this presentation discusses the PRENATAL diagnostic tests and protocols available

1. PRENATAL DIAGNOSIS
YESTERDAY TODAY AND TOMORROW
Narendra Malhotra
Jaideep Malhotra
Neharika Malhotra Bore
Rishabh Bora
Keshav Malhotra
RAINBOW FETAL MEDICINE FOUNDATION,AGRA
info@rainbowhospitals.org

2. “I get by with a little help from my
friends”
Dr Ashok Khurana
Dr Prashant Acharya
Dr Pratima Radhakrishnan
Dr Deepika Deka
Dr Anita Kaul
Dr S Suresh
Dr.Raju Sahetya
Dr Manjeet mehta
Dr Kuldeep Singh
THANK YOU FRIENDS FOR HELPING AND CONTRIBUTING TO THIS PRESENTATION

3. Generation X
Generation Y
1940 1950 1960 1970 1980 1990 2000 2010
Silent generation
Baby boom generation
Net generation
Different generations – different values
- different learning needs
Paradigm Shift Prenatal Diagnosis

4. The beliefs of great phylosopher Socrates….
“THE SECRET OF CHANGE IS
TO FOCUS ALL OF YOUR
ENERGY,
NOT ON FIGHTING THE OLD,
BUT ON BUILDING THE NEW.”
~ SOCRATES

6. Prenatal Diagnosis
The quest for a less invasive approach
to Prenatal Diagnosis
has been the focus of much research
over recent decades.

7. Prenatal Diagnosis
• In late 70s, the introduction of ultrasound and the
possibility to visualize the fetus in utero.
A true revolution in two respects.
• For doctors, it allowed to diagnose in fetal life
problems, that until then, had been only known in
the newborn.
• For parents, it facilitated the recognition of the
fetus as a person.

8. The Fetus as a Person
This resulted in the development of a new concept
“The Fetus as a Patient”
and with it, a sub-speciality
“Fetal Medicine”.

9. Maternal-Fetal medicine (MFM)
• is the branch of obstetrics that
focuses on
the medical and surgical managemen
t of high-risk pregnancies.
• Management includes monitoring
and treatment including
comprehensive ultrasound, chorionic
villus sampling,
genetic amniocentesis, and fetal
surgery or treatment. Obstetricians
who practice maternal-fetal medicine
are also known asperinatologists.
• This is a subspecialty to obstetrics
and gynecology mainly used for
patients with high-risk pregnancies.
FETAL MEDICINE SPECIALISTS

10. The Fetal Medicine Team
maternal fetal medicine specialists
Obstetrician
Manages pregnancy and invasive diagnostic tests
Maternal-Fetal Specialist – a Perinatologist
Diagnosis and treatment of High-Risk Pregnancy.
Geneticist or Genetic Counselor
Prenatal genetic counselling, diagnosis, prognosis,
prepare the to be parent, for the consequences
Neonatologist
Know Prenatally about expectations and outcomes
and prepares to care for the baby after birth.
Pediatric surgeon
Who, with the neonatologist,
makes the fetal/Newborn treatment plan
Obstetric sonologist
Fetal diagnosis and its severity and guiding
diagnostic and Intrauterine therapeutic procedures.
Pediatric super-specialists
Cardiologists, Cardiothoracic Surgeons,
Neurosurgeons, and Urologists, as per the defect.

11. • Maternal-fetal medicine specialists are
obstetrician-gynecologists who undergo an
additional 2–3 years of specialized training in the
assessment and management of high-risk
pregnancies.
• As a result, they are able to take care of pregnant
women who have special medical problems (e.g.
heart or kidney disease, hypertension, diabetes,
and thrombophilia), pregnant women who are at
risk for pregnancy-related complications
(e.g. preterm labor, pre-eclampsia, and twin or
triplet pregnancies), and pregnant women with
fetuses at risk.
• Fetuses may be at risk because of chromosomal
or congenital abnormalities, maternal disease,
infections, genetic diseases, and growth
restriction.

12. Maternal-fetal
specialists
• Maternal-fetal medicine specialists
have training in
obstetric ultrasound,
invasive prenatal diagnosis using
amniocentesis and chorionic villus
sampling,
and the management of high-risk
pregnancies.
• Some of them are further trained in
the field of fetal diagnosis and
prenatal therapy where they become
competent in advanced procedures
such as
targeted fetal assessment using
ultrasound and Doppler,
fetal blood sampling and transfusion,
fetoscopy,
and open fetal surgery

13. Addition of GENETISIST
to the
TEAM MFM
• The field of maternal-fetal
medicine is one of the most
rapidly evolving fields in medicine
especially in what concerns the
fetus. Research is being carried on
in the field of fetal gene and stem
cell therapy in hope to provide
early treatment for genetic
disorders, open fetal surgery for
the correction of birth defects like
congenital heart disease, and the
prevention of preeclampsia

14. MFM specialists
• MFM subspecialists are now required to do
a minimum of 12 months clinical rotation
and 18 month research activities.
• They are encouraged to use simulation and
case-based learning incorporated in their
training, a certification in advanced cardiac
life support (ACLS) is required, they are
required to develop in-service examination
and expand leadership training.
• As Maternal-fetal medicine subspecialists
improve their work ethics and knowledge of
this advancing field, they are capable of
reducing the rate of maternal mortality and
maternal morbidity.

15. Routine Antenatal Care 1990s…….
Early scan to diagnose
pregnancy & dating
Fetal defects
22-24 wks
Anomaly scan

16. Routine Antenatal Care 2005
11-14 wks
Fetal defects
20-23 wks
P I P I P
The great
Ob syndrome

17. Routine Antenatal Care 2010
11-13+6 wks
Fetal defects
Chemical markers Major
Cardiac defects
Uterine artery Doppler
20-23 wks
Anomaly scan

18. Magnitude of Problem
Congenital & Genetic Disorders
Disorder Frequency
at birth in stillbirths in NN Deaths
Congenital 2-5% (1:50 , 6 lacs/yr) 13.6 % 7 – 10 %
Malformations
Chromosomal 0.5% ( DS – 1:916 , 22,000 / yr )
Single Gene 0.6% ( B- thal – 1:2700 , 9000/ yr ; SCD - 5,200 /yr ;
DMD + SMA - 4,500 / yr )
( IC Verma , Preventive Genetics ,2006 )

20. GENETIC DIAGNOSIS – YESTERDAY, TODAY & TOMORROW
Testing of chromosomes
Down Syndrome
Many more conditions can
be tested
As causative gene is now
known
Advances in
technology
Larger panels can be
tested
Epigenetics /
Expression
Gene Therapy
Gene Modification

21. For the busy practitioner,
a) Genetics is viewed as a
complex nuisance that interferes
with clinical management
b) Family histories are often
inaccurate, and may violate
privacy of other family members
c) Limited Genetic Counseling
support is available nationally
d) Commercial Laboratories with
financial interest often provide
genetic support

22. Cut off by AGE
• Screening for open neural tube defects (ONTDs) by
MSAFP began in the 1970s and screening for fetal
chromosomal anomalies began with amniocentesis in
the mid-1960s.
• Maternal age, ≥ 35 years at the expected date of
delivery, was used as the lower threshold of who
should be offered prenatal diagnostic testing for
chromosomal anomalies.
• This age was chosen as it was the point at which the
risk of a pregnancy loss was less than the chance of
identifying a pregnancy with a significant
chromosomal abnormality.

23. Most problems actually
happen in the so called “Low risk” group

24. PAST
Invasive Procedures and Karyotype
Late Maternal Age
Positive History
Recent Past
Ultrasonography
Nuchal Translucency
Serum Screening
Invasive Procedures
Metaphase Karyotype

25. Inverted Pyramid of Care and Triage
effectively in 1st Trimester

26. Historical Perspective
Prenatal Screening and Diagnosis
• 1980 : Amniocentesis / CVS (advanced maternal age)
• 1990 : Triple / Dual screening (T21, T18 and T13)
• 2000 : First trimester screening (T21, T18 and T13)
• 2012 : First trimester screening + NIPT (T21, T18 and T13)
• 2015 : NIPT (more extensive genetic screening)

27. Screening tests
• Maternal serum screening enabled women of
all ages to pursue screening for chromosomal
abnormalities.
• It also provided women who were concerned
about the chance of miscarriage associated with a
diagnostic test the opportunity to obtain
information without the added risk.
• Screening provides women with an individual
risk by adjusting their age related risk (in the case
of chromosomal abnormalities) or population risk
(open neural tube defects) with the
biochemical results specific to their pregnancy.

28. • Prenatal screening programs are
applicaple to the population as a whole.
• Diagnostic testing programs
are targeted to specific high risk
populations.
• Screening cut-offs are one way to
define who is considered at high risk
and who should be offered diagnostic
testing.

29. Down syndrome – Serum screening
• Dual screening ( > 1990)
– Maternal age
– Serum : free B-HCG, PAPP-A
• Combi test ( > 2000)
– Maternal age
– Nuchal translucency (NT)
– Serum : free B-HCG, PAPP-A

32. Screening is VOLUNTARY
Screening should be made UNIVERSAL
 SCREENING SHOULD BE OFFERED TO
EVERYONE
 Screening should be DONE only to women willing
for the same
 Those who decline screening may not be offered this
tests and decision should be documented.

33. Biochemical Screening
NOW ONLY

34. Watch
carefully the
picture

35. Where do all these markers come
from?
(adrenals)
(liver)

36. Various Integrated screening in
strategies (1st and 2nd trim)
Main strategies:
• Fully Integrated
• Step-wise sequential
• Contingent screening
1st trimester:
NT, PAPP-A
No risk estimate
2nd trimester:
Fb-hCG, AFP, uE3, (± Inhibin)
Final risk estimate:
All markers
1st trimester:
NT, PAPP-A, Fb-hCG
Risk estimate
2nd trimester:
Fb-hCG, AFP, uE3, (± Inhibin)
Final risk estimate:
All markers
CVS
NIPT
High risk
Low risk
1st trimester:
NT, PAPP-A, Fb-hCG
Risk estimate
2nd trimester:
Fb-hCG, AFP, uE3, (± Inhibin)
Final risk estimate:
All markers
CVS
NIPT
HR
Borderline risk
No further
screening
LR

37. First trimester screening FOR
CHROMOSOMAL ANOMALIES

38. Screening in the 1st trimester
• Time window: 8 - 14 weeks
• Ultrasound Marker:
– NT
• Biochemical markers:
– PAPP-A
– Fb-hCG
• Marker combination:
– Combined test: NT, PAPP-A, Fb hCG

39. Screening for Trisomy 21 at 11- 14 weeks
2-stage (contingency) screening- UK system
USG (N.T.) AND DUAL MARKER FOR ALL
Fetal NT and
free BhCG and
PAPP-A at 12 wks
Very high risk
Very low risk
CVS
Reassure
Borderline
risk
Further
screening
Nasal bone
DV, TR

40. Screening for Trisomy 21 at 11- 14 weeks for
India
2-stage (contingency) screening proposed
RISK ESTIMATE BY ONLY USG N.T. AND OTHER MARKERS
Fetal NT
Nasal bone and
ductus venosus
tricuspid
regurgitation at 12
wks
Very high risk
Very low risk
CVS
Reassure
Borderline
risk
Further
screening
Free B hCG
PAPP-A
THIS WILL SAVE
TIME
MONEY
OPTIMUM USE OF OUR
SKILL
DOUBLE MARKER
TEST
Scan 20w
NIPT

41. • MA + NIPT(OPTIONAL)
• Dual Marker
• NT + NB + TR + DV
First
trimester
• Quad Marker
• Genetic Sonogram
Second
trimester
Integrated 1st and 2nd trimester screening
DR – 97%
FPR – 2.5%

42. 99

43. Greater opportunity to secure a healthy pregnancy and healthy child,
with 1st Trimester evaluation
WHY CHOOSE BE HAPPY FIRST
TRIMESTER PREGNANCY CHECK?
 All the advantages of 1T screening:
Earlier reassurance and early
intervention if required
 Enhanced performance for 1T
combined aneuploidy screening:
To enable to higher DR or lower FPR
 4 marker biochemistry:
Aneuploidy risk on par with 2T Quad
 Some significant Fetal structural
abnormalities
 Pre-term Pre-eclampsia:
Identify a high risk group, who would
benefit from Aspirin

44. “Life is not free of problems, but happiness is having a path forward”-
 Earlier assessment enables more options and in the case of pre-eclampsia – the
opportunity to prevent.
 Not just aneuploidy, also includes maternal pre-eclampsia and structural defects
 Pregnancy MUST be Ultrasound dated with CRL (ideally 8-10 wks)
 Range of options: from Biochemistry only to a full portfolio, with multiple ultrasound
markers
What is BE HAPPY First Trimester Pregnancy Check?

46. Biochemistry based screening protocol for
Trisomy 21 + PE + ONTDs
in first trimester
Viability Scan: 6-8 weeks
HCG
AFP
PAPP-A
PLGF
Your NT scan
11-14 weeks
FIRST TRIMESTER
BIOCHEMISTRY

48. New Screening Protocol for Trisomy 21 + PE + ONTDs
in Indian Scenario
Viability Scan: 6-8 weeks
LOW RISK HIGH RISK
NIPT
Anomaly scan at 19 weeks
with genetic sonogram
Amniocentesis
CVS
LOW RISKHIGH RISKLOW RISK HIGH RISK

50. Ultrasonography
• First trimester Ultrasonic ‘Soft Markers’
Increased Fetal Nuchal Translucency
Absence of Fetal Nasal Bone
In common use to enable detection at increased risk of
Down Syndrome fetus
• Ultrasound can also assist
Prenatal Diagnostic Procedures
Amniocentesis, Chorionic Villus Sampling, Cordocendesis
Fetal therapy
Intra Uterine Transfusion and placements of Shunts

51. One Picture is Worth a Thousand Words

52. The Late First Trimester Scan
• Screen for Aneuploidies (Nicolaides)
• Assess structural defects
• Screen for Neural tube defects (Chaoui)
& Cleft Palate (Sepulveda)
• Screen for Pre-eclampsia
• Screen for Pre-term labor
What can ultrasound assess ?

53. The 11-13 weeks 6 days Scan
• Nuchal translucency (NT)
• Nasal bone (NB)
• Ductus Venosus (DV)
• Tricuspid regurgitation (TR)
Major ultrasound parameters for trisomy 21

54. When should the scan be performed?
• “18-22 weeks”
• Earlier scans date better
• Earlier scans require equipment, expertise & time
• Later scans see better – confirm earlier findings
• Later scans see more – heart, kidneys, palate, etc
• Local legislation – 20 wks to 24 wks
The mid-trimester fetal ultrasound scan

55. 3D Scan

56. Non Invasive Prenatal Testing
The game changer!

57. NIPT: The future is here already
● Conventional
screening
● Essentials of NIPT
● The Changed
Scenario

58. Non Invasive Prenatal Testing (NIPT)
• NIPT – 9 weeks onwards
• At least 4% fetal fraction to be identified
• Twin Pregnancy – confusing results
• Vanishing twin – confusing results
• If positive – CVS / Amniocentesis
• >99 % accuracy but still not accepted as a
DIAGNOSTIC TEST

59. NIPT: Emerging Trends
Misuse

60. NIPT: Emerging Trends
Future status
• Single gene disorders
• Triploidy
• Multifetal pregnancy
• Deletions/Duplications
• Cost
• Shorter turn around
times

61. Screening Tests
Why do these change/evolve?
• To increase the detection rate
• To reduce the false positive rate
• Risks of invasive testing
• Unreliable Serum Screening in the Indian scenario
• Unreliable NT assessment in the Indian Scenario

62. Good rapport: family listens !!!
First to identify cases : requiring evaluation by Geneticist
Reinforcing the Genetic Counselling
Evaluate still-borns  Good records of examination and autopsy are essential
If parents are not ready for any investigations for untreatable
conditions….
– Store all medical records
– Take clinical photograph ….and
– STORE FETAL DNA …for future testing…. ….take help of a clinical
geneticist!
They may change their minds in future…
Obstetrician: first point of contact

63. Invasive Prenatal Diagnostic Tests

64. CVS
• 11-14 weeks
• Transcervical
• Check for chorionic
villi under
microscope
• Risk of miscarriage
1%
• Need is obviated
now due to NIPT
????

65. Amniocentesis
• 15-18 weeks
• Risk of miscarriage < 1%
• To confirm diagnosis in positive
integrated screen and/or positive
NIPT
• To screen known carriers for
chromosomally abnormal fetus

66. ASSESMENT OF VILLI QUALITY
 Distinctive frond like appearance
 Bud - like projections
 Blood vessels coursing along the surface
 Quantity 15 to 20 mgs. Weight when Wet

67. Invasive Diagnostic Techniques
Amniocentesis

68. When Invasive Prenatal Diagnostic
Techniques ??
Patient’s Age
Obstetric History
Family History
Screening tests - High Risk
NIPT Positive

69. Metaphase Karyotype

70. TYPES OF GENETIC TESTING WITH APPLICATIONS
Chromosome Testing – for trisomies, balanced translocations
FISH – for rapid aneuploidy testing, microdeletion syndromes
PCR – Beta Thalassemia
Sequencing – BRCA testing
Next Generation Sequencing – Autism, Epilepsy
Chromosomal Microarrays – unknown cause

71. ASSAY GENETIC ABNORMALITY
Karyotyping Detect structural, numerical defects chromosomal Syndromes
FISH syndromes where gene locus is
known
Aneuploidies, Microdeletions,
22q del. , 9;22 in CML
Microarray
Based on RNA
analysis
Deletion and duplication at higher
resolution. Cannot detect
inversions and balanced
translocations
Intellectual disability,
Structural defects, physical/ USG
Biochemical tests
(chromatography
techniques)
Examines proteins (blood, urine ,
CSF) instead of the gene,
metabolite or protein structure.
Disrupts key metabolic pathway
IEM
PCR Detect specific disease-causing
mutations
Triple repeats fragile -X
Molecular testing
•Sanger, NGS,
Exome, Whole
genome
Single gene mutations including
point mutations , deletions ,
duplications within the gene
Holt-oram syndrome (TBX5)
Alagille syndrome(JAG1)
Char syndrome (TFAP2B)
Noonan syndrome (PTPN11)
DIAGNOSTIC
GENETIC TESTING

72. TODAY PRENATAL DIAGNOSIS HAS
GONE TO A NEW ERA OF FISH AND
CHIPS
• SCREENING FROM MATERNAL BLOOD FOR
FETAL DNA WILL PROBABLY REVOLUTIONIZE
THE PRENATAL DIAGNOSIS

73. FISH & CHIPS
Microarray / NGS Chips
Fluorescent in-situ
Hybridization

74. Testing by Individual Methods
FISH
PCR
Sensitive
Specific
Should have strong clinical suspicion
Need to know what we’re looking out for

75. Fluorescent In-Situ Hybridization
(FISH)
WHAT IS FISH?
Fluorescent probes
Bind to a specific site in the Genome
Enumeration (Trisomy) / Specific chromosome
band – Microdeletion
Observed under specialized microscope
Look out for dots in the cell – count them

76. Rapid Testing – Results are available in 24–48 hrs
Target Specific – looks at that region only
Operator dependent – ask who is reporting
Visual Interpretation, Cannot rule out other
conditions
FISH – Advantages & Limitations

77. WHOLE
GENOME

78. What is PCR
Polymerase Chain Reaction
Very Sensitive
Very Specific
Specific ONLY to Target region

79. Next generation sequencing (NGS)
Also known as high-throughput sequencing,
Massively parallel or deep sequencing
An entire human genome can be sequenced within a single day
Technology which has revolutionized genomic research
Chromosomal microarray analysis (CMA)
CMA is a new laboratory test used to detect chromosomal
imbalance at a higher resolution than current techniques.
CMA can find chromosome problems with more detail
WHOLE GENOME CHIPS

80. NEXT GENERATION SEQUENCING (NGS) USING CHIPS
Method: Sequencing of several genes at the
same time
Advantages:
High speed, Low cost
Great accuracy
Applications:
Multigenic conditions (ASD, CM)
Benefits:
Improved diagnosis of disease
Earlier detection of genetic predispositions
to disease
Personalized, custom drugs

81. CHROMOSOMAL MICROARRAY (CMA) USING CHIPS
High-resolution whole-genome screening
Comprehensive coverage
Can even identify submicroscopic abnormalities, that are too small to be
detected by karyotyping
Advantages (over conventional karyotype):
1. much higher resolution, which yields more genetic information
2. DNA can be obtained from uncultured specimens
3. Results are available more quickly than with karyotyping
4. No problem of culture failure / contamination
5. Involves computerized analysis, less subjective / prone to human error
VUS
in
PND
Hunter’s Syndrome X-linked disorder caused by deficiency of the lysosomal exohydrolase – iduronate-2-sulphatase (IDS).
There is accumulation of the mucopolysaccharides dermatan sulphate and heparan sulphate, in the brain and other tissues, often results in death before adulthood.

82. Once the index case is evaluated and exact cause is
identified, risk estimation and prenatal screening
and diagnosis can be offered in next pregnancy
Which method to use for Prenatal Diagnosis
Karyotyping?
FISH?
Sequencing?
NGS / CMA?

84. BUT STILL…….

85. Ultrasound
CVS
Amniocentes
is
NIPT
Combined Test
Other Screening Test

86. ADD SCREENING FOR N.C.D.HERE
THE PYRAMID OF ANTENATAL CARE
Post delivery continued screening

90. CAN WE SCREEN BEFORE
IMPLANTATION…………..

91. PGS/PGD SIMPLIFIED
Dr KESHAV MALHOTRA
DIRECTOR RAINBOW IVF

92. 18

93. 18
21
13
X
Y

94. QF-PCR

97. NGS

98. Reproductivechoicesforcouplesatgeneticrisk
• No testing
• Prenatal testing: CVS or amniocentesis
• Preimplantation Genetic Diagnosis (PGD)
• Donor egg/sperm or embryo
• Adoption

99. High Risk Couple
Conceiving Naturally
Non Invasive Prenatal Testing

100. 113
Carrier Couple/Couple with a known genetic disorder
High risk Couple Unable to conceive naturally

101. Benefits
•Healthy baby
•No termination
•Reduced miscarriage risk
•End disease in families
•Relieves anxiety and
heartache

102. Future
Sequencing of maternal plasma DNA for
the whole Fetal Genome
Use of the Transcriptome to develope
Novel Therapies

105. Fetal Transcriptome
• The power of cell-free fetal RNA reports on the
development of the living fetus in real time.
• Examination of these transcripts on a genome-wide basis
has led to new insights into the prenatal pathophysiology
of multiple, Genetic Developmental and Environmental
diseases.
• Analysis of the fetal transcriptome in normal and
abnormal development has led to novel approaches for in
utero prenatal treatment.

112. DNA alone is not our destiny:
Epigenomics- Future medicine
Epigenome: is a multitude of chemical
compounds that can tell the genome what to
do.
Epigenetic factors: methylation (promoter
regions) and histone modifications
(Accessibility of DNA).
Methylation interact with the promoters of the
genes to control their expression.
Small portion of Epigenome is inherited
Environmental factors such as smoking, diet,
infections and stress can change epigenome
Epigenetic changes cause diseases including
cancer and psychiatric diseases.

113. • Human body - 10 trillion cells, but we carry over
90 trillion microbes
• It is unique to every individual
• We die with more DNA than we are born with
• Study of microbiome is important for
understanding both human health and disease
Nature 2010
MICROBIOME
The tale of our other Genome
• Symbiotic bacteria are essentially a human organ since they
produce anti inflammatory factors, pain relieving compounds
antioxidant and vitamins to protect human body cells
• Harmful bacteria can produce toxins that mutate DNA, affecting
nervous, immune systems and cause obesity, diabetes and cancer

115. The most critical period of human development is the 1000 days from
pregnancy to a child’s second birthday, a period known as the 1000-day
window
(United Nations Standing Committee on Nutrition, 2010)
The in utero environment that a foetus is
exposed to can cause direct epigenetic
effects in the foetus, resulting in the
offspring being predisposed to a number
of conditions including cardiovascular
disease, diabetes, obesity and reduced
lifespan.

117. THALESSEMIA
(carrier
/diagnostic)
HUNTINGTON’S
(predective/diagno
stic)
NEW BORN
SCREENING
FRAGILE X
(carrier
/diagnostic)
BREAST
CANCER
(presymptomati
c)
PRENATAL
SCREENING
Mater
nal
SERU
M
Fetal
DNA

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updated in
FETAL-MATERNAL MEDICINE