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Prenatal screening and diagnosis of aneuploidy in multiple pregnancies
Best Practice & Research Clinical Obstetrics and Gynaecology 28 (2014) 285–294
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Prenatal screening and diagnosis of aneuploidy in multiple pregnancies Alain Gagnon, MD, Clinical Professor a, *, Francois Audibert, MD, MSc, Professeur Titulaire b a b
University of British Columbia, Vancouver, British Columbia, Canada Université de Montréal, Montréal, Québec, Canada
Keywords: prenatal screening Down syndrome aneuploidy multiple pregnancies amniocentesis chorionic villus sampling
Prenatal screening for aneuploidy has changed significantly over the last 30 years, from being age-based to maternal serum and ultrasound based techniques. Multiple pregnancies present particular challenges with regards to screening as serum-based screening techniques are influenced by all feti while ultrasoundbased techniques can be fetus specific. Tests currently available tend to not perform as well in multiple compared to singleton pregnancies. Considerations must be given to these variations when discussing and performing screening for aneuploidy in this situation. Prenatal invasive diagnosis techniques in multiple pregnancies bring their own challenges from a technical and counselling point of view, in particular with regards to sampling error, mapping and assignment of results and management of abnormal results. This review addresses these particular challenges and provides information to facilitate care. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction Multiple pregnancies present specific challenges with regards to prenatal screening and diagnosis. Although some of the usual components of screening such as previous history and family history remain an important feature of the screening process for aneuploidy, the various pregnancy specific screening methods present particularities when it comes to multiple pregnancies. Most of the
* Corresponding author. Division of Maternal-Fetal Medicine, BC Women’s Hospital, B241-4500 Oak Street, Vancouver, BC, Canada V6H 3N1. Tel.: þ1 6048753174. E-mail address: [email protected] (A. Gagnon). 1521-6934/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bpobgyn.2013.12.010
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information available on the topic covers twin pregnancies whereas the literature is much sparser on higher order multiples. The following paper reviews the literature available to facilitate counselling and health care resources planning on this topic. Background Zygosity and chorionicity Zygosity is determined by the number of fertilized ova that resulted in the multiple pregnancy. It is assumed that monozygotic multiples (originating from a single fertilized ovum) have the same genetic make-up and consequently, should be concordant for aneuploidy. For screening purposes, this assumption is applied, although rare exceptions to this rule exist [1], described in case reports of heterokaryotypic monozygotic twins. Feti in a dizygotic or polyzygotic multiple pregnancy will have as many different genetic make-ups as there are zygotes (or fertilized ova). Consequently, these feti may be concordant or more frequently discordant for aneuploidy. As zygosity cannot be assessed directly without invasive testing during pregnancy, some inferences based on ultrasound findings must be applied, through the assessment of the chorionicity. Chorionicity refers to the type of placentation and specifically to the number of chorions or functional placentas. It can be assessed by ultrasound, most successfully in the first trimester when the accuracy is between 96–100% [2–4]. Monochorionic multiple pregnancies are essentially always monozygotic. Most dichorionic or polychorionic multiple pregnancies are dizygotic or polyzygotic, with as many zygotes as there are chorions; less than 10% of them [2–4] will arise from a single zygote that divided within the first 3 days post-fertilization. As the majority of polychorionic multiple pregnancies are polyzygotic, it is assumed that they have different genetic make-ups and are most likely discordant for aneuploidy. These concepts are particularly important when assessing the risks through screening and counselling on invasive procedures. Prenatal screening in multiple pregnancies Risk assessment based on maternal age, periconceptional factors and chorionicity It is easy to conceive that the overall risk of aneuploidy is higher in multiple pregnancies compared to singletons. This is in part due to increased maternal age in multiple pregnancies (either spontaneous or conceived through fertility treatments) [5]. The maternal age to be used for counselling is the age of the egg at the time of retrieval for pregnancies arising from frozen embryo transfer or egg donation. A monochorionic multiple pregnancy spontaneously conceived or conceived without the use of intracytoplasmic sperm injection (ICSI) carries a similar overall risk of aneuploidy compared to a spontaneously conceived singleton pregnancy for the same maternal age. Both twins will either be affected or not, with only rare exceptions as described above. A polychorionic multiple pregnancy has an increased risk of aneuploidy as it is most likely polyzygotic and it is the number of zygotes that determines the level of risk for aneuploidy. Each fetus has, in theory, its own risk of aneuploidy, relatively independent of the risk for the other(s). Consequently, it has been suggested that the risks are additive. [6] For example, the risk of aneuploidy would be double that of a singleton in dichorionic multiple pregnancies, triple in trichorionic pregnancies and so on. This concept has been recently challenged [7] as the observed prevalence of Trisomy 21 in twin pregnancies is much less than the theoretical risk based on the above assumptions and as such, this simple rule must be applied with caution when assessing risk based on history alone. This is likely related to a variety of environmental and parental genetic factors that are not completely independent of each other and thus influenced all zygotes similarly rather than independently. In view of the challenges described above and the trends towards using comprehensive screening rather than maternal age alone, as recommended by multiple national bodies such as Canada and Singapore [8,9], maternal age alone should be discouraged as a screening method for multiple pregnancies, unless no other methods are available. In that case, chorionicity should be considered, and the
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counseling should include the best possible risk assessment based on the information available together with the risk of the invasive procedure. The use of ICSI has been associated with an increased risk of aneuploidy, particularly in the sex chromosomes pair [10]. In these cases, the rates of aneuploid counts for the X and Y chromosomes are estimated at 0.75%. This should be taken into consideration in the counselling regarding the risks, screening methods and invasive testing. Most screening tests are not well designed for assessment of sex chromosomes anomalies beyond the possible detection of 45,XO or Turner Syndrome when nuchal translucency is used as part of the screening methods (see below). First trimester ultrasound including nuchal translucency Over the last 20 years, the use of first trimester ultrasound including nuchal translucency (NT) has become a significant component of aneuploidy screening. It has been the main component in multiple pregnancy as it can provide a fetus specific risk of aneuploidy, when combined with maternal age [10]. The detection rates using NT alone with maternal age in twin pregnancies are similar to those observed in singletons [11–13] because the distribution of NT measurements is similar for feti from a singleton or multiple pregnancy. In monochorionic (assumed monozygotic) multiple pregnancies, the NT are measured for each fetus sharing a chorion and the average of all NTs is used to provide a single pregnancy risk of aneuploidy [17]. In polychorionic multiple pregnancies, each fetus not sharing a chorion is considered separately, and an individual risk is typically calculated for each one based on the established curves in singletons, using the NT measurement and the crown-rump length for each fetus as well as maternal age [13]. The risk is usually additive and the total risk for the pregnancy is the sum of all risks for each fetus. This calculation method leads to an increase in the false-positive rate for those polychorionic pregnancies who are monozygotic (as the risk should have been averaged for those, as described above), but as they represent less than 10% of polychorionic pregnancies and cannot be differentiated from the polyzygotic polychorionic multiples pregnancies, this does not affect significantly the performance of the test [14]. It is felt to be appropriate to calculate pregnancy specific risks rather than solely fetus specific risks as most women will elect to test all fetuses should they undergo invasive testing and most of the data available on the risk of invasive testing in multiple pregnancies accounts for testing of all fetuses. By combining NT measurements with maternal age in a twin pregnancy population and calculating a fetus specific risk for each of them, Sebire et al. described a detection rate of 88% with a false-positive rate of 7.3% [13]. A higher prevalence of increased NT and thus positive screening tests was noted in the monochorionic twins (8.4% vs 5.4%), which was later suspected to potentially be an early sign of twinto-twin transfusion syndrome [15,16]. This is why Vandercruys et al. later suggested using an average risk for monochorionic twins after studying 769 monochorionic twin pairs [17]. They described with this technique a 100% detection rate for a 4.2% false-positive rate in a monochorionic population, a significant improvement over the fetus specific risk calculation. Although such detection rates are not consistently reproduced [13,14,17–22] this is the current standard recommended by the Fetal Medicine Foundation as it does provide the best screening performance [23]. There is a paucity of studies looking at a more elaborate panel of ultrasound markers in the first trimester, including nasal bone assessment and ductus venosus. Krantz et al. reported on the mathematical modelling using nuchal translucency and nasal bone assessment in 3187 twin pregnancies and 219 triplet pregnancies [24]. They described a decrease in the positive rate by combining the two ultrasound markers from 9.0% to 4.7% in twins and 13.0% to 11.9% in triplets. In triplet pregnancies, using a simulation model assuming a 10% positive rate, the detection rate was estimated to be 93% using the two markers. First trimester ultrasound combined with serum markers In singleton pregnancies, the use of nuchal translucency is ideally combined with 2 or more serum markers in order to improve the detection rate and/or decrease the false positive rate. There are a few studies describing this technique in twin pregnancies only. It has not been studied in higher order multiples. Although nomograms for the various analytes (b-hCG, PAPP-A, AFP, Estriol, Inhibin-A) have
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been established for twin pregnancies, the key challenge is that compared to ultrasound based markers which are fetus specific, serum markers are always a mixture, influenced by all feti and placentas present. Consequently, in polyzygotic pregnancies, the effect of the abnormal fetus on the serum analytes values is diluted and brought closer to the normal range by the normal fetus or feti. This effect is particularly noted in higher order multiple pregnancies. Consequently, it is usually recommended to use serum analytes only in singleton and twin pregnancies. With regards to first trimester screening, free b-hCG and PAPP-A have been noted to be approximately twice as high as in twin pregnancies compared to singleton pregnancies. More specifically, bhCG is around 2.023 times that of singleton pregnancies whereas PAPP-A is 2.192 and 1.788 times higher than singletons in dichorionic and monochorionic twin pregnancies, respectively [25]. As there is not enough data to know the distribution of these values in pregnancies affected by trisomy 21, a pseudo-risk is usually calculated. Wald et al described this technique where the value of each analytes is divided by the correction factor suggested above and then used to calculate the risk for each fetus as if it was a singleton. With this technique, they reported an improved detection rate in monochorionic twins from 73% to 84% for a 5% false positive rate, using NT and 2 first trimester analytes. The same improvements were not noted in dichorionic twin pregnancies where the detection changed from 68% to 70% using the same technique [14]. Table 1 provides a summary of the studies available on first trimester combined screening (NT, PAPP-A, b-hCG and maternal age). The only study reporting on triplet pregnancy using NT and biochemistry did not notice any improvements in performance by adding the biochemistry compared to NT alone [24]. Wald et Rish published performance estimations of an integrated screening test combining NT ultrasound with PAPP-A in the first trimester and AFP, b-hCG, estriol and inhibin-A in the second trimester, providing a single risk at the end of the process [26]. Using many assumptions to perform their calculations, they estimated the detection rate to be 93% in monochorionic twins and 78% in dichorionic twins, for an overall detection rate of 80%, at a 5% positive rate. Maternal serum screening alone Even though more and more women have access to quality first trimester ultrasound, in many parts of the world, maternal serum markers remain the mainstay of prenatal screening for aneuploidy. Not all jurisdictions offer a first trimester ultrasound routinely and consequently, many multiple pregnancies are still only identified in the second trimester, after the window of opportunity for nuchal translucency assessment. In view of this reality, serum based screening in multiples has been studied. As described above, the challenges with the interpretation of serum analytes in higher order multiples are such that serum based screening is only validated in twin pregnancies. Even then, the paucity of data in affected pregnancies is such that only pseudo risks can be calculated [26]. It is also important to note that the interpretation of serum markers must provide a risk for the whole pregnancy when, in the majority of cases of affected pregnancies, only one fetus will be affected. Finally, it is important to be aware of the chorionicity of the pregnancy, for the same reasons as described above. This is unfortunately less reliable after 14 weeks.
Table 1 Summary of studies on combined first trimester screening in twins using Nuchal translucency, PAPP-A, free b-hCG and maternal age. Reference
Term cut-off Number of twin Number of affected Detection rate for False positive risk pregnancies feti Trisomy 21 (%) rate (%)
Spencer, 2000 [18] NA Sebire et al., 2000 [16] NA Spencer and Nicolaides, 2003 [20] 1:300 Vandercruys et al., 2005 [17] 1:300 Wald and Rish, 2005 [26] Theoretical Chasen et al., 2007 [22] 1:198 1:300
159 Theoretical 206 769 Theoretical 535
NA Theoretical 4 6 Theoretical 7
80.1 73 (MZ) 43 (DZ) 75 72 72 100 100
5.0 5.0 NA 5.0 5.0 5.0 7.0
Legend: NA: Not available; MZ: Monozygotic; DZ: Dizygotic; Theoretical: data based on modeling, not actual patients.
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Spencer et al. and Muller et al. studied the double test (AFP and free b-hCG combined with maternal age) and reported a 51–63% detection rate for a 5–10.8% positive rate, with the best performance being described when serum values were corrected for chorionicity [27–29]. Of note, these results represent an improvement over the use of maternal age alone (27.3% detection rate for a 6.6% positive rate in one studied population) [28]. The use of the triple test (AFP, free b-hCG and estriol combined with maternal age) was studied in a population of 60 twin pregnancies by Maymon et al. [30] To detect the one case of trisomy 21 in their population required a 15% positive rate. Cuckle studied both the triple test and the quadruple test (AFP, free b-hCG, inhibin-A and estriol combined with maternal age) and reported only marginal improvement in the detection rate from 41% with dual test to 44% with the triple test and 47% for the quadruple when a 5% positive rate was used [7]. In summary, although serum screening alone performs better than maternal age alone, it is outperformed by screening methods incorporating ultrasound findings such as NT. Non-invasive prenatal testing (NIPT) The last few years have seen the emergence of NIPT as a new tool to assess pregnancies for aneuploidy. Very little information is available on the use of this tool in multiple pregnancies. Only 3 studies have reported on this topic, all using cell-free DNA technology [31]. The first one reported on a population at high risk of chromosomal anomaly based on screening and included 3 pairs of twins with discordant karyotypes (2 with trisomy 21 and 1 with trisomy 13), 5 pairs concordant for trisomy 21, 17 euploid twin pregnancies and 2 euploid triplet pregnancies [32]. All were correctly classified. The second study included both low risk and high risk pregnancies and included one pregnancy with twins discordant for trisomy 21 and 11 euploid twin pregnancies [33]. Here again, all were correctly classified. Finally, Gil et al. recently reported on 207 samples previously collected and 68 samples collected prospectively in twin pregnancies [34]. A result could be provided in 92.7% of samples. 13 out of 14 pregnancies (92.9%) with at least one trisomic fetus were correctly identified with no reported false positive results. More studies are required to better define the performance, positive and negative predictive values of such test in a multiple pregnancy population. Second trimester ultrasound screening Despite ultrasound in the second trimester conferring the theoretical advantage of being fetus specific and thus having the potential to be a better screening tool for the reasons described above, limited data is available on the efficacy of second trimester ultrasound in multiple pregnancies to detect aneuploidy. It is well known that obtaining a detailed assessment is more challenging in multiple pregnancies due to the limited field of vision associated with the presence of one or more feti around the fetus being imaged. One study reported a detection of 5 out of 9 feti with trisomy 21 using nuchal thickness assessment [35]. The other markers studied in that publication were less efficacious. If these markers were to be considered as a significant part of a screening protocol, further studies would be required to better assess the performance of ultrasound in this context. Invasive prenatal testing in multiple pregnancies When to offer prenatal testing? When it comes to invasive procedures, non-directive counselling is recommended, allowing patients to make the best decision possible for themselves. Cut-offs used to call a screen test positive should take into account the cut-off used in the singleton population but also the risk associated with procedures in multiple pregnancies, as described below. It is also important to keep in mind the background risk of spontaneous pregnancy loss in a multiple pregnancy, estimated at 6–7% in twins and up to 10% in higher order multiples, between 11 weeks and 24 weeks [36]. Considerations prior to proceeding with testing Some key elements must be considered and discussed prior to proceeding with invasive testing. A thorough ultrasound examination is required, in particular if chorionicity has not been already
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determined, as it has implications for the interpretation of the screening test and the potential results of the procedure as well as the management should a result be abnormal. Mapping of the location of each fetus to be tested is also paramount. All possible elements identifying each fetus (location of the sacs, placental location, cord insertion, fetal genitalia, discordant anomalies) should be noted clearly using text and/or diagrams [37]. This will facilitate assignment of the correct result to each fetus. This becomes of particular importance when results are discordant and pregnancy management is altered for that fetus (such as selective reduction) [38] or when the sex chromosomes do not match the genitalia phenotype (in particular when concordant karyotypes are associated with discordant phenotypes, thus raising the suspicion of improper sampling). Such possibilities should also be discussed prior to proceeding with invasive testing. When chorionicity is unclear by ultrasound, consideration should be given to assessing zygosity by molecular testing in complex multiple pregnancies (such as those with discordant anomalies), as polyzygosity would be associated with polychorionicity and the pregnancy could be managed as such. Amniocentesis As in singleton pregnancies, amniocentesis is typically performed after 15 weeks in multiple pregnancies, in view of the increased risk associated with earlier gestational ages noted in singletons [39]. Three techniques have been described to address the particular challenges posed with this procedure in multiple pregnancies. They have not been compared in a randomised fashion and consequently, one cannot be recommended over the other, but they can be described with their pros and cons. The most widely used technique is the multiple punctures technique where multiple different needles are inserted in different locations but in each separate sac, using ultrasound guidance to identify the various locations. In twins, the risk of sampling the same sac twice using this technique has been estimated to be around 1.8% in a Canadian study of 260 twin pregnancies [40]. Dye was not instilled in the first punctured sac in this study. Correlation between karyotype results and fetal genitalia appearance may help identify pregnancies where the same sac was punctured twice. Because of this issue, a modification was suggested, using a coloured dye to mark the sac(s) that have been tapped and expecting a dye free sample when sampling the next sac. The first dye used was methylene blue. As it has since been associated with small bowel atresia and fetal death, its use is now considered contraindicated [41–45]. Indigo carmine has since been used and, although no risks of congenital anomalies above the background risk have been reported [46,47], the report of at least 4 cases of jejunal atresia in exposed newborns [44,47,48] raises some concerns. In this context, most experts suggest using dye to identify the various gestational sacs only when ultrasound visualization is poor or in the case of higher order multiples [38,49,50]. Another modification used to ensure sampling on both sides of a membrane is a single puncture technique where the first sac is entered with a needle in close proximity to the dividing membrane. Once the sample is collected, the needle is advanced through the membrane, the first 1–2 ml of amniotic fluid collected is discarded and then a sample is collected on the other side of the membrane, thus ensuring both sacs are sampled [51–55]. Potential challenges described include tenting of the intertwin membrane, contamination of the second sample with amniotic fluid from the first sac [49] and the creation of iatrogenic monoamnionicity by disrupting the integrity of the intertwin membrane [56]. These complications have not been described in 2 studies reporting on a total of 77 amniocentesis performed in this fashion in twins, yet to this day, this technique has not gained widespread acceptance [49]. The third modification used in order to decrease the risk of sampling the same sac twice is the simultaneous visualisation technique where both needles are inserted at the same time and visualised on each side of the intertwin membrane [57]. Despite the theoretical advantage from a sampling confirmation point of view, the challenges in obtaining such visualization and the time required are such that this technique is seldom used [49]. Many experts have rightly suggested that in monochorionic multiple pregnancies (which are typically monozygotic), a single sample could be taken from only one sac. Since multiple case reports exist describing discordant karyotypes in monochorionic multiple pregnancies [1,58] and as
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chorionicity is less reliable if identified after 14 weeks [2–4,12], many will still advocate sampling of both sacs, unless the feti are concordant for growth, anatomy and nuchal translucency and chorionicity was determined before 14 weeks [49,59]. As multiple pregnancies carry a higher spontaneous loss rate compared to singletons, it is important to assess the procedure related loss rate specifically in multiple pregnancies. The only data available is on twin pregnancies. Taking into considerations as many confounding factors as possible, the most recent studies describe an attributable loss rate between 0.3% and 2.2% [60–62]. A recent meta-analysis used pooled data from four case-control studies and estimated the loss rate post amniocentesis to be 2.59% compared to 1.53% in the control group [72]. This would provide a procedure related risk of 1%. The risk does not appear to be significantly different from the background risk when amniocentesis follows a multifetal reduction [63,64]. Chorionic villus sampling (CVS) Chorionic Villus Sampling in multiple pregnancies carry increased complexity compared to singletons. Issues specific to multiple pregnancies include challenges with accurate intrauterine mapping of the placentas, their relationship to each fetus and the need for sampling away from the margins of the placenta to avoid contamination with chorionic villi from another placenta [49]. It has also been noted that the amount of filling of the bladder can significantly change the apparent position of the uterus and the relative position of each fetus/placenta [65]. Because of these challenge, some experts have suggested that weekly ultrasound be performed to follow the position of each fetus between the time of the CVS and the time when the results are available to ensure correct assignment of results. Transabdominal and transcervical techniques have been described to perform CVS in multiple pregnancies either as the sole method or in combination [65–71]. In reviewing this literature, the largest proportion of follow-up amniocentesis required due to inconclusive results was in a population where all samples were obtained transcervically (15 out of 38), although this rate seemed to decrease with increasing experience [66]. In monochorionic pregnancies, a single sample will be collected from the single placenta. The loss rate following a CVS in multiple pregnancy has not been well established. Nine studies were included in a meta-analysis on post-procedural loss rate [72]. The overall loss rate in a total of 632 twin pregnancies undergoing CVS was 3.84%. No good studies with an appropriate control group to assess the procedure related risk is available, but based on previously reported spontaneous loss rate, this risk is estimated around 1%. Sampling errors (sampling the same placenta twice or collecting a sample from both placenta at the same time) are best identified when concordant sexes are expected from the CVS results but discordant sex twins are noted on ultrasound or at birth. Most recent studies report a rate of sampling error between 2% and 4% [37,50,69,70,73], whereas fetal-fetal contamination has been noted in up to 11.5% of samples [73]. Wapner and Jenkins have suggested that collecting the sample close to the cord insertion, avoiding the area close to the dividing membrane and using a combination of transabdominal and transcervical approach can minimize sampling error. Choosing between amniocentesis and CVS Only 2 studies are available to compare the loss rates between the two procedures in twin pregnancies. Overall loss rates were similar, both studies reporting a non-significant 0.3% difference in the loss rate between both procedures (CVS: 3.2% and 4.5%; Amniocentesis: 2.9% and 4.2%) [50,68]. It is important to note that these studies were not powered to assess such differences, report cohorts followed during a similar time period, were not randomized and the choice of procedure was based on patient’s and provider’s preferences. A recently published meta-analysis calculated an overall loss rate of 3.84% after CVS and 3.07% after amniocentesis, respectively [72]. They concluded that there was no current evidence that the rate of procedure related loss was different between amniocentesis and CVS and that this rate was estimated around 1%. Consequently, equivalence cannot be inferred or denied and other criteria such as those described above must be used to make this decision together with the pregnant woman.
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Summary Prenatal screening in multiple pregnancies carries some specific challenges, as many of the techniques used in singleton pregnancies are not as accurate in multiple pregnancies. Techniques using an ultrasound based approach, combined with serum markers, seem to be the most reliable in twin pregnancies whereas in higher order multiple pregnancies, ultrasound based screening performs better. Newer techniques using cell-free DNA still require to be studied in multiple pregnancies to assess their efficacy. Chorionic villous sampling and amniocentesis present specific challenges in multiple pregnancies including the risk of sampling the same sac or placenta twice, higher pregnancy loss rate and the challenge of assigning the right result to the right fetus. Specialists with expertise in those procedures should perform them to ensure the most reliable information is collected and provided to women undergoing these tests.
Practice points Chorionicity assessment is important to improve screening performance in multiple pregnancies. Screening methods including an ultrasound component perform better then serum based only methods in multiple pregnancies and should be the standard of care. Non Invasive Prenatal Testing is promising in multiple pregnancies but there is currently only limited information on the performance of this test. Amniocentesis technique must be adapted to a multiple pregnancy. The risk of sampling the same sac twice is around 1–2% and should be discussed in advance. Chorionic villus sampling carries additional challenges in polychorionic multiple pregnancies. Sampling technique must be modified accordingly. The risk of sampling the same placenta twice is around 2–4% and should be discussed in advance. Chorionic Villous Sampling and Amniocentesis seem to carry a similar rate of procedure related loss in multiple pregnancies.
Research agenda Screening performance of integrated ultrasound and maternal serum markers in multiple pregnancies. Accuracy of non-invasive prenatal testing (NIPT) using cell-free DNA in multiple pregnancies. Procedure related loss rate in prenatal invasive testing in multiple pregnancies.
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First-trimester screening in triplets. Am J Obstet Gynecol 2011 Oct;205(4):364.e1–5. *[25] 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. [26] Wald NJ, Rish S. Prenatal screening for Down syndrome and neural tube defects in twin pregnancies. Prenat Diagn 2005; 25:740–5. [27] Garchet-Beaudron A, Dreux S, Leporrier N, et al. Second-trimester Down syndrome maternal serum marker screening: a prospective study of 11 040 twin pregnancies. Prenat Diagn 2008;28:1105–9. [28] Muller F, Dreux S, Dupoizat H, et al. Second-trimester Down syndrome maternal serum screening in twin pregnancies: impact of chorionicity. Prenat Diagn 2003;23:331–5. [29] Spencer K, Salonen R, Muller F. Down’s syndrome screening in multiple pregnancies using alpha-fetoprotein and free beta hCG. Prenat Diagn 1994;14:537–42. [30] Maymon R, Dreazen E, Rozinsky S, et al. Comparison of nuchal translucency measurement and second-trimester triple serum screening in twin versus singleton pregnancies. Prenat Diagn 1999;19:727–31. [31] Benn P, Cuckle H, Pergament E. Non-invasive prenatal testing for aneuploidy: current status and future prospects. Ultrasound Obstet Gynecol 2013;42:15–33. [32] Canick JA, Kloza EM, Lambert-Messerlian GM, et al. DNA sequencing of maternal plasma to identify Down syndrome and other trisomies in multiple gestations. Prenat Diagn 2012;32:1–5. *[33] Lau TK, Jiang F, Chan MK, et al. Non-invasive prenatal screening of fetal Down syndrome by maternal plasma DNA sequencing in twin pregnancies. J Matern Fetal Neonatal Med 2013;26:434–7. [34] Gil MM, Quezada MS, Bregant B, et al. Cell-free DNA analysis for trisomy risk assessment in first-trimester twin pregnancies. Fetal Diagn Ther Nov 15, 2013:1–8. Online first. [35] Lynch L, Berkowitz GS, Chitkara U, et al. Ultrasound detection of Down syndrome: is it really possible? Obstet Gynecol 1989;73:267–70. [36] Yaron Y, Bryant-Greenwood PK, Dave N, et al. Multifetal pregnancy reductions of triplets to twins: comparison with nonreduced triplets and twins. Am J Obstet Gynecol 1999;180:1268–71. [37] Jenkins TM, Wapner RJ. The challenge of prenatal diagnosis in twin pregnancies. Curr Opin Obstet Gynecol 2000;12: 87–92. [38] Taylor MJ, Fisk NM. Prenatal diagnosis in multiple pregnancy. Baillieres Best Pract Res Clin Obstet Gynaecol 2000;14:663–75. [39] The Canadian Early and Mid-trimester Amniocentesis Trial (CEMAT) Group. Randomised trial to assess safety and fetal outcome of early and Midtrimester amniocentesis. Lancet 1998;351:242–7. *[40] Delisle MF, Brosseuk L, Wilson RD. Amniocentesis for twin pregnancies: is alpha-fetoprotein useful in confirming that the two sacs were sampled? Fetal Diagn Ther 2007;22:221–5. [41] Kidd SA, Lancaster PA, Anderson JC, et al. A cohort study of pregnancy outcome after amniocentesis in twin pregnancy. Paediatr Perinat Epidemiol 1997;11:200–13. [42] Wapner RJ. Genetic diagnosis in multiple pregnancies. Sem Perinatol 1995;5:361–2. [43] Kidd SA, Lancaster PAL, Anderson JC. Fetal death after exposure to methylene blue dye during mid-trimester amniocentesis in twin pregnancy. Prenat Diagn 1996;16:39–47. [44] van der Pol JG, Wolf H, Boes K, et al. Jejunal atresia related to the use of methylene blue in genetic amniocentesis in twins. Br J Obstet Gynaecol 1992;99:141–3. [45] McFadyen I. The dangers of intra-amniotic methylene blue. Br J Obstet Gynaecol 1992;99:89–90.
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[46] Cragan JD, Martin ML, Khoury MJ, et al. Dye use during amniocentesis and birth defects. Lancet 1993;341:1352. [47] Pruggmayer MR, Jahoda MG, van der Pol JG, et al. Genetic amniocentesis in twin pregnancies: results of a multicenter study of 529 cases. Ultrasound Obstet Gynecol 1992;2:6–10. [48] Brandenburg H. The use of synthetic dyes for identification of the amniotic sacs in multiple pregnancies. Prenat Diagn 1997;17:281–2. [49] Weisz B, Rodeck C. Invasive diagnostic procedures in twin pregnancies. Prenat Diagn 2005;25:751–8. [50] Antsaklis A, Souka AP, Daskalakis G, et al. Secondtrimester amniocentesis vs. chorionic villus sampling for prenatal diagnosis in multiple gestations. Ultrasound Obstet Gynecol 2002;20:476–81. [51] Jeanty P, Shah D, Roussis P. Single-needle insertion in twin amniocentesis. J Ultrasound Med 1990;9:511–7. [52] Buscaglia M, Ghisoni L, Bellotti M, et al. Genetic amniocentesis in biamniotic twin pregnancies by a single transabdominal insertion of the needle. Prenat Diagn 1995;15:17–9. [53] van Vugt JM, Nieuwint A, van Geijn HP. Single-needle insertion: an alternative technique for early second-trimester genetic twin amniocentesis. Fetal Diagn Ther 1995;10:178–81. [54] Cirigliano V, Cañadas P, Plaja A, et al. Rapid prenatal diagnosis of aneuploidies and zygosity in multiple pregnancies by amniocentesis with single insertion of the needle and quantitative fluorescent PCR. Prenat Diagn 2003;23:629–33. [55] Sebire NJ, Noble PL, Odibo A, et al. Single uterine entry for genetic amniocentesis in twin pregnancies. Ultrasound Obstet Gynecol 1996;7:26–31. [56] Megory E, Weiner E, Shalev E, et al. Pseudomonoamniotic twins with cord entanglement following genetic funipuncture. Obstet Gynecol 1991;78:915–7. [57] Bahado-Singh R, Schmitt R, Hobbins JC. New technique for genetic amniocentesis in twins. Obstet Gynecol 1992;79: 304–7. [58] Shalev SA, Shalev E, Pras E, et al. Evidence for blood chimerism in dizygotic spontaneous twin pregnancy discordant for Down syndrome. Prenat Diagn 2006;26:782–4. [59] Morin L, Lim K. Ultrasound for twin pregnancies. J Obstet Gynaecol Can 2011;33:643–56. *[60] Cahill AG, Macones GA, Stamilio DM, et al. Pregnancy loss rate after mid-trimester amniocentesis in twin pregnancies. Am J Obstet Gynecol 2009;200:257.e1–6. [61] Ghidini A, Lynch L, Hicks C, et al. The risk of second trimester amniocentesis in twin gestations: a case-control study. Am J Obstet Gynecol 1993;169:1013–6. [62] Yukobowich E, Anteby EY, Cohen SM, et al. Risk of fetal loss in twin pregnancies undergoing second trimester amniocentesis. Obstet Gynecol 2001;98:231–4. [63] McLean LK, Evans MI, Carpenter Jr RJ, et al. Genetic amniocentesis following multifetal pregnancy reduction does not increase the risk of pregnancy loss. Prenat Diagn 1998;18:186–8. [64] Stephen JA, Timor-Tritsch IE, Lerner JP, et al. Amniocentesis after multifetal pregnancy reduction: is it safe? Am J Obstet Gynecol 2000;182:962–5. [65] Rochon M, Stone J. Invasive procedures in multiple gestations. Curr Opin Obstet Gynecol 2003;15:167–75. [66] Casals G, Borrell A, Martinez JM, et al. Transcervical chorionic villus sampling in multiple pregnancies using a biopsy forceps. Prenat Diagn 2002;22:260–5. [67] Appelman Z, Furman B. Invasive genetic diagnosis in multiple pregnancies. Obstet Gynecol Clin North Am 2005;32:97–103. [68] Wapner RJ, Johnson A, Davis G, et al. Prenatal diagnosis in twin gestations: a comparison between second-trimester amniocentesis and first-trimester chorionic villus sampling. Obstet Gynecol 1993;82:49–56. [69] Brambati B, Terzian E, Tognoni G. Randomized clinical trial of transabdominal versus transcervical chorionic villus sampling methods. Prenat Diagn 1991;11:285–93. [70] Pergament E, Schulman JD, Copeland K, et al. The risk and efficacy of chorionic villus sampling in multiple gestations. Prenat Diagn 1992;12:377–84. *[71] De Catte L, Liebaers I, Foulon W. Outcome of twin gestations after first trimester chorionic villus sampling. Obstet Gynecol 2000;96:714–20. *[72] Agarwal K, Alfirevic Z. Pregnancy loss after chorionic villus sampling and genetic amniocentesis in twin pregnancies: a systematic review. Ultrasound Obstet Gynecol 2012;40:128–34. [73] Fiddler M, Frederickson MC, Chen PX, et al. Assessment of fetal status in multiple gestation pregnancies using interphase FISH. Prenat Diagn 2001;21:196–9.
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Best Practice & Research Clinical Obstetrics and Gynaecology journal homepage: www.elsevier.com/locate/bpobgyn
9
Prenatal screening and diagnosis of aneuploidy in multiple pregnancies Alain Gagnon, MD, Clinical Professor a, *, Francois Audibert, MD, MSc, Professeur Titulaire b a b
University of British Columbia, Vancouver, British Columbia, Canada Université de Montréal, Montréal, Québec, Canada
Keywords: prenatal screening Down syndrome aneuploidy multiple pregnancies amniocentesis chorionic villus sampling
Prenatal screening for aneuploidy has changed significantly over the last 30 years, from being age-based to maternal serum and ultrasound based techniques. Multiple pregnancies present particular challenges with regards to screening as serum-based screening techniques are influenced by all feti while ultrasoundbased techniques can be fetus specific. Tests currently available tend to not perform as well in multiple compared to singleton pregnancies. Considerations must be given to these variations when discussing and performing screening for aneuploidy in this situation. Prenatal invasive diagnosis techniques in multiple pregnancies bring their own challenges from a technical and counselling point of view, in particular with regards to sampling error, mapping and assignment of results and management of abnormal results. This review addresses these particular challenges and provides information to facilitate care. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction Multiple pregnancies present specific challenges with regards to prenatal screening and diagnosis. Although some of the usual components of screening such as previous history and family history remain an important feature of the screening process for aneuploidy, the various pregnancy specific screening methods present particularities when it comes to multiple pregnancies. Most of the
* Corresponding author. Division of Maternal-Fetal Medicine, BC Women’s Hospital, B241-4500 Oak Street, Vancouver, BC, Canada V6H 3N1. Tel.: þ1 6048753174. E-mail address: [email protected] (A. Gagnon). 1521-6934/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bpobgyn.2013.12.010
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information available on the topic covers twin pregnancies whereas the literature is much sparser on higher order multiples. The following paper reviews the literature available to facilitate counselling and health care resources planning on this topic. Background Zygosity and chorionicity Zygosity is determined by the number of fertilized ova that resulted in the multiple pregnancy. It is assumed that monozygotic multiples (originating from a single fertilized ovum) have the same genetic make-up and consequently, should be concordant for aneuploidy. For screening purposes, this assumption is applied, although rare exceptions to this rule exist [1], described in case reports of heterokaryotypic monozygotic twins. Feti in a dizygotic or polyzygotic multiple pregnancy will have as many different genetic make-ups as there are zygotes (or fertilized ova). Consequently, these feti may be concordant or more frequently discordant for aneuploidy. As zygosity cannot be assessed directly without invasive testing during pregnancy, some inferences based on ultrasound findings must be applied, through the assessment of the chorionicity. Chorionicity refers to the type of placentation and specifically to the number of chorions or functional placentas. It can be assessed by ultrasound, most successfully in the first trimester when the accuracy is between 96–100% [2–4]. Monochorionic multiple pregnancies are essentially always monozygotic. Most dichorionic or polychorionic multiple pregnancies are dizygotic or polyzygotic, with as many zygotes as there are chorions; less than 10% of them [2–4] will arise from a single zygote that divided within the first 3 days post-fertilization. As the majority of polychorionic multiple pregnancies are polyzygotic, it is assumed that they have different genetic make-ups and are most likely discordant for aneuploidy. These concepts are particularly important when assessing the risks through screening and counselling on invasive procedures. Prenatal screening in multiple pregnancies Risk assessment based on maternal age, periconceptional factors and chorionicity It is easy to conceive that the overall risk of aneuploidy is higher in multiple pregnancies compared to singletons. This is in part due to increased maternal age in multiple pregnancies (either spontaneous or conceived through fertility treatments) [5]. The maternal age to be used for counselling is the age of the egg at the time of retrieval for pregnancies arising from frozen embryo transfer or egg donation. A monochorionic multiple pregnancy spontaneously conceived or conceived without the use of intracytoplasmic sperm injection (ICSI) carries a similar overall risk of aneuploidy compared to a spontaneously conceived singleton pregnancy for the same maternal age. Both twins will either be affected or not, with only rare exceptions as described above. A polychorionic multiple pregnancy has an increased risk of aneuploidy as it is most likely polyzygotic and it is the number of zygotes that determines the level of risk for aneuploidy. Each fetus has, in theory, its own risk of aneuploidy, relatively independent of the risk for the other(s). Consequently, it has been suggested that the risks are additive. [6] For example, the risk of aneuploidy would be double that of a singleton in dichorionic multiple pregnancies, triple in trichorionic pregnancies and so on. This concept has been recently challenged [7] as the observed prevalence of Trisomy 21 in twin pregnancies is much less than the theoretical risk based on the above assumptions and as such, this simple rule must be applied with caution when assessing risk based on history alone. This is likely related to a variety of environmental and parental genetic factors that are not completely independent of each other and thus influenced all zygotes similarly rather than independently. In view of the challenges described above and the trends towards using comprehensive screening rather than maternal age alone, as recommended by multiple national bodies such as Canada and Singapore [8,9], maternal age alone should be discouraged as a screening method for multiple pregnancies, unless no other methods are available. In that case, chorionicity should be considered, and the
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counseling should include the best possible risk assessment based on the information available together with the risk of the invasive procedure. The use of ICSI has been associated with an increased risk of aneuploidy, particularly in the sex chromosomes pair [10]. In these cases, the rates of aneuploid counts for the X and Y chromosomes are estimated at 0.75%. This should be taken into consideration in the counselling regarding the risks, screening methods and invasive testing. Most screening tests are not well designed for assessment of sex chromosomes anomalies beyond the possible detection of 45,XO or Turner Syndrome when nuchal translucency is used as part of the screening methods (see below). First trimester ultrasound including nuchal translucency Over the last 20 years, the use of first trimester ultrasound including nuchal translucency (NT) has become a significant component of aneuploidy screening. It has been the main component in multiple pregnancy as it can provide a fetus specific risk of aneuploidy, when combined with maternal age [10]. The detection rates using NT alone with maternal age in twin pregnancies are similar to those observed in singletons [11–13] because the distribution of NT measurements is similar for feti from a singleton or multiple pregnancy. In monochorionic (assumed monozygotic) multiple pregnancies, the NT are measured for each fetus sharing a chorion and the average of all NTs is used to provide a single pregnancy risk of aneuploidy [17]. In polychorionic multiple pregnancies, each fetus not sharing a chorion is considered separately, and an individual risk is typically calculated for each one based on the established curves in singletons, using the NT measurement and the crown-rump length for each fetus as well as maternal age [13]. The risk is usually additive and the total risk for the pregnancy is the sum of all risks for each fetus. This calculation method leads to an increase in the false-positive rate for those polychorionic pregnancies who are monozygotic (as the risk should have been averaged for those, as described above), but as they represent less than 10% of polychorionic pregnancies and cannot be differentiated from the polyzygotic polychorionic multiples pregnancies, this does not affect significantly the performance of the test [14]. It is felt to be appropriate to calculate pregnancy specific risks rather than solely fetus specific risks as most women will elect to test all fetuses should they undergo invasive testing and most of the data available on the risk of invasive testing in multiple pregnancies accounts for testing of all fetuses. By combining NT measurements with maternal age in a twin pregnancy population and calculating a fetus specific risk for each of them, Sebire et al. described a detection rate of 88% with a false-positive rate of 7.3% [13]. A higher prevalence of increased NT and thus positive screening tests was noted in the monochorionic twins (8.4% vs 5.4%), which was later suspected to potentially be an early sign of twinto-twin transfusion syndrome [15,16]. This is why Vandercruys et al. later suggested using an average risk for monochorionic twins after studying 769 monochorionic twin pairs [17]. They described with this technique a 100% detection rate for a 4.2% false-positive rate in a monochorionic population, a significant improvement over the fetus specific risk calculation. Although such detection rates are not consistently reproduced [13,14,17–22] this is the current standard recommended by the Fetal Medicine Foundation as it does provide the best screening performance [23]. There is a paucity of studies looking at a more elaborate panel of ultrasound markers in the first trimester, including nasal bone assessment and ductus venosus. Krantz et al. reported on the mathematical modelling using nuchal translucency and nasal bone assessment in 3187 twin pregnancies and 219 triplet pregnancies [24]. They described a decrease in the positive rate by combining the two ultrasound markers from 9.0% to 4.7% in twins and 13.0% to 11.9% in triplets. In triplet pregnancies, using a simulation model assuming a 10% positive rate, the detection rate was estimated to be 93% using the two markers. First trimester ultrasound combined with serum markers In singleton pregnancies, the use of nuchal translucency is ideally combined with 2 or more serum markers in order to improve the detection rate and/or decrease the false positive rate. There are a few studies describing this technique in twin pregnancies only. It has not been studied in higher order multiples. Although nomograms for the various analytes (b-hCG, PAPP-A, AFP, Estriol, Inhibin-A) have
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been established for twin pregnancies, the key challenge is that compared to ultrasound based markers which are fetus specific, serum markers are always a mixture, influenced by all feti and placentas present. Consequently, in polyzygotic pregnancies, the effect of the abnormal fetus on the serum analytes values is diluted and brought closer to the normal range by the normal fetus or feti. This effect is particularly noted in higher order multiple pregnancies. Consequently, it is usually recommended to use serum analytes only in singleton and twin pregnancies. With regards to first trimester screening, free b-hCG and PAPP-A have been noted to be approximately twice as high as in twin pregnancies compared to singleton pregnancies. More specifically, bhCG is around 2.023 times that of singleton pregnancies whereas PAPP-A is 2.192 and 1.788 times higher than singletons in dichorionic and monochorionic twin pregnancies, respectively [25]. As there is not enough data to know the distribution of these values in pregnancies affected by trisomy 21, a pseudo-risk is usually calculated. Wald et al described this technique where the value of each analytes is divided by the correction factor suggested above and then used to calculate the risk for each fetus as if it was a singleton. With this technique, they reported an improved detection rate in monochorionic twins from 73% to 84% for a 5% false positive rate, using NT and 2 first trimester analytes. The same improvements were not noted in dichorionic twin pregnancies where the detection changed from 68% to 70% using the same technique [14]. Table 1 provides a summary of the studies available on first trimester combined screening (NT, PAPP-A, b-hCG and maternal age). The only study reporting on triplet pregnancy using NT and biochemistry did not notice any improvements in performance by adding the biochemistry compared to NT alone [24]. Wald et Rish published performance estimations of an integrated screening test combining NT ultrasound with PAPP-A in the first trimester and AFP, b-hCG, estriol and inhibin-A in the second trimester, providing a single risk at the end of the process [26]. Using many assumptions to perform their calculations, they estimated the detection rate to be 93% in monochorionic twins and 78% in dichorionic twins, for an overall detection rate of 80%, at a 5% positive rate. Maternal serum screening alone Even though more and more women have access to quality first trimester ultrasound, in many parts of the world, maternal serum markers remain the mainstay of prenatal screening for aneuploidy. Not all jurisdictions offer a first trimester ultrasound routinely and consequently, many multiple pregnancies are still only identified in the second trimester, after the window of opportunity for nuchal translucency assessment. In view of this reality, serum based screening in multiples has been studied. As described above, the challenges with the interpretation of serum analytes in higher order multiples are such that serum based screening is only validated in twin pregnancies. Even then, the paucity of data in affected pregnancies is such that only pseudo risks can be calculated [26]. It is also important to note that the interpretation of serum markers must provide a risk for the whole pregnancy when, in the majority of cases of affected pregnancies, only one fetus will be affected. Finally, it is important to be aware of the chorionicity of the pregnancy, for the same reasons as described above. This is unfortunately less reliable after 14 weeks.
Table 1 Summary of studies on combined first trimester screening in twins using Nuchal translucency, PAPP-A, free b-hCG and maternal age. Reference
Term cut-off Number of twin Number of affected Detection rate for False positive risk pregnancies feti Trisomy 21 (%) rate (%)
Spencer, 2000 [18] NA Sebire et al., 2000 [16] NA Spencer and Nicolaides, 2003 [20] 1:300 Vandercruys et al., 2005 [17] 1:300 Wald and Rish, 2005 [26] Theoretical Chasen et al., 2007 [22] 1:198 1:300
159 Theoretical 206 769 Theoretical 535
NA Theoretical 4 6 Theoretical 7
80.1 73 (MZ) 43 (DZ) 75 72 72 100 100
5.0 5.0 NA 5.0 5.0 5.0 7.0
Legend: NA: Not available; MZ: Monozygotic; DZ: Dizygotic; Theoretical: data based on modeling, not actual patients.
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Spencer et al. and Muller et al. studied the double test (AFP and free b-hCG combined with maternal age) and reported a 51–63% detection rate for a 5–10.8% positive rate, with the best performance being described when serum values were corrected for chorionicity [27–29]. Of note, these results represent an improvement over the use of maternal age alone (27.3% detection rate for a 6.6% positive rate in one studied population) [28]. The use of the triple test (AFP, free b-hCG and estriol combined with maternal age) was studied in a population of 60 twin pregnancies by Maymon et al. [30] To detect the one case of trisomy 21 in their population required a 15% positive rate. Cuckle studied both the triple test and the quadruple test (AFP, free b-hCG, inhibin-A and estriol combined with maternal age) and reported only marginal improvement in the detection rate from 41% with dual test to 44% with the triple test and 47% for the quadruple when a 5% positive rate was used [7]. In summary, although serum screening alone performs better than maternal age alone, it is outperformed by screening methods incorporating ultrasound findings such as NT. Non-invasive prenatal testing (NIPT) The last few years have seen the emergence of NIPT as a new tool to assess pregnancies for aneuploidy. Very little information is available on the use of this tool in multiple pregnancies. Only 3 studies have reported on this topic, all using cell-free DNA technology [31]. The first one reported on a population at high risk of chromosomal anomaly based on screening and included 3 pairs of twins with discordant karyotypes (2 with trisomy 21 and 1 with trisomy 13), 5 pairs concordant for trisomy 21, 17 euploid twin pregnancies and 2 euploid triplet pregnancies [32]. All were correctly classified. The second study included both low risk and high risk pregnancies and included one pregnancy with twins discordant for trisomy 21 and 11 euploid twin pregnancies [33]. Here again, all were correctly classified. Finally, Gil et al. recently reported on 207 samples previously collected and 68 samples collected prospectively in twin pregnancies [34]. A result could be provided in 92.7% of samples. 13 out of 14 pregnancies (92.9%) with at least one trisomic fetus were correctly identified with no reported false positive results. More studies are required to better define the performance, positive and negative predictive values of such test in a multiple pregnancy population. Second trimester ultrasound screening Despite ultrasound in the second trimester conferring the theoretical advantage of being fetus specific and thus having the potential to be a better screening tool for the reasons described above, limited data is available on the efficacy of second trimester ultrasound in multiple pregnancies to detect aneuploidy. It is well known that obtaining a detailed assessment is more challenging in multiple pregnancies due to the limited field of vision associated with the presence of one or more feti around the fetus being imaged. One study reported a detection of 5 out of 9 feti with trisomy 21 using nuchal thickness assessment [35]. The other markers studied in that publication were less efficacious. If these markers were to be considered as a significant part of a screening protocol, further studies would be required to better assess the performance of ultrasound in this context. Invasive prenatal testing in multiple pregnancies When to offer prenatal testing? When it comes to invasive procedures, non-directive counselling is recommended, allowing patients to make the best decision possible for themselves. Cut-offs used to call a screen test positive should take into account the cut-off used in the singleton population but also the risk associated with procedures in multiple pregnancies, as described below. It is also important to keep in mind the background risk of spontaneous pregnancy loss in a multiple pregnancy, estimated at 6–7% in twins and up to 10% in higher order multiples, between 11 weeks and 24 weeks [36]. Considerations prior to proceeding with testing Some key elements must be considered and discussed prior to proceeding with invasive testing. A thorough ultrasound examination is required, in particular if chorionicity has not been already
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determined, as it has implications for the interpretation of the screening test and the potential results of the procedure as well as the management should a result be abnormal. Mapping of the location of each fetus to be tested is also paramount. All possible elements identifying each fetus (location of the sacs, placental location, cord insertion, fetal genitalia, discordant anomalies) should be noted clearly using text and/or diagrams [37]. This will facilitate assignment of the correct result to each fetus. This becomes of particular importance when results are discordant and pregnancy management is altered for that fetus (such as selective reduction) [38] or when the sex chromosomes do not match the genitalia phenotype (in particular when concordant karyotypes are associated with discordant phenotypes, thus raising the suspicion of improper sampling). Such possibilities should also be discussed prior to proceeding with invasive testing. When chorionicity is unclear by ultrasound, consideration should be given to assessing zygosity by molecular testing in complex multiple pregnancies (such as those with discordant anomalies), as polyzygosity would be associated with polychorionicity and the pregnancy could be managed as such. Amniocentesis As in singleton pregnancies, amniocentesis is typically performed after 15 weeks in multiple pregnancies, in view of the increased risk associated with earlier gestational ages noted in singletons [39]. Three techniques have been described to address the particular challenges posed with this procedure in multiple pregnancies. They have not been compared in a randomised fashion and consequently, one cannot be recommended over the other, but they can be described with their pros and cons. The most widely used technique is the multiple punctures technique where multiple different needles are inserted in different locations but in each separate sac, using ultrasound guidance to identify the various locations. In twins, the risk of sampling the same sac twice using this technique has been estimated to be around 1.8% in a Canadian study of 260 twin pregnancies [40]. Dye was not instilled in the first punctured sac in this study. Correlation between karyotype results and fetal genitalia appearance may help identify pregnancies where the same sac was punctured twice. Because of this issue, a modification was suggested, using a coloured dye to mark the sac(s) that have been tapped and expecting a dye free sample when sampling the next sac. The first dye used was methylene blue. As it has since been associated with small bowel atresia and fetal death, its use is now considered contraindicated [41–45]. Indigo carmine has since been used and, although no risks of congenital anomalies above the background risk have been reported [46,47], the report of at least 4 cases of jejunal atresia in exposed newborns [44,47,48] raises some concerns. In this context, most experts suggest using dye to identify the various gestational sacs only when ultrasound visualization is poor or in the case of higher order multiples [38,49,50]. Another modification used to ensure sampling on both sides of a membrane is a single puncture technique where the first sac is entered with a needle in close proximity to the dividing membrane. Once the sample is collected, the needle is advanced through the membrane, the first 1–2 ml of amniotic fluid collected is discarded and then a sample is collected on the other side of the membrane, thus ensuring both sacs are sampled [51–55]. Potential challenges described include tenting of the intertwin membrane, contamination of the second sample with amniotic fluid from the first sac [49] and the creation of iatrogenic monoamnionicity by disrupting the integrity of the intertwin membrane [56]. These complications have not been described in 2 studies reporting on a total of 77 amniocentesis performed in this fashion in twins, yet to this day, this technique has not gained widespread acceptance [49]. The third modification used in order to decrease the risk of sampling the same sac twice is the simultaneous visualisation technique where both needles are inserted at the same time and visualised on each side of the intertwin membrane [57]. Despite the theoretical advantage from a sampling confirmation point of view, the challenges in obtaining such visualization and the time required are such that this technique is seldom used [49]. Many experts have rightly suggested that in monochorionic multiple pregnancies (which are typically monozygotic), a single sample could be taken from only one sac. Since multiple case reports exist describing discordant karyotypes in monochorionic multiple pregnancies [1,58] and as
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chorionicity is less reliable if identified after 14 weeks [2–4,12], many will still advocate sampling of both sacs, unless the feti are concordant for growth, anatomy and nuchal translucency and chorionicity was determined before 14 weeks [49,59]. As multiple pregnancies carry a higher spontaneous loss rate compared to singletons, it is important to assess the procedure related loss rate specifically in multiple pregnancies. The only data available is on twin pregnancies. Taking into considerations as many confounding factors as possible, the most recent studies describe an attributable loss rate between 0.3% and 2.2% [60–62]. A recent meta-analysis used pooled data from four case-control studies and estimated the loss rate post amniocentesis to be 2.59% compared to 1.53% in the control group [72]. This would provide a procedure related risk of 1%. The risk does not appear to be significantly different from the background risk when amniocentesis follows a multifetal reduction [63,64]. Chorionic villus sampling (CVS) Chorionic Villus Sampling in multiple pregnancies carry increased complexity compared to singletons. Issues specific to multiple pregnancies include challenges with accurate intrauterine mapping of the placentas, their relationship to each fetus and the need for sampling away from the margins of the placenta to avoid contamination with chorionic villi from another placenta [49]. It has also been noted that the amount of filling of the bladder can significantly change the apparent position of the uterus and the relative position of each fetus/placenta [65]. Because of these challenge, some experts have suggested that weekly ultrasound be performed to follow the position of each fetus between the time of the CVS and the time when the results are available to ensure correct assignment of results. Transabdominal and transcervical techniques have been described to perform CVS in multiple pregnancies either as the sole method or in combination [65–71]. In reviewing this literature, the largest proportion of follow-up amniocentesis required due to inconclusive results was in a population where all samples were obtained transcervically (15 out of 38), although this rate seemed to decrease with increasing experience [66]. In monochorionic pregnancies, a single sample will be collected from the single placenta. The loss rate following a CVS in multiple pregnancy has not been well established. Nine studies were included in a meta-analysis on post-procedural loss rate [72]. The overall loss rate in a total of 632 twin pregnancies undergoing CVS was 3.84%. No good studies with an appropriate control group to assess the procedure related risk is available, but based on previously reported spontaneous loss rate, this risk is estimated around 1%. Sampling errors (sampling the same placenta twice or collecting a sample from both placenta at the same time) are best identified when concordant sexes are expected from the CVS results but discordant sex twins are noted on ultrasound or at birth. Most recent studies report a rate of sampling error between 2% and 4% [37,50,69,70,73], whereas fetal-fetal contamination has been noted in up to 11.5% of samples [73]. Wapner and Jenkins have suggested that collecting the sample close to the cord insertion, avoiding the area close to the dividing membrane and using a combination of transabdominal and transcervical approach can minimize sampling error. Choosing between amniocentesis and CVS Only 2 studies are available to compare the loss rates between the two procedures in twin pregnancies. Overall loss rates were similar, both studies reporting a non-significant 0.3% difference in the loss rate between both procedures (CVS: 3.2% and 4.5%; Amniocentesis: 2.9% and 4.2%) [50,68]. It is important to note that these studies were not powered to assess such differences, report cohorts followed during a similar time period, were not randomized and the choice of procedure was based on patient’s and provider’s preferences. A recently published meta-analysis calculated an overall loss rate of 3.84% after CVS and 3.07% after amniocentesis, respectively [72]. They concluded that there was no current evidence that the rate of procedure related loss was different between amniocentesis and CVS and that this rate was estimated around 1%. Consequently, equivalence cannot be inferred or denied and other criteria such as those described above must be used to make this decision together with the pregnant woman.
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Summary Prenatal screening in multiple pregnancies carries some specific challenges, as many of the techniques used in singleton pregnancies are not as accurate in multiple pregnancies. Techniques using an ultrasound based approach, combined with serum markers, seem to be the most reliable in twin pregnancies whereas in higher order multiple pregnancies, ultrasound based screening performs better. Newer techniques using cell-free DNA still require to be studied in multiple pregnancies to assess their efficacy. Chorionic villous sampling and amniocentesis present specific challenges in multiple pregnancies including the risk of sampling the same sac or placenta twice, higher pregnancy loss rate and the challenge of assigning the right result to the right fetus. Specialists with expertise in those procedures should perform them to ensure the most reliable information is collected and provided to women undergoing these tests.
Practice points Chorionicity assessment is important to improve screening performance in multiple pregnancies. Screening methods including an ultrasound component perform better then serum based only methods in multiple pregnancies and should be the standard of care. Non Invasive Prenatal Testing is promising in multiple pregnancies but there is currently only limited information on the performance of this test. Amniocentesis technique must be adapted to a multiple pregnancy. The risk of sampling the same sac twice is around 1–2% and should be discussed in advance. Chorionic villus sampling carries additional challenges in polychorionic multiple pregnancies. Sampling technique must be modified accordingly. The risk of sampling the same placenta twice is around 2–4% and should be discussed in advance. Chorionic Villous Sampling and Amniocentesis seem to carry a similar rate of procedure related loss in multiple pregnancies.
Research agenda Screening performance of integrated ultrasound and maternal serum markers in multiple pregnancies. Accuracy of non-invasive prenatal testing (NIPT) using cell-free DNA in multiple pregnancies. Procedure related loss rate in prenatal invasive testing in multiple pregnancies.
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