Limitations of Prenatal Screening and Invasive Procedures In ART Pregnancies Prof.Dr. Mehmet Zeki TANER Gazi University Department of Obstetrics and Gynecology Division of Perinatal Medicine and High Risk Pregnancies

The Main Problems Increased background risk Altered biochemical parameters even in singleton Increased NT measurement Multiple pregnancy Zygosity

- The average age of women using ART services in 2005 was The group of women using ART services younger than 35, representing 40% of all ART cycles. - In the other world the remaning 60% over 35 years old age group.

Risks of Having Multiple-Fetus Pregnancy and Multiple- Infant Live Birth from ART Cycles, % Multiple

In Turkey ART report of 3628 live births-2006 –37.11% were 35 years old –36,5% twin or high order multiple pregnancy

Factors Affecting Biochemical Parameters Weight Smoking Diabetes Race Diet habitus –Vegetarian Culture media Orasanu et al 2006

Possible Causes of Adverse Outcomes Culture Media Formulations Viable singleton pregnancies at 12 weeks gestation (day 3 ET) Orasanu et al ’06 RBMOnline 12: hCG on day 15 post-ET ( mIU/ml) P1 IVF-500 G1.2 G1.3 N = a b a,c d P<0.05

Alteration of Biochemical Parameters In ART Pregnancies: Free and total hCG increased ( multiple corpus luteum, undiagnosed implantation, vanishing twin, exogenous drugs) PAPP-A, Unconjugated estriol and AFP decreased Increased FPR Increased Invasive Procedure Increased Pregnancy Loss Probability Psicologic and emotional stress

Multiple marker screens are particularly problematic in multiple gestations because one must rely on a single maternal serum value to provide information about multiple fetuses

Serum screening in triplets Although second trimester maternal serum screening for Down syndrome in triplets has been evaluated in a research setting, this is not currently used in clinical practice.

The criticisms associated with using serum analytes in twins in the second trimester also apply to the first trimester. It is still uncertain if the combinationof NT and serum in the first trimester is superior to using NT only. Antony O, 2003

Benefits and pitfalls of prenatal screening in a twin pregnancy A.M. Wray *, H.J. Landy, J.M. MeckInternational Journal of Gynecology and Obstetrics (2005) 91, 170—171

NT In patients with multiples, NT combined with maternal age appears to be a promising modality for aneuploidy screening. Because the NT distribution does not differ significantly in singletons compared with twins, the Down syndrome detection rate in multiples is similar to that in singletons using this modality. Conception with ART does not affect the results, and NT can be performed in triplets and high-order multiples with similar accuracy as in singletons.

Zygosity and Aneuploidy Risk Determination of zigosity is the first step in screening of twin pregnancies. 1) In monozygotic twins, a single fertilized ovum splits into two distinct individuals after a variable number of divisions. These twins are almost always genetically identical and of the same sex. On occasion, mutations can cause genetic discordance resulting in phenotypic and chromosomal dissimilarities. 2)Dizygotic twins result when two separate ova are fertilized. These individuals are genetically distinct and most often discordant for chromosomal anomalies. In dizygotic twins, In other words, the individual risk estimates for each fetus are summed to obtain the pregnancy-specific risk. Meyers et al.,1997

Zygosity and Aneuploidy Risk Counseling patients with triplets and high-order multiples about the risk for aneuploidy is even more complex. Estimates of pregnancy risk for fetal aneuploidy in trichorionic pregnancies can be made by multiplying the singleton risk by the number of fetuses. This technique assumes that each fetus is genetically distinct. Since assisted conception has been associated with increased rates of monozygosity, and the majority of triplets and high-order multiples are conceived following infertility procedures, this method of risk assessment is really not precise Jenkins TM, 200

Advanced Maternal Age in Multiple Pregnancies With twin, triplets and high-order multiples, “advanced maternal age” is thought to be younger than 35 years. In twins AMA is defined as a 31 years old. In high order pregnnacies due to small numbers of patients, charts have not been created which calculate age-related risks for fetal aneuploidy in this patient population

Donor eggs Of note, in women who have pregnancies conceived with donor eggs, the age of the ovum donor should be used to determine the pregnancy’s age-related risk for aneuploidy.

DR and FPR SingletonDRFPR –First trimester screen 60-65%5% –NT and MA76.8%4.2% –Combined test87-89%5% –PAPP-A38%5% –PAPP-A and MA48%5% –Triple test65%5% –Quad screen81%5% –Integrated test94% 5% Twin –First trim screning50%5% –Second trimester50%5% –Quad screen47%5% –NT and MA88%9.3% –First trim combined79%9%

Benefits and pitfalls of prenatal screening in a twin pregnancy A.M. Wray *, H.J. Landy, J.M. MeckInternational Journal of Gynecology and Obstetrics (2005) 91, 170—171

Clinical application of nazal bone,tricuspid flow, ductus venosus, facial angle findings If the risk is more than 1 in 50 and – tricuspid flow is normal –Nazal bone present –Ductus venosus normal –Facial angle normal the risk does not change. If the risk is 1 in 50 to 1 in 1,000 and the –tricuspid flow is normal –Nazal bone present –Ductus venosus normal –Facial angle normal the risk is usually reduced. –If there is tricuspid regurgitation –Nazal bone absent –Ductus venosus abnormal –Facial angle wide the risk is always increased.

CONCLUSION-1 For singleton the policy is the same as in non-ART pregnancy For multiples: There are many problems. Down syndrome risk assessment in multiple gestation using first- or second-trimester serum analytes is less accurate than in singleton pregnancies, For twin NT may be used only or combined with serum analytes in the first trimester followed by second trimester ultrasonographic survey. In the first trimester screening the combined risk calculation but, not the biochemical risk sholud be used. For high order multiple pregnancy the only one choice is ultrasonographic evaluation

CONCLUSION-2 Adding NT Nazal bone tricuspid regurtitaion, Ductus venosus blood flow, and facial angle may increase the sensitivity and decrase the FPR Experience with maternal serum screening in patients with a multiple gestation during the second trimester has been limited, and many centers do not offer this test to these patients. Sonographic risk adjustment is important At this time, there are no clear standards for screening for aneuploidy in patients with multiples.

ACOG Practice Bulletin 2007 How does screening for aneuploidy differ in multifetal gestations? Serum screening tests are not as sensitive in twin or triplet gestations, in part because data from multiple gestations that include an aneuploid fetus is so scarce that expected analyte levels must be estimated by mathematical modeling. In addition, analytes from both the normal and the affected fetuses enter the maternal serum and are in effect averaged together, thus masking the abnormal levels of the affected fetus. In monochorionic twin pregnancies, the median nuchal translucency values are larger in 38% of twin pairs destined to develop severe twin–twin transfusion syndrome.

ACOG Practice Bulletin 2007 How does screening for aneuploidy differ in multifetal gestations? Furthermore, counseling is more complex because women must consider a different set of options in the event that only one of the fetuses is affected. Nuchal translucency screening in the first trimester with the option of a CVS and earlier selective reduction may be desirable for some women. Experience is limited with triplet gestations, but studies suggest that nuchal translucency measurement is feasible. Until further studies are done, however, risk assessment in multiple gestations should be performed judiciously, and patients who are at increased risk of aneuploidy should be counseled regarding diagnostic testing

Invasive Procedure In Multiples

Invasive screening tests have the advantage of providing a definitive diagnosis, but with increased risk to the pregnancy over non-invasive screening.

Chorionicity Knowledge of chorionicity is critical for all invasive procedures in multiple pregnancies, and is best established by ultrasound in the first trimester. Before 13 weeks, the correct identification of the lambda sign (a triangular projection of echodense placental chorionic villi between two sacs) can correctly identify dichorionicity in all twin pregnancies. Brambati B,et al. 2001

Amniocentesis/CVS in Monochorionic Twins Tests for prenatal diagnosis in monochorionic pregnancies should require only one sample, as the fetuses should be chromosomally identical. Due to the small but present risk of post- zygotic mutations, testing of both fetuses in a monochorionic pregnancy should be considered.

Discordant Monochorionic Twins However, many case reports have described ‘Identical Twins’ with discordant karyotypes. The causes of the discordance are varied and include: – mosaicism, –skewed X-inactivation, –differential gene-imprinting and – small scale mutations (Machin, 1996). Cases of monozygotic twins with discordant karyotypes include mosaicism for –Turner syndrome, –Down syndrome, –Patau syndrome, –trisomy 1 and 22q11 deletion syndrome (Hausknecht et al., 1981; Rogers et al., 1982; Watson et al., 1990; Kaplowitz et al., 1991; Beattie et al., 1993; Lespinasse et al., 1998; Nieuwint et al., 1999; Schmid et al., 2000; Wachtel et al., 2000).

Amniocentesis in Monochorionic Twins The exact prevalence of heterokaryotypic monozygotic pregnancies is unknown, but it is a rare phenomenon. General opinion is, –if one, or both, of the fetuses has an ultrasound abnormality (or marker of aneuploidy), both sacs should be sampled, even if the twins are apparently monochorionic. When monochorionicity is certain, neither fetus has an anatomical abnormality and fetal growth is not severely discordant, sampling a single amniotic sac is sufficient. Nieuwint et al., 1999

Detailed Placental Location and Gestational Sac Description A detailed verbal description of placental locations and gestational sacs should be included in the procedure note, The following is an example of such a description: –triplet A is located lowermost with a posterior placenta and was tested with transcervical CVS; – triplet B is located on the maternal right with an anterior placenta and was tested with transabdominal CVS/AC; –triplet C is located on the maternal left with a postero-lateral placenta and was tested with transabdominal CVS/AC. The relative filling of the bladder should also be noted.

Since any woman undergoing genetic amniocentesis is unfortunately a potential candidate for fetocide, incorrect documentation, sampling or labeling might bring about a disastrous termination of the wrong fetus (Taylor and Fisk, 2000).

Chorionic villus sampling When more than one placenta is tested, separate needles are used for each sampling so as to avoid cros- contamination

AMNIOCENTESIS Three techniques have been described for amniocentesis in twins. –1)The most common technique is the use of two different needles. ( Misdiagnosis using such a technique has been reported and might involve up to 3.5% of samples,van der Pol et al., 1992; Jenkins and Wapner, 2000). –2)The single needle technique. –3) The third technique, even less widely used, is the insertion of two different needles with simultaneous ultrasound visualization of both sides of the dividing membranes

CONCLUSİON A detailed description of placental locations and gestational sacs should be included in the procedure note, In dizygotic pregnancy all fetusus sholud be sampled. In monozygotic pregnancies without abnormalities only one, if abnormalities present or suspected both/all fetuses should be sampled.