ppt related to prenatal genetic screening

1. By Dr Sreelasya Kakarla
Dept of OBG,
SSMC, Tumkur

2.  Congenital defects may be anatomical or
functional.
 Causes of these defects may be preconceptional or
because of post conceptional exposure
 Chromosomal abnormalities : 0.2%
 Single gene defects : 0.4%
 Multifactorial : 0.7%
 Unknown : 0.6%
 Anomalies due to exposure to teratogen : 0.1%

3.  Prenatal diagnosis is the science of identifying
structural and functional abnormalities in the
developing fetus.

4.  Diagnostic evaluation typically involves 3 major
categories :
1. Fetuses at a high risk for a genetic or congenital
disorder.
2. Fetuses at high risk for common congenital
abnormalities.
3. Fetuses discovered ultrasonographically to have
structural or functional abnormalities.
 High risk describes ‘a risk greater than the chance of
fetal death associated with the diagnostic procedure
considered’.

5.  Increased risk for fetal chromosomal abnormalities
based on advanced maternal age, previous
pregnancy affected by fetal chromosomal
abnormality
 Family history of a known genetic condition or if
the couple is a known carrier of gene mutation or
balanced translocation.
 Either of the couple affected by congenital
disorder : eg ; congenital cardiac defects
 Previous history of a pregnancy affected by
congenital anomalies

6.  Family history of congenital abnormalities
 Maternal medical condition : diabetes,
autoimmune diseases, hypertension,
hypothyroidism etc.,
 Abnormal ultrasound findings-soft markers.
 Medications : anticonvulsants, oral anticoagulants,
chemotherapeutic agents.
 Congenital infections : rubella, cytomegalovirus,
chickenpox, syphilis.

7. NON INVASIVE
 Ultrasound
 Fetal MRI
 Free fetal DNA
INVASIVE
 Amniocentesis
 Chorionic villus sampling
 Fetal blood sampling
 Fetal biopsy
 Fetal surgery

8. Chromosome abnormalities
 Karyotype by conventional chromosome analysis
 Fluoroscent in situ hybridization (FISH)
 Multiplex ligation dependent probe amplification (MLPA)
 Comparative genomic hybridization (CGH)
Single gene analysis
 Sanger sequencing
 Next generation sequencing(NGS)
 Others : amplification refractory mutations system(ARMs)

9. NEURAL TUBE DEFECTS :
 Screening for NTDs is recommended if the
following RISK FACTORS are present
 Family history of neural tube defects
 Exposure to certain environmental agents
 Diabetes
 Hyperthermia
 Drugs : anticonvulsants
isotretinoin
 Antifolate receptor antibodies

10.  Glycoprotein
 Synthesized early in gestation by fetal yolk sac ;
later by fetal gastro intestinal tract and liver
 Concentration increases steadily in both fetal
serum and amniotic fluid until 13 weeks, after
which these levels rapidly decrease.
 Passes into maternal circulation by diffusion across
the placental membranes and may also be by
placental circulation.

11.  NUCHAL TRANSLUCENCY
 Anechoic stripe visible just internal to the skin
stripe at the level of back of the fetal neck.
 Consequent to the subcutaneous accumulation of
fluid in the fetal neck in the 1st trimester.
 Incidence of chromosomal abnormalities and
structural anomalies is related to the thickness
rather than the appearance.
 The translucency usually resolves in the 2nd
trimester but may persist as a cystic hygroma or
nuchal oedema.

13.  Chromosomal abnormalities are found in 20-30%
of fetuses with increased nuchal translucency.
 50% of these are trisomy 21,
 Rest are contributed by trisomy 13, 18, turners
syndrome.
 Majority of the cases, NT < 4.5 mm

14. Aetiology of increased nuchal
translucency
Multifactorial
 cardiac failure
 Superior mediastinal compression causing
venouscongestion,
 Altered composition of extracellular matrix,
 Abnormal or delayed development of lymphatic
system
 Consequent to decreased fetal movements,
 Fetal anemia

15.  Fetus to be in true sagital section
 Ideal image includes : nasal skin, echogenic tip of
nose, nasal bone, palate in rectangular shape, the
translucent diencephalon in the centre and the
nuchal translucency posteriorly in the same image.
 It should definitely not include any part of the
zygoma between the nose and the palate.
 CRL should range between 45 and 84 mm.
 It is important to exclude the presence of umbilical
cord near the fetal neck.

16.  Nasal bone is absent or hypoplastic in around 69% of
fetuses with trisomy 21 in 11-13 weeks.
 Technically ,the section for assessment and
measurement is same as for nuchal translucency.
 Transducer should be parallel to the direction of the
nose.
 3 lines are evident :
 skin represented by the top line ,
 Echogenic nasal bone just below this which is thicker
than overlying skin and
 A line in front of the nose which represents tip of the
nose

19.  Done between 14-22 weeks
 Measured in ng/dl
 Reported as multiples of the median (MoM)
 Weight, race, diabetic status, gestational age,
number of fetuses influence the level.
 2.0-2.5 MoM : upper limit of normal.
 2.5-3.5 MoM : indiscriminate zone
 >3.5 MoM : increased fetal risk .
 Sensitivity : 90%
 PPV: 2-6%

20. ELEVATED LEVELS :
 Neural tube defects.
 Pilonidal cysts
 Esophageal or intestinal obstruction
 Liver necrosis
 Cystic hygroma
 Sacrococcygeal teratoma
 Abdominal wall defects : omphalocele,
gastroschisis
 Multifetal gestation
 Undetermined gestation.

21.  LOW LEVELS :
 Chromosomal trisomies
 Gestational trophoblastic disorders
 Fetal death
 Overestimated gestational age.

22.  A combination of the MSAFP test +
Ultrasonography detects almost all cases of
anencephaly and most cases of spina bifida.
 Also, a NTD can be distinguished from other fetal
defects, such as abdominal wall defects, by the use
of an acetylcholinesterase test carried out on
amniotic fluid.
 If the level of acetylcholinesterase rises along with
AFAFP, it is suspected as a condition of a NTD.
 However, the MSAFP levels also increase with
gestational age, gestational diabetes, twins,
pregnancies complicated by bleeding, and in
association with intrauterine growth retardation.

23.  Depending on the gestational age at pregnancy
booking,testing options that can be offered include
 Combined screening (11-13 weeks) : NT + serum
PAPPA & free B-Hcg
 Quadruple screening (15-18 weeks) : serum
AFP,uE3, free B-Hcg & inhibin A
 Integrated screen: NT + serum PAPPA+Quadruple
screening

24.  Stepwise screening: Combined screening+
Quadruple marker test in all patients with down
syndrome risk (DSR) <1 in 30 on the Combined
screen.
 Contingent screen: Combined screening+
Quadruple marker test only if the DSR is 1 in 30 to
1500

25.  Femur length < 0.91MoM
 Echogenic intracardiac focus
 Increased nuchal skinfold thickness ≥ 6mm
 Humerus length < 0.89 MoM
 Moderate or marked echogenic bowel
 major malformations
 Pyelectasis ≥3mm
 ventriculomegaly

26.  MOST COMMONLY USED
 Amniocentesis
 Chorionic villus sampling (CVS)
 Cordocentesis

27.  Usually performed between 16-20 weeks of gestation.
 Procedure performed using ultrasound guidance and
sterile technique.
 Typically performed by two operators.
 The main operator performs the invasive procedure
while the assistant performs the ultrasound examination
and guides the needle insertion.
 Pre procedure ultrasound examination is performed to
identify the placental location and fetal position in an
attempt to avoid both during the needle insertion.

28.  The desired area of the maternal abdomen is
cleaned, sterilized and draped with sterile drapes.
 Ultrasound probe covered by sterile sleeve an
continuous ultrasound guidance is provided during
the procedure.
 Ultrasound probe held vertically and the desired
target is centered on the screen.
 Needle guide is attached to the probe laterally ,
which provides a needle track ,at a 45◦ angle to the
horizontal plane.

29.  Alternative :
 Free hand needle insertion can be done , the needle
is inserted 3 cm lateral to the probe, in the same
plane and at 45◦ angle.
 The guide increases the ease of needle insertion &
reduces the risks of failed attempts and
complications.
 5 inch length 22 gauge spinal needle is used.
 Rarely 7 inch length needle is used in obese
patients.

30.  Amniotic sac is entered and fluid is aspirated using
sterile syringes.
 The first 1-2ml of the amniotic fluid may be
contaminated by maternal cells and can be
discarded.
 Fluid subsequently aspirated can be sent for fetal
chromosomal analysis after tissue culture or direct
fluorescent insitu hybridization techniques.
 Amount required for chromosomal analysis : 15-
20 ml.

32.  Pregnancy loss rate : 1 in 200
 Complications :
 Infection
 Inadvertent trauma to the fetus or placenta
 Leakage of amniotic fluid
 Miscarriage.
 Feto maternal hemorrhage,
 Isoimmunization may occur in Rh negative women
and it should be covered by prophylactic antiD in
non sensitized women.

33.  12-14 WEEKS
 Done in order to obtain the results earlier in
gestation
 Increase in risk of talipes equinovarus.
 For patients desiring earlier diagnosis ,
transabdominal CVS should be preferred over
early amniocentesis.

34. Early prenatal diagnosis
First successful CVS was reported in 1983.

35. TECHNIQUE
 Performed between 10-12 weeks
 Later weeks preferred as the thicker placenta
increases the success and the ease of the
procedure.
 A semi full or a full bladder is essential.
 Ultrasound guided sterile technique can be
performed using the needle guide or the free hand.
 After sterilizing the skin over the maternal
abdomen, local anaesthesia is administered.

36.  A 19 or 20 gauge needle is used
 Insertion done at 45◦angle and the path of the tip is
being continuously visualized on the ultrasound
moniter.
 Once the tip of the needle has reached the target,
the stillete is removed & a 10 ml luer-lock syringe
is attached to the needle hub for an air tight seal.
 The tissue is aspirated by applying the negative
pressure in the syringe.

37.  Due to more solid nature of the CVS sample, tip of
the needle has to be moved back and forth 5-10
times while applying continuous negative pressure
with the syringe to get adequate amount of tissue.

39.  Ultrasound guidance provided abdominally
 Patient placed in lithotomy position,
 Vulva cleaned and sterilized.
 Speculum inserted into the vaginal canal and the
cervix is exposed
 1.5mm diameter plastic catheter which has been
threaded over a solid malleable aluminum stylet is
introduced into the cervical canal under the
ultrasound guidance.
 Once the target tissue reached, aluminum obturator
is withdrawn carefully, avoiding the tear or
puncture of the plastic catheter.

40.  A syringe is attached to the hub of the catheter and
suction applied.
 A single aspiration will typically provide adequate
amount of sample for chromosomal analysis.

41.  In both the transabdominal and the transcervical
CVS, extreme caution is taken to avoid injury to
amnion and chorion.
 Injury will increase risk of amniotic fluid leakage
and repeated pregnancy loss.
 It is important to avoid losing the negative
pressure which can occur, if the negative pressure
applied is too high and the piston of the syringe
comes off.

43.  Cordocentesis or percutaneous umbilical blood
sampling (PUBS).
 It was initially described for fetal transfusion of
red blood cells in the setting of anemia from
alloimmunization
 Fetal blood sampling is also performed for fetal
karyotype determination, particularly in cases of
mosaicism identified following amniocentesis or
CVS.
 Fetal blood karyotyping can be accomplished
within 24 to 48 hours.

45.  Under direct sonographic guidance using a 22- or
23-gauge spinal needle into the umbilical vein, and
blood is slowly withdrawn into a heparinized
syringe.
 Adequate visualization of the needle is essential.
 Fetal blood sampling is often performed near the
placental cord insertion site, where it may be easier
to enter the cord if the placenta is anterior .
 Alternatively, a free loop of cord may be
punctured.

46.  Arterial puncture is avoided, because it may result in
vasospasm and fetal bradycardia. After the needle is
removed, fetal cardiac motion is documented, and the
site is observed for bleeding.
 fetal loss rate is approximately 1.4 %
 Other complications – cord vessel bleeding in 20 to 30
% of cases,
 fetal-maternal bleeding in 40 % of cases in which the
placenta is traversed
 fetal bradycardia in 5 to 10 %
 Most complications are transitory, with complete
recovery, but some result in fetal loss.

47.  Fluorescent in situ hybridization
 Chromosomal microarray analysis
 Free fetal DNA

48.  Involves detection of aneuploidies using
probes derived from specific sub regions of
the chromosomes in uncultured amniocytes.
 Results can be available within 24-48 hrs.

50.  CMA can detect the abnormality when the genetic
abnormality involves more than 300 kilo bases.
 limitations
 It cannot detect balanced translocations
 Point mutations
 Low level mosaicism
 Previously not described gene deletions
/duplications involving <300 kb
 There is also a possibility of an abnormality
detected on CMA which have no clinical
implications.

52.  Genetic testing performed on oocytes or embryos
before implantation in vitro fertilization (IVF),
may provide valuable information regarding the
chromosomal complement and single-gene
disorders.
1. polar body analysis
2. blastomere biopsy
3. trophectoderm biopsy

53.  Maternally inherited genetic disorder.
 The first and second polar bodies are extruded
from the developing oocyte.
 Sampling does not affect fetal development
 Disadvantages : paternal genetic contribution is
not evaluated.

55.  Done at the 6- to 8-cell stage
 limitation : mosaicism of the blastomeres may not
reflect the chromosomal complement of the
developing embryo.
 The technique is associated with a 10%reduction
in the pregnancy rate.

57.  5 to 7 cells from a 5- to 6-day old blastocyst
Advantage
 no embronyal cells are removed as
trophectoderm cells give rise to the
trophoblast.
Disadvantage
 performed later in development.