Free Beta Human Chorionic Gonadotropin

Only free beta subunit is used for screening. It increases at the end of first trimester and continues during second trimester, making it useful for both trimester screening. Maternal serum free beta hCG levels are increased in Trisomy 21 (Down Syndrome) and decreased in trisomy 13 and 18. Its levels are normal in fetus with sex chromosomal abnormality. The mean level of free beta hCG in first trimester Down syndrome pregnancies is elevated to 1.98 MoM

Pregnancy Associated Plasma Protein A

Level increases with gestational age and is lower in pregnancies with aneuploidy (trisomy 2 13,18). The median MoM of PAPP-A is 0.5 in aneuploid pregnancies. When used alone, it detects 40% of fetuses with aneuploidy, increases to 48% when combined with maternal age. Mean level of PAPP-A. is reduced to approx. 0.43 MoM in Down syndrome (DS) pregnancies.

Combined Screening Test

It is most effective method of screening of trisomy 21, achieved by a combination of matern age, fetal NT and maternal serum free beta hCG and PAPP-A concentrations at 11 to 13 wks. 6 days. It detects 90% Trisomy21 with false positive rate of 5%.

Dual Test                  11-l4weeks               Free beta HCG, PAPP-A

Triple test                 15-20 weeks            Free beta HCG, MDAPL, U-estriol

Quadruple test         15-20 weeks            Tripe test + insulin A

Sonography             18-20 weeks

ADVANTAGES OF FIRST TRIMESTER SCREENING

Detection rate is better than second trimester screening. Majority of the patients can be reassured early in gestation of the normalcy of pregnancy and those found to have an affected fetus and who choose to terminate the pregnancy could have it done by a procedure much safer than that used in later gestation.

Screening for fetal aneuploidy Detection of structural abnormalities:

•                          Some major structural anomalies are detectable as early as 12 weeks. These include anencephaly, holoprosencephaly, abdominal wall defects, and major limb defects.

•                          Detection rates are dependent on experience in obstetric ultrasound.

Multiple pregnancies:

•                          Chorionicity is optimally determined by either visualization or absence of the lambda sign.

•                          Twin-twin transfusion syndrome is more likely in monochorionic pregnancies with increased NT thickness.

After undergoing combined fetal NT and maternal serum free beta hCG and PAPP-A screening, patients are assigned to high risk category with a risk of 1 in 100 or more, a low risk category with a risk estimate of less than 1 in 1000 or an intermediate risk category with a risk estimate between 1 in 101 and 1 in 1000. Those in intermediate risk category, has further assessment of risk by first trimester US to determine the presence/absence of Nasal Bone, normal/abnormal Doppler velocity waveform in Ductus Venosus, or the presence/ absence of TR. CVS is offered if their adjusted risk become 1 in 100 or more.

SECOND TRIMESTER SCREENING OF FETAL ABNORMALITIES

o   Maternal biochemical analytes like MSAFP, free beta hCG, free unconjugated estriol (uE3) and Inhibin A.

o   Ultrasound screening method is called as comprehensive method or genetic ultrasound.

AFP

MSAFP screening is done between 15 to 20 wks. Using maternal serum AFP level of 2.0 to 2.5 MoM as the upper limit of normal.

Factors influencing the MSAFP levels-

o   Maternal weight

o   Gestational age - MSAFP increases by 15% per week during second trimester, so accurate gestation is essential.

o   Race - African - American women have 10 times higher MSAFP despite having a lower NTD risk.

o   Diabetes

o   Multifetal gestation-a higher screening threshold value is used in twin pregnancies.

·       Elevated Levels Of Maternal Serum AFP (MSAFP) was seen in Underestimated gestational age, Multifetal gestation, Fetal death, Neural tube defects, Omphalocoele and gastroschisis, Low maternal weight, Renal agenesis, oligohydraminos, Placental abruption, chorioangioma of placenta, Low birth weight, Maternal hepatoma or teratoma, Preeclampsia, Esophageal or intestinal obstruction, Cystic hygroma, sacrococcygeal teratomd, Urinary obstruction,

·       Low levels of AFP seen in Obesity, Diabetes; Chromosomal trisomies (T21), Gestational  trophoblastic disease, Overestimated gestational age.

·       In Neural Tube Defects (NTDs), after attempting amniocentesis we determine amniotic fluid level of AFP and it is raised. Then an assay of acetylcholinesterase is performed, if it is also positive, it is diagnostic of NTDs

Free Beta hCG levels increased to twice the normal value during the second trimester in fetuses with aneuploidy.

Unconjugated Estriol (UE3)

The concentration of free estriol in the second trimester is decreased about 25% in aneuploid pregnancies, making it valuable for screening.

Inhibin A: inha levels are not discriminatory in first trimester between fetuses with trisomy 21 and normal ones, but in second trimester affected fetuses show higher concentrations than normal ones.

Triple test: it includes msafp, beta hhcg and ue3. The three variable are independent predictors of genetic risk and in combination with maternal age generate a patient specific risk of having fetus with down syndrome. The gestational age to perform it is between 15 to 21 weeks.

Quadruple test: it includes msafp, beta - hcg, free estriol and inhubin a to assess the risk for down syndrome. The addition of inhibin a improves the detection rate of down syndrome.

Genetic sonogram: ultrasound markers play important role in aneuploidy screening & ideally between 18-20wks. The most powerful markers of aneuploidy are the absence of nasal bone, increased nuchal fold, and cardiac abnormalities. The prior risk is based on serum screening results/maternal age.

Aneuploidy soft markers

1.                Structural anomalies, including cardiac (four-chamber and outflow tracts)

2.                Short femur (observed to expected <10th percentile)

3.                Short humerus (observed to expected <10th percentile)

4.                Pyelectasis (anteroposterior diameter of renal pelvis ≥4 mm)

5.                Nuchal fold thickening (≥6 mm)

6.                Echogenic bowel (similar to echogenicity of iliac bones)

7.                Choroid plexus cysts (>10 mm)

8.                Hypoplastic middle phalanx of the fifth digit

9.                Wide space between the first and second toes (sandal gap)

10.            Two-vessel umbilical cord

11.              Echogenic intracardiac focus, short tibia, short fibula, short ear

12.            Absent nasal bone

Second trimester sonoraphic signs in NTDs

•                          Lemon sign -frontal bone scalloping

•                          Banana sign- bowing of cerebellum with effacement of cisterna magna

•                          Ventriculomegaly

•                          Small biparietal diameter

Screening Test Sensitivity for Down Syndrome (%)

Age, triple-screen results                                           60%-70%

Age, quad-screen results                                            67%-75%

Invasive procedures for prenatal diagnosis

•                          Chorion Villous Sampling (CVS), Fetal Blood Sampling (FBS), Amniocentesis, Fetal Biopsy allow testing of fetal materials for chromosomal, genetic, and biochemical abnormalities. All invasive procedures should be done after informed consent of the couple by practitioner with high level of training in obstetric USG under complete asepsis. 300g Anti-D injection intramuscular after invasive procedures is recommended to prevent alloimmunization in Rh negative mothers.

CHORIONIC VILLUS SAMPLING (CVS)

•           CVS is performed from 11weeks onwards for the diagnosis of chromosomal and genetic conditions.

•           It is associated with more than 50% of diagnosis of chromosomal abnormality because of first trimester screening.

•           It can be done transabdominally (preferred) or transcervically

Indications

•           Early detection of chromosomal disorders

•           Rapid diagnosis

•           Fetal karyotype is decided by qf-pcr (quantitative fluorescent polymerase chain reaction) of chromosomes 13, 18, 21 and y or by fish (fluorescent in situ hybridisation)

Complications of cvs

Fetal loss rate - 1 %, Severe limb defects - seen with first trimester CVS (done as early as 6-7wk). These defects are not seen after 11 wks gestation.

FETAL BLOOD SAMPLING

Cordocentesis / Percutaneous umbilical blood sampling (PUBS) Indications

•                          Chromosomal abnormalities

•                          Single gene defects

•                          Fetal anemia

•                          Prior to intrauterine transfusion

•                          Thrombocytopenia

•                          Hypoxia and acidosis

•                          Infection -viral/ bacterial culture

•                          Monitoring of transplacental therapy

•                          Pcr/ metabolic and hematological studies              -

Complications and risks

•                          Fetal Bradycardia is most common complication of cordocentesis but is usually transient.

•                          Fetal Loss 1.4%.

•                          Hemorrhage or hematoma

•                          Chorioamnionitis

•                          PROM

•                          Placental abruption

•                          Infection transmission (hepatitis, HIV)

•                          Maternalisoimmunization in rhesus negative mothers

•                          Maternal intra-abdominal infection and bleeding

•                          Needle injury to maternal organs like bowel or vessels.

Fetoscopy: Fetoscopy is performed during the second trimester (after 16 weeks' gestation). In this technique, a fine-caliber endoscope is inserted into the amniotic cavity through a small maternal abdominal incision, under sterile conditions and ultrasound guidance, for the visualization of the embryo to detect the presence of subtle structural abnormalities.

•                          It is also used for fetal blood and tissue sampling. Fetoscopy is associated with a 3-5% risk of miscarriage, therefore, it is superseded by detailed ultrasound scanning.

FETAL TISSUE BIOPSY FETAL LIVER BIOPSY FETAL MUSCLE BIOPSY

Preimplantation biopsy of blastocysts obtained by In vitro fertilization:- These techniques will be helpful for selective transfer and implantation of those pregnancies into the uterus that are not affected by a specific genetic disorder. Place and referral for different intervention:

Place and Referral for different interventions

Level -1 (Sub-center, Community Health Centre, and PHC): Screening of all pregnant women at risk for chromosomal and structural fetal malformation.

Level - II (Civil Hospital): Ultrasound at 11 -13 weeks for structure defects/Genetic ultrasound or level -Ill ultrasound at 18-20 weeks.

Level -III (Medical Colleges/ Institutes / Tertiary Care): Aneuploidy Screening: - First Trimester/Double Screen at 11-14 weeks, Free β-hCG and PAPP-A test.

Second Trimester Screen: Triple test (15-18 weeks) MSAF-P, Free β-hCG, uncogugated oestriol.

Quadruple screen (15-20 weeks) - MSAFP, free βhCG, UE3, inhibin test.

Level -IV: Advanced centres for fetal medicine with expert ultrasonologist. All invasive procedures CVS, Cordocentesis, Amniocentesis, Fetal Biopsy.