1. Dr. Joseph Har-Toov Lis Maternity Hospital Tel-Aviv, Israel
Amniocentesis and CVS
Dr. Joseph Har-Toov
Lis Maternity Hospital
Tel-Aviv, Israel

2. Methods of chromosomal evaluation
Non invasive:
Fetal cells from maternal blood
preimplantation embryos (PGD)
Invasive:
amniotic fluid (amniocentesis)
placenta (chorionic villus tissue)
Fetal blood

3. Invasive techniques Amniocentesis: Chorionic villus sampling (CVS)
Late – second trimester after 15 weeks
Early – earlier than 15 weeks
Chorionic villus sampling (CVS)
Abdominal
Trans cervical
Trans vaginal
Fetal blood sampling

4. karyotype
fish
PCR

5. What can be evaluated? Chromosomal aberrations:
Trisomy,
Monosomy,
Polyploidy,
Marker chromosome,
Deletion, duplication, inversion, translocation, ring chromosome .
Genetic aberrations (DNA)
Infectious disease
Biochemical markers (AFP)

6. Amniocentesis
First introduced by Serr and Fuchs and Riis in the 1950s for fetal sex determination
Only at the late 70th a static ultrasound was used to locate the placenta and amniotic fluid pocket
Only In 1983, Jeanty reported a technique of amniocentesis ’’under ultrasound vision’’

7. Mid Trimester Amniocentesis
Per coetaneous
20-23g needle
Ultrasound guided
Usually 20cc amniotic fluid
Results – 2 to 3 weeks

10. complications Pregnancy loss 0.3-1.0%. Increase risk:
Needle larger than 18g
Multiple needle insertion
Discoloration of the fluid
High AFP, multiple late abortions, previous vaginal bleeding
Placental perforation – recent studies didn’t find correlation

11. Complications
Leakage of amniotic fluid (better prognosis than spontaneous leakage)
Amnionitis
Vaginal bleeding
Needle puncture of the fetus
Long term complications:
Respiratory distress??
Isoimmunization??

12. Amniocentesis and HIV positive women
Increased rate of vertical transmission
Chemoprophylaxis previous to amniocentesis appears to be beneficial in preventing vertical transmission

13. Multiple Gestation Three methods:
Indigo carmine injection to the first sac
A single needle puncture sampling technique (Jeanty 1990)
Simultaneous visualization of two needles on each side of the separating membrane (Bahado-Singh 1992)
Abortion risk – probably higher
Detailed description of fetus position and placental location

14. Early Amniocentesis: 9-14 weeks
Introduced at late 80th
10-14 weeks gestation
Only the amniotic (inner) sac should be aspirated
Approximately 1 cc for gestational age
Higher rate of pregnancy loss, talipes equinovarus, and post procedural amniotic fluid leakage
laboratory failure op to 20%

15. Chorionic villus sampling
Was developed in the 80th
percutaneous transabdominal with 19-20g needle

16. Chorionic villus sampling
Was developed in the 80th
percutaneous transabdominal
transvaginal
transcervical

19. 15-30mg each aspiration
20mg ideal for cytogenetic testing
30-40mg for cytogenetic and other direct molecular and biochemical tests

20. CVS results
Direct analysis examines the trophoblast cells of the placenta (very rapidly dividing cells)
Results in few hours
greater vulnerability to mitotic error
Cultured analysis examines the fibroblast like cells of the villus stroma or mesenchymal core.
Approximately 7-10 days
Accurately reflect the chromosomes of the fetus. 

21. Risk of invasive procedure
Early amniocentesis:
High pregnancy loss
High fetal malformations
High rate of multiple needle insertions (4.7%)
High rate laboratory failures (1.8%)
Late amniocentesis:
“Low” pregnancy loss (0.3-1%)
Low rate of multiple needle insertions (1.7%)
Low rate laboratory failures (0.2%)

22. Risk of invasive procedure - CVS
Transabdominal CVS as safe as second trimester amniocentesis
Trans abdominal and transcervical CVS are equally safe and efficacious, provided that centers have expertise with both approaches
In approximately 3–5% of cases, clinical circumstances will support one approach over the other
Limb reduction – not after 9 weeks

23. mosaicism
True chromosomal mosaicism is when two or more abnormal cells lines are detected in two or more culture flasks from the same individual.
Pseudomosaicism is a term used to describe two abnormal cell lines that are found in only one culture flask (not reported to the patient)

24. mosaicism Most often involving trisomic cell and normal cells
1-2% of pregnancies undergoing CVS
0.1% of pregnancies undergoing amniocentesis
Clinical outcome of chromosomal mosaicism is strongly dependent on the specific chromosome involved and the number of trisomic cells in both the placenta and the fetus

25. Mosaicism (trisomic cells) in CVS
Option of an additional prenatal diagnostic procedure (amniocentesis or fetal blood sampling)

26. Mosaicism (trisomic cells) in CVS
Four possible conditions:
Mosaicism only in the placenta not affecting the fetus or placental function.
Mosaicism only in the placenta not affecting the fetus but alter placental function (IUGR)
Trisomy cells are both in the placenta and in the fetus
Trisomy cells in the placenta and uniparental disomy in the fetus

27. Mosaicism (trisomic cells) in amniotic fluid
Probably there are trisomic cells in the fetus
The true level and distribution of trisomic cells cannot be accurately assessed with any prenatal procedure
Ultrasound is often the best judge of how a baby is developing

28. Uniparental Disomy
Arises when an individual inherits two copies of a chromosome pair from one parent and no copy from the other parent
Maternal UPD – two copies from the mother
Paternal UPD – two copies from the father

29. How does UPD happen?
Loss of a chromosome from a trisomic zygote, "trisomic rescue"
Duplication of a chromosome from a monosomic zygote, "monosomic rescue"
Fertilization of a gamete with two copies of a chromosome by a gamete with no copies of the same chromosome, called gamete complementation.

30. Trisomic rescue following an error in meiosis
heterodisomy

31. Trisomic rescue followed an error in meiosis II
isodisomy

32. UPD - health concerns in people for two possible reasons:
Parental imprinting in the case of heterodisomy and isodisomy
Unmasking of recessive conditions in some cases of isodisomy

33. Clinical consequences of UPD
molecular UPD testing should be considered for certain chromosomes (including 6, 7, 11, 14, 15) that are known to have adverse phenotypic imprinting effects. 

34. Factors considered when trying to predict the outcome of mosaicism
the chromosome involved
A mosaic finding 18 or 21 is likely to have worse implications
mosaic finding for trisomy 15 or 16 is likely to have less implications (trisomy 15 or 16 cells cannot survive )

35. Factors considered when trying to predict the outcome of mosaicism
The tissues affected and level of trisomy in those tissues
The tissue affected cannot be evaluated
The level of trisomy can be only estimated   

36. Factors considered when trying to predict the outcome of mosaicism
method of ascertainment  
CVS shows that the placenta is affected
Amniotic fluid suggests that at least one fetal tissue may be affected
Fetal blood sampling confirms the diagnosis of chromosomal mosaicism

37. Factors considered when trying to predict the outcome of mosaicism
ultrasound findings  
presence/absence of uniparental disomy  
number of previous case reports known in the literature  

38. Thank you