1. Challenges of screening for fetal abnormalities
Prof.Dr.S.Cansun Demir
Past/Vice President of TSOG

2. Multiple pregnancies constitute 1-2 % of all pregnancies and approximately 98 % are twins.
Use of ART has been responsible for increased prevalance of multiple pregnancies.
Maternal age is higher in these cases.
These parents are less inclined to opt for any invasive testing because of the fear of pregnancy loss.

3. Numbering or identification of twins Assessing risk of aneuploidy
Prenatal diagnostic procedures are more complicated in multiple pregnancies because of issues relating to:
Chorionicity
Numbering or identification of twins
Assessing risk of aneuploidy

4. Chorionicity and Zygosity
2/3 of twins are dizygotic (DZ) and /3 monozygotic (MZ)
ART increases not only DZ twins , but also MZ twins increase 2 to 3 fold.

5. In all DZ pregnancy each zygote develops its own placenta (dichorionic),
Whereas in MZ pregnancies, the twins share the same placenta (monochorionic) and amniotic sac (monoamniotic).

8. Diamniotic DiChorionic Separate placentae
Diamniotic DiChorionic fused placentae
Diamniotic MonoChorionic single placentae
Monoamniotic MonoChorionic single placentae
Frequency: 35%
27%
36% 2%
Mortality: 13%
11%
32%
44%

9. All monochorionic twin fetuses have virtually the same genetic composition (monozygotic),
Whereas the majority of dichorionic fetuses are dizygotic.

10. Estimating the risk of aneuploidy
The risk of chromosomal abnormality is directly related to zygosity.
The risk of individual fetus is independent of the other, and therefore the risk of at least one fetus being affected is approximately twice that of a singleton pregnancy.
Monochorionic fetuses have the same karyotype and the risk that both have aneuploidy is the same as that of a singleton.

11. Estimating the risk of aneuploidy
As chorionicity not zygosity can be established on early USG, parental counselling should be done.
90 % of dichorionic pregnancies are dizygotic.
10 % of dichorionic pregnancies will be monozygotic.

12. Screening for Down syndrome
 Monozygotic twins are thought to have the same Down syndrome risk per pregnancy as maternal age–matched singletons, and dizygotic twin pregnancies are thought to have twice the risk of at least one affected fetus as maternal age–matched singleton pregnancies.
However, observed rates of Down syndrome are lower than expected, possibly due to an increased frequency of early fetal loss .

13. Monochorionic twins – the risk of a T21 birth from a monochorionic pregnancy is lower than that from a singleton pregnancy due to a higher fetal loss rate among affected pregnancies.
Dichorionic twins – the risk of a T21 birth of at least one baby from a dichorionic twin pregnancy is higher than that from a singleton pregnancy.

14. Nuchal Translucency in Twins
Because the nuchal translucency distribution does not differ significantly between twins and singletons, the Down syndrome detection rate in multiples is similar to that of singletons.

15. Using first trimester NT and maternal age, in a study with 448 twin pregnancies, specific risk for Down syndrome for each twin;
The DR was 88% for a 7.3% FPR. Importantly, the prevalence of increased NT was higher in women with monochorionic pregnancies than in those with dichorionic pregnancies (8.4% vs. 5.4%), suggesting that increased NT in monochorionic twins may be an early manifestation of the twin–twin transfusion syndrome.
Sebire NJ, Snijders RJ, Hughes K, Sepulveda W, Nicolaides KH. Screening for trisomy 21 in twin pregnancies by maternal age and fetal nuchal translucency thickness at 10–14 weeks of gestation. Br J Obstet Gynaecol 1996;103:999–1003.

17. Fetal nuchal translucency combined with maternal age is an acceptable first trimester screening test for aneuploidies in twin pregnancies. (II-2)
Prenatal Screening for and Diagnosis of Aneuploidy in Twin Pregnancies JOINT SOGC-CCMG CLINICAL PRACTICE GuIDELINE No. 262 (Replaces No. 187, February 2007)

18. Combined test
Offering Down syndrome screening with the first-trimester combined test, can provide fetus-specific risk assessment.
Increased nuchal translucency at >10 and <14 weeks of gestation is a marker for Down syndrome, other aneuploidies, congenital malformations, and development of twin-twin transfusion syndrome (TTTS).
  Twin pregnancy: Prenatal issues Stephen T Chasen, MD, Frank A Chervenak, MD Up To Date Feb.2017

19. In a recent study of 1914 sets of twins, Spencer et al
In a recent study of 1914 sets of twins, Spencer et al. examined the impact of chorionicity on first trimester serum markers.
In general, for free β-hCG, this should be by dividing the observed corrected multiples of the median by
For PAPP-A, 2 different factors are required: in dichorionic twins and in monochorionic twins.
Spencer and Nicolaides reported a 75% DR of Down syndrome for a 9% FPR using NT and first trimester serum markers in 206 twin pregnancies, including 4 twins discordant for Down syndrome.The DR by nuchal translucency and maternal age alone was similar.
Spencer K, Kagan KO, Nicolaides KH. Screening for trisomy 21 in twin pregnancies in the first trimester: an update of the impact of chorionicity on maternal serum markers. Prenat Diagn 2008;28:49–52.

20. First trimester serum screening combined with nuchal translucency may be considered in twin pregnancies. It provides some improvement over the performance of screening by nuchal translucency and maternal age by decreasing the false-positive rate. (II-3)
Prenatal Screening for and Diagnosis of Aneuploidy in Twin Pregnancies JOINT SOGC-CCMG CLINICAL PRACTICE GUIDELINE No. 262 (Replaces No. 187, February 2007)

21. 73% and 84% in monochorionic pregnancies,
Wald et al. found that for a fixed 5% FPR, the detection rates of nuchal translucency alone or in combination with first trimester serum markers were respectively:
73% and 84% in monochorionic pregnancies,
68% and 70% in dichorionic pregnancies, and
69% and 72% in all twin pregnancies.
The combination of NT and first trimester biochemistry may provide detection rates in twins similar to those in singletons, especially in monochorionic pregnancies.
Wald NJ, Rish S, Hackshaw AK.Combining nuchal translucency and serum markers in prenatal screening for Down syndrome in twin pregnancies. Prenat Diagn 2003;23:588–92.

23. Maternal serum marker interpretation is problematic in twin pregnancies since both twins contribute to the marker concentration levels may be affected by early loss of one or more embryos of a multiple gestation .
Measurement of nuchal thickness can improve the detection rate and help identify which fetus is affected

24. test sensitivity in monochorionic twins was 87 percent (95% CI 53-98)
In a 2014 systematic review of first trimester combined risk assessment (nuchal translucency and maternal serum markers) in twin pregnancies,
test sensitivity in dichorionic twins was 86 percent (95% CI 73-94) and
test sensitivity in monochorionic twins was 87 percent (95% CI 53-98)
Prats P, Rodríguez I, Comas C, Puerto B. Systematic review of screening for trisomy 21 in twin pregnancies in first trimester combining nuchal translucency and biochemical markers: a meta-analysis. Prenat Diagn 2014; 34:1077

25. The false-positive rate of nuchal translucency screening is higher in monochorionic than in dichorionic twins because increased nuchal translucency can be an early manifestation of TTTS as well as a marker of aneuploidy
In vitro fertilization affects analyte values used in Down syndrome screening and may be considered by some laboratories when calculating screening results in twins conceived by this method.

26. An additional factor complicating prenatal diagnosis of monozygotic twins is that rarely these twins have different genotypes due to fetal mosaicism or confined placental mosaicism .
They can also be discordant for X-inactivation in females, differential gene imprinting, and smaller-scale genetic abnormalities, such as microdeletion .

27. Maternal Serum Screening
The use of maternal serum screening in twin pregnancies raises a number of complications and unresolved issues.
First, serum marker levels in twins are approximately twice those found in singleton pregnancies, but there are wide variations across studies because the numbers of cases and controls available are much smaller than for singletons.
Second, the interpretation of the markers necessarily relates to the entire pregnancy, while ultrasound markers such as NT are specific to each twin.
Third, the role of zygosity and chorionicity in the risk estimation is similar to their role in NT screening.

28. Muller et al. evaluated second trimester maternal serum screening for Down syndrome in 3292 twin pregnancies.30 Pregnancy outcomes were available in 3043 cases.
Median AFP levels were similar between dichorionic and monochorionic twins, free β-hCG levels were higher in monochorionic pregnancies.
Down syndrome detection rates and screen positive rates were, respectively,
27.3% and 6.6% using maternal age alone,
54.5% and 24.6% using maternal age corrected for chorionicity,
54.5% and 7.75% using observed AFP and free β-hCG divided by 2,

29. Twin pregnancies: second trimester screening for Down’s syndrome (T21)
When is the quadruple screening test offered?
There is the choice of quadruple screening in twin pregnancies for:
l women who present for the first time in the second trimester
l where the nuchal translucency (NT) could not be measured in the first trimester
Quadruple screening can be offered between 14 weeks and 2 days, and 20 weeks and 0 days. The ideal time to screen is around 16 weeks of pregnancy.
Monochorionic twins: detection rate is 80% for a screen positive rate of 3%.
Dichorionic twins: detection rate of 40-50% for a screen positive rate of 3%.

30. Integrated Screening
In singleton pregnancies, integrated testing has been proposed to combine the benefits of first and second trimester screening.
The method, combining nuchal translucency and serum markers in the first and second trimesters, has been validated in singleton pregnancies in large prospective studies, showing high detection rates and low false-positive rates.
To date there are no prospective studies of the performance of integrated screening in twins.

31. Noninvasive screening using cell free DNA
Noninvasive prenatal screening for Down syndrome using cell free DNA is challenging because the fetal cell free DNA in the maternal circulation derives from each fetus.
Testing is commercially available for trisomies 13, 18, and 21, although less validation data are available from twin gestations than from singletons.

32. It is impossible to determine which twin is abnormal based on cell free DNA analysis alone, results are reported for the entire pregnancy, and invasive testing is required to distinguish which twin, if either one, is affected.
An additional challenge in twin pregnancy is that the amount of cell free DNA contributed by each twin is lower than in a singleton pregnancy and may be quite different for the two fetuses in dizygotic twins

33. Failure rate of the cfDNA test at first sampling was 1
Failure rate of the cfDNA test at first sampling was 1.7% in singletons and 5.6% in twins.
Of those with a test result, the median fetal fraction in twins was 8.7% (range, 4.1–30.0%), which was lower than that in singletons (11.7% (range, 4.0–38.9%))
Follow-up was available in 351 (68.2%) of the twin pregnancies and comprised 334 with euploid fetuses, 12 discordant for trisomy 21 and five discordant for trisomy 18.
In all 323 euploid cases with a result, the risk score for each trisomy was < 1:10 000. In 11 of the 12 cases with trisomy 21 and in the five with trisomy 18, the cfDNA test gave a high-risk result, but in one case of trisomy 21, the score was < 1:10 000.
Bevilacqua, E., Gil, M. M., Nicolaides, K. H., Ordoñez, E., Cirigliano, V., Dierickx, H., Willems, P. J. and Jani, J. C. (2015), Performance of screening for aneuploidies by cell-free DNA analysis of maternal blood in twin pregnancies. Ultrasound Obstet Gynecol, 45: 61–66. doi: /uog.14690

34. Performance of screening for aneuploidies by cell‐free DNA analysis of maternal blood in twin pregnancies
Relationship of maternal and pregnancy characteristics (pregnancy status (a), mode of conception (b) and maternal weight (c)) with failure of cell‐free DNA (cfDNA) testing of maternal blood for aneuploidies. IVF, in‐vitro fertilization.
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Bevilacqua, E., Gil, M. M., Nicolaides, K. H., Ordoñez, E., Cirigliano, V., Dierickx, H., Willems, P. J. and Jani, J. C. (2015), Performance of screening for aneuploidies by cell-free DNA analysis of maternal blood in twin pregnancies. Ultrasound Obstet Gynecol, 45: 61–66. doi: /uog.14690

35. Ultrasonographic Screening for congenital anomalies
  We suggest an anatomic survey at 18 to 23 weeks of gestation . The incidence of congenital anomalies is three- to fivefold higher in monozygotic twins than in singletons or dizygotic twins, and higher in monochorionic monozygotic twins than in dichorionic monozygotic twins.
Twins are not predisposed to any specific type of congenital anomaly, although congenital heart disease is more prevalent in monochorionic twins, particularly those with TTTS .

36. The diagnosis of a congenital anomaly in one twin is especially problematic since decisions regarding monitoring, therapy, and delivery affect both fetuses. Expectant management, pregnancy termination, and selective feticide should all be discussed, if appropriate for the type of abnormality and gestational age. Women who choose to continue the pregnancy should understand how the anomalous fetus might affect the co-twin's outcome (eg, preterm birth, organ damage), including the role of chorionicity.
Selective termination of the anomalous fetus with dichorionic placentation is a safe and effective option in expert hands, although there is a risk of miscarriage or preterm delivery of the normal co-twin.

37. INVASIVE PRENATAL DIAGNOSIS FOR TWIN PREGNANCIES
Offering Invasive Testing in Multiple Pregnancies
AMNIOCENTESIS
Two-puncture technique
One-puncture technique
CHORIONIC VILLuS SAMPLING

38. Diagnostic test
The risks of miscarriage and other procedure related complications are higher, around 1 in 50, in twin pregnancies.
If one fetus is affected, selective reduction may be an option.
The method of diagnostic testing depends on the clinician performing the procedure.
Although both chorionic villus sampling (CVS) and amniocentesis can be performed in a twin pregnancy, there is evidence that a double amniocentesis has a lower risk of sampling the same fetus twice (known as contamination) compared to double CVS.

39. Diagnostic test
It is essential that any invasive diagnostic test is performed in a unit with experience in invasive procedures in multiple pregnancies and preferably by the same person who will be responsible for a selective feticide if required.
The pregnancy should be clearly mapped using ultrasound scan prior to the procedure, using features such as placental localization, fetal sex, fetal biometry and any structural features, such that each fetus can be specifically identified at a later stage if selective feticide is required.

40. Invasive Prenatal diagnosis
Selection of the procedure depends on reliability of test and procedure related risk of fetal loss.
Prenatal diagnosis of inheritable disease
in multiples is complicated by the possibility of obtaining discordant results.
Selective termination or loss of a healthy co-fetus in case of pregnancy loss might not be acceptable to some parents and the decision of invasive testing might be modified.

41. Pregnancy loss after twin amniocentesis is similar to that of a singleton in a cohort of 176 dichorionic twin pregnancies.
Sebire et al.Ultrasound in Obstetrics & Gynecology 1996
Fetal loss after twin amniocentesis was higher (2.73%) than twin controls (0.63%) and singleton amniocentesis (0.6%) in a study of 476 dichorionic twin pregnancy.
Yukobowich et al.Obstetrics and Gynecology 2001

42. The counselling of genetic amniocentesis is more complicated in women with multiple pregnancy.
Additional issues include :
The need for multiple sampling,
The possibility of sampling error,
Management options in case one of the fetuses is abnormal.
Such counselling should preferably be provided by those with extensive experience in prenatal diagnosis.

43. The main issues in the management of twin pregnancies with fetal trisomies
When both fetuses are chromosomally abnormal, the parents usually choose termination of pregnancy.
In pregnancies discordant for chromosomal abnormalities, the main options are either selective fetocide or expectant management.

44. The main issues in the management of twin pregnancies with fetal trisomies
3.Selective fetocide can result in spontaneous abortion or severe preterm delivery, which may occur several months after the procedure
4.In pregnancies discordant for trisomy 21, the usual choice is selective fetocide, because with expectant management the majority of affected babies would survive

45. SCREENING IN MULTIPLE PREGNANCIES
Data on multiple pregnancies with one aneuploid fetus are limited; therefore, when performing screening tests, analyte levels must be estimated.
Additionally, analytes from all the fetuses will enter the mother's serum and will be averaged, which could hide the abnormal levels of the aneuploid fetus.
Therefore, serum screening is not as sensitive in multiple pregnancies as it is in single pregnancies.
Counseling also could prove more difficult because women who are pregnant with one or more normal fetuses and one aneuploid fetus have different screening and diagnostic options.
Until further evaluation is performed, assessing risk in multiple pregnancies should be done cautiously.
ACOG Releases Guidelines on Screening for Fetal Chromosomal Abnormalities 2007