Determination of Fetal Sex from Maternal Serum

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Determination of Fetal Sex from Maternal Serum

• Dr/Salwa Hassan Teama
• M.D.clinical pathology Oncologic Laboratory
  Medicine (N.C.I)
• Fellow/Ain Shams University

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Fetal DNA

• Molecular analysis of serum DNA during human
  pregnancy has led to the discovery that maternal
  serum contains fetal DNA. This valuable source of
  fetal DNA opens up new possibilities for
  noninvasive prenatal diagnosis and detection of
  fetal sex, fetal rhesus D status and other
  inherited genetic
• diseases.

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Fetal DNA

• The discovery of free nucleic acids in the
  blood stream was first reported by Mandel and
  Metais 1948. In 1997, Lo et al., described the
  presence of fetal circulating DNA in maternal
  plasma and serum as early as eight weeks of
  amenorrhea and showed that fetal DNA
  concentration increased as pregnancy progressed
  (Lo et al., 1998), and can be detected using a
  variety of molecular methods.

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Fetal DNA

• Abnormal fetal DNA concentrations in maternal
  plasma and serum have been found in common
  pregnancy-associated disorders, including preterm
  labor and pre-eclampsia, patients with premature
  separation of the placenta and placenta previa as
  well as in pregnancies complicated by fetal
  trisomy 21.

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• Fetal male DNA can be identified in maternal
  serum by PCR amplification of Y-specific
  sequences, which provides a useful non-invasive
  procedure for fetal sex determination.

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Y- chromosome specific sequences

• Including
• DYZ1
• Sex determining region Y(SRY)
• Zinc finger protein encoded (ZFY)
• DYS14 ( Maps Yp11.2)

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Aim of the work

• The objective of the study is to determine the
  availability of determination of fetal gender by
  PCR analysis of the maternal serum in early
  pregnancy.

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Subjects and Methods

• Prospective observational study will include 25
  pregnant women, during the first trimester (10
  17 weeks), attending the outpatient antenatal
  Care Clinic, Ain Shams University hospital.
• Five healthy men and five non pregnant women
  were used as a negative controls.

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All cases will be subjected to

• Full history, stress on date of last menstrual
  period and menstrual history.
• In addition to routine antenatal investigations,
  the following was done
• Pelvic Ultrasound scan confirming the
  gestational age at the time of selection.
  Another U/S at 21 weeks gestation to determine
  fetal gender.
• Molecular investigation
• Isolation of fetal DNA from maternal serum and
  PCR analysis to detect y- specific chromosome
  sequences (DYS 14).

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• The data obtained from PCR analysis of Fetal
  DNA will be compared with the follow up U/S which
  confirm the fetal gender.

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Methods

• Informed consent were taken from the patients
  after discussing the aim of the study with them.
• Sample collection
• 6ml of peripheral blood was collected into
  vacutainer tube with no anticoagulant for serum
  separation.

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Methods

• DNA was extracted from 800 ?L of each serum
  sample. To detect the Y- chromosome specific
  sequences DYS14 in maternal serum, 40 cycles of
  PCR were carried out for each DNA extract. The
  PCR products were analyzed by 2.5 agarose gel
  electrophoresis and ethidium bromide staining,
  and the result were compared with the results of
  U/S .

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• Polymerase Chain Reaction

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Polymerase Chain Reaction

• It is a technique for the in vitro
  amplification of specific DNA sequences by the
  simultaneous primer extension of complementary
  strands of DNA.
• Starting materials for gene analysis may be
• Genomic DNA
• RNA
• Nucleic acid from archival specimens
• Cloned DNA
• PCR products

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Polymerase Chain Reaction

• The Mechanism of PCR
• The polymerase chain reaction is a test tube
  system for DNA replication that allows a "target"
  DNA sequence to be selectively amplified, or
  enriched, several million fold in just a few
  hours. Within a test tube, PCR uses just one
  indispensable enzyme - DNA polymerase - to
  amplify a specific fraction of the genome.

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Polymerase Chain Reaction

• During PCR, high temperature is used to separate
  the DNA molecules into single strands, and
  synthetic sequences of single-stranded DNA (20-30
  nucleotides) serve as primers. Two different
  primer sequences are used to bracket the target
  region to be amplified. One primer is
  complementary to one DNA strand at the beginning
  of the target region a second primer is
  complementary to a sequence on the opposite DNA
  strand at the end of the target region.

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Detection and analysis of the reaction product

• Detection and analysis of the reaction product
• The PCR product should be a fragment or fragments
  of DNA of defined length. The simplest way to
  check for the presence of these fragments is to
  load a sample taken from the reaction product,
  along with appropriate molecular-weight markers,
  onto an agarose gel which contains 0.8-4.0
  ethidium bromide. DNA bands on the gel can then
  be visualized under ultraviolet
  trans-illumination. By comparing product bands
  with bands from the known molecular-weight
  markers, you should be able to identify any
  product fragments which are of the appropriate
  molecular weight.

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Gel Electrophoresis

• The principle for the operation of gel
  electrophoresis lies in the charge associated
  with most molecules.  DNA, for example, is
  composed of a negatively charged phosphate
  group.  The apparatus uses a positive and a
  negative charged pole generated by electrical
  currents.  The DNA is loaded in on the negatively
  charged pole and pulled through the gel toward
  the positive pole. 

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Gel Analysis
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Advantages of PCR

• Useful non- invasive procedure.
• Simplicity of the procedure.
• Sensitivity of the PCR.

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Disadvantages of PCR

• False positive results (cross contamination).
• False negative results (rare of circulating fetal
  cells).

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Result

• The result of PCR analysis of all maternal serum
  participating in the study were identical to the
  result obtained by U/S scan and the results of
  both methods were confirmed by results obtained
  after delivery. DYS 14 gene was detected in serum
  of pregnant women carrying male fetus and not
  detected in pregnant women carrying female fetus.

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Near Future

• maternal plasma and serum may play an important
  and powerful role in noninvasive prenatal
  diagnosis.

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Near Future

• With quantitative analysis of fetal DNA, we may
  also assess the prognosis of abnormal pregnancies
  e.g. (pre-eclampsia, preterm labour) as well as
  the presence of fetal chromosomal abnormalities.

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Near Future

• Recently, fetal RNA has also been found in
  maternal plasma. Such fetal RNA has been shown to
  originate from the placenta and to be remarkably
  stable. The use of microarray-based approaches
  has made it feasible to rapidly generate new
  circulating RNA markers. It is hoped that further
  developments in this field will make the routine
  and widespread practice of noninvasive nucleic
  acidbased prenatal diagnosis for common
  pregnancy-associated disorders feasible in the
  near future.
• (J Histochem Cytochem 53293296, 2005)

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Conclusion

• PCR analysis of maternal serum can be used to
  diagnose fetal gender.

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• REFRENCES
• Lo YMD, Corbetta N, Chamberlain PF, Rai V,
  Sargent IL, Redman CWG, Wainscoat JS. Presence of
  fetal DNA in maternal plasma and serum. Lancet
  1997350485-487
• Lo YMD, Patel P, Wainscoat JS, Sampietro M,
  Gillmer MDG, Fleming KA. Prenatal sex
  determination by DNA amplification from maternal
  peripheral blood. Lancet 198921363-1365.
• Lo YMD, Tein MSC, Lau TK, Haines CJ, Leung TN,
  Poon PMK, et al. Quantitative analysis of fetal
  DNA in maternal plasma and serum implications
  for noninvasive prenatal diagnosis. Am J Hum
  Genet 199862768-775.
• VanWaijk IJ,Van Vugt JM,Mulders MA, Konst AA.
  Enrichment of fetal trophoblast cells from
  maternal fetal blood followed by detection of
  fetal deoxyribonucleic acid with a nested x/y
  polymerase chain reaction.Am JObeset Gy n1996
  174871-878