Aneuploidy screening Aneuploidy screening

Aneuploidy screening

1. ANTENATAL SCREENING FOR FETAL ANEUPLOIDY Dr RADHA REEDY DrNB NEONATOLOGY RESIDENT

2. INTRODUCTION Study of chromosomes (structure , function , inheritance and abnormalities ) – CYTOGENETICS. Chromosomal abnormalities are very common and occur in appr 1-2% of live births 5% still births 50% early fetal losses in I trimester .

3. INCIDENCE SEX CHROMOSOME ANEUPLOIDY - 1/360 males & 1/580 females AUTOSOMAL ANEUPLOIDY - 1/700 LB STRUCTURAL ABNORMALITIES – 1/375 LB ALL CHROMOSOME ABNORMALITIES – 1/154 Prenatal diagnosis of chromosomal abnormalities through amniocentesis.In Milunsky A editor: Genetic disorders and fetus , ed 4 Batilmore , 1998 , Johns Hopkins University Press,PP179-248. Recent studies show prevalence of 1:580

4. Human cells contain – 23 chromosomes . HAPLOID CELL (n=23) – ovum /sperm DIPLOID (n=46) EUPLOID – Multiples of 23 POLYPLOID – euploid cells with more than normal diploid (n=46) KARYOTYPE – P icture of all chromosomes in a cell arranged in pairs

5. Chromosomal Abnormalities There are two types of fetal chromosomal abnormalities – result of errors during cell division . Aneuploidy, which is an abnormal number of chromosomes Aberrations of chromosome structure , such as deletions, rings, translocations, and duplications

6. Somatic cells Germ cells DNA duplication in interface of S phase It reduces chromosome number from diploid to haploid so that on fertilization a diploid number is restored and it allows genetic recombination

7. WHAT IS ANEUPLOIDY ? Abnormal cells that do not contain a multiple of haploid number of chromosomes . ANEUPLOIDY – most common and clinically significant abnormality ,occurring in atleast 3-4% of all clinically recognized pregnancies . The antepartum detection of fetal aneuploidy is one of the major goals of prenatal screening programs.

8. WHAT CAUSES ANEUPLOIDY ?

9. 2 common ERRORS resulting in abnormal number of chromosome NONDISJUNCTION ANAPHASE LAG

10. NONDISJUNCTION 2 chromosomes fail to separate during meiosis I/II or mitosis and thus migrate together into 1 of the new cell . 1 cell with 2 copies and the other with no copies Maternal meiosis I is most common .

11. ANAPHASE LAG Chromatid / chromosome is lost during mitosis because it fails to move quickly enough during anaphase to become incorporated into 1 of the new daughter cells.

12. PRENATAL SCREENING FOR ANEUPLOIDY

13. DILEMMAS Universal Screening Vs High Risk population (screening ) Vs High Risk population (Diagnostic tests) CURRENT RECOMMONDATIONS is to provide ROUTINE (Universal ) Antenatal Screening for Aneuploidy to all pregnant females . Current guidelines from ACMG and ACOG recommend that all pregnant women, regardless of age, have the option to undergo invasive diagnostic testing for fetal aneuploidy.

14. High Risk Population is defined as : Maternal age >35 yrs (singleton) ; >31 yrs (multiple) gestations Previous autosomal trisomy birth . Patient /partner is carrier of chromosome translocation /inversion or has aneuploidy H/O triploidy Major fetal structural defect by sonography.

15. SPECTRUM OF PRENATAL TESTING

16. WHAT DOES SCREENING RESULTS TELL US ??? Screening results offer estimates of risk . Negative or low risk results may be misinterpreted to mean the fetus is normal positive results , based on results may lead to invasive procedures . WITH ANY TYPE OF TESTING THERE IS POSSIBILITY OF FALSE POSITIVE AND FALSE NEGATIVE RESULTS

17. BIOCHEMICAL SCREENING FOR ANEUPLOIDIES FIRST TRIMESTER SCREENING COMBINED TESTS – PAPP A , Fb HCG +NT OTHERS – PIGF and AFP SECOND TRIMESTER SCREENING The full integrated test   - UE3 , AFP , Fb HCG , INHIBIN A Cell Free DNA Screening

18. DOUBLE TEST - AFP, hCG TRIPLE TEST - AFP, hCG , and unconjugated estriol, QUAD TEST - AFP, hCG , and unconjugated estriol, INHIBIN A COMBINED - PAPP A , Fb HCG +NT INTEGRATED - I-trimester NT, [PAPP-A], and II trimester [AFP], unconjugated estriol [uE3], [ hCG ], and inhibin A . SERUM INTEGRATED – Integrated test Without NT

19. The combined test detects approximately 85 % of Down syndrome ( ie , detection rate [DR] = sensitivity = 85 %) with a false-positive rate (FPR) of 5 % The combined test performs slightly better than the second-trimester quadruple test  FULL INTEGRATED TEST -  is the most efficient serum-based screening test (high detection rate [DR], low false-positive ratio [FPR]) .

20. NON INVASIVE PRENATAL TESTING (NIPT) Cell Free DNA ( cf DNA) -- Reported by Lo, et al. in 1997 Fetal cf DNA released through apoptosis trophoblastic cells of placents Released into blood stream as small DNA fragments (150-200 bp ) Reliably detected after 7+ weeks gestation. Meta-analysis - 99% detection and a false positive rate <1%),

21. METHODS OF NIPT Whole Genome sequencing , also K/N as massively parallel or short gun sequencing Chromosome Selective (targeted) sequencing Single Nucleotide Polymorphism (SNP) analysis NIPS Screening using next Generation Sequencing.

22. LIMITATION is that the circulating DNA originates from placental trophoblast , which can have isolated placental mosaicism and does not always reflect the true fetal phenotype. INTERPRETATION

23. ADVANCING NIPT

24. The ACOG now recommends that -- “no one test is superior for all test characteristics and not every test is available at all centers . Each test has advantages and disadvantages that should be discussed with each patient, with the appropriate test offered based on her concerns, needs, and values.”

25. SONOGRAPHIC FINDINGS SOFT MARKERS DOPPLER MARKERS STRUCTURAL ANOMALIES FGR

26. SOFT MARKERS - often associated with normal fetuses have no clinical sequelae, and are transient, resolving with advancing gestation . do carry an increased risk for fetal aneuploidy after birth in correlation with biochemical risk status . - Isolated soft markers are identified in 11 to 17 percent of normal fetuses Breathnach FM, Fleming A, Malone FD. The second trimester genetic sonogram. Am J Med Genet C Semin MedGenet 2007;145C:62.

27. WHAT ARE THOSE SOFT MARKERS ? Increased nuchal translucency Absent nasal bone Echogenic bowel Pyelectasis Shortened long bones ( humerus , femur) Echogenic intracardiac focus Choroid plexus cysts

28. INCREASED NUCHAL TRANSLUCENCY The space that can be visualized between the fetal skin and the soft tissues covering the cervical spine . Performed on fetuses at 10 – 14 wks gestation when the CRL corresponds to 36-84mm. Increased thickness strongly associated with fetal aneuploidy. The most commonly used thresholds the 95 th  and 99 th  percentiles of GA .

29. The ACOG recommends that patients with a fetal nuchal trans- lucency of 3.5 mm Detection of Down syndrome with nuchal translucency alone is 70% Not specific for aneuploidies. This hypoechoic space is presumed to represent mesenchymal edema and is often associated with distended jugular lymphatics.

30. The NUCHAL FOLD second trimester measurement between the outer edge of the occipital bone to the outer margin of the skin and is taken in the axial plane. Increase – associated with aneuploidy . Seen in 20 to 33 % with Down syndrome and 0.5 to 2 % of euploid fetuses Agathokleous M, Chaveeva P, Poon LC, et al. Meta-analysis of second-trimester markers for trisomy 21. Ultrasound Obstet Gynecol 2013; 41:247.

31. PATHOGENESIS : Trisomy 21 - the collagen content of the dermis is abnormal; - its hydrophilic properties may lead to the accumulation of subcutaneous fluid. Turner syndrome - lymphatic dysplasia may lead to increased nuchal fluid. Congenital heart disease - mutations in genes encoding for endothelium. Abnormal nuchal lymphatic development, distension of the jugular lymphatic sacs, accumulation of fluid in the nuchal region, and retrograde increases in venous pressure may occur.

32. HOW TO MEASURE ? The margins of the NT edges should be clear with the angle of insonation perpendicular to the NT line. The fetus should be in the midsagittal plane, with visualization of the tip of the nose, palate, and diencephalon. The fetal neck should be in a neutral position. The amnion should be seen as separate from the NT line.

33. CYSTIC HYGROMA It is a congenital malformation resulting from lymph accumulation in the jugular lymphatic sacs due to obstruction of the lymphatic system in the fetal neck. Cystic hygromas may be septated or simple. Prenatal diagnosis - USG in first trimester Single or multilocular fluid-filled structure in the nuchal region or extending along the entire length of the fetus . Fetal genetic analysis should be offered to any patient with a first-trimester cystic hygroma or significantly enlarged NT

34. Three-dimensional image of a first-trimester fetus with a cystic hygroma (arrows) Cystic hygroma extending the entire length of the fetus with associated generalized subcutaneous fluid and pleural effusion

35. The risk of the following anomalies associated with - Hydrocephalus - Agenesis, hypoplasia, and dysplasia of the lung - Atresia and stenosis of the small intestine - Osteodystrophies . - Diaphragm anomalies

36. ABSENT NASAL BONE The optimum time for nasal bone assessment is at crown rump length of 65 to 74 mm (13 to 13.5 weeks of gestation) Indicates – delayed maturation . In the second trimester The nasal bone is absent in approximately 30 to 40 % of Down syndrome fetuses and 0.3 to 0.7 % of euploid fetuses . Hypoplastic in approximately 50 to 60 percent of Down syndrome fetuses and 6 to 7 % of euploid foetuses.   https:// www.uptodate.com /contents/sonographic-findings-associated-with-fetal-aneuploidy/abstract/23

37. An absent nasal bone is less common in fetuses with trisomy 18 or 13 than in those with Down syndrome In a euploid fetus , case reports have described an association between a hypoplastic or absent nasal bone and Binder syndrome (nasomaxillary hypoplasia or maxilla-facial dysplasia) and fragile X syndrome

38. ECHOGENIC BOWEL Fetal echogenic bowel refers to increased echogenicity (brightness) of the fetal bowel  More commonly identified in second trimester. 1-2% in normal fetus 13-21% in down syndrome 4-25% in chromosomal abnormalities 0-10% congenital infection. 0-5% cystic fibrosis

39. PYELECTASIS Aneuploidy is present in 0.3 to 0.9 %of fetuses with isolated pyelectasis . When pyelectasis is identified in an otherwise normal second trimester fetus , a normal cell-free DNA test for fetal aneuploidy can be very reassuring 

40. VENTRICULOMEGALY Mild ventriculomegaly is detected in 4 to 13 percent of fetuses with Down syndrome and 0.1 to 0.4 percent of euploid foetuses. The risk of abnormal outcome, such as Down syndrome, increases with the degree of ventriculomegaly, progression of ventriculomegaly, and presence of other anomalies Ventriculomegaly is diagnosed when the atrial diameter is ≥10 mm. mild: 10 to 12 mm moderate: 13 to 15 mm severe ≥16 mm

41. SHORTENED LONG BONES A shortened humerus appears to be a better predictor of Down syndrome than a shortened femur  severely shortened (<5th percentile) or abnormal appearing long bones may be a sign of a skeletal dysplasia or early onset fetal growth restriction Agathokleous M, Chaveeva P, Poon LC, et al. Meta-analysis of second-trimester markers for trisomy 21. Ultrasound Obstet Gynecol 2013; 41:247.

42. ECHOGENIC INTRACARDIAC FOCUS  echogenic intracardiac foci is brightness. echogenic intracardiac foci usually occur as a single focus in the left ventricle, but multiple foci, biventricular foci equivalent to that of bone .  In second trimester 21 to 28 % of fetuses with Down syndrome 3 to 5 percent of normal Autopsy studies – seen more commonly in trisomy 13 than trisomy 21

43. CHOROID PLEXUS CYSTS Result from filling of the neuroepithelial folds with CSF . When isolated choroid plexus cyst(s) are detected in an otherwise low risk patient , the risk of amniocentesis (1/250 chance of pregnancy loss) is higher than the risk that the fetus has trisomy 18 (< 1/374). Restricting amniocentesis only to patients with additional sonographic abnormalities or high risk factors . Transverse image of the fetal head of an early second trimester fetus . The arrow demonstrates a unilateral choroid plexus cyst, which is anechoic and surrounded by the more brightly echogenic choroid plexus.

44. FINDING SENSITIVITY FALSE POSITIVE RATE LIKELYHOOD RATIO Absent or hypoplastic nasal bone 48.9 - 69.9% 1.9 – 4.0 6.58 Abberent right subclavian artery 17.9-47.4 % 1.0 – 2.1 3.94 Ventriculomegaly 4.2 – 12.9% 0.1 – 0.4 3.81 Increased Nuchal fold 20.3 – 32.9 % 0.5 – 1.9 3.79 Hyperechoic bowel 13.4 – 20.7 % 0.8 – 1.5 1.65 Pyelectasis 11.2 – 17.2 % 1.4 – 2.0 1.08 Echogenic Intracardiac focus 20.9 - 28.2 % 3.4 – 4.5 0.95 Short Humerus 17.1 – 47.9 % 2.8 – 7.4 0.78 Short Femur 19.3 – 38.1 % 4.7 – 8.8 0.61

45. DOPPLER MARKERS Doppler velocimetry studies are not generally performed in the first trimester. Safety has not yet established . Reversed end-diastolic flow - the observation of persistent reversed end-diastolic umbilical artery or ductus venosus flow (REDV) in the fetus with increased nuchal translucency appears highly predictive of aneuploidy . Abnormal flow in the ductus venosus   Tricuspid regurgitation Florjański J, Fuchs T, Zimmer M, et al. The role of ductus venosus Doppler flow in the diagnosis of chromosomal abnormalities during the first trimester of pregnancy. Adv Clin Exp Med 2013; 22:395.

46. STUCTURAL ANOMALIES Frequency of chromosomal abnormalities is increased in fetuses with sonographic evidence of structural anomalies. FIRST TRIMESTER - Some anomalies, such as holoprosencephaly, abdominal wall defects, and major abnormalities of fetal contour, should be identifiable ( 11 to 14 weeks was 51 percent). Other anomalies are detected in 2 nd trimester scan only . In retrospective series, an isolated fetal anomaly was associated with chromosome abnormalities in 2 to 18 percent of cases; multiple anomalies were associated with a fetal chromosome abnormality in 13 to 35 percent of cases  Staebler M, Donner C, Van Regemorter N, et al. Should determination of the karyotype be systematic for all malformations detected by obstetrical ultrasound? Prenat Diagn 2005; 25:567.

47. FETAL GROWTH RESTRICTION Although the most common etiologies for small fetal size are uteroplacental insufficiency and constitutional factors, aneuploidy has been detected in 20 percent of pregnancies  Early (<24 weeks), severe (<5 th  percentile), and symmetrical. FGR with major fetal structural abnormalities. FGR with soft ultrasound markers

48. SINGLE UMBLICAL ARTERY SUA has increased risk of aneuploidy when associated with other fetal malformations . The rate of aneuploidy with isolated SUA is not known

49. SONOGRAPHIC FEATURES OF SELECTED ANEUPLOIDIES

50. TRISOMY 21 Slightly shortened humerus Slightly shortened femur Echogenic intracardiac foci Echogenic bowel Pyelectasis Hypoplastic or absent nasal bone Hypoplasia of the middle phalanx of the fifth digit Clinodactyly Separation of the great toe (sandal gap toe) Widened iliac angle Short ear length Short frontal lobe

51. TRISOMY 18 Limb abnormalities ( eg , upper limb reduction, clenched hands with overlapping index finger, clubbed feet, rocker bottom feet) Nuchal thickening or cystic hygroma Choroid plexus cyst(s) Abnormal cisterna magna, absent corpus callosum, and/or cerebellar hypoplasia Neural tube defects (NTDs) Ventriculomegaly Strawberry shaped calvarium (pointed front and a flat occiput) Facial defects ( eg , clefts, micrognathia, low-set ears, microphthalmus ) Cardiovascular defects ( eg , septal and valvular defects) Gastrointestinal defects ( eg , omphalocele, diaphragmatic hernia) Urogenital defects ( eg , horseshoe kidney, hydronephrosis) Two-vessel umbilical cord, umbilical cord cysts

52. TRISOMY 13 lobar holoprosencephaly Severe midline facial abnormalities ( eg , cyclopia, midline facial clefts, anophthalmia, hypoplastic nose) Polycystic kidneys Posterior fossa anomalies Agenesis of the corpus callosum Ventriculomegaly Neural tube defects Cardiac defects ( eg , septal defects, tetralogy of Fallot, hypoplastic left ventricle, echogenic intracardiac foci) Skeletal defects ( eg , polydactyly) Nuchal thickening Urogenital defects ( eg , enlarged echogenic kidneys, horseshoe kidney) Gastrointestinal defects ( eg , omphalocele, diaphragmatic hernia)

53. TRIPLOIDY Major facial defects Central nervous system anomalies ( eg , ventriculomegaly, holoprosencephaly, Dandy-Walker anomaly) Neural tube defects Renal anomalies Syndactyly of the third and fourth digits Clubbed feet Cardiac anomalies Absent gallbladder Clenched hands Two vessel umbilical cord Nuchal thickening

54. TURNER SYNDROME Large septate cystic hygromata , usually noted in the second trimester Total body lymphangiectasia ( fetal hydrops) Cardiac defects ( eg , coarctation) Nuchal thickening Short femur

55. CURRENT DIAGNOSTIC OPTIONS - KARYOTYPE AMNIOCENTESIS CHORIONIC VILLOUS SAMPLING CORDOCENTESIS / PUBS

56. AMNIOCENTESIS It is a diagnostic technique of withdrawing amniotic fluid from the uterine cavity using a needle via a transabdominal approach . Timing – 15+0 to 17+6 weeks <14 weeks – high complication rates , orthopaedic malformation . >22 weeks - provokes labor or rupture of membranes

57. INDICATIONS - For prenatal genetic studies for evaluation of fetal lung maturity Infection, degree of hemolytic anemia , blood or platelet type, hemoglobinopathy, and neural tube defects a therapeutic procedure to remove excess amniotic fluid.

58. An obstetric ultrasound examination is initially performed to determine fetal viability, position, biometry, location of the placenta, and anatomic survey. The presence and location of an amniochorionic separation, which may be seen as late as 16 to 17 weeks of gestation, should also be noted if present.  AVOID placenta if possible . Levels of AFP and AChE in the amniotic fluid can help identify fetuses at high risk for NTDs .

59. COMPLICATIONS – Leakage of amniotic fluid Chorioamniotic separation Direct /indirect fetal injury. Fetal loss

60. CHORIONIC VILLOUS SAMPLING CVS involves procuring a small sample of the placenta for genetic diagnosis Performed between 10 and 13 weeks Using ultrasound guidance, the placental villi can be obtained through a transcervical or transabdominal approach depending on placental location. Maternal alloimmunization is a relative contraindication  complications – damage to the fetus and pregnancy loss. -Increased risk of transverse limb and oromandibular defects

61. When the villi have been obtained, the medium is placed onto a tissue culture dish to evaluate the sample. If the sample appears inadequate, the operator may opt to take a second pass to obtain more villi. Blood clots and maternal decidua can be separated from the villi, and these cleaned villi can be transferred to different media for further evaluation, including FISH studies and long-term culture.

62. CORDOCENTESIS / PERCUTANEOUS UMBILICAL BLOOD SAMPLING Involves puncturing the umbilical vein under ultrasound guidance to obtain fetal blood cells for genetic analysis. After 18 weeks, Procedure-related pregnancy loss rate is approximately 1 in 100 ( fetomaternal hemorrhage ) A 20 to 22 gauge spinal needle is generally used for FBS.

63. Identify a fixed segment of the cord, preferably where the cord inserts into the placenta since the stability of the abdominal insertion site is at risk if the fetus moves.  A free loop of cord can be used if it can be stabilized by the uterine sidewall . The major disadvantage of using the placental insertion site is the possibility of contamination by maternal blood.

64. Diagnostic evaluation of fetal disorders. Should be limited to situations where amniocentesis , CVS doesn’t provide sufficient information . Confirmation of severe fetal anemia suspected because of elevated middle cerebral artery peak systolic velocity (>1.5 multiples of the median for gestational age) is a common indication for FBS

65. TAKE HOME MESSAGE SCREENING to be offered to ALL pregnant women at appropriate gestational age . Types of screening and diagnostic tests , components of the biochemical screens. Combined test is the method of choice in 1 st trimester RAISED NT next step karyotype/chromosomal microarray if normal ECHO AND LEVEL II USG 1 st trimester scan is to be done in all pregnant women irrespective of their willingness to undergo NIPT . NIPT is only a screening test with very high detection rate and low false positive rate .