Prenatal and neonatal clinical analyses


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      Prenatal clinical analyses are most often concerned with genetic disorders of the foetus. In principle several fluids of foetal origin can be obtained and submitted to chemical, biochemical and microbiological analysis, e.g. amniotic fluid and blood. This however is technically difficult and of limited diagnostic value since these fluids equilibrate with maternal blood, thus eventual pathological alterations may be concealed.

      Obtaining samples of foetal tissues or fluids always entails some risk of abortion (e.g. in the case of amniocentesis estimates range between 0.05% and 0.5%; in villocentesis between 0.5 and 1%), thus the indications must be critically evaluated. Risk factors that constitute strong indications are:
- maternal age > 35 years
- history of miscarriages or neonatal deaths
- exposure to theratogenic agents or infectious diseases during pregnancy
- hereditary diseases in the paternal or maternal lineages
- abnormal nuchal translucency or ultrasound finding
- positive tri-test. The triple test is carried out on maternal blood (starting from the 14th week of gestation) and relies on the measurement of the serum concentrations of (i) chorionic gonadotropin, (ii) alpha fetoprotein, and (iii) non conjugated estriol. The tri-test is positive if (i) is elevated while (ii) and (iii) are decreased; positivity is associated to an increased risk of Down syndrome of the foetus (predictive value = 60%)

      Foetal cells can be obtained from the amniotic fluid (amniocentesis), from the chorionic villi (villocentesis, chorionic villus sampling) or from foetal blood (cordocentesis).
Amniocentesis is possible from the 16th to the 22nd week of gestation, and is effected using a specifically designed syringe, under echographic control.
Villocentesis (biopsy of the chorionic villi of the placenta) is possible starting from the 10th-12th week of gestation and is not used after the 15th because at that time amniocentesis (which has the same diagnostic indications less risk) becomes possible.
Cordocentesis (percutaneous umbilical cord blood sampling, PUBS) cannot be performed before the 17th week of gestation and entails a 1-2% risk of miscarriage.

      The foetal cells obtained by amniocentesis or villocentesis are cultured in artificial media and, when confluent, they are treated with colchicine to block all mitoses in the metaphase. The samples are then stained using quinacrine or Giemsa, and observed under a microscope. Metaphase chromosomes are well formed and easy to visualize; dedicated computer softwares for image analysis are available.
      Pathological conditions that may be diagnosed with this method include aneuploidies (alterations in the number of the chromosomes and/or structural anomalies):

1. Alterations in the number of the sex chromosomes
1a. Monosomy: kariotype X0 (Turner syndrome)
1b. Sexual trisomies: kariotypes XXX, XXY (Klinefelter syndrome) and XYY

2. Alterations in the number of the chromosomes
Trisomies: 21 (Down syndrome); 18 (Edwards syndrome); 13 (Patau syndrome)

3. Translocations (exchanges of genetic material between different chromosomes)
3a. Balanced translocations
3b. Unbalanced translocations

4. Partial deletions or partial duplications

5. Somatic mosaicism (some but not all of the cells present any of the aneuploidies listed above)

      In the presence of familiarity for hereditary diseases, selected genes can be amplified using the Polymerase Chain Reaction method and sequenced, to screen for mutations.
      The Polymerase Chain Reaction is carried out by adding to the genetic material to be tested the RNA primers corresponding to the beginning of the gene of interest (of bothDNA strands), a heat-resistant DNA polymerase, and an excess of deoxyribonucleotides tri-phosphate. A thermal cycle is then started in which the mixture is heated to promoted the dissociation of the complementary DNA strands, and cooled to allow the DNA polymerase to synthesize the segment (gene) that follows the chosen primer. After a chosen number of cycles the DNA segment (gene) of interest has been greatly amplified and can be submitted to the detection of its nucleotide sequence (sequencing).
      Examples of hereditary genetic diseases that can be diagnosed by selective amplification and sequencing include:
Disease Frequency at birth Hereditary mechanism Gene/protein involved
Familial hypercholesterolemia 1:500 autosomal dominant LDL receptor
Polycistic kidney disease 1:1,200 autosomal dominant  
Marfan syndrome 1:4,000 autosomal dominantFibrillin-1
Sickle cell anemia 1:600 (USA) autosomal recessive Hemoglobin beta subunits
Beta thalassemia 1:500 autosomal recessiveHemoglobin beta chain
Cystic fibrosis 1:2,000 autosomal recessive Chloride membrane transporter CFTR
Hemophilia A 1:10,000 X-linked Coagulation factor VIII

      All the mitochondria of the foetus (or of the adult) cells come from the ovum, i.e. they are of maternal origin. Several genetic diseases due to mitochondrial defects are known and are maternally inherited. They can be diagnosed by studying the mitocondrion DNA extracted from any cell (including those obatined by amniocentesis, or villocentesis). The most common mitochondrial diseases are myopathies and neuropathies.
      Foetal infections may be diagnosed from the presence of bacteria in the (normally sterile) amniotic fluid. They are severe, life-threatening conditions and should be promptly treated with antibiotics administered to the mother or directly in the amnios.
      Lung maturity can be estimated by the lecithin/sphingomyelin ratio (normal value >2:1) and the surfactant/albumin ratio (normal value >55) in the amniotic fluid.
      Rh incompatibility and risk of foetal erythroblastosis.


      Several clinical analysis are routinely carried out on neonatal blood and urine. Technically these are not different from the equivalent analyses one could carry out on the adult; but some diseases must be diagnosed immediately after birth because they require prompt therapy. Examples of these include:
Congenital hypothyroidism
Phenylketonuria and other metabolic defects
      The reason why diagnosis is so urgent is that the foetus suffering of any of these conditions is normal at birth, because the metabolism of the mother (if she is healthy) compensates for the defect. After birth the disease becomes evident and usually begins to produce organ damage (most often in the brain).

Questions and exercises:
1) At which gestational age the amniocentesis is indicated:
11th - 15th week
16th - 22nd week
after the 18th week

2) The diseases that can be diagnosed from the fetal cells obtained by the procedures described in question 1 are:
Monogenic hereditary diseases, chromosomal abnormalities, mitochondrial diseases
Monogenic hereditary diseases, chromosomal abnormalities, skeletal deformities
Mitochondrial diseases, chromosomal abnormalities, hydrocephalus

3) The prenatal diagnosis of inherited enzymatic defects is achieved by
PCR followed by gene sequencing
Protein sequencing
Detection of abnormal concentrations of the substrate of the affected enzyme

4) To select the gene to be amplified by PCR one needs:
the appropriate DNA primers
the appropriate gene promoter
the appropriate RNA primers

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Thank you Professor (lecture on bilirubin and jaundice).

The fourth recorded part, the one on hyper and hypoglycemias is not working.
Bellelli: I checked and in my computer it seems to work. Can you better specify
the problem you observe?

This Presentation (electrolytes and blood pH) feels longer than previous lectures
Bellelli: it is indeed. Some subjects require more information than others. I was
thinking of splitting it in two nest year.

Bellelli in response to a question raised by email: when we compare the blood pH
with the standard pH we do not mean to compare the "normal" blood pH (7.4)
with the standard pH. Rather we compare the actual blood pH of the patient, with
the pH of the same blood sample equilibrated under standard conditions.
Thus, if we say that standard pH is lower than pH we mean that equilibriation with
40 mmHg CO2 has caused absorption of CO2 and has lowered the pH with respect
to its value before equilibration.

(Lipoproteins) Is the production of leptin an indirect cause of type 2 diabetes since
it works as a stimulus to have more adipose tissue that produces hormones?
Bellelli: in a sense yes, sustained increase of leptin causes the hypothalamus to adapt
and to stop responding. Obesity ensues and this in turn may cause an increase in the
production of resistin and other insulin-suppressing protein hormones produced by the
adipose tissue. However, this is quite an indirect link, and most probably other factors
contribute as well.

(Urea cycle) what is the meaning of "dissimilatory pathway"?
Bellelli: a dissimilatory pathway is a catabolic pathway whose function is not to produce
energy, but to produce some terminal metabolyte that must be excreted. Dissimilatory
pathways are necessary for those metabolytes that cannot be excreted as such by the
kidney or the liver because they are toxic or poorly soluble. Examples of metabolytes
that require transformation before being eliminated are heme-bilirubin, ammonia,
sulfur and nitrogen oxides, etc.

Talking about IDDM linked neuropathy can be the C peptide absence considered a cause of it??
Bellelli: The C peptide released during the maturation of insulin, besides being an indicator
of the severity of diabetes, plays some incompletely understood physiological roles. For
example it has been hypothesized that it may play a role in the reparation of the
atherosclerotic damage of the small arteries. Thus said, I am not aware that it plays a direct
role in preventing diabetic polyneuropathy. Diabetic neuropathy has at least two causes: the
microvascular damage of the arteries of the nerve (the vasa nervorum), and a direct
effect of hyperglycemia and decreased and irregular insulin supply on the nerve metabolism.
Diabetic neuropathy is observed in both IDDM and NIDDM, and requires several years to
develop. Since the levels of the C peptide differ in IDDM and NIDDM, this would suggest
that the role of the C peptide in diabetic neuropathy is not a major one. If you do have
better information please share it on this site!

In acute intermitted porphyria and congenital erythropoietic porphyria why do the end product
of the affected enzymes accumulate instead of their substrate??
Bellelli: First of all, congratulations! This is an excellent question.
Remember that a condition is which the heme is not produced is lethal in the foetus; thus
the affected enzyme(s) must maintain some functionality for the patient
to be born and to come to medical attention. All known genetic defects of heme
biosynthesis derange but do not block this metabolic pathway.
Congenital Erythropoietc Porphyria (CEP) is a genetic defect of uroporphyrinogen
III cosynthase. This protein associates to uroporphyrinogen synthase (which is present
and functional in CEP) and guarantees that the appropriate uroporphyrinogen isomer is produced
(i.e. uroporphyrinogen III). In the absence of a functional uroporphyrinogen III
cosynthase other possible isomers of uroporphyrinogen are produced together with
uroporpyrinogen III, mostly uroporphyrinogen I. The isomers of uroporphyrinogen
that are produced differ because of the positions of propionate and acetate side chains,
and this in turn is due to the pseudo symmetric structure of porphobilinogen. Only
isomer III can be further used to produce protoporphyrin IX. Thus in the
case of CEP we observe accumulation of abnormal uroporphyrinogen derivatives, which, as
you correctly observed are the products of the enzymatic synthesis operated by
uroporphyrinogen synthase.
The case of Acute Intermittent Porphyria (AIP) is similar, although there may be variants
of this disease. What happens is that either the affected enzyme is a variant that does not
properly associate with uroporphyrinogen III cosynthase or presents active site mutations
that impair the proper alignement of the phoprphobilinogen substrates. In either case
abnormal isomers of uroporphyrinogen are produced, as in CEP.
Also remark that in both AIP and CEP we observe accumulation of the porphobilinogen
precursor: this is because the overall efficiency of the biosynthesis of uroporphyrinogens is
reduced. Thus: (i) less uroporphyrinogen is produced, and (ii) only a fraction of the
uroporphyrinogen that is produced is the correct isomer (uroporphyrinogen III).

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