
• This statistical algorithm is known as multivariate Gaussian
distribution analysis.
 Influenced by machine and reagents used, gestational age, maternal weight, ethnicity,

smoking status and method of conception

 In Black women, the PAPP-A level is about 60% higher than in White women.

Failure to take into account ethnic origin would result in substantial underestimate of
the true risk

 Smoking and conceiving by IVF- decreased PAPP-A: increase in false positive rates
In trisomy 21 pregnancies

 Maternal serum free ß-hCG is about twice as high and PAPP-A is

reduced to about half compared to chromosomally normal pregnancies

Performance of screening for trisomy 21 by

maternal age and serum free ß-hCG and PAPP-A:

 Detection rate 65%

 False positive rate 5%

 Combined screening

 The overall performance of combined screening is better at 11 than

at 13 weeks and may be best at 10 weeks

 Ultrasound scanning for fetal abnormalities is better at 12 than at 11

weeks and much better than at 10 weeks

 A good way of achieving a high performance of screening for trisomy

21 and diagnosing major fetal defects by ultrasound is to carry out the

blood test at 10 or 11 weeks and the ultrasound scan at 12 weeks
 OTHER ANEUPLOIDIES

There are differences between the three trisomies:

 Fetal NT is higher in trisomies 18 and 13 than in trisomy 21

 Serum PAPP-A is lower in trisomies 18 and 13 than in trisomy 21

 Serum free ß-hCG in trisomy 21 is high whereas in trisomies 18 and 13 this is low

 Fetal heart rate in trisomy 13, unlike trisomies 21 and 18, is high

 The use of specific algorithms for trisomy 18 and trisomy 13, in addition to the use of the algorithm

for trisomy 21, improves the detection of trisomies 18 and 13 from about 75% to 95% with a minor
increase in the total false positive rate from 3% to 3.1%
 ULTRASOUND SCREENING FOR ANEUPLOIDIES

 The least effective primary screening with only 50-60% detection rate

 Should not be used in isolation to diagnose or exclude Down

 If negative screening or diagnostic test done already then USG should not be used again to

screen

 The ‘genetic sonogram’ at 15–20 weeks

 Applied to adjust risk assignment for aneuploidy by identification of either major

malformations or ‘soft markers’.

 WHAT IS THE ROLE OF USG IN SCREENING?

 ‘Soft markers’-

- Sonographic findings widely regarded as variants of normal

- Do not confer any clinical ill-effects in their own right
- Commonly seen to resolve as pregnancy advances
- Known to have an association with aneuploidy.
- Association most marked when multiple markers are observed
 Absence of USG markers decreases a woman’s advanced age related risk of aneuploidy by
>80%
 Higher risk if markers are identified
 Provides a refined, individualized risk-estimate to facilitate decision making on invasive
 Trisomy 21 Trisomy 18 Trisomy 13

Structural Cardiac abnormalities Cardiac abnormalities, Esophageal atresia Cardiac abnormalities Diaphragmatic
abnormalities Duodenal atresia Strawberry-shaped head hernia, Omphalocele Holoprosencephaly
Brachycephaly Diaphragmatic hernia Facial clefting, Cyclopia
Hydrocephalus Omphalocele, Umbilical cord cyst Agenesis corpus callosum Talipes,
Clinodactyly Meningomyelocele, Agenesis corpus callosum Rocker-bottom foot, Polydactyly
Cystic hygroma & hydrops Facial clefting, Talipes, Rocker-bottom foot Cystic hygroma & hydrops
Radial aplasia, Overlapping digits
Cystic hygroma & hydrops

Soft markers Nuchal fold thickening Choroid plexus cysts Echogenic intracardiac foci
Ventriculomegaly Enlarged cisterna magna Enlarged cisterna magna
Short femur or humerus Ventriculomegaly Ventriculomegaly
Hypoplastic nose Short femur or humerus Pyelectasis
Echogenic bowel Hypoplastic nose Single umbilical artery
Pyelectasis Echogenic bowel
Sandal gap toes Pyelectasis
Single umbilical artery
ISOLATED VERSUS MULTIPLE MARKERS FOR TRISOMY
21

Trisomy 21 Normal LR

No markers 31% 87% 0.4

One marker 23% 11% 2

Two markers 15% 2% 10

≥ 3 markers 15% 0.1% 115

• Controversial area
• In UK, the current policy is not to adjust a Down’s risk derived from a nationally
recognized screening program on the basis of soft markers
• Among soft markers, 3rd tri FU in only isolated renal pelvis dilatation, echogenic
bowel or short humerus/femur.
• If CPC or EIF and screening negative- no further evaluation needed
• If EB, Incresed NF, Absent/Hypoplastic NB- further counselling needed
• If isolated marker found then screening be offered if not already done
• If increased NT and normal chromosomes on diagnostic testing- still at risk of various
Genetic syndromes, CHD, Abd wall defects, CDH
 ANEUPLOID CONDITIONS OTHER THAN TRISOMY 21

 CPCs in 1/3rd of T18 fetuses in 2nd trimester in contrast to 1–2% of euploid

Triploidy

 Severe, early onset asymmetrical IUGR is the hallmark

 Oligohydramnios and small placenta if maternally derived

 Partial molar or hydropic placenta if paternally derived

 Ventriculomegaly, DWM, ACC, cardiac anomalies, micrognathia, echogenic bowel, renal

malformations, thickened nuchal fold, NTD, talipes and syndactyly of 3rd and 4th digits
SINGLE SCREENING TESTS

1ST TRIMESTER

 When CRL is 45 to 84mm (10 0/7 TO 13 6/7)

 NT+f or t HCG+PAPPA
QUAD SCREEN

 15 0/7 to 22 6/7 weeks

 Does not require specialized USG for NT and has added advantage of

screen for NTD

 Best time is 16-18wks

 PENTA SCREEN

 Includes Hyperglycosylated HCG(Invasive Trophoblast Antigen) in

addition to Quad screen markers

 2nd Tri- Limited availability and limited data

 APPROACHES FOR SCREENING FOR DOWN SYNDROME
Name Description

Triple Screen MSAFP, UE3, and hCG

Quad Screen MSAFP, UE3, hCG, and inhibin A

Combined FTS NT, maternal serum PAPP-A, and free b-hCG

Integrated Screen NT and MS-PAPP-A in 1st trimester, and quad screen in 2nd trimester;
Final result is disclosed at the completion of all the tests

Serum-Only Integrated Maternal serum PAPP-A in 1st trimester, and quad screen in 2nd Trimester

Stepwise Sequential Combined 1st trimester screen and quad screen with results disclosed after each test;
If positive, early diagnostic test. If negative, 2 nd tri quad screen and final risk incorporating both

Contingent Sequential Combined 1st trimester screen with disclosure of the result.
Intermediate risk- 1:30 and 1:1500- continues to quad screen with the final risk assessment
incorporating first and second results.
Negative risk- <1:1500 and no further screening is applied.
DOWN SYNDROME SCREENING TESTS AND DETECTION RATES (5% SCREEN POSITIVE
RATE)

Screening Test Detection Rate (%)

First Trimester NT 64–70
NT+PAPP-A+ free or total β-hCG 82–87
Second Trimester Triple screen (MSAFP, hCG, UE3) 69
Quadruple screen (MSAFP, hCG, UE3, inhibin A) 81
First Plus Second Integrated (NT, PAPP-A, quad screen) 94–96
Trimester
Serum integrated (PAPP-A, quad screen) 85–88
Stepwise sequential First-trimester test result 95
screen Positive: diagnostic test offered
Negative: second-trimester test offered
Final: risk assessment incorporates first

Contingent sequential First-trimester test result 88–94

screen Positive: diagnostic test offered
 NASAL BONE AND FACIAL ANGLE

 11+0-13+6 weeks and the CRL 45-84 mm

 Magnification, mid-sagittal view
 Three distinct lines:
 Top line- skin
 Bottom- nasal bone
 Line in front of the bone and at a higher level than the skin represents the tip of the nose
 At 11-13 weeks the nasal bone is absent in about:
 Euploid fetuses 1-3%
 Fetuses with trisomy 21 60%
 Fetuses with trisomy 18 50%
 Fetuses with trisomy 13 40%
 FIRST-TRIMESTER DOPPLER ASSESSMENT OF THE DUCTUS VENOSUS

 Reversed flow associated both with aneuploidy and with congenital heart disease
 Abnormal ductus venosus flow in 82% of aneuploid fetuses, false positive rate of 5%
 11+0-13+6 weeks and the CRL 45-84 mm
 At 11-13 weeks reversed a-wave is found in about:
Euploid fetuses 3%
Fetuses with trisomy 21 65%
Fetuses with trisomy 18 55%
Fetuses with trisomy 13 55%
 FIRST-TRIMESTER TRICUSPID REGURGITATION

 Has to occupy at least half of systole and reach a velocity of over 80 cm/s.
 Validated in high-risk groups only

At 11-13 weeks TR is found in about:

 Euploid fetuses 1%

 Fetuses with trisomy 21 55%

 Fetuses with trisomy 18 30%

 Fetuses with trisomy 13 30%

CHARACTERISTICS AND LIMITATIONS OF VARIOUS TESTS:

1ST TRIMESTER:
2ND TRIMESTER:
 Combined NT+Serum
 Screen for Open NTD
 Advantages:
 Quad detection rate similar to 1st tri-80% with
 Slightly higher but not sig different detection
5% FP
rate
 Additional screen for SLOS and Placental
 Earlier detection
Sulphatase deficiency
 Earlier screen for fetal n placental disorders  Triple test- lesser detection -69%
 Disadvantages  Penta screen –appears to perform well but no
 Cannot test for Open NTD valid data
 Availability of Certified sonographer
 DOES SCREENING IN MULLTIFETAL GESTATIONS DIFFER?

 Risk of aneuploidy is affected by the number of fetuses and Zygosity

 Limited data

 In Dizygous- each fetus carriers a risk similar to mother’s age adjusted risk

 Mother carries increased risk of having Aneuploid fetus because of carrying 2 fetuses

 Monozygous- usually will have same karyotype

 No method of screening is as accurate as in singleton

 More complex because of diminished effectiveness and no clarity on how to manage if

one fetus found to be aneuploidy

 Diagnostic testing is less acceptable because of increased loss rate

 NT allows each fetuses to be assessed separately and can be used in even higher order

preg

 Distribution of NT does not differ significantly from singleton and hence same standard

cut-offs may be used

 NT and FTS detection rate of 75% with FP of 9%, more reliance should be placed on NT
 Increased NT in Monochorionic twins of Discordant size could be due to TTTS and hence FU

 Data not available for serum screening of higher order pre hence limited to Singleton OR Twin

 1ST Tri, Quad and Combined analyte can be used- few data from prospective studies

 2nd Tri serum screening can identify 50% of fetuses with Down sy with a 5% FP rate

 CfDNA limited evidence of efficacy and hence should not be used.

 If Fetal demise or anomaly detected in one fetus then serum screening should not be done- significantly

inaccurate results
 FEW WORDS ON NIPT
 Conventional FTS for aneuploidy is done by USG(NT) and biochemical

Double marker test

 These have a sensitivity of up to 95% but also have a high False

positive rate of 3-5%

 A positive screen leads to many invasive procedures to diagnose a

single case of aneuploidy.

 Invasive procedures have a miscarriage risk of 0.1 to 1% hence

placing many normal foetuses at risk

 Newer method of screening - NIPT

 Sequencing technology to provide risks for aneuploidy

 NIPT analyzes cell-free fetal DNA circulating in maternal blood

 Testing can be done any time after 10 weeks; typically between 10-22

weeks.

36
 Most of commercially available NIPTs have a sensitivity and

specificity of 99% with a very low False positive and False

negative rates in the range 0.1 to 0.5%.

 Hence Non-invasive prenatal testing (NIPT) offers an inter-

mediate step between serum screening and invasive diagnostic

testing

 As of 2014, commercially available NIPT identifies aneuploidies in

chromosomes 13, 16, 18, 21, 22, X, and Y

 Some commercially available NIPT also tests for abnormalities on chromosomes

1p, 5p, 15q, 22q, 11q, 8q, and 4p

 NIPT was first released in Hong Kong in August 2011 and soon after was

introduced commercially in the US in October 2011.

 As of 2014, five for-profit companies offer NIPT

 MaterniT21 Plus: detects trisomies 13, 16, 18, 21, and 22, certain sex chromosome aneuploidies and some

micro deletions

 Verifi: basic test detects trisomies 13, 18, and 21 expanded test also reports sex chromosome aneuploidies.

 HarmonyTM : tests for trisomies 13, 18, and 21

 PanoramaTM: detects trisomies 13, 18, and 21 as well as some sex chromosome aneuploidies and some

microdeletions.

 InformaSeq: detects trisomies 13, 18, and 21, and additional optional testing can detect fetal sex/sex

chromosome aneuploidies.

 Four of these tests can detect fetal sex, but physicians need to request this information. 39
 1 in 400 pregnancies have Down syndrome
4000 women screened
Only 10 have Down syndrome

Quad test
• 80% Sensitive 3792 report low risk
208 report high risk • 5% Test positive
rate
• 5% False positive
8 of Down test 200 do not have Down• 4% PPV 2 of them will have Down
positive syndrome
Invasive test on 208
• 8 will have Down
• Other 200 will be told they are
normal

• Invasive procedures have 0.5% abortion

risk
• 1 among 200 normal pregnancy abort
due to procedure
 LOW RISK AVERAGE HIGH RISK
RISK

Nothing else need to 20% chance of missing Down 96% are false positive
be done .Out of 200
procedures,only 10 will
have Down
Previously not offered
any test. NIPT can
pick up the missed Previously used to
cases undergo Invasive
test. NIPT
Reassured if significantly reduces
Negative unnecessary
procedures

Invasive test if
Positive
RISK CUT-OFF LEVELS IN DOWN’S SYNDROME SCREENING
 Nuchal translucency (NT) is the sonographic appearance of a collection of fluid under the skin behind

the fetal neck in the first trimester of pregnancy

 The term translucency is used, irrespective of whether it is septated or not and whether it is confined to

the neck or envelopes the whole fetus

 The incidence of chromosomal and other abnormalities is related to the size, rather than the appearance

of NT

 During the second trimester, the translucency usually resolves and, in a few cases, it evolves into either

nuchal edema or cystic hygromas with or without generalized hydrops