Prof. Ilona Hromadnikova, PhD.

Department of Molecular Biology and Cell Department of Molecular Biology and Cell Pathology, Third Faculty of MedicinePathology, Third Faculty of Medicine

Noinvasive: Biochemical screening

Prevention of severe genetic disorders Examination of serum Screening in the 1st vs 2nd trimester

Ultrasound examination

Invasive Chorionic villus sampling (I. trimester) Amniocentesis (II. trimester) Cordocentesis (III. trimester)

Examination Synthesis

Embryo a foetus

Alpha-fetoprotein (AFP)

Yolc sac, gastrointestinal system, liver of embryo and foetus, kidneys and placenta (in lower amount)

Placenta

hCG Syncytiotrophoblast (whole gestation)

PAPP-A Syncitiotrophoblast (till the 20th week of gestation)

Uncojugated estriol (uE3)

Feto-placental unit (whole gestation)

Hájek Z, Kulovaný E, Macek M. Grada Publishing, spol. s. r. o., 2000

Synthesis detectable from the 29th day of gestation Synthesis in yolc sac, gastrointestinal system, liver of embryo

and foetus, less in kidneys and placenta AFP levels: rapid increase till 10. – 13. week, rapid decrease

after 16. and 32. – 34. w. of gestation- amniotic fluid- follows synthesis in the foetus

- serum – slow increase till 28. – 32. w., followed by rapid decrease(↑ permeability of placenta for AFP during the gestation)

Functions: immunomodulation, protection against IS of mother

AFP is the only marker, that directly shows defects in fetal AFP is the only marker, that directly shows defects in fetal metabolism and in placental developmentmetabolism and in placental development

2 subunits: α a β Diagnosis: β-hCG subunit degradation products

„nicked hCG – partial unfoldment of β subunit. → β-core hCG – final product of degradation

Synthesis: syncytiotrophoblast hCG - ↑ till 11. w., 80% decrease – this level till the end of

g. Levels of both free hCG subunits - 0,5% of hCG levels Alpha-subunit – increase during the whole gestation β-subunit – ↑ till 10. t., ↓ till 22. t., slow ↑ till 32. w., then

decrease till the end of gestation Function: keeps synthesis of progesterone in

corpus luteum, supports synthesis of testosterone in fetal testis

Examination in serum, as well as in urine of pregnant women

The most perspective marker for first-trimester screening

Synthesis: syncytiotrophoblast Levels: ↑ from 5. till 18. w.

Synthesis by feto-placental unit, shows metabolic activity of f. u.

mother´s cholesterol crosses to placenta→ in placenta and foetus metabolised to pregnenolone →

in adrenal glands – dehydroepiandrosterone → in liver hydroxylated to estriol

Most of estriol – conjugation in mother´s liver Diagnostics: uncojugated fraction (~ 10% of

estriol), 60 – 70% is protein binded (sex hormone binding globulin)

Smoking decreases uE3 levels up to 15%

MoM (multiples of median)Evaluation of biochemical markers during normal pregnancies

→ foetus with defect – evaluation of standard deviation, expressed in multuples of median (MoM)

Percentiles – comparison of individual value with reference populational values

F.e..: probability 0,98 (98. percentile) – in reference population, there are only 2 % of individuals with higher values

The amount of fluid behind the neck of the fetus = nuchal translucency (NT)

Presence/absence of nasal bone Shorter femur bone Change of the 5th finger Almost 50% - heart disease

Figure.: Nuchal scan is the most important feature for Down syndrome (http://www2.ocn.ne.jp/~baba/us-NT.htm).

Chromosomal disorder Miscarriage (%)

Trisomy 21 30

Trisomy 18 68

Monosomy X 75

47, XXX 0

47, XXY 8

47, XYY 3

Structural aberrations 3

Data about pregnancies where parents decided to continue pregnancy although the foetus suffered from chromosomal disorders (Hook et al., 1978)

Screening in the 1st trimester and early stages of the 2nd trimester (11.-13. w., only specialised centers) AFP, free beta-hCG, newly PAPP-A Decrease of stress for women

Screening in the 2nd trimester Triple test: AFP, hCG, uE3

Determination of Down sydromeDetermination of Down sydrome ↑ ↑ ββ-hCG, the most valuable marker till 14. w. (serum, urine)-hCG, the most valuable marker till 14. w. (serum, urine) ↓ PAPP-A, till 14. w., combination with maternal age – up to PAPP-A, till 14. w., combination with maternal age – up to

71 % detection 71 % detection ↓ ↓ AFP – detected in 18% pregnancies with Down syndrome, AFP – detected in 18% pregnancies with Down syndrome,

larger dispersal of levelslarger dispersal of levels

Markers Diagnostics effectivity(%)

AFP 32

uE3 36

β-hCG 57

PAPP-A 50

Diagnostic effectivity of biochemical markers for detection of Down syndrome, 11. – 14. week of gestation

Combination of positive screening of Combination of positive screening of PAPP-A and PAPP-A and ββ-hCG with -hCG with maternal agematernal age

detection up to 62 – 82% cases with Down syndromedetection up to 62 – 82% cases with Down syndrome(with 5% false positive limit)(with 5% false positive limit)

+ combination with increased nuchal scan+ combination with increased nuchal scanDetection up to 90% pregnancies with severe chromosomal Detection up to 90% pregnancies with severe chromosomal

disorders (11. – 14. w.)disorders (11. – 14. w.)

Example: Detection of Down syndrome in the 13th week of Example: Detection of Down syndrome in the 13th week of gestationgestation

↑ ↑ NT…………………………… 2,5 mm NT…………………………… 2,5 mm >> 97. 97. percentile percentile

↑↑Beta-hCG………………… IU/ml = 95. percentileBeta-hCG………………… IU/ml = 95. percentile↓↓PAPP-A …………………. 5 mg/ml = 5. percentile PAPP-A …………………. 5 mg/ml = 5. percentile

Trisomy 18Trisomy 18 ↓ PAPP-APAPP-A ↓ ↓ ββ-hCG (serum, urine), x Down syndrome-hCG (serum, urine), x Down syndrome

Trisomy 13; 47, XXX and triploidiesTrisomy 13; 47, XXX and triploidies ↓ PAPP-APAPP-A

More reliable than screening in the 1st trimesterMore reliable than screening in the 1st trimester Usage of screening softwaresUsage of screening softwares

Estimation of aneuploidy risk:Estimation of aneuploidy risk: Triple test between 15. - 20. week of gestationTriple test between 15. - 20. week of gestation

↓ ↓ AFP AFP ↑ ↑ hCGhCG ↓ uEuE33

+ maternal age → Detection up to 60,2% + maternal age → Detection up to 60,2% ±± 7,4% 7,4%

Down syndrome:Down syndrome: ↑ ββ-hCG in serum-hCG in serum ↑ ββ-core hCG in urine– most sensitive between 17. – 21. t., -core hCG in urine– most sensitive between 17. – 21. t.,

detection up to 80% of pregnancies with Down syndromedetection up to 80% of pregnancies with Down syndrome

Other aneuploidiesOther aneuploidies ↓ ↓ AFP AFP ↑ hCG ↑ β-hCG - triploidies, Turner syndrome

Trisomy 18 ↓ uE3- the most effective test ↓ β-hCG ↓ β-core hCG in urine

Open nOpen neural tube defectseural tube defects– one of the most common – one of the most common genetic disordersgenetic disorders Spina bifida, Spina bifida, Anencephaly, exencephaly, iniencephaly, Anencephaly, exencephaly, iniencephaly,

hydrocephalus,.. hydrocephalus,.. Frequency: 1-8/1000 newbornsFrequency: 1-8/1000 newborns Multifactorial disorder (condition of mother, nutrition, Multifactorial disorder (condition of mother, nutrition,

medicaments) with genetic risk about 3%medicaments) with genetic risk about 3% Often accompanied by chromosomal disordersOften accompanied by chromosomal disorders

Abdominal wall defectsAbdominal wall defects

↑ ↑ AFP in serum – pathological increase of feto-placental AFP in serum – pathological increase of feto-placental barrier permeability, barrier permeability, combination with ultrasound combination with ultrasound examinationexamination

Optimal in the 16. w. (16. – 18. w.) Optimal in the 16. w. (16. – 18. w.) Prevention: vitamin D, riboflavin, vitamin C and vitamin B9Prevention: vitamin D, riboflavin, vitamin C and vitamin B9

CNS defects – usually visible before 24th w. CNS defects – usually visible before 24th w. Anencephaly – the most common (1 - 4:1000), defect in Anencephaly – the most common (1 - 4:1000), defect in

closing of neural tubeclosing of neural tube Heart disordersHeart disorders Sceletal defects and many othersSceletal defects and many others

PPregnancy-induced hypertension in association with regnancy-induced hypertension in association with proteinuria and edema after 20th week of gestationproteinuria and edema after 20th week of gestation

OOccurs in as many as ccurs in as many as 77% of pregnancies % of pregnancies Diagnosis usually after manifestation of clinical symptomsDiagnosis usually after manifestation of clinical symptoms ! Early diagnos! Early diagnosisis!! Etiology:Etiology:

1. Defect in expression of cytotrophoblast adhesion molecules → insufficient invasion of trophoblast into maternal spiral arteries → decrease of uteroplacental perfusion

2. Ischaemia of placenta injures foetus, released toxic substances and regulation factors injures mother too

Diagnosis of preeclampsiaDiagnosis of preeclampsia

↓ ↓ PP-13 (placental protein 13) – most promising marker of PP-13 (placental protein 13) – most promising marker of placental insufficiency predictionplacental insufficiency prediction

↓ ↓ ADAM 12ADAM 12 ↓↓PAPP-A PAPP-A

PP13 serum levels in the Ist trimester (Chafetz PP13 serum levels in the Ist trimester (Chafetz et al., et al., 2007)2007)

Early diagnosis of Early diagnosis of preeclampsiapreeclampsia

Angiogenesis – pathological oxidative stress is Angiogenesis – pathological oxidative stress is connected with inhibition of angiogenesisconnected with inhibition of angiogenesis

Key molecule: VEGF-A (Vascular endothelial growth factor A), Key molecule: VEGF-A (Vascular endothelial growth factor A), activation of 2 tyrosinkinase receptors:activation of 2 tyrosinkinase receptors:

VEGFR1 = sFlt-1 – ↑ production after oxygen lack,antagonist of angiogenic molecules VEGF (vascular endothelial growth factor) and PIGF (placental growth factor)

VEGFR2 = oncological

Preeclampsia – ↑sFlt-1, ↓PIGF detected up to several weeks (including 1st trimester) before the onset of disease when compared to normal pregnancies

Early diagnosis of Early diagnosis of preeclampsiapreeclampsia

Acquirement of biological material for examination Chromosomal disorders Neural tube defects Biochemical disorders, infections, HDN

Risk of 0,5 – 1% miscarriage Indications for cytogenetic examination:

Maternal age over 35 Birth of child with chromosomal disorder Positive biochemical screening in the I. and II. trimester Increased NT in the 10. – 14. w. and other ultrasound pathologies Unexplained reproduction problems Repeated miscarriages Birth of child with multiple congenital disorders Pregnancy associated placental inssuficiencies, IUGR Exposure of parents to mutagens Birth of child with fragile X mental retardation syndrome

Extraction of amniotic fluid from amnionAmniotic fluid consist of

Maternal plasma, direct filtration Fetal plasma, release from fetal skin Secretion from tracheobronchial tree Kidneys of foetus Filtration from the cord blood

Source of cells for cytogenetical analysis: skin of the foetus Basic procedure of PD, 90,2% procedures in 2002-2003

(Víšková a Calda, 2004) Usually between the 16. – 18. week, early between 11. – 14.

week Transabdominally under ultrasound control

Amniocentesis in the 2nd trimester under ultrasound control.(http://amniocentesis.upmc.com/Images/Sub/ImageAmnio.jpg)

Indication: At risk of X-linked diseases Risk of single gene disorders

From the 10th – 13th week Transabdominal CVS, safer

and more practical then previously used transcervical CVS

http://www.pregnancy-calendars.net/images/cvs.jpg

Sampling of fetal blood from umbilical cord (v. umbilicalis) Usually after 20. w., not convenient after 34. w., under

ultrasound control Diagnostics indications:

Rapid karyotyping of the foetus (IUGR, failure of cytodiagnostics after CVS and AMC, suspect chromosomal aberrations)

Suspect infection of the foetus (parvovirus, syphylis, HIV) Severe Rh isoimmunization of the foetus - (diagnostics of fetal

anemia: determination of Rh status, blood count) – often continues to therapeutic procedure

Therapeutic indications: Intraumbilical transfusion in the cases of severe Rh

isoimmunization Application of medicaments (sedatives during

therapeutic procedure, after intravascular transfusion)

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Main proportion of cffDNA is in fractionsMain proportion of cffDNA is in fractions 100-300 bp and 300-500 bp100-300 bp and 300-500 bp

Concentration of fetal DNA in different fractionsConcentration of fetal DNA in different fractionsfraction 100-300 bp 4,2x (0,8 – 16,7x) fraction 100-300 bp 4,2x (0,8 – 16,7x) fraction 300-500 bp 2,5x (0-8,1x)fraction 300-500 bp 2,5x (0-8,1x)

Separation using agarose gel electrophoresis is not Separation using agarose gel electrophoresis is not suitable for clinical purpose – risk of contamination, suitable for clinical purpose – risk of contamination, time consumingtime consuming

Experimentally – usage for nonivasive detection of Experimentally – usage for nonivasive detection of fetal point mutations (achondroplasy, fetal point mutations (achondroplasy, ββ-thalassemy)-thalassemy)

Fetal DNA (~1% of total DNA)

Maternal DNA(~99% of total DNA)

Fetal DNA (no change)

Maternal DNA (digested)

Ng et al., 2003

Routine (CR, France, GB)Routine (CR, France, GB)

Detection of Rh status of the fetus- RHD a RHCE genotyping: at risk of fetal

erythroblastosis and haemolytic disease of the newborn at alloimunised pregnancies (anti-D, anti-c, anti-C, anti-E)

Fetal sex determination - Detection of SRY gene: pregnancies at risk of X-

linked diseases and congenital adrenal hyperplasia

Experimental NIPD of chromosomal aneuploidies

- Trisomy 21, Trisomy 18, Trisomy 13

Detection of diseases associated with monogene mutation

- paternally AD or AR inheritance: achondroplasia, β-thalassemia, Huntington disease)

Quantitave analysis of extracellular nucleic acids

- higher amount of extracellular DNA in the cases of placental dysfunctions (preeclampsia, IUGR) and other pregnancy-related complications